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The role of endoplasmic reticulum chaperone protein GRP78 in breast cancer
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The role of endoplasmic reticulum chaperone protein GRP78 in breast cancer
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Content
THE ROLE OF ENDOPLASMIC RETICULUM CHAPERONE PROTEIN GRP78 IN
BREAST CANCER
by
Chun-Chih Tseng
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
December 2019
Copyright 2019 Chun-Chih Tseng
ii
Dedication
To my parents, Chao-Ching Tseng and Chun-Yu Chen
and to my wife, Nai-Ling Chang and daughters, Eunice Tseng and Ellie Tseng, for their
support and love along the way.
iii
Acknowledgments
First and foremost, I would like to thank my advisor, Dr. Amy S. Lee for her guidance and
support during my graduate study at USC. Amy has demonstrated great dedication and
commitment toward research. I especially thank her for enlightening me to think experimental
results in different ways and providing an effective environment for collaborations. I also want to
thank the Lee lab alumnus, Yi Zhang, for his guidance in biochemical experiments and the current
members, Pu Zhang for her support in research, and Dat Ha and Daisy Flores Rangel for their
contribution of reagents and discussion in science.
I am thankful to my committee members, Drs. Cheng-Ming Chuong, Fabien Pinaud and Min
Yu for their helpful discussions and collaborations throughout the years. I want to thank Dr.
Chuong for his support and insightful ideas to make science a fun park since I joined his lab as a
master student. The research trainings and experience that I obtained in Chuong lab pushed my
eyes to the next level and guided me into deep beauty of nature, and these had established a strong
foundation for my doctoral research and future scientific career. I also thank Drs. Mei Kong, Kevin
Kelly, Adrienne W. Paton, James C. Paton, Parkash S. Gill for providing reagents and materials,
and Ramunas Stanciauskas, Dennis Woo, Mari B. Ishak Gabra and Dr. Kaijin Wu for assistance
and helpful discussions in experiments. I am grateful for the technical support from USC core
facilities: Michelle MacVeigh-Aloni from Cell and Tissue Imaging Core at USC Liver Center,
Ernesto Barron and Anthony Rodriguez from Cell and Tissue Imaging Core at USC Norris
Comprehensive Cancer Center, and Seth Ruffins from Optical Imaging Facility at USC Stem Cell
Center.
iv
Last but not least, I would like to thank my wife, daughters, parents, parents-in-law (Chao-Pin
Chang and Yueh-Pei Wu), sisters (Tzu-Ting Tseng and Chi-Wen Tseng), brother-in-law (Shu-I
Chang), and my beloved grandma (Chun-Mei Lin Lu, passed away during my doctoral research)
for their greatest support, encouragement and joyful moments to keep me on the path on scientific
journey.
v
Table of Contents
Dedication
Acknowledgments
List of Tables
List of Figures
Abstract
Chapter 1: Overview and Introduction
1.1 Endoplasmic reticulum stress and unfolded protein response in breast cancer
1.2 Mechanisms of GRP78 cell surface translocation
Chapter 2: GRP78 binds CD44v and regulates its membrane homeostasis and
signaling in aggressive breast cancer
2.1 Introduction
2.2 Materials and methods
2.3 Results
2.3.1 GRP78 knockdown suppresses cancer cell migration and F-actin
integrity
2.3.2 Identification of CD44v as a novel partner protein of csGRP78
2.3.3 CD44 can directly bind to GRP78 in vitro
2.3.4 CD44 containing exon v3 is a major mediator of CD44 functions in
aggressive breast cancer
2.3.5 Dual-color single particle tracking reveals the interaction and co-
confinement of GRP78 and CD44v in plasma membrane nanodomains
2.3.6 Antibody against GRP78 alters F-actin dynamics and cell polarity in
breast cancer cells
2.3.7 Targeting csGRP78 by antibody suppresses polarized distribution of
IQGAP1 and active CDC42
2.3.8 Antibody and bacterial toxin targeting csGRP78 reduce CD44 protein
level and suppress CD44 downstream signaling
2.4 Discussion
ii
iii
vii
viii
x
1
1
5
9
9
11
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33
40
44
48
51
56
60
70
vi
Chapter 3: The C-terminal proline-rich region of GRP78 is a key regulator of
its cell surface expression and viability of tamoxifen-resistant breast cancer
cells
3.1 Introduction
3.2 Materials and methods
3.3 Results
3.3.1 GRP78 co-localizes with CD44v in MCF7-LR breast cancer cells
3.3.2 The C-terminal PRR of GRP78 is essential for forming complex with
CD44v in vitro
3.3.3 The C-terminal PRR of GRP78 is critical for its cell surface expression
and can modulate STAT3 signaling
3.3.4 The C-terminal polyproline sequence of GRP78 is evolutionally
conserved in higher eukaryotic organisms
3.3.5 Introduction of short peptide encoding the PRR of GRP78 reduced cell
viability and promoted apoptosis in tamoxifen-resistant breast cancer
3.4 Discussion
Chapter 4: Conclusions and Perspectives
Bibliography
73
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75
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84
86
89
91
94
97
101
110
List of Tables
Table 2.1: LC-MS/MS identifies cell migration- and metastasis-related proteins
forming complex with cell surface GRP78
59
vii
viii
List of Figures
Figure 2.1: GRP78 knockdown suppresses cancer cell migration and F-actin
integrity
Figure 2.2: Identification and verification of CD44v expression in breast cancer
cells
Figure 2.3: Cell surface GRP78 co-localizes with CD44v in breast cancer cells
Figure 2.4: More examples that show csGRP78 co-localizes ex vivo with CD44v in
circulating tumor cells
Figure 2.5: The anti-GRP78 antibody (clone A-10) reproduces immunofluorescent
staining pattern of the other anti-GRP78 antibody (MAb159)
Figure 2.6: CD44v can directly bind to GRP78 in vitro
Figure 2.7: The lower molecular weight CD44s can directly bind to GRP78 in vitro
Figure 2.8: Co-localization and distribution of CD44 isoforms in breast cancer cells
Figure 2.9: Stable knockdown of CD44 disrupted F-actin integrity, cell spreading
and cell attachment in MDA-MB-231 cells
Figure 2.10: Dual-color single particle tracking reveals the interaction and co-
confinement of GRP78 and CD44v in plasma membrane nanodomains
Figure 2.11: Antibody against GRP78 suppresses MCF7-LR breast cancer cell
adhesion, spreading and matrix degradation
Figure 2.12: Antibody against GRP78 suppresses MDA-MB-231 breast cancer cell
motility, migration and alters cell polarity
Figure 2.13: Targeting csGRP78 by antibody suppresses polarized distribution of
IQGAP1 and active CDC42
Figure 2.14: Targeting csGRP78 by antibody results in reduction of CD44 protein
level
Figure 2.15: Targeting csGRP78 by antibody leads to modest increase of CD44v
endocytosis and up-regulation of CD44v RNA expression
Figure 2.16: Targeting csGRP78 by antibody suppresses CD44 downstream
pSTAT3 signaling
32
34
36
38
39
41
43
45
47
50
53
55
57
61
63
64
ix
Figure 2.17: The 76-E6 antibody targets HSP70 family proteins
Figure 2.18. Antibody specifically targeting csGRP78 reduces CD44v level and cell
attachment
Figure 2.19. The bacterial cytotoxin specifically targeting csGRP78 suppresses cell
spreading and induces cell aggregation
Figure 3.1: GRP78 co-localizes with CD44v in MCF7-LR breast cancer cells
Figure 3.2: The C-terminal proline-rich region of GRP78 is essential for forming
complex with CD44v in vitro
Figure 3.3: The C-terminal proline-rich region of GRP78 is critical for its cell
surface expression and can modulate STAT3 signaling in MCF7-LR breast cancer
cells
Figure 3.4: Comparisons of the proline-rich regions of GRP78
Figure 3.5: Introduction of short peptide encoding the proline-rich region of GRP78
reduced cell viability and promoted apoptosis in MCF7-LR breast cancer cells
66
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90
93
96
x
Abstract
The 78 kD glucose-regulated protein (GRP78) is a major chaperone protein in the
endoplasmic reticulum (ER). It conducts protein folding and quality control and regulates
unfolded protein response (UPR) under ER stress. The expression and cell surface
translocation of GRP78 was elevated in aggressive tumors. Cell surface GRP78 (csGRP78)
majorly exists as a peripheral protein and exerts novel signaling functions beyond its traditional
role as a chaperone protein in the ER. However, the underlying mechanisms enabling GRP78
to be expressed on the cell surface and conduct novel signaling functions at the plasma
membrane are just emerging.
In this dissertation, we discovered that the type-I transmembrane protein CD44 is a novel
partner protein of GRP78 on the cell surface and in the cytosol of breast cancer cells. Studies
have shown that CD44 is an important regulator of breast cancer progression. Using
conventional confocal microscopy and super-resolution dual-color single particle tracking, we
found that GRP78 interacts with CD44 variant isoform (CD44v) in plasma membrane
nanodomains of breast cancer cells. We further showed that this interaction regulates CD44v
membrane homeostasis and multifaceted cell signaling impacting cell adhesion, spreading,
migration and polarity. Thus, this work provides new mechanistic insights on how the
GRP78/CD44v axis regulates cell migration in aggressive breast cancer.
Furthermore, we mapped the region of GRP78 that is important for forming complex with
CD44v by the in vitro GST pull-down assay and discovered that the C-terminal proline-rich
region (PRR) containing the PPP polyproline sequence is essential for the complex formation.
Mutation of the PPP polyproline sequence of GRP78 suppresses its cell surface expression in
xi
the tamoxifen-resistant breast cancer cells and inhibits STAT3 signaling, suggesting novel
functions of PPP polyproline sequence in regulation of csGRP78 expression and cell signaling.
We further designed an expression plasmid encoding a secretory signal peptide and 20 amino
acids spanning the PPP polyproline sequence of GRP78, and we showed that overexpression
of this plasmid reduced cell viability and led to an increase in apoptotic markers in tamoxifen-
resistant breast cancer cells. Our study uncovered a previously unidentified interaction between
two major players in cell and cancer biology, GRP78 and CD44, and discovered novel
functions of the C-terminal proline-rich region of GRP78 in breast cancer cells.
1
Chapter 1
Overview and Introduction
1.1 Endoplasmic reticulum stress and unfolded protein response in breast cancer
The endoplasmic reticulum (ER) is a major organelle for folding and post-translationally
processing of secreted and membrane-bound proteins, and it is also the largest intracellular store
of calcium ions (English, Zurek, and Voeltz 2009, Lee 2014). The homeostasis of ER is sensitive
to the reduction–oxidation reaction, the rate of protein synthesis as well as the alterations of
calcium ion, glucose and oxygen levels (Luo and Lee 2013, Ma and Hendershot 2004, Schroder
and Kaufman 2005, Ellgaard and Helenius 2003, Chakrabarti, Chen, and Varner 2011, Shen,
Zhang, and Kaufman 2004). Disruption of ER homeostasis leads to a condition of accumulation
of misfolded proteins in the ER lumen which is known as ER stress. ER stress transiently turns on
the unfolded protein response (UPR) to alleviate the ER stress by activating signaling pathways
leading to attenuation of protein synthesis, increase of protein folding capacity, and degradation
of misfolded proteins through ER-associated degradation (ERAD) and autophagy (Ma and
Hendershot 2004, Luo and Lee 2013, Schroder and Kaufman 2005, Ellgaard and Helenius 2003,
Chakrabarti, Chen, and Varner 2011, Shen, Zhang, and Kaufman 2004). Prolonged and severe
activation of UPR will result in cell death (Hetz and Papa 2018). Solid tumors often experience
short of oxygen and nutrient supply and these can lead to non-lethal and chronic activation of UPR
in tumor cells as well as cells in the tumor microenvironment (Urra et al. 2016). The chronic UPR
allows tumor cells to adapt stressful conditions and develop therapeutic resistance (Chevet, Hetz,
and Samali 2015, Wang and Kaufman 2014).
UPR is a result of activation of three ER transmembrane proteins, double-stranded RNA-
activated protein kinase-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1
2
(IRE1) and activating transcription factor 6 (ATF6) (Schroder and Kaufman 2005). Under non-
stressed conditions, 78 kD glucose-regulated protein (GRP78) binds to and inhibits activation of
these ER stress signaling transducers. Under ER stress, GRP78 binds to misfolded proteins and
detaches from these sensors resulting in their activation. Activated PERK dimerizes and inhibits
eukaryotic initiation factor 2α (EIF2α) resulting in suppression of general protein translation.
Activating transcription factor 4 (ATF4) can escape the translational block and facilitate the
transcription of Grp78 (Luo et al. 2003) and signaling pathways controlling apoptosis, autophagy,
and metabolism of redox, cholesterol and amino acids (Fusakio et al. 2016, Schroder and Kaufman
2005, Shen, Zhang, and Kaufman 2004, Chakrabarti, Chen, and Varner 2011). Active IRE1 splices
the mRNA of transcription factor X-box-binding protein 1 (XBP-1). Spliced XBP-1 (XBP-1s)
promotes transcription of genes associated with chaperone function and ERAD pathways (Yoshida
et al. 2001). ATF6 released from ER is then activated by proteolytic cleavage at Golgi complex.
Active ATF6 promotes transcription of XBP-1, Grp78 and pro-survival genes (Yoshida et al. 2001,
Li et al. 2000, Wang et al. 2000).
Breast cancer consists of heterogeneous populations of cancer cells, each population shows
distinct molecular signature, histology, physiology, prognosis and therapeutic strategy (Network
2012, Perou et al. 2000, Sotiriou and Pusztai 2009, Koren and Bentires-Alj 2015). Subtypes of
breast cancer are classified according to the histological expression of estrogen receptor (ESR1,
also referred to as ERα), progesterone receptor (PR) and human epidermal growth factor receptor
2 (HER2, also referred to as ERBB2), and the “intrinsic” molecular expression pattern of 50 genes
(PAM50) (Parker et al. 2009). Luminal A and luminal B subtypes express gene signatures of
luminal epithelial cells and are usually ESR1+ and PR+. They usually respond to ESR1 modulators
such as tamoxifen. Breast tumors overexpressing HER2 can be targeted by anti-HER2 antibodies.
3
Triple negative breast cancers (TNBC) do not exhibit HER2 overexpression and do not express
ESR1 and PR. Targeted therapy is lacking for TNBC patients and prognosis is relatively poor
compared to other subtypes (Perou et al. 2000, Weigelt and Reis-Filho 2009, Foulkes, Smith, and
Reis-Filho 2010).
UPR is usually lacking or transiently activated in normal cells but chronically active in tumor
cells. Thus, UPR is a good target to develop new therapeutic strategy for breast cancer, especially
for TNBC, which lacks targeted therapy. It was reported that PERK promotes breast cancer cell
proliferation by limiting oxidative DNA damage and loss of PERK arm blocks tumor growth
(Bobrovnikova-Marjon et al. 2010). PERK and its downstream ATF4 mediated LAMP-3,
lysosomal associated membrane protein 3, signaling pathway support breast cancer cell migration,
invasion and radioresistance (Nagelkerke, Bussink, Mujcic, et al. 2013, Nagelkerke, Bussink, van
der Kogel, et al. 2013). The Expression of active PERK as indicated by the level of phosphorylated
PERK is elevated in TNBC cell lines compared with the luminal cell lines (Chen et al. 2014) and
in human breast ductal carcinoma in situ compared with normal breast tissues (Avivar-Valderas et
al. 2011). Breast cancer cells stimulated with 17β-estradiol (E2), the major estrogen during human
reproductive years, exhibited specific upregulation of XBP-1, a downstream effector of IRE1 (Lin
et al. 2007, Wang et al. 2004). The co-expression of ESR1 and XBP-1 was observed in luminal
breast cancers (Lacroix and Leclercq 2004). Interestingly, it was found that both spliced and
unspliced XBP-1 could induce homodimerization of ESR1 in an estrogen-independent manner
(Lacroix and Leclercq 2004), leading to transcription of ESR1 target genes including XBP-1 (Ding
et al. 2003, Fang et al. 2004, Hu et al. 2015). It was also reported that XBP-1 promotes anti-
estrogen resistance in breast cancer in a NF- κB dependent manner (Hu et al. 2015). Notably,
activation of NF-κB signaling represents early malignant transformation of mammary cells (Kim
4
et al. 2000), and it also controls breast cancer cell proliferation and invasion by regulating the
expression of CD44, a multi-faceted regulator of breast cancer progression (Smith and Cai 2012,
Smith, Lyu, and Cai 2014). The importance of XBP-1 in TNBC breast cancers has been shown in
many in vivo and in vitro studies. It was reported that the spliced XBP-1 was displayed throughout
primary mammary tumorigenesis in an in vivo mouse model (Spiotto et al. 2010). Overexpression
of XBP-1s in the CD44
low
/CD24
high
non-stem cell-like cells derived from the TNBC patients
promoted the tumor formation in a mouse xenograft model (Chen et al. 2014). Mechanistically,
XBP-1 was able to directly interact with key transcriptional regulators in breast cancer including
hypoxia inducible factor 1α (HIF1α) (Chen et al. 2014) and proto-oncogene MYC (Zhao et al.
2018). Ablation of RNase activity of IRE1 by RNAi or small molecule inhibitors, which leads to
decreased levels of XBP-1s, reduced cytokine production and suppressed a stem cell-like
phenotype of TNBC cells (Logue et al. 2018). Little is known about the roles of ATF6 in breast
cancer. However, it was reported that knockdown of ATF6 reduced in vivo tumor growth and
angiogenesis in a mouse model of breast cancer (Ruan et al. 2013) and suppressed 17β-estradiol
(E2)-induced cell proliferation (Andruska et al. 2015).
GRP78, as a master regulator of unfolded protein response, can also be regulated by all three
arms of UPR signaling, and its mRNA and protein expression levels are often elevated upon
activation of UPR (Li et al. 2000, Luo et al. 2003, Wang et al. 2000, Yoshida et al. 2001). GRP78
is upregulated in human breast carcinoma cell lines compared to normal breast epithelial cells
(Gazit, Lu, and Lee 1999). Knockdown of GRP78 inhibits lung metastasis of triple-negative MDA-
MB-231 breast cancer cells and blocks in vitro migration and invasion of MCF7 breast cancer cells,
while overexpression of GRP78 increases in vitro migration and invasion of MCF7 cells (Chang
et al. 2016). The heterozygosity of Grp78 suppresses transgene-induced mammary tumor growth
5
and promotes apoptosis of tumor cells (Dong et al. 2008). The tumor associated angiogenesis is
blocked by Grp78 heterozygosity in endothelial cells, and the normal organ development and
vasculature are not affected (Dong et al. 2011). Furthermore, studies have shown that GRP78 plays
pivotal roles in promoting anti-estrogen resistance of ESR1+ breast cancer cells by stimulating
pro-survival autophagy and inhibiting apoptosis (Cook et al. 2012), and modulating lipid
metabolism and innate immunity (Cook et al. 2016). Interestingly, secretion and atypical
translocation of GRP78 to the cell surface, cytosol, mitochondria and nucleus were observed in a
diverse range of cancer cells under ER stress conditions. Cell surface GRP78 (csGRP78) mediates
novel signaling function independent of its intracellular function as a foldase in the ER
(Pfaffenbach and Lee 2011, Ni, Zhang, and Lee 2011, Zhang et al. 2010, Zhang et al. 2013, Lee
2014). Targeting csGRP78 has shown great efficacy in blocking cancer progression including
breast cancer (Lee 2014) and the mechanisms underlying its cell surface translocation are just
emerging.
1.2 Mechanisms of GRP78 cell surface translocation
Growing evidences have shown how GRP78 relocalizes from the ER to the cell surface. First,
recent studies revealed that csGRP78 largely exists as a peripheral protein at the plasma membrane,
where it associates with transmembrane or GPI-anchored proteins for its anchor on the cell surface
(Tsai et al. 2015). Second, GRP78 harboring a mutation in the T453D substrate binding domain
exhibited about 80% reduction of its cell surface expression in HeLa cells, suggesting substrate
binding function of GRP78 is required for its cell surface expression (Tsai et al. 2015). Third, it
has been reported that the GRP78 co-chaperone MTJ-1, a transmembrane protein, also referred to
as DnaJC1 (DnaJ homolog subfamily C member 1), was required for cell surface expression of
6
GRP78 in mouse macrophages (Misra et al. 2005). However, GRP78 harboring R197H DnaJ
binding mutation (Awad et al. 2008) showed similar level of cell surface expression as wild-type
GRP78 in human HeLa cervical cancer cells (Tsai et al. 2015). These results suggest that the
requirement of MTJ-1 or the human homolog, HTJ-1, for csGRP78 expression is cell context-
dependent.
The C-terminal KDEL sequence of the ER resident chaperones, such as GRP78, GRP94 and
protein disulphide isomerase (PDI), served as a retrieval signal to recycle these chaperone proteins
from Golgi complex back to ER (Pelham 1988, Gao et al. 2014, Capitani and Sallese 2009).
Chaperone proteins trafficked newly synthesized membrane or secretory proteins from ER to cis-
Golgi for further Golgi complex to plasma membrane (Golgi-PM) anterograde transportation
through secretory vesicles. Chaperone proteins bind to KDEL receptors localized at cis-Golgi
membrane through the KDEL motif and are transported back to the ER through the retrograde
Golgi-to-ER transport of KEDL receptors (Gao et al. 2014, Capitani and Sallese 2009). The
deletion of the KDEL sequence of GRP78 resulted in secretion of GRP78 protein into the culture
medium (Munro and Pelham 1987). Of note, GRP78 bearing a deletion of KDEL motif could still
be translocated to the cell surface, indicating the interaction of KDEL motif with the KDEL
receptor is dispensable for the cell surface expression of GRP78 (Zhang et al. 2010). GRP78 and
KDEL-bearing chaperone proteins need to “escape” the KDEL retrieval machinery for them to
reach the plasma membrane, and multiple mechanisms have been demonstrated to support the idea.
First, engagement of the KDEL motif to the KDEL receptors will activate Src family kinases
(SFKs) on the Golgi complex (Sallese, Giannotta, and Luini 2009, Pulvirenti et al. 2008, Asp and
Nilsson 2008, Giannotta et al. 2012). There are two major outcomes of this SFKs activation
(Capitani and Sallese 2009, Wiersma et al. 2015): 1) progression of intra-Golgi (Giannotta et al.
7
2012) and Golgi-PM (Sallese, Giannotta, and Luini 2009, Pulvirenti et al. 2008, Weller et al. 2010)
anterograde transport of cargos through secretory vesicles; 2) promotion of retrograde KDEL
receptor recycling (Bard et al. 2003, Tsai et al. 2018). The former facilitated transportation and
cell surface expression/secretion of cargo proteins, and the latter led to redistribution/dispersion of
KDEL receptors out of Golgi and slower rate of retrograde Golgi-to-ER transport of cargos. The
key processes in common among these pathways are active promotion of fission of vesicles from
Golgi apparatus by Src-activated Golgi-localized GTPases and elevation of cell surface expression
of cargo proteins/KDEL-bearing chaperones. Notably, Src was reported to be also activated by
UPR signaling transducer IRE-1α under the ER stress condition (Tanjore et al. 2011, Tsai et al.
2018). Second, the binding of KDEL receptors to their ligands could be saturated by excessive
ligands under transient expression (Dean and Pelham 1990, Zhang et al. 2010) or induction of ER
stress (Zhang et al. 2010, Tsai et al. 2015), and the non-bound fraction of ligands may escape the
KDEL retrieval mechanism and then arrive at the plasma membrane or extracellular space. Third,
it is possible that KDEL receptors can directly transport their ligands to the cell surface, as the
KDEL receptor and its ligand, mesencephalic astrocyte-derived neurotrophic factor (MANF), was
found associated with each other on the cell surface (Henderson et al. 2013). Moreover, the
clustering of KDEL receptors on the cell surface could be stimulated by a ligand of KDEL receptor,
a fluorescent variant of ricin toxin A chain (Becker et al. 2016). However, there is no study to the
best of our knowledge, to support that GRP78 can be associated with KDEL receptors on the cell
surface. Taken together, current studies suggest that cell surface expression of GRP78 is dependent
on its capacity to form complex with transmembrane proteins, GPI-anchored proteins, co-
chaperones and likely folding substrates, and the complexes are more efficiently transported to the
8
cell surface through Src-dependent vesiculation of Golgi complex and/or ER-Golgi independent
mechanisms (Tsai et al. 2015) under stress conditions.
9
Chapter 2
GRP78 binds CD44v and regulates its membrane homeostasis and signaling in aggressive
breast cancer
2.1 Introduction
GRP78 (78 kD glucose-regulated protein, also referred to as BiP or HSPA5) belongs to the
heat-shock protein-70 (HSP70) family and is a major endoplasmic reticulum (ER) chaperone
protein that facilitates protein folding, quality control and regulates the unfolded protein response
(UPR) (Ni and Lee 2007, Luo and Lee 2013, Lee 2014). Overexpression of GRP78 is associated
with cancer cell growth, invasion and multi-drug resistance (Lee 2007, 2014). Atypical
translocation of GRP78 to cell surface was observed in various cancer cells and further elevated
under stress conditions (Ni, Zhang, and Lee 2011, Zhang et al. 2013, Tsai et al. 2015). Cell surface
GRP78 (csGRP78) has emerged as a novel regulator of cell surface signaling, beyond the
traditional protein foldase activity as a chaperone protein in the ER (Ni, Zhang, and Lee 2011,
Zhang et al. 2013, Tsai et al. 2018). Previous studies have highlighted the importance of csGRP78
in cancer cell-matrix adhesion, motility, invasion and proliferation; however, the underlying
mechanisms are just emerging (Kelber et al. 2009, Li et al. 2013, Misra, Deedwania, and Pizzo
2005). Since GRP78 exists on the cell surface primarily as a peripheral protein (Tsai et al. 2015),
the identification of transmembrane protein partners of csGRP78 is critical towards understanding
how it is anchored on the cell surface and mediates its signaling functions.
Recently, it was reported that GRP78 facilitated chemo-radioresistance and invasion in head
and neck cancer (HNC) cells exhibiting molecular characteristics (CD24
-
CD44
+
) of HNC stem
cells (Chiu et al. 2013). In addition, GRP78 knockdown in HNCs suppressed stem cell regulatory
10
proteins, Oct-4 and Slug, and transformed cell morphology into rounder cell shapes (Chiu et al.
2013). CD44 is a type I transmembrane glycoprotein known to facilitate cell adhesion, spreading,
migration, invasion, ROS defense and multi-drug resistance in a variety of cancer types (Misra,
Ghatak, and Toole 2005, Ishimoto et al. 2011, Zoller 2011, Montgomery et al. 2012, Hiraga, Ito,
and Nakamura 2013). It is widely used as a cancer stem cell marker in subtypes of cancers
including breast (Al-Hajj et al. 2003, Liu et al. 2010, Yan, Zuo, and Wei 2015) and serves as the
major receptor of hyaluronan (Ghatak et al. 2010). It can also bind to a wide range of extracellular
matrix (ECM) components, including metalloproteinases, collagen, laminin, chondroitin sulfate
and fibronectin (Zoller 2011). CD44 is a highly heterogeneous glycoprotein, it can be regulated by
alternative splicing and post-translational modifications (Zoller 2011, Yae et al. 2012). CD44
variant isoforms are created by alternative splicing through incorporation of variable exons into
the extracellular juxtamembrane region. CD44 standard isoform (CD44s) lacks variable exons. In
addition to its role as a cell surface receptor, CD44 variant isoforms can function as a co-receptor
that binds FGF2, HGF, VEGF and osteopontin, and present them to their receptors (Zoller 2011).
It has been reported that breast cancer stem-like cells expressing CD44 variant isoforms exhibited
enhanced metastatic capacity (Yae et al. 2012). CD44 containing variable exon 3 to 10 (CD44v3-
10) instead of CD44v8-10 or CD44s correlated with poor prognosis of breast cancer patients (Hu
et al. 2017). Collectively, CD44 is a critical regulator of cytoskeletal dynamics, cell motility,
migration, invasion and polarity in normal development and cancer progression (Senbanjo and
Chellaiah 2017).
Here, we studied the interactions between GRP78 and CD44 and investigated function and cell
signaling mediated by their interplay in aggressive breast cancer cells using biochemical analyses,
conventional confocal microscopy and time-lapse TIRF/PALM (total internal reflection
11
fluorescence/photoactivated localization microscopy) imaging. We discovered that CD44v3-10
(hereinafter CD44v) is a novel partner protein of GRP78. CD44v and GRP78 co-localized at
plasma membrane of tamoxifen-resistant MCF7 cells (MCF7-LR), metastatic MDA-MB-231 cells,
patient-derived circulating tumor cells and MDA-MB-231 tumor xenografts. Co-diffusion and co-
confinement of CD44v and GRP78 were observed in the plasma membrane nanodomains in
MCF7-LR cells. We further showed that GRP78 can directly bind to the extracellular region of
CD44v in vitro. An antibody against GRP78 (clone: 76-E6), which may also have affinity for
HSP70, potently disrupted the integrity of F-actin network, inhibited cell motility, altered cell
polarity and perturbed CD44 membrane homeostasis. The antibody specifically against GRP78
(clone: MAb159) reduced CD44v protein level and stability, and the ability of MCF7-LR cells to
attach to the culture matrix. The bacterial subtilase cytotoxin A subunit (SubA), specifically
cleaving csGRP78 suppressed cell spreading of MCF7-LR cells. Our study uncovers a new
mechanism for GRP78 in modulating behaviors of aggressive breast cancer cells via its regulation
of CD44v signaling pathway.
2.2 Materials and methods
Antibodies
We used the following primary antibodies for the proteins listed: CD44v3 from Thermo Fisher
Scientific (BMS144, Waltham, MA), CD44 from GeneTex (GTX102111, Irvine, CA), GRP78
from Abcam (ab25192, Cambridge, MA), Santa Cruz Biotechnology, Inc. (sc-1050 and sc-1051,
Dallas, TX) and Dr. Parkash S. Gill at USC (MAb159, gift from Dr. Parkash Gill at USC), IQGAP1
from Sigma-Aldrich (SAB4200079, St. Louis, MO), HA tag from Santa Cruz Biotechnology, Inc.
12
(sc-805, Dallas, TX), GST tag from Santa Cruz Biotechnology, Inc. (sc-138, Dallas, TX), Annexin
II from BD Biosciences (610068, San Jose, CA), GAPDH from Santa Cruz Biotechnology, Inc.
(sc-32233, Dallas, TX), β-actin from Sigma-Aldrich (A5316, St. Louis, MO), Phospho-
STAT3(Y705) from Cell Signaling Technology (9145, Danvers, MA), STAT3 from BD
Biosciences (610190, San Jose, CA), Phospho-FAK(Y397) from Cell Signaling Technology (8556,
Danvers, MA), FAK from BD Biosciences (610087, San Jose, CA), Phospho-SRC (Y419) from
Cell Signaling Technology (2101, Danvers, MA), SRC from Cell Signaling Technology (2110,
Danvers, MA), and HSP70 from Santa Cruz Biotechnology, Inc. (sc-66048, Dallas, TX).
Secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY), Santa Cruz
Biotechnology (Dallas, TX), Inc., and LI-COR Biosciences (Lincoln, NE).
Plasmids and cloning
The hemagglutinin (HA)-tagged full-length CD44 containing v3-v10 exons (CD44v-HA)
expression plasmid was produced by PCR amplification from cDNA library of MCF7-LR cells
using the reverse primer containing HA tag sequence. The PCR product was inserted in-frame into
pcDNA3 expression vector (Thermo Scientific, Waltham, MA) at KpnI and ECoRI sites. The
pGEX-4T-1 bacterial expression plasmid for glutathione S-transferase (GST) tag was obtained
from GE Healthcare (Chicago, IL). The GST-tagged full-length human GRP78 (a.a. 19-654) and
the truncated GRP78 mutants containing the N-terminal half (a.a. 19-407), the C-terminal half (a.a.
413-654) and large segments (a.a. 19-196, 19-511, 19-526, 19-541, 19-556, 19-571, 19-581 and
582-654) were generated by PCR amplification of the GRP78 coding sequencing from FLAG-
tagged human GRP78 (wild-type) expression plasmid (Zhang et al. 2013) and then inserted in-
frame into pGEX-4T-1 expression plasmid at BamHI and XhoI sites. The PATagRFP-GRP78 was
produced by PCR amplification of the PATagRFP coding sequencing from an ABP-PATagRFP
13
expression plasmid using the forward primer containing GRP78 ER signal sequence (78ERSS).
The caveolin-1 coding sequence in caveolin-1-SNAP expression plasmid was then replaced by the
78ERSS-PATagRFP sequence at ECoRI and SacII sites. This generated an intermediate 78ERSS-
PATagRFP-SNAP expression plasmid. Then the PATagRFP-GRP78 expression plasmid was
produced by PCR amplification of GRP78 coding sequencing from FLAG-tagged human GRP78
(wild-type) expression plasmid using the forward primer containing a linker sequence. The SNAP
coding sequence in 78ERSS-PATagRFP-SNAP expression plasmid was then replaced by the
GRP78 coding sequence at SacII and KpnI sites. The SacII restriction site was destroyed after
cloning. The CD44v-PAGFP expression plasmid was produced by PCR amplification of the
CD44v3-10 coding sequence from the CD44v-HA expression plasmid. The caveolin-1 coding
sequence in caveolin-1-SNAP expression plasmid was then replaced by the CD44v3-10 coding
sequence at ECoRI and SacII sites. This generated an intermediate CD44v3-10-SNAP expression
plasmid. Then the CD44v-PAGFP expression plasmid was produced by PCR amplification of
PAGFP coding sequencing from PAGFP-CD4 expression plasmid using the forward primer
containing a linker sequence. The SNAP coding sequence in CD44v3-10-SNAP expression
plasmid was then replaced by the PAGFP coding sequence at SacII and KpnI sites.
We used the following primers: CD44v3-10-HA 5’-
CGGGGTACCATGGACAAGTTTTGGTGGCACGCAGCCT-3’ and 5’-CCGGAATTCTT
AAGCGTAATCTGGAACATCGTATGGGTACACCCCAATCTTCATGTCCACATTCT-3’;
GST-GRP78 (FL) 5’-CGCGGATCCATGGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTACAACTCATCTTTTTCTGCT-3’; GST-GRP78 (N) 5’-CGCGGAT
CCATGGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-CCGCTCGAGCTAAC
CAGAGAGCACACCAGC-3’; GST-GRP78 (C) 5’-CGCGGATCCATGG
14
ACCTGGTACTGCTTGATGTA-3’ and 5’-CCGCTCGAGCTACAACTCATCTTTTTCTGCT-
3’; GST-GRP78 (a.a. 19-496) 5’-CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and
5’-CCGCTCGAGCTACTGTGGGACCCCACGAG-3’; GST-GRP78 (a.a. 19-511) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTACACTCGAAGAATACCATTCACATCTATC-3’; GST-GRP78 (a.a. 19-526)
5’-CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTAGATTGTGATCTTATTTTTGTTCCCTGTACC-3’; GST-GRP78 (a.a. 19-
541) 5’-CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTACATCCTTTCGATTTCTTCAGGTGTC-3’; GST-GRP78 (a.a. 19-556) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTACTTGAGCTTTTTGTCTTCCTCAGC-3’; GST-GRP78 (a.a. 19-571) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTAAGAATAGGCATAGCTTTCCAACTCA-3’; GST-GRP78 (a.a. 19-581) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGG-3’ and 5’-
CCGCTCGAGCTACTTTTCTTTATCTCCAATCTGATTC-3’; GST-GRP78 (a.a. 582-654) 5’-
CGCGGATCCCTGGGAGGTAAACTTTCCTCTG-3’ and 5’-
CCGCTCGAGCTACAACTCATCTTTTTCTGCTG-3’; GRP78 ER signal sequence-PATagRFP
5’-
CCGGAATTCATGAAGCTCTCCCTGGTGGCCGCGATGCTGCTGCTGCTCAGCGCGGCG
CGGGCCATGAGCGAGCTGATTAAGGAGAAC-3’ and 5’-TCCCCGCGGA
TTAAGCTTGTGCCCCAGTTTGC-3’; Linker-GRP78 5’-TCCCCGCGGTCCGGAGCGGC
CGCTGCAGGAGGCAGCCAAAAAGAGGAGGAGGACAAGAAGGAG-3’ and 5’-
CGGGGTACCCTACAACTCATCTTTTTCTGCTGTATC-3’; CD44v3-10 5’-CCGGAATT
15
CATGGACAAGTTTTGGTGGCACGCAGCCT-3’ and 5’-TCCCCGCGGCACCCC
AATCTTCATGTCCACATTCT-3’; Linker-PAGFP 5’-TCCCCGCGGTCCGGAGCGGCC
GCTGCAGGAGGCAGCCAAAAAATGGTGAGCAAGGGCGAGGAG-3’ and 5’-
CGGGGTACCTTACTTGTACAGCTCGTCCATGC-3’.
Short hairpin RNA (shRNA) that target either standard exon (sh44-2) as described (Godar et
al. 2008) or variable exon 3 (sh44v3) in CD44 mRNA were cloned into pLKO.1 lentiviral vector
at AgeI and EcoRI sites. Scrambled controls have same ATCG composition as either sh44-2
orsh44v3. All constructs were verified by sequencing. We used the following primers: shCD44-2
5’-
CCGGTGTAACACCTACACCATTATCCTCGAGGATAATGGTGTAGGTGTTACATTTTT
G-3’ and 5’-AATTCAAAAATGTAACA
CCTACACCATTATCCTCGAGGATAATGGTGTAGGTGTTACA-3’; shCD44v3 5’-
CCGGCATTGATGATGATGAAGATTTCTCGAGAAATCTTCATCATCATCAATGTTTTTG
-3’ and 5’-AATTCAAAAACATTGATGATGATGAAGATTTCTCGAGAAATCTTCATCA
TCATCAATG-3’; shCtrl-1 5’-CCGGGCTATCATACTCCTCTAAACACTCGAGTG
TTTAGAGGAGTATGATAGCTTTTTG-3’ and 5’-AATTCAAAAAGCTATCATA
CTCCTCTAAACACTCGAG TGTTTAGAGGAGTATGATAGC-3’; shCtrl-2 5’-CCGGGAA
TTAAGCGGATTGTATTTA CTCGAGTAAATACAATCCGCTTAATTCTTTTTG-3’ and 5’-
AATTCAAAAAGAATTAAGCGGATTGTATTTACTCGAGTAAATACAATCCGCTTAATT
C-3’
Gene knockdown
For short interfering RNA (siRNA) knockdown, cells were transfected with Lipofectamine™
RNAiMAX Reagent (Thermo Fisher Scientific, Waltham, MA) containing siRNA (Dharmacon
16
Inc., Lafayette, CO) to the final concentration of 60 pM. We used the following siRNAs: siGrp78
5’-GGAGCGCAUUGAUACUAGAdTdT-3’; sictrl 5’-GAGAUCGUAUAGCAACGGUdTdT-3’.
The shRNA plasmids (sh44-2, sh44v3 and two scrambled controls) were transfected into
MDA-MB-231 cells with BioT transfection reagent (Bioland Scientific, Paramount, CA). Because
the CD44 knockdown cells were sensitive to common antibiotic selection (Godar et al. 2008), we
performed short-term selection in the presence of puromycin (1 mg/mL) for 24 h. Then, cells were
cultured in normal culture media for two weeks to allow proper cell recovery. Cells then were
seeded by limited dilution in the 96-well culture plate (BD Biosciences, San Jose, CA). Only cells
grew from single colonies were selected to establish stable CD44 knockdown cells.
Real-time quantitative reverse transcription –polymerase chain reaction
The MCF7-LR cells were treated with the 76-E6 antibody or the control IgG for 24 h, then
RNA was extracted from the treated cells with the TRI Reagent® (Sigma-Aldrich, St. Louis, MO).
The reverse transcription was performed using 1.5 µg extracted total RNA per condition, random
primers (New England Biolabs, Ipswich, MA) and the SuperScript II Reverse Transcriptase
(Thermo Fisher Scientific, Waltham, MA). The cDNA samples were analyzed with the SYBR
Green Supermix (Quanta Biosciences, Gaithersburg, MD) according to manufacturer's instructions
using the Mx3005P thermocycler (Stratagene, San Diego, CA). We used the following primers:
18S rRNA 5’- GCTTAATTTGACTCAACACGGGA-3’ and 5’-
AGCTATCAATCTGTCAATCCTGTC-3’; CD44t 5’- TCCAACACCTCCCAGTATGACA-3
and 5’- GGCAGGTCTGTGACTGATGTACA-3’; CD44s 5’- AGCAGCGGCTCCTCCAGTGA-
3’ and 5’- CCCACTGGGGTGGAATGTGTCT-3’; CD44v3 5’- GCAGGCTGGGAGCCAAAT-
3’ and 5’- GAGGTGTCTGTCTCTTTCATCTTCATT-3’; CD44v6 5’-
GGAACAGTGGTTTGGCAACAG-3’ and 5’- TTGGGTGTTTGGCGATATCC-3’.
17
Cell culture
Endocrine-responsive MCF7-L breast cancer cells were gifts from Dr. Rachel Schiff (Baylor
College of Medicine, TX) and cultured in phenol-red free RPMI 1640 medium containing 5%
charcoal-stripped fetal bovine serum, 200 mM glutamine, 2.5 µg/ml fungizone, 10 IU/ml penicillin
and 10 µg/ml streptomycin. Tamoxifen-resistant MCF7 (MCF7-LR) cells (gift from Dr. Rachel
Schiff) were cultured in phenol-red free RPMI 1640 medium described above and supplemented
with 100 nM 4-hydroxy tamoxifen. MDA-MB-231 cells were originally obtained from Dr.
Shyamala Maheswaran (Harvard Medical School, MA) and cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum, 4 mM L-glutamine, 4.5 g/L glucose,
100 IU/mL penicillin, and 100 μg/mL streptomycin. HEK 293T cells were cultured in DMEM
medium described above. Patient-derived circulating tumor cells (CTCs) were cultured as
described (Yu et al. 2014). All non-CTCs were authenticated by STR DNA profiling analysis at
the reagent core facility in the USC Norris Comprehensive Cancer Center. Only mycoplasma-
negative cells were used.
For functional studies, cells were treated with 50 µg/mL anti-GRP78 antibodies in antibiotic-
free culture media containing 1% serum. SubA and SubAA272 were used at the concentration of 0.2
µg/mL in antibiotic-free culture media containing 1% serum. Osteopontin was used at the
concentration of 10 µg/mL in calcium- and magnesium-free HBSS (Hank's balanced salt solution).
Plasmid transfection
Cells were transfected with BioT transfection reagent (Bioland Scientific, Paramount, CA)
according to the manufacturer’s instruction. Media were freshly replaced 5 h post-transfection.
18
Cells were collected 48 h post-transfection for Western blot analysis or purification of recombinant
proteins.
Scratch-wound migration assay
Cells were transfected with non-targeting scrambled siRNA control (sictrl) or siRNA targeting
Grp78 (siGrp78) and grown for 72 h to confluence in triplicate in 6-well plates. Cells were
incubated with 10 μg/ml mitomycin C (Roche, Indianapolis, IN) for 4 h prior to the scratch assay.
Cells were then scratched by a 200 µL pipette tip, followed by a brief wash. Wounds were imaged
at three different locations per well immediately (time=0 h) and in 24 h (time=24 h) at the same
position. Images were photographed on a bright-field microscope. Wound closures were calculated
using the FIJI-ImageJ software.
Cell adhesion and spreading assay
MCF7-LR cells were treated with the 76-E6 antibody or control IgG for 24 h, and then equal
number of cells were seeded in triplicate on 96-well plates coated with 100 µg/mL collagen I. After
7 h, cells were washed and then fixed with freezer-cold methanol at RT for 10 min and visualized
by crystal violet staining. Cells were imaged on a bright-field microscope, and the edge of
individual cell was manually outlined using the FIJI-ImageJ software. Cell spreading area,
circularity and roundness were then calculated by the same software.
For adhesion assay of stable CD44-knockdown and control MDA-MB-231 cells, equal number
of cells were seeded in triplicate on 48-well plates coated with 100 µg/mL collagen I. After 13 min,
cells were washed and then fixed with freezer-cold methanol at RT for 10 min and visualized by
crystal violet staining. Crystal violet was recovered by adding 200 µl of 100% methanol to each
well. Extracted crystal violet (100 µl) from each well was then transferred to 96-well plates, and
19
absorbance was measured at 590 nm. The spreading area was calculated by the FIJI-ImageJ
software according to the cell area occupied by F-actin. Briefly, the cells were seeded on the
collagen I-coated coverslip, stained with rhodamine phalloidin and then visualized on a Leica TCS
SP8 confocal microscope equipped with Leica Application Suite 10 Software (Leica Microsystems,
Wetzlar, Germany).
Gelatin matrix degradation assay
Fluorescein isothiocyanate (FITC)-conjugated Gelatin (Anaspec, Fremont, CA) was coated
onto the inner well (ø12 mm) of PELCO
®
glass bottom dish (Ted Pella Inc., Redding, CA) as
described (Martin et al. 2012). MCF7-LR cells were pre-treated with reversible metalloproteinase
inhibitors GM6001 (10 µM) for 24 h and then seeded onto the coated glass bottom dishes in the
presence of GM6001 one day before the 76-E6 antibody or control IgG treatment. Then, cells were
thoroughly and gently washed to remove GM6001, followed by incubation of the 76-E6 antibody
or isotype-matched control IgG at the concentration of 50 µg/mL for 24 h. Nuclei were then stained
by Hoechst
®
33342. Live cell imaging was performed on a Zeiss LSM 510 confocal microscope
(Carl Zeiss, Oberkochen, Germany) equipped with an environmental chamber. The percentage of
degraded gelatin from five random views was analyzed by the FIJI-ImageJ software. Total number
(N) of cells analyzed in the image areas as determined by the number of nuclei visualized by
Hoechst
®
33342: N=367 (IgG), N=405 (76-E6).
Western blotting
Cell lysates were obtained using radioimmunoprecipitation (RIPA) buffer containing 50 mM
Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease
and phosphatase inhibitor cocktail. Proteins were analyzed by 10% SDS-PAGE and transferred at
20
4°C overnight on nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes
were blocked by Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk
at room temperature (RT) for 1 h and then incubated with primary antibody at 4°C overnight. After
3 washes with TBS containing 0.05% Tween-20, membranes were incubated with HRP-
conjugated secondary antibodies (Enzo Life Sciences, Farmingdale, NY and Santa Cruz
Biotechnology, Dallas, TX) or fluorescent IRDye-labeled antibodies (LI-COR Biosciences,
Lincoln, NE). HRP signal was detected by an ECL chemiluminescent substrate (Thermo Fisher
Scientific, Waltham, MA) and quantified with Image Lab software (Bio-Rad Laboratories,
Hercules, CA). Fluorescent IRDye signal was detected by Odyssey (LI-COR Biosciences, Lincoln,
NE).
Immunofluorescence and confocal microscopy
For detection of endogenous GRP78 and CD44 containing v3 exon on the cell surface of
MCF7-LR and MDA-MB-231 cells, cells were grown for 48 h to subconfluence on sterile
coverslips. Coverslips were sequentially coated with 50 μg/ml poly-L-lysine in ultrapure water
(Sigma-Aldrich, St. Louis, MO) at RT for 1 h and then 100 µg/mL collagen I from rat tail (Corning
Inc., Corning, NY) in 0.02% acetic acid at RT for 2 h. The cells were fixed in 4% paraformaldehyde
(Electron Microscopy Sciences, Hatfield, PA) in Dulbecco's Phosphate-Buffered Saline (DPBS)
at RT for 10 min and blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline
(PBS) at RT for 1 h. The primary antibody against GRP78 (MAb159, a kind gift from Dr. Parkash
Gill at USC) was incubated with the cells at 4°C overnight in blocking buffer, followed by staining
with AlexaFluor-594 or AlexaFluor-568 secondary antibody (Thermo Scientific, Waltham, MA)
at RT for 1 h. Then, the cells were treated with M.O.M.™ Mouse Ig Blocking Reagent (Vector
Laboratories, Burlingame, CA) at RT for 2 h to block mouse immunoglobulin from the primary
21
mouse anti-GRP78 antibody. The cells were then incubated with the primary antibody against
CD44 variable exon 3 (Thermo Scientific, Waltham, MA) at 4°C overnight in blocking buffer,
followed by staining with AlexaFluor-488 or AlexaFluor-647 secondary antibody (Thermo
Scientific, Waltham, MA) at RT for 1 h. Each step was followed by 4 washes in PBS. Coverslips
were rinsed once with ultrapure water (Sigma-Aldrich, St. Louis, MO) before mounting with
Vectashield anti-fade medium containing DAPI (Vector Laboratories, Burlingame, CA). Z-stack
images were obtained on a Zeiss LSM 510 confocal microscope equipped with a Plan-Apochromat
100x, 1.4 NA oil DIC objective lens, a Hamamatsu R6357 photomultiplier, and LSM 510 version
4.2 SP1 acquisition software (Carl Zeiss, Oberkochen, Germany).
For detection of endogenous GRP78 and CD44 containing v3 exon on the cell surface of BRx-
68 and BRx-07 patient-derived circulating tumor cells (CTCs), cells were stained in suspension
using a similar protocol described above. The cells were rinsed once with ultrapure water (Sigma-
Aldrich, St. Louis, MO) and then applied to Superfrost
®
Plus Micro Slide (VWR International).
The cells were semi-dried before mounting with Vectashield anti-fade medium containing DAPI
(Vector Laboratories, Burlingame, CA). Z-stack images were obtained on a Zeiss LSM 510
confocal microscope equipped with a 63x, 1.4 NA oil DIC objective lens, a Hamamatsu R6357
photomultiplier, and LSM 510 ZEN 2009 acquisition software (Carl Zeiss, Oberkochen, Germany).
For detection of endogenous GRP78 and CD44 containing v3 exon in tumor xenografts, MDA-
MB-231 xenografts (Pan et al. 2016) were frozen sectioned (7 µm) and placed onto Superfrost
®
Plus Micro Slide (VWR International), followed by similar staining protocol described above. Z-
stack images were obtained on a Leica TCS SP8 confocal microscope equipped with a 63x, 1.4
NA oil DIC objective lens and Leica Application Suite 10 Software (Leica Microsystems, Wetzlar,
Germany).
22
For detection of Rab5 and CD44 containing v3 exon in MCF7-LR cells, cells were grown for
24 h to subconfluence on sterile coverslips and then treated with the 76-E6 antibody or isotype-
matched control IgG for another 24 h. Coverslips were coated as described above before seeding
the cells. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA)
in Dulbecco’s Phosphate-Buffered Saline (DPBS) at RT for 10 min and then permeabilized by
0.3% saponin in PBS at RT for 15 min. Then, the cells were treated with M.O.M.™ Mouse Ig
Blocking Reagent (Vector Laboratories, Burlingame, CA) at RT for 2 h to block potential
background from the antibody treatment, followed by incubation with blocking buffer containing
4% BSA and 0.01% saponin in PBS at RT for 1 h. The primary antibody against CD44 variable
exon 3 was incubated with the cells at 4°C overnight in blocking buffer, followed by staining with
the AlexaFluor-647 secondary antibody (Thermo Scientific, Waltham, MA) at 37°C for 40 min.
Then, the cells were treated with M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories,
Burlingame, CA) at 37°C for 2 h to block mouse immunoglobulin from the primary antibody
against CD44 variable exon 3. The cells were then incubated with the primary antibody against
Rab5 at 4°C overnight in blocking buffer, followed by staining with the AlexaFluor-488 secondary
antibody (Thermo Scientific, Waltham, MA) at 37°C for 40 min. Each step was followed by 4
washes in PBS containing 0.01% saponin. Coverslips were rinsed once with ultrapure water
(Sigma-Aldrich, St. Louis, MO) before mounting with Vectashield anti-fade medium containing
DAPI (Vector Laboratories, Burlingame, CA). Z-stack images were obtained on a Leica TCS SP8
confocal microscope equipped with a 63x, 1.4 NA oil DIC objective lens and Leica Application
Suite 10 Software (Leica Microsystems, Wetzlar, Germany).
23
Intra-molecular Raichu-Cdc42 FRET imaging
For detection of spatial distribution of active Cdc42, MCF7-LR cells were transfected with
Raichu-Cdc42 biosensor and then treated with the 76-E6 antibody or isotype-matched control IgG
for 24 h before imaging. Forty-eight h after transfection, hyaluronan (100 ug/mL; a CD44 ligand)
or DPBS vehicle control was added into culture media for 30 min, and then cells were immediately
imaged according to the protocol developed by Dr. Michiyuki Matsuda (Aoki and Matsuda 2009)
on a LSM 780 confocal microscope equipped with a motorized xyz stage, a temperature and CO 2
controlled environmental chamber (pre-equilibrated for 4 h before imaging) and definite focus
function. For Raichu-Cdc42 ratio imaging, 458 nm laser line was used to excite cells and emission
signals were collected between 470-500 nm for CFP and 520-560 nm for FRET. Cells were also
sequentially excited with 514 nm laser line and YFP signal (520-560 nm) was collected for
reference. Four independent areas per condition were imaged per min for 1 h through the motorized
stage, and signals were collected with definite focus through a 40x, 1.4 NA oil immersion objective
lens. After background subtraction, time-lapse ratiometric FRET/CFP images were created by the
ratio plus plug-in in the FIJI-ImageJ software.
Cell tracking and track analysis
MDA-MB-231 cells were seeded in low density in the inner well (ø12 mm) of collagen I (100
µg/mL) coated glass bottom dish (Ted Pella Inc., Redding, CA) 24 h before treatment of the 76-
E6 antibody or isotype-matched control IgG for another 24 h. Time-lapse differential interference
contrast (DIC) images were then captured on a LSM 510 confocal microscope equipped with a
10x (7 h cell tracking) or 20x (2 h imaging) objective lens. Culture medium was covered with
mineral oil (Sigma-Aldrich, St. Louis, MO) and under temperature and CO2 control during time-
lapse imaging. Images were captured every 5 min for 7 h cell tracking and per min for 2 h time-
24
lapse imaging. Pinhole was fully opened to minimize the use of laser power and maximize the
depth of view. Mitotic or apoptotic cells were excluded from analysis. Straightness and velocity
were analyzed by MTrackJ plug-in in the FIJI-ImageJ software.
Single particle tracking and diffusion analysis
For dual-color single particle tracking by photoactivated localization microscopy (PALM),
MCF7-LR cells were seeded on Marienfeld-Superior precision coverslips (thickness no. 1.5H)
coated with collagen I (100 µg/mL) and transfected with PATagRFP-GRP78 and CD44v-PAGFP
expression plasmids 24 h after seeding. For sptPALM of CD44v-PAGFP with GRP78 knockdown,
MCF7-LR cells seeded on collagen I-coated Marienfeld coverslips were co-transfected with the
CD44v-PAGFP expression plasmid and siRNA using BioT transfection reagent (Bioland
Scientific, Paramount, CA) for 5 h. The culture media was replaced by media containing
Lipofectamine™ MessengerMAX™ Reagent (Thermo Fisher Scientific, Waltham, MA) and
siRNA oligos. Forty-eight h after initial transfection, imaging was performed by TIRF on an
inverted Nikon Eclipse Ti-E microscope, equipped with a 100X/1.49 NA objective (Nikon,
Melville, NY), two iXon EMCCD cameras (Andor Technology, Belfast, United Kingdom), a dual
camera light path splitter (Andor Technology, Belfast, United Kingdom), an axial stabilizing
system (Perfect Focus System) and laser lines at 405, 488 and 561 nm (Agilent Technologies,
Santa Clara, CA). A multiband pass ZET405/488/561/647x excitation filter (Chroma Technology,
Bellows Falls, VT), a quad-band ZT405/488/561/647 dichroic mirror (Chroma Technology,
Bellows Falls, VT), an emission FF560-FDi01 dichroic mirror (Semrock, Rochester, NY) and
appropriate emission filters for simultaneous sptPALM imaging of PATagRFP-GRP78 (600/50
nm, Chroma Technology, Bellows Falls, VT) and CD44v-PAGFP (525/50 nm, Chroma
Technology, Bellows Falls, VT) were used. In both emission channels, images were acquired
25
continuously at a frame rate of 30 ms/frame. Precise image alignment of both RFP and GFP
channels was performed using 40 nm TransFluoSphere fiducials (488/685 nm, Thermo Fisher
Scientific, Waltham, MA) spread around the cells.
Single particle localization and tracking were performed using SlimFast, a single-molecule
detection and tracking software written in Matlab (MathWorks, Natick, MA). Localizations were
done by 2D Gaussian fitting of the point-spread function of each activated PATagRFP-GRP78 and
CD44v-PAGFP in each frame. Diffusion trajectories were built by linking individual localized
positions from one frame to the other, taking into account blinking statistics and local particle
densities. Only trajectories with at least 3 steps were kept for diffusion analyses based on the mean
square displacement (MSD) of individual trajectories or on the probability distribution of square
displacement (PDSD) (Fernandez et al. 2017) of all trajectories. Diffusion coefficients from
individual MSD were determined by fitting MSD curves over the first three time lags using a free
Brownian diffusion model with measurement error:
𝑟 2
= 4𝐷𝑡 + 4𝜎 2
(1)
where σ is the position error and D is the diffusion coefficient.
Diffusion coefficients from PDSD were determined by fitting each Pr
2
curve over the first 10
time lags with the general model:
𝑃 (𝑟⃗
2
, 𝑡 ) = 1 − ∑ 𝛼 𝑖 (𝑡 )𝑒 −𝑟 2
/𝑟 𝑖 2
(𝑡 )
𝑛 𝑖 =1
(2)
∑ 𝛼 𝑖 (𝑡 ) = 1
𝑛 𝑖 =1
26
where ri
2
(t) and αi(t) are the square displacement and the fraction corresponding to i numbers of
diffusive behaviors at each time lag t, respectively. For both PATagRFP-GRP78 and CD44v-
PAGFP, the Pr
2
distributions were best fit with i = 2 behaviors. Error bars for each ri
2
in ri
2
(t)
curves were determined using
𝑟 𝑖 2
√𝑁 , where N is the number of data points used to build each
probability distribution function. Diffusion coefficients were obtained by fitting ri
2
(t)curves with
an Origin software (OriginLab, Northampton, MA) and using the free Brownian diffusion model
with localization error in equation (1) or using a circularly confined diffusion model with
measurement error:
𝑟 2
= 𝑅 2
(1 − 𝐴 1
𝑒 −
4𝐴 2
𝐷𝑡
𝑅 2
) + 4𝜎 2
(3)
where R is the confinement radius, σ is the position error, D is the diffusion coefficient, A1= 0.99
and A2= 0.85 (Pinaud et al. 2009).
All the diffusion coefficients Dare reported in micrometer squared per sec ± standard deviation
of the fit value.
Flow cytometry
Cells were collected using non-enzymatic cell dissociation solution (Sigma-Aldrich, St. Louis,
MO) and incubated with blocking buffer (DPBS, 3% FBS and 0.1% sodium azide) on ice for 1 h.
For co-expression analysis, 1x10
6
cells were aliquoted and incubated with the primary antibody
against GRP78 (MAb159, 10 µg/mL) or isotype-matched control IgG (BioLegend, San Diego, CA)
on ice for 1 h in blocking buffer, followed by staining with the AlexaFluor-488 secondary antibody
(1:200; Thermo Scientific, Waltham, MA) on ice for 1 h. Then, the cells were treated with
M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories, Burlingame, CA) at RT for 1 h to
27
block mouse immunoglobulin from the primary mouse anti-GRP78 antibody. The cells were then
incubated with the primary antibody against CD44 variable exon 3 (10 µg/mL, Thermo Scientific,
Waltham, MA) or isotype-matched control IgG (BioLegend, San Diego, CA) on ice for 1 h in
blocking buffer, followed by staining with the AlexaFluor-647 secondary antibody (1:200; Thermo
Scientific, Waltham, MA) on ice for 40 min. Each step was followed by 3 washes in blocking
buffer. For single staining in antibody treatment experiments, 1x10
5
cells were aliquoted, and then
sequentially blocked with M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories,
Burlingame, CA) and 3% FBS in DPBS. Then, cells were incubated with the anti-CD44v3 primary
antibody (10 µg/mL, Thermo Scientific, Waltham, MA) or isotype-matched control IgG
(BioLegend, San Diego, CA) followed by the AlexaFluor-647 secondary antibody (1:200). Cells
were resuspended in blocking buffer containing 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI,
Sigma-Aldrich, St. Louis, MO) and subjected to flow cytometry. The data was acquired by either
LSR II (co-expression analysis) or FACSVerse (single staining) flow cytometer (Becton Dickinson,
Franklin Lakes, NJ) and then analyzed with FlowJo v10 software.
Purification of GST-tagged recombinant proteins
Full-length GRP78 and truncated mutants were subcloned into pGEX-4T-1 vector using the
BamHI and XhoI restriction sites and transformed into Escherichia coli (BL21). Small volume (40
ml) of overnight starter broth culture in Luria-Bertani (LB) medium was added into 400 ml pre-
warmed LB broth culture until optical density measured at a wavelength of 600 nm (OD600) reaches
0.5. Then, expression of the GST fusion proteins was induced with 4 mM isopropyl-β-D-
thiogalactoside (IPTG) at 37°C and 200 rpm for 4 h. Bacterial cells were lysed in Tris-buffered
saline (TBS) containing 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mg/ml
lysozyme and protease and phosphatase inhibitor cocktails (Thermo Scientific, Waltham, MA).
28
After 30 min incubation on ice, bacterial cells were sonicated for 4 min with 20 sec on and 20 sec
off and then centrifuged at 4°C and 11500 rpm for 1 h. Supernatant was collected and incubated
with glutathione-Sepharose 4B beads (GE Healthcare, Chicago, IL) at 4°C for 12 h. Recombinant
GST-tagged protein was eluted with freshly prepared reduced glutathione (10 mM, Sigma-Aldrich,
St. Louis, MO) at 4°C for 12 h. Then, the solution containing recombinant proteins was buffer-
exchanged to TBS. For long-term storage, recombinant proteins in TBS containing 15% glycerol
was snap-frozen in liquid nitrogen and then stored at -80°C.
Purification of polyhistidine-tagged recombinant proteins
Extracellular region of carboxyl terminal 6x histidine-tagged CD44v3-10 was subcloned into
pcDNA3 vector using the KpnI and ECoRI restriction sites and then transfected into mammalian
HEK 293T cells. Forty-eight h after transfection, cells were lysed in PBS buffer containing 50 mM
sodium phosphate, 150 mM NaCl, 1% NP-40, and protease and phosphatase inhibitor cocktails
(Thermo Scientific, Waltham, MA). Clarified cell lysate was pooled with concentrated conditional
media and then incubated with TALON
®
cobalt resin (Clontech Laboratories, Mountain View, CA)
at 4°C for 12 h with gentle rotation. Then, the beads were washed 3 times with 100x bed volume
of PBS buffer supplemented with 40 mM imidazole (Sigma-Aldrich, St. Louis, MO). The beads
were then transferred to gravity flow column and washed 3 times with 40x bed volume of PBS
buffer supplemented with 40 mM imidazole. Polyhistidine-tagged protein was eluted with PBS
buffer containing 250 mM imidazole at 4°C for 12 h. Then, the solution containing the recombinant
protein was buffer-exchanged to TBS. For long-term storage, the recombinant protein in TBS
containing 15% glycerol was snap-frozen in liquid nitrogen and then stored at -80°C.
29
In vitro GST pull-down assay
GST tag (5 µg) or GST-tagged GRP78 (5 µg; FL, N and C) recombinant proteins were coupled
to Glutathione Sepharose
®
4B beads (GE Healthcare, Chicago, IL) at 4°C for 4 h. Then, the
conjugated GST or GST-tagged GRP78 was incubated with 1 mg whole cell lysate from HEK
293T cells overexpressing HA-tagged full-length CD44v3-10 at 4°C overnight in IP lysis buffer
(Thermo Fisher Scientific, Waltham, MA; 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1
mM EDTA, 5% glycerol). The beads were then washed 6 times with IP lysis buffer. Bound
proteins were eluted from the beads with equal volume of 2x SDS sample buffer.
In vitro direct binding assay
GST tag (2.5 µg) or GST-tagged full-length GRP78 (2.5 µg) recombinant proteins were
coupled to Glutathione Sepharose
®
4B beads (GE Healthcare, Chicago, IL) at 4°C for 4 h. Then,
the conjugated GST or GST-tagged GRP78 (FL) was incubated with 1.5 µg recombinant
polyhistidine-tagged extracellular region of CD44v3-10 at 4°C overnight in the binding buffer (50
mM Tris-HCl, pH 7.5, 150 mM NaCl). The beads were then washed 4 times with the binding
buffer containing 0.05% Tween-20. Bound proteins were eluted from the beads with equal volume
of 2x SDS sample buffer.
Purification of cell surface proteins for Western blot analysis
Experiments were performed as previously described (Tsai et al. 2015). Briefly, cell surface
proteins were biotinylated with 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher
Scientific, Waltham, MA) at 4°C for 30 min. Excessive biotin was quenched by 100 mM glycine
in cold PBS. Cells were then lysed with IP lysis buffer (Thermo Fisher Scientific, Waltham, MA).
The biotinylated cell surface proteins in cell lysates were captured on high capacity NeutrAvidin
30
agarose resin (Thermo Fisher Scientific, Waltham, MA).
Purification of cell surface proteins for co-immunoprecipitation and mass spectrometry
Cell surface proteins were biotinylated with 0.5 mg/ml EZ-link Sulfo-NHS-LC-Biotin
(Thermo Fisher Scientific, Waltham, MA) at 4°C for 30 min. Protein lysate was obtained with IP
lysis buffer (Thermo Fisher Scientific, Waltham, MA). The cell lysate was subjected to monomeric
avidin (Thermo Fisher Scientific, Waltham, MA) pull-down as previously described (Zhang et al.
2010). Co-immunoprecipitation (co-IP) was performed as described (Zhang et al. 2013). Briefly,
5 µg anti-GRP78 antibody (MAb159, gift from Dr. Parkash Gill at USC) or isotype-matched
control IgG was added into equal amount of pre-cleared cell surface proteins (Thermo Fisher
Scientific, Waltham, MA), followed by incubation with 50 µL Dynabeads protein G (Thermo
Fisher Scientific, Waltham, MA) at 4°C for 2 h. The beads were washed with IP lysis buffer and
boiled for 5 min in 1X SDS sample buffer. Then, the samples were subjected to 10% SDS-PAGE
and colloidal coomassie G-250 staining with PageBlue™ Protein Staining Solution (Thermo
Fisher Scientific, Waltham, MA). Gel slices were submitted to mass spectrometry.
Mass spectrometry
LC-MS/MS analysis was performed as previously described (Zhou et al. 2011) with a modified
in-gel digestion protocol at the Proteomic Core Facility in University of Southern California.
Statistical analysis
Data are presented as means ± S.E.M. P values were calculated via two-tailed unpaired
Student's t test or Kolmogorov-Smirnov test. Statistical significance was represented as *P≤0.05,
**P<0.01 and ***P<0.001.
31
2.3 Results
2.3.1 GRP78 knockdown suppresses cancer cell migration and F-actin integrity
Tamoxifen-resistant MCF7 (MCF7-LR) breast cancer cells showed elevated expression of
GRP78 (Zhang et al. 2013, Cook and Clarke 2015) and exhibited increased cell spreading (Fig.
2.1 A) and proteomic signature of enhanced migration (Hiscox et al. 2006, Zhou et al. 2012)
compared to the parental cells. We first tested whether GRP78 plays critical roles in cell migration
in MCF7-LR cells using siRNA knockdown. To validate this approach, we treated the cells with
siRNA against GRP78 and observed decreased protein levels of GRP78 by about 60 to 70% in the
whole cell lysate (WCL) and the biotinylated cell surface fraction (Fig. 2.1 B). Downregulation of
GRP78 by siRNA suppressed directional cell migration in MCF7-LR and triple-negative
metastatic MDA-MB-231 breast cancer cells as demonstrated by scratch-wound assay (Fig. 2.1, C
and D). Single cell morphological analysis showed inhibition of cell spreading in MCF7-LR and
MDA-MB-231 cells (Fig. 2.1, E and F). As dynamic assemblies of filamentous actin (F-actin)
network controls cell shape change, we asked if GRP78 can regulate F-actin organization. To
address this issue, we visualized F-actin structure by fluorescent F-actin staining and confocal
microscopy. Knockdown of GRP78 by siRNA resulted in disorganized F-actin in MCF7-LR cells
(Fig. 2.1 G). Taken together, GRP78 contributes to directional cell migration and F-actin integrity
of human breast cancer cells.
32
Figure 2.1. GRP78 knockdown suppresses cancer cell migration and F-actin integrity. (A) Bright-field
microscopic images of tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) and parental cells (MCF7-L). Scale
bar, 20 µm. (B) Western blot analysis of MCF7-LR cells transfected with control (sictrl) or Grp78 (siGrp78) siRNA.
Annexin II and β-actin are loading controls for cell surface fraction and whole cell lysate (WCL), respectively.
Numbers below the GRP78 bands represent relative levels of GRP78 and are derived from the ratio of GRP78 to β-
actin in WCL and Annexin II in cell surface fraction, respectively. Experiments were repeated twice with similar
results. (C and D) Scratch-wound assay. Left: Representative images of MCF7-LR (C) and MDA-MB-231 (D) breast
cancer cells transfected with sictrl or siGrp78 (si78). Cell migration was monitored over 24 h on a light microscope.
Right: Percent area of migration was measured by the FIJI-ImageJ software. Data represents means ± S.E.M. (n=9 for
each group). ***P < 0.001 by Student’s t test. Scale bars, 100 µm. (E and F) Bright-field microscopic images of
A B
C D
E F
MCF7-L MCF7-LR
MCF7-LR
sictrl siGrp78
0 h 24 h
0
0.2
0.4
0.6
0.8
1
1.2
sictrl si78
%Area of migration
***
G
MDA-MB-231
sictrl siGrp78
0 h 24 h
0
0.2
0.4
0.6
0.8
1
1.2
sictrl si78
%Area of migration
***
MCF7-LR
F-actin
DAPI
sictrl siGrp78
MCF7-LR
sictrl siGrp78 sictrl siGrp78
MDA-MB-231
Cell surface
sictrl siGrp78 sictrl siGrp78
WCL
GRP78
Annexin II
β-actin
1.00 0.40 1.00 0.30
80
30
46
(kD)
33
MCF7-LR (E) and MDA-MB-231 (F) cells transfected with sictrl or siGrp78. The arrows indicate the cell protrusions.
Scale bars, 20 µm. (G) Immunofluorescence micrographs showing the morphology and F-actin organization of MCF7-
LR cells transfected with sictrl or siGrp78. Boxed regions were enlarged in the lower panels. The arrows indicate the
F-actin structure. Green, F-actin stained by ActinGreen
TM
488; blue, nuclei stained by DAPI. Scale bar, 20 µm.
2.3.2 Identification of CD44v as a novel partner protein of csGRP78
Because csGRP78 has been shown to facilitate cancer cell migration (Li et al. 2013, Misra,
Deedwania, and Pizzo 2005) and the mechanisms are still largely unknown, we next sought to
identify novel partner proteins of csGRP78 that potentially can mediate the signaling of csGRP78
to cytoskeleton. Previous studies have demonstrated that acquired tamoxifen-resistance of MCF7
cells is accompanied with increased CD44 expression (Hiscox et al. 2012, Bellerby et al. 2016),
and GRP78 is an important regulator of stemness and cell morphology in CD24
-
CD44
+
HNC cells
(Chiu et al. 2013). As CD44 is a transmembrane protein and plays important roles in cytoskeletal
dynamics, cell morphology and migration, we hypothesize that CD44 could act as a partner protein
of GRP78 on the cell surface. To test this notion, we first determined that MCF7-LR cells
expressed CD44v, containing variable exons 3 to 10, using RT-PCR and DNA sequencing (Fig.
2.2 A). At the protein level, the expression of CD44v was confirmed by Western blotting using
the monoclonal antibody specifically against CD44 variable exon 3, in ex vivo cultured breast
cancer patient-derived circulating tumor cells (CTCs), BRx-68 and BRx-07, and in vitro cultured
breast cancer cells including parental MCF7-L cells, tamoxifen-resistant MCF7-LR cells and
metastatic MDA-MB-231 cells. The observed molecular sizes of CD44v ranging from 100 to 250
kD were like those previously reported (Hu et al. 2017) (Fig. 2.2 B). We further determined that
the expression level of csGRP78 was positively correlated with CD44v in both MCF7-LR and
34
MDA-MB-231 breast cancer cells by flow cytometry, with the specificity for the signals confirmed
by the absence of primary antibody and isotype control stainings (Fig. 2.2 C).
Figure 2.2. Identification and verification of CD44v expression in breast cancer cells. (A) Gel electrophoresis of
RT-PCR products from MCF7-LR cells. The bands were cloned into pcDNA3 vector and subjected to DNA
sequencing. The arrow indicates the position of CD44 containing variable exons 3 to 10 (CD44v3-10). The asterisks
denote non-specific bands. M, marker. (B) Western blot analysis of whole cell lysates from breast cancer cell lines
indicated on top using the antibody specifically targeting CD44v3 exon. β-actin is loading control. (C) Flow cytometry
dot plots representing immunofluorescent double staining results of endogenous levels of csGRP78 and CD44v in
MCF7-LR and MDA-MB-231 cells. GRP78 and CD44v were detected by MAb159 and anti-CD44v3 antibody,
respectively. Thresholds were set according to control stainings. No CD44v3 primary controls were performed with
the same protocol as double-stained cells but lack primary antibody targeting CD44v. The cells in isotype IgG controls
were incubated with control IgG and Alexa Fluor
®
488 or Alexa Fluor
®
647-conjugated secondary antibodies. Cell
number (n) analyzed in this assay: 100,000 (MCF7-LR) and 50,000 (MDA-MB-231) for doubled-stained cells; n is
about 10,000 for each control.
A
C
No CD44v primary Isotype ctrl-A488 Isotype ctrl-A647
MCF7-LR
Double staining
MDA-MB-231
GRP78-Alexa 488
CD44v-Alexa 647 (anti-CD44v3 antibody)
B
CD44v3-10
MCF7-LR
3.0
2.0
1.5
1.0
(kb)
*
*
M
245
135
100
80
58
β-actin
WB:
CD44v
46
(kD)
35
We next investigated the spatial distribution of csGRP78 and CD44v using immunofluorescent
(IF) staining and confocal microscopy. As shown in Fig. 2.3 A, endogenous csGRP78 and CD44v
exhibited prominent punctate co-localization in non-permeabilized migratory MCF7-LR and
MDA-MB-231 breast cancer cells. (Fig. 2.3 A). To extend our studies in patient-derived cells, we
utilized CTCs which are considered as metastatic precursors (Alix-Panabieres et al. 2007, Aceto
et al. 2014). Non-permeabilized breast cancer patient-derived ex vivo cultured CTCs, BRx-68 and
BRx-07 showed punctate staining and co-localization of GRP78 and CD44v at the plasma
membrane (Fig. 2.3 B, arrows and Fig. 2.4 A, arrows). The co-localization of GRP78 and CD44v
in each cell type from multiple Z-stack images covering whole cells was analyzed by the Coloc 2
plug-in (Mander’s overlap coefficient) in the FIJI-ImageJ software (Fig. 2.3 C), and the results
showed that substantial amounts of csGRP78 co-localized with CD44v (intermediate to high M1
value), and CD44v partially co-localized with GRP78 (intermediate M2 value). In addition, we
observed multiple co-localizations of csGRP78 and CD44v in vivo from frozen sections of MDA-
MB-231 tumor xenografts (Fig. 2.3 D, arrows).
36
Figure 2.3. Cell surface GRP78 co-localizes with CD44v in breast cancer cells. (A) Immunofluorescence and
confocal images showing the distribution and co-localization of GRP78 (red) and CD44v (green) on the cell surface
of non-permeabilized unipolar MCF7-LR and MDA-MB-231 breast cancer cells. GRP78 and CD44v were detected
by MAb159 and anti-CD44v3 antibody, respectively. DIC: differential interference contrast. Arrows indicate direction
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
MCF7-LR
MDA-MB-231
BRx-68
BRx-07
MCF7-LR
MDA-MB-231
BRx-68
BRx-07
M1 M2
Mander's overlap coefficient
A
MCF7-LR MDA-MB-231
DIC csGRP78 csCD44v Merge Merge-CZ
B
GRP78 CD44v DAPI Merge
MDA-MB-231 xenograft
BRx-68
DIC csGRP78 csCD44v DAPI Merge
BRx-07
Patient-derived CTCs
C D
37
of migration. Thickness of single immunofluorescent image section: 0.8 µm. CZ: compressed z-stacks. Cell
peripheries were outlined with white lines. Scale bars, 20 µm. (B) Immunofluorescence and confocal images
representing the distribution and co-localization of GRP78 (red) and CD44v (green) on the cell surface of non-
permeabilized breast cancer patient-derived BRx-68 and BRx-07 circulating tumor cells. GRP78 and CD44v were
detected by MAb159 and anti-CD44v3 antibody, respectively. Representative co-localizations were indicated by
arrows. Thickness of image section: 0.8 µm. The nuclei were stained by DAPI in blue. Scale bars, 5 µm. (C) Co-
localization of GRP78 and CD44v was analyzed by the Coloc 2 plug-in with the FIJI-ImageJ software. Mander’s
overlap coefficient: M1 is the contribution of GRP78 to the co-localized area while M2 is the contribution of CD44v.
Data represents means ± S.E.M. Number of analyzed independent image areas (A) and cells (N): A/N=9/23.5 (MCF7-
LR); 11/14 (MDA-MB-231); 5/70 (BRx-68); 5/307 (BRx-07) (D) Immunofluorescence and confocal images showing
the distribution and co-localization of GRP78 (red) and CD44v (green) in the tissue section of the MDA-MB-231
xenograft. Boxed region indicates the area enlarged in lower panels. Representative co-localizations were indicated
by arrows. GRP78 and CD44v were detected by MAb159 and anti-CD44v3 antibody, respectively. Thickness of image
section: 0.3 µm. The nuclei were stained by DAPI in blue. Scale bars, 20 µm (upper panels); 5 µm (enlarged images
in lower panels).
The control stainings for immunocytochemistry ruled out background from first primary and
secondary antibodies (Fig. 2.4, B and C). Similar immunofluorescent staining results could be
reproduced using commercially available anti-GRP78 antibody (clone: A-10, Santa Cruz
Biotechnology, Dallas, TX; Fig. 2.5) These results suggest CD44v could act as a major cell surface
partner protein of csGRP78 in a variety of breast cancer cells.
38
BRx-68
DIC
csGRP78 csCD44v DAPI Merge
BRx-07
B
C
csGRP78
No CD44v
primary DAPI Merge
MCF7-LR MDA-MB-231 BRx-68 BRx-07
MDA-MB-231
xenograft
BRx-68 BRx-07
No primary
control
MCF7-LR
MDA-MB-231
xenograft MDA-MB-231
A
DIC
39
Figure 2.4. More examples that show csGRP78 co-localizes ex vivo with CD44v in circulating tumor cells. (A)
Immunofluorescence and confocal images representing the distribution and co-localization of GRP78 (red) and
CD44v (green) on the cell surface of non-permeabilized breast cancer patient-derived BRx-68 and BRx-07 circulating
tumor cells. GRP78 and CD44v were detected by MAb159 and anti-CD44v3 antibody, respectively. Representative
co-localizations were indicated by arrows. DIC: differential interference contrast. Thickness of image section: 0.3 µm.
The nuclei were stained by DAPI in blue. Scale bars, 20 µm. (B) Mouse-on-mouse (M.O.M.) control stainings for Fig.
2.3 and supplementary Fig. 2.4 A. Cells were stained using the same protocol as double-stained cells but lacking
primary antibody targeting CD44v. Cell surface GRP78 was shown in red and background signal of CD44v (no CD44v
primary control) was displayed in green for non-permeabilized MCF7-LR, MDA-MB-231, BRx-68 and BRx-07 cells
and the tissue section of the MDA-MB-231 xenograft. The nuclei were stained by DAPI in blue. Scale bars, 20 µm
except for the BRx-68 cells (5 µm). (C) No primary control stainings for Fig. 2.3 and supplementary Fig. 2.4 A.
Immunofluorescence and confocal micrographs showing MCF7-LR, MDA-MB-231, BRx-68 and BRx-07 cells and
the tissue section of the MDA-MB-231 xenograft stained without the primary antibodies but with secondary antibodies.
The nuclei were stained by DAPI in blue. Scale bars, 20 µm.
DIC csGRP78 (A-10) csCD44v Merge
No anti-CD44v primary DIC csGRP78 (A-10) Merge
MCF7-LR M.O.M control
40
Figure 2.5. The anti-GRP78 antibody (clone A-10) reproduces immunofluorescent staining pattern of the other
anti-GRP78 antibody (MAb159). CD44v was detected by anti-CD44v3 antibody. GRP78 was stained with the
mouse monoclonal IgM (A-10) antibody from Santa Cruz Biotechnology (red, Dallas, TX) prior to CD44v staining
(green). Cell peripheries were outlined with white lines. DIC: differential interference contrast. Thickness of
immunofluorescent image section: 0.8 µm. Scale bars, 20 µm. M.O.M. control (ctrl) staining only lacked primary
antibody targeting CD44v.
2.3.3 CD44 can directly bind to GRP78 in vitro
Next, we used GST pull-down to map domains of GRP78 for CD44v binding. We first
constructed bacterial expression plasmids for GST-tagged full-length GRP78 (FL) and mutants
containing only the amino (N) or the carboxyl (C) half of GRP78 (Fig. 2.6 A) and mammalian
expression plasmid for HA-tagged CD44v (CD44v-HA) (Fig. 2.6 B). Then, we prepared
recombinant GRP78 proteins from E. coli (BL21) cells and confirmed the purity and identity of
these recombinant proteins by colloidal blue staining and Western blot analysis, respectively (Fig.
2.6 C). The identity of CD44v-HA was confirmed by Western blot analysis (Fig. 2.6 D), and the
observed 130 kD molecular size of CD44v-HA in 293T cells was different from the breast cancer
cells (Fig. 2.2 B) likely due to cell type-specific post-translational modifications. We then
performed GST pull-down assay and showed that CD44v-HA expressed in the WCL of 293T cells
formed complexes with GRP78 FL and C, but only minimally with N (Fig. 2.6 E), suggesting that
CD44v binds the carboxyl-half of GRP78.
To further determine whether CD44v can directly bind to GRP78, we constructed mammalian
expression plasmid for polyhistidine-tagged extracellular region of CD44v (CD44v-EC-His) (Fig.
2.6 B) and prepared the recombinant CD44v-EC-His protein from 293T cells. The purity of the
41
recombinant protein was confirmed by colloidal blue staining (Fig. 2.6 F). We then conducted in
vitro direct binding assay and the binding of CD44v-EC-Histo GST-GRP78 FL was observed but
not GST alone (Fig. 2.6 G). These results indicate that the carboxyl half of GRP78 can directly
bind to the extracellular domain of CD44v in cell free systems.
Figure 2.6. CD44v can directly bind to GRP78 in vitro. (A) Schematic representation of human GRP78, GST-
tagged GRP78 wild-type and deletion mutants. SBD, substrate binding domain. G, GST. a.a., amino acids. (B)
Schematic representation of human CD44 containing variable exon 3 to 10 and expression constructs including HA-
tagged CD44v (CD44v-HA) and His-tagged extracellular region of CD44v (CD44v-EC-His). EC, extracellular; TM,
transmembrane; IC, intracellular. a.a., amino acids. (C) Left: SDS-PAGE analysis followed by colloidal blue staining
of recombinant GST and GST-tagged full-length GRP78 (FL) and deletion mutants (N and C). Two µg was loaded
for each protein. Right: Western blot analysis of recombinant GST and GST-tagged GRP78 FL, N and C using
antibodies against GRP78 (76-E6 and then N-20). (D) Western blot analysis of whole cell lysate from 293T cells
expressing CD44v-HA using antibody against CD44 (102111). (E) Western blot analysis of samples from in vitro
GST pull-down assay. GST or GST-tagged GRP78 wild-type and mutant proteins purified from E. coli (BL21) were
A
D
C
G
654
GRP78
1 18
ER signal KDEL
ATPase SBD
125 280 400 500
GST-GRP78-C
654 413
GST-GRP78-N
407
19
GST-GRP78-FL
654 19
(a.a.) 700 1
CD44v3-10
ER signal TM
EC IC
21 607 629 (a.a.)
6XHis
607 1
CD44v-EC-His
700 1
CD44v-HA HA
N G FL
WB: HA
N C G vHA FL
WB: GST
C
80
60
50
30
25
40
100
230
80
60
50
30
25
40
100
230
(kD)
(kD)
WB: GST
G
100
80
58
30
25
46
135
FL
(kD)
FL
WB: CD44
CD44v-EC-His
FT
100
80
58
46
135
30
25
FL G G
Bound
(kD)
B
E
100
80
58
32
25
46
135
Colloidal Blue
G FL N C
(kD)
N
100
80
58
32
25
46
135
WB: GRP78
G FL C
(kD)
WB: CD44
CD44v-HA
100
80
58
32
25
46
135
190
(kD)
58
46
80
175
CD44v-EC-His
Colloidal
Blue
30
(kD)
F
G
G
G
42
incubated with 293T whole cell lysate containing overexpressed CD44v-HA. Input GST and GST-tagged GRP78
proteins were shown in the left panel. Input CD44v-HA was shown in the leftmost lane in the right panel. Experiments
were repeated three times with similar results. (F) SDS-PAGE analysis followed by colloidal blue staining of
recombinant CD44v-EC-His. Two µg was loaded. (G) Western blot analysis of samples from in vitro direct binding
assay. GST or GST-tagged full-length GRP78 (FL) proteins purified from E. coli (BL21) were incubated with freshly
prepared recombinant CD44v-EC-His purified from 293T cells. Input GST and GRP78 proteins were shown in the
left panel. The arrow indicates CD44v-EC-His. Flow through (FT) shows unbound fraction. CD44v-EC-His was
detected by the anti-CD44 (102111) antibody. Experiments were repeated four times with similar results.
Growing evidences have highlighted the isoform and cell type-specific functions of CD44
(reviewed in (Zoller 2011)), and CD44s was reported to mediate the invadopodia activity in the
MDA-MB-231 cells (Zhao et al. 2016). Therefore, we tested if CD44s could form complex and
directly bind to GRP78. We constructed the expression plasmid containing HA-tagged CD44s
(CD44s-HA, Fig. 2.7 A) and verified its identity by SDS-PAGE and Western blot analysis using
the antibody against CD44 (clone:102111, Fig. 2.7 B). We found two major isoforms of CD44s-
HA with observed molecular weights between 45 kD and 60 kD in 293T cells (Fig. 2.7 B). We
then performed GST pull-down assay and showed that CD44s-HA expressed in the WCL of 293T
cells formed complexes with GRP78 FL, N and C, but N exhibited lower affinity to CD44s-HA
(Fig. 2.7 C). To further determine whether CD44s can directly bind to GRP78, we constructed
mammalian expression plasmid for polyhistidine-tagged extracellular region of CD44s (CD44s-
EC-His) (Fig. 2.7 A) and prepared the recombinant CD44v-EC-His protein from 293T cells. The
purity of the recombinant protein was confirmed by colloidal blue staining (Fig. 2.7 D). We then
conducted in vitro direct binding assay and the binding of lower molecular weight CD44s-EC-His
to GST-GRP78 FL was observed but not GST alone (Fig. 2.7 E). These results indicate that the
lower molecular weight CD44s can form complex with GRP78 through direct binding in the in
43
vitro cell free systems, however, the higher molecular weight CD44s likely forms complex with
GRP78 through indirect means.
Figure 2.7. The lower molecular weight CD44s can directly bind to GRP78 in vitro. (A) Schematic representation
of human CD44 standard isoform (CD44s) and expression constructs including HA-tagged CD44s(CD44s-HA) and
His-tagged extracellular region of CD44s (CD44s-EC-His). EC, extracellular; TM, transmembrane; IC, intracellular.
a.a., amino acids. (B) Western blot analysis of whole cell lysate from 293T cells expressing CD44s-HA using antibody
against CD44 (102111). (C) Western blot analysis of samples from in vitro GST pull-down assay. GST (G) or GST-
tagged full-length GRP78 (FL) and deletion mutants (N and C) purified from E. coli (BL21) were incubated with
293T whole cell lysate containing overexpressed CD44s-HA. Input GST and GST-tagged GRP78 proteins were shown
in the left panel. Input CD44s-HA (sHA) was shown in the leftmost lane in the right panel. Experiments were repeated
two times with similar results. (D) SDS-PAGE analysis followed by colloidal blue staining of recombinant CD44s-
EC-His. Two µg was loaded. (E) Western blot analysis of samples from in vitro direct binding assay. GST or GST-
tagged full-length GRP78 (FL) proteins purified from E. coli (BL21) were incubated with freshly prepared
recombinant CD44s-EC-His purified from 293T cells. Input GST and GRP78 proteins were shown in the left panel.
80
60
50
40
WB: HA
N C G sHA FL C
WB: GST
100
80
58
32
25
46
135
N G FL
361
CD44s
1
HA CD44s-HA
6XHis
268 1
L
CD44s-EC-His CD44s
268 1
CD44s
ER signal TM
IC EC
361 290 (a.a.) 21
100
80
25
135
WB: GST
FL G
(kD)
Input: sEC
FL G FL G
58
46
(kD)
WB: CD44 WB: CD44
(Flow thru)
58
46
80
100
CD44s-EC-His
Colloidal Blue
(kD)
80
60
50
40
100
135
CD44s-HA
WB: CD44
(kD)
B C D
A
E
(kD) (kD)
44
CD44s-EC-His was detected by the anti-CD44 (102111) antibody. Flow through (thru) shows unbound fraction.
Experiments were repeated two times with similar results.
2.3.4 CD44 containing exon v3 is a major mediator of CD44 functions in aggressive breast
cancer
The CD44v isoform, CD44v3-10 (250 kD), identified in this study has been correlated with
poor prognosis of breast cancer patients (Hu et al. 2017). We next sought to examine the
importance of CD44 containing variable exon 3. We first investigated the CD44 protein expression
patterns in MCF7-LR and MDA-MB-231 breast cancer cells using the antibodies targeting the
common epitope (A1351) or specifically on variable exon 3 (BMS144) by co-immunostaining and
confocal microscopy (Fig. 2.8, A and B). We found substantial co-localization of A1351 and
BMS144 signals, indicating CD44 containing variable exon 3 is the major isoform in MCF7-LR
and MDA-MB-231 cells (Fig. 2.8, A and B). Of note, we observed that a significant population of
BMS144 signal (CD44v) did not co-localize with the A1351 signal in MCF7-LR cells (Fig. 2.8
A). This is likely due to differential post-translational modifications, as CD44 is one of the most
heavily post-translationally modified protein at the plasma membrane.
45
Figure 2.8. Co-localization and distribution of CD44 isoforms in breast cancer cells. (A and B)
Immunofluorescence and confocal images showing the distribution and co-localization of CD44 (A1351, red) and
CD44v (BMS144, green) on the cell surface of non-permeabilized MCF7-LR (A) and MDA-MB-231 (B) breast cancer
cells. Anti-CD44 antibody (A1351) targets common region of all CD44 isoforms. CD44v were detected by the anti-
CD44v3 antibody (BMS144). No primary control stainings lacked primary antibodies but with Alexa Fluor
®
647 or
Alexa Fluor
®
568-conjugated secondary antibodies. Thickness of image sections: 0.3 µm. Scale bars, 20 µm.
A
CD44(A1351) CD44v(BMS144) DAPI Merge
Alexa 647 Alexa 568 DAPI Merge
MDA-MB-231
No primary control
CD44(A1351) CD44v(BMS144) DAPI Merge
Alexa 647 Alexa 568 DAPI Merge
MCF7-LR
No primary control
B
46
Next, we explored the functional significance of CD44 containing variable exon 3. As CD44
has been shown to play important roles in regulating cytoskeletal dynamics and cell adhesion, we
evaluated the functions of CD44 containing variable exon 3 using cell morphology, F-actin
structure, cell spreading and attachment in CD44-knockdown cells (Fig. 2.9 A-D). We developed
pLKO.1-shRNAs targeting the common region of CD44 isoforms (sh44-2) (Godar et al. 2008) or
specifically on variable exon 3 (sh44v3). The non-targeting shRNA controls included two
scrambled sequences matched the ATCG compositions of the individual targeting sequence
(shCtrl-1 and shCtrl-2). Because the control group of MDA-MB-231 cells showed normal growth
properties compared to MCF7-LR cells during our selection procedure, we used MDA-MB-231
cells to establish stable CD44-knockdown cells. Knockdown of CD44 by both sh44-2 and sh44v3
effectively reduced CD44v levels as demonstrated by flow cytometry (Fig. 2.9 A). While the cells
stably transfected with control shRNAs displayed intact F-actin networks, both stable CD44-
knockdown cells showed similar extent of disorganized F-actin (Fig. 2.9 B). In addition, both
CD44-knockdown cells exhibited 70-80% decrease of cell spreading (Fig. 2.9 C) and 50-60%
reduction of cell adhesion compared to the scrambled control (Fig. 2.9 D). These results are
consistent with the critical roles of CD44 in regulating F-actin reported in other cell types
(Bourguignon et al. 2005, Acharya et al. 2008). Importantly, knockdown of CD44 containing
variable exon 3 resulted in similar phenotypes as knockdown of CD44 through targeting the
common epitope. This indicates that CD44 containing v3 exon plays major roles in regulating F-
actin network regarding the cellular functions of cell morphology, spreading and adhesion in the
MDA-MB-231 cells. Of note, both knockdown of CD44v and GRP78 (Fig. 2.1 G) resulted in F-
actin disorganization in the breast cancer cells, suggesting that GRP78 may regulate cytoskeletal
dynamics in part via partnership with CD44v.
47
Figure 2.9. Stable knockdown of CD44 disrupted F-actin integrity, cell spreading and cell attachment in MDA-
MB-231 cells. (A) Flow cytometry histograms representing the levels of CD44v on the cell surface of stable MDA-
MB-231 cells transfected with shRNAs targeting common sequence of CD44 (sh44-2), specifically on variable exon
3 (sh44v3) or non-targeting scrambled controls (shCtrl-1 and shCtrl-2). % of max: percentage of maximum staining
intensity. CD44v was detected by the anti-CD44v3 antibody. Cyan, Anti-CD44v3; Red, Isotype IgG. Cell number
analyzed in each group is about 30,000. (B) Upper: Bright-field micrographs of stable MDA-MB-231 cells in culture.
Lower: Immunofluorescence and confocal images showing the morphology and F-actin organization of stable MDA-
MB-231 cells seeded on collagen I-coated coverslips. F-actin was stained by rhodamine phalloidin in red and the
nuclei were stained by DAPI in blue. Scale bars, 20 µm. (C) Quantification of cell spreading area of rhodamine
phalloidin-stained stable MDA-MB-231 cells using the FIJI-ImageJ software. Data represents means ± S.E.M. ***P<
0.001 by Student’s t test. (D) Cell attachment assay. Non-enzymatically dissociated stable MDA-MB-231 cells were
re-seeded on collagen I-coated culture plate for 13 min and then stained by crystal violet. Crystal violet staining was
0
0.2
0.4
0.6
0.8
1
1.2
Cell attachment (OD
595
)
0
20
40
60
80
100
Spreading area (μm
2
)
***
***
A
B
sh44v3 shCtrl-2 sh44-2
CD44v-Alexa 647
shCtrl-1
% of max
Anti-CD44v3
Isotype IgG
shCtrl-2 sh44-2 sh44v3
F-actin
DAPI
shCtrl-1
D
C
***
***
48
measured by spectrophotometer at O.D. 595 from three experimental repeats. Data represents means ± S.E.M. ***P<
0.001 by Student’s t test.
2.3.5 Dual-color single particle tracking reveals the interaction and co-confinement of
GRP78 and CD44v in plasma membrane nanodomains
To characterize further the interaction of GRP78 and CD44v with high spatial resolution, we
employed single molecule tracking and total internal reflection fluorescence (TIRF) microscopy.
We constructed two expression plasmids with GRP78 fused to the photoactivatable Tag-red
fluorescent protein (PATagRFP-GRP78) and CD44v fused to the photoactivatable green
fluorescent protein (CD44v-PAGFP) for dual-color single particle tracking by photoactivated
localization microscopy (sptPALM) (Subach et al. 2010) (Fig. 2.10 A). Proper cell expression of
both fusions was confirmed by Western blot analysis (Fig. 2.10, B and C). When imaged by TIRF
on live MCF7-LR cells, lateral diffusion and partial co-localization of GRP78 and CD44vwere
observed in the plasma membrane as shown in the conventional TIRF images and the images of
super-resolved positions for both proteins (Fig. 2.10 D).
The distribution of diffusion coefficients determined by the analysis of individual mean square
displacements for CD44v and GRP78 indicated that both proteins display rather heterogeneous
lateral mobility at the cell surface, with GRP78 diffusing significantly more slowly than CD44v
(Fig. 2.10 E). A more detailed diffusion analysis by probability distribution of the squared
displacement (PDSD) revealed two equal populations of fast (49%) and slow (51%) diffusing
CD44v, respectively undergoing free diffusion at the cell surface (D1-CD44v = 0.213 ± 0.004 µm
2
/s)
or confined diffusion (D2-CD44v = 0.016 ± 0.001 µm
2
/s) in membrane nanodomains having a mean
radius size of 74 nm (Fig. 2.10 F). These diffusion coefficients and the respective fractions of fast
49
and slow diffusing populations are in good agreement with previous single particle tracking and
fluorescence recovery after photobleaching of CD44 in other cells (Jacobson, O'Dell, and August
1984, Wang et al. 2014) and with its ability to cluster in glycosphingolipid-rich plasma membrane
domains (Wang et al. 2014, Ilangumaran, Briol, and Hoessli 1998).
Using the same PDSD analysis on GRP78, we also identified two confined diffusive behaviors
of GRP78. A minority fraction of fast diffusing GRP78 (24%, D1-GRP78 = 0.104 ± 0.006 µm
2
/s)
was confined in membrane domains 280 nm in radius, while a majority fraction (76%) diffused
more slowly (D2-GRP78 = 0.00095 ± 0.0006 µm
2
/s) in 65 nm radius nanodomains, a size similar to
the confinement domain of slow diffusing CD44v (Fig. 2.10 F). From an observation of GRP78
and CD44v diffusion trajectories, we detected cases where both fluorescent protein fusions
undergo correlated lateral diffusion as well as co-confinement in the same nanodomains within the
plasma membrane (Fig. 2.10 G), consistent with the interactions and binding between GRP78 and
CD44v observed in biochemical assays. The similarity in nanodomain size for both slow diffusing
populations of GRP78 and CD44v (65 nm vs. 74 nm) also suggested that the proteins might indeed
co-diffuse in shared membrane nanodomains. To further assess how GRP78 influences the
dynamics and the confinement of CD44v, endogenous GRP78 was knocked down by siRNA and
CD44v was again tracked at the plasma membrane of MCF7-LR cells. As shown in Fig. 2.10, H
and I, reduced expression of GRP78 led to significantly faster diffusion of the free and fast
diffusing subpopulation of CD44v (56%, Fig. 2.10 H). Interestingly, GRP78 knockdown resulted
in significantly slower diffusion for the slow diffusing subpopulation of CD44v (44%, Fig. 2.10
H). In MCF7-LR cells treated with a control siRNA, no significant differences in CD44v diffusion
coefficients were observed compared to non-treated cells (Fig. 2.10 H). Together, these data
50
suggest that GRP78 interacts to CD44 at the plasma membrane nanodomains, and the expression
of GRP78 impacts the diffusive behavior and the interaction of CD44 with nanometer size domains.
D
H
F
E
G I
NT sictrl si78
Diffusion coefficient
(X0.1µm
2
/s)
n.s.
**
Fast population
NT sictrl si78
Diffusion coefficient
(X0.01µm
2
/s)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 n.s.
**
Slow population
CD44v-PAGFP
Displacement squared r
2
2
(X0.001µm
2
)
3
4
5
6
7
D
2
: 0.00095±0.0006 µm
2
/s
r
2
: 65nm
Time (X0.01 s)
Displacement squared r
1
2
(X0.01µm
2
)
D
1
: 0.104±0.006 µm
2
/s
r
1
: 280nm
Time (X0.01 s)
1
3
4
5
6
8
7
2
Slow population (76%)
Fast population (24%)
PATagRFP-GRP78
Displacement squared r
2
2
(X0.001µm
2
)
2
3
4
5
6
7
D
2
: 0.016±0.001 µm
2
/s
r: 74nm
Time (X0.01 s)
Displacement squared r
1
2
(X0.01µm
2
)
D
1
: 0.213±0.004 µm
2
/s
2
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Time (X0.01 s)
10
15
20
30
0
25
5
Slow population (51%) Fast population (49%)
CD44v-PAGFP
PATagRFP-GRP78
CD44v-PAGFP
Cumulative probability
0
20
40
60
80
100
1 10
10
-1
10
-2
10
-3
10
-4
10
-5
Diffusion coefficient (µm
2
/s)
**
PATagRFP-GRP78
CD44v-PAGFP
Conventional TIRF imaging
PATagRFP-GRP78
CD44v-PAGFP
Super-resolved image
PATagRFP-GRP78
CD44v-PAGFP
1.00 0.28
sictrl siGrp78
β-actin
GRP78
80
46
(kD)
B A
PAGFP CD44v3-10
CD44 ER signal
CD44v-PAGFP
GRP78 PATagRFP
GRP78 ER signal KDEL
PATagRFP-GRP78
CD44v-PAGFP
WB: CD44
100
80
58
30
25
46
135
190
(kD)
100
80
58
46
135
190
WB: GRP78
PATagRFP-GRP78
Endogenous GRP78
(kD)
C
51
Figure 2.10. Dual-color single particle tracking reveals the interaction and co-confinement of GRP78 and
CD44v in plasma membrane nanodomains. (A) Schematic illustration showing the constructs of PATagRFP-
GRP78 and CD44v-PAGFP. (B) Western blot analysis of whole cell lysate (WCL) from MDA-MB-231 cells
overexpressing PATagRFP-GRP78 using antibody against GRP78 (MAb159). (C) Western blot analysis of WCL
from 293T cells overexpressing CD44v-PAGFP using antibody against CD44 (102111). (D) Conventional TIRF
(upper panels) and super-resolved (lower panels) images of PATagRFP-GRP78 (red) and CD44v-PAGFP (green) in
MCF7-LR cells. Boxed regions were enlarged in the right panels. Dashed circles highlight co-localized area. Scale
bars, 5 µm. (E) The distribution of diffusion coefficients was determined by the analysis of individual mean square
displacements for CD44v-PAGFP (n=51540 trajectories) and PATagRFP-GRP78 (n=37269 trajectories). **P<0.01
by Kolmogorov-Smirnov test. (F) Diffusion analysis by probability distribution of the squared displacement (PDSD)
showing quantifications of square displacement per second (D) and radius of nanodomain (r) in each fast and slow
population of CD44v-PAGFP and PATagRFP-GRP78. nm, nanometer. (G) Examples of CD44v-PAGFP (green) and
PATagRFP-GRP78 (red) co-diffusion at the plasma membrane. Circles represent the localization error at each position
for individual CD44v-PAGFP (light green) and PATagRFP-GRP78 (light red) along their respective path of diffusion.
Yellow areas indicate effective co-localization within position error. Scale bar, 100 nm. (H) The diffusion coefficients
of CD44v-PAGFP showing fast and slow populations in non-treated (NT) cells and cells treated with control (sictrl)
or Grp78 (si78) siRNA. Trajectory number (n) analyzed in the study: n=102957 (sictrl) n=164741 (si78), **P<0.01
(Student's t test) n.s., not significant. (I) Western blot analysis of MCF7-LR cells transfected with control (sictrl) or
Grp78 (siGrp78) siRNA for the diffusion tracking analysis. β-actin is loading control. Numbers below the GRP78
bands represent relative levels of GRP78 and are derived from the ratio of GRP78 to β-actin.
2.3.6 Antibody against GRP78 alters F-actin dynamics and cell polarity in breast cancer cells
To further investigate if csGRP78 can regulate F-actin dynamics and cellular functions, we
treated MCF7-LR cells with a panel of commercially available anti-GRP78 antibodies and
identified a rat monoclonal anti-GRP78 antibody (76-E6) that has the highest potency in altering
F-actin structure and cell morphology in 24 h after treatment (Fig. 2.11 A). We then performed
52
cell adhesion and spreading assay and found that treatment of cells with 76-E6 led to about 60%
reduction in cell adhesion, as calculated by the ratio of the cells left on the culture plate (Fig. 2.11
B; upper panel; 76-E6/IgG= 294/754), and decreased cell spreading (Fig. 2.11 B; lower panel) in
MCF7-LR cells. Statistical analyses of the individual cell morphology by the FIJI-ImageJ software
showed that 76-E6 treatment resulted in less cell protrusions (higher circularity score) and reduced
cell polarity (higher roundness score) (Fig. 2.11 C). We then used the CD44 ligand, hyaluronan,
to stimulate F-actin formation and further showed that treatment of MCF7-LR cells with 76-E6
caused disorganized F-actin (Fig. 2.11 D, arrows) and rounder cell periphery (Fig. 2.11 D,
arrowheads) compared to the IgG control. At 48 h post 76-E6 treatment, we observed bigger and
rounder cells compared to the control (Fig. 2.11 E). Furthermore, we found that treatment of
MCF7-LR cells with 76-E6 inhibited cell invasion as measured by degradation of fluorescent
matrix (Fig. 2.11 F). We then tested if 76-E6 treatment led to cell death within the timeframe of
our experiments, and we found no significant difference between 76-E6 and control IgG at 24 and
48 h post treatment (Fig. 2.11 G).
53
B D
IgG 76-E6
IgG
76-E6
FITC-gelatin DAPI Merge
IgG 76-E6
MCF7-LR
0
20
40
60
80
100
120
IgG
76-E6
Gelatin degradation (% of IgG)
***
Roundness
0
0.5
1
0 40008000 12000
Circularity
Whole cell area (px
2
)
IgG
0 4 8 12
0
0.5
1
0 4000 800012000
Whole cell area …
76-E6
0 4 8 12
0
0.5
1
0 4000 8000 12000
Whole cell …
Whole cell area
(x1000 px
2
)
Whole cell area
(x1000 px
2
)
0
0.5
1
0 4000 800012000
Whole cell area …
Whole cell area
(x1000 px
2
)
Whole cell area
(x1000 px
2
)
0 4 8 12 0 4 8 12
IgG 76-E6
Hyaluronan
DPBS
MCF7-LR
F-actin
DAPI
E C
IgG 76-E6
MCF7-LR
Goat IgG Rat IgG N-20 C-20 76-E6
654
KDEL
GRP78
76-E6
1
ER Signal
N-20 C-20
18
ATPase SBD
280 125 400 500 651
A
F
0
5
10
15
20
24 48
Cell death (%)
IgG
76-E6
MCF7-LR
n.s.
n.s.
(h)
G
54
Figure 2.11. Antibody against GRP78 suppresses MCF7-LR breast cancer cell adhesion, spreading, and matrix
degradation. (A) Upper: Schematic illustration of the target regions of the antibodies targeting GRP78. Lower:
Immunofluorescence micrographs showing the morphology and F-actin organization of MCF7-LR cells treated with
the indicated antibodies and the corresponding control IgG. Green, F-actin stained by ActinGreen
TM
488; blue, nuclei
stained by DAPI. Scale bar, 20 µm. (B and C) (B) Cell adhesion and spreading assay. Representative light
micrographs showing crystal violet-stained MCF7-LR cells treated with rat anti-GRP78 monoclonal antibody (clone:
76-E6) or control IgG for 24 h and then re-seeded onto collagen I-coated culture plates for 7 h. Representative
individual cells were enlarged in lower panels with same magnification. Scale bar, 20 µm. (C) Circularity and
roundness of individual cells from (A) were quantified by the FIJI-ImageJ software. Circularity= 4π × Area/Perimeter
2
.
Roundness= 4 × Area/(π × Major axis
2
). Cell numbers (n) analyzed in the study: n=754, IgG n=294, 76-E6. (D)
Immunofluorescence micrographs showing the morphology and F-actin organization of MCF7-LR cells treated with
76-E6 or control IgG. The cells were treated with hyaluronan or DPBS control for 4 h. The arrows indicate F-actin.
The arrowheads highlight cell edges. Boxed regions were enlarged in the right panels. Green, F-actin stained by
ActinGreen
TM
488; blue, nuclei stained by DAPI. Scale bar, 20 µm. (E) Bright-field micrographs showing the
morphology of MCF7-LR cells treated with 76-E6 antibody or control IgG for 48 h. Double arrowheads indicate
protrusion-like cell edges. Scale bar, 20 µm. (F) Matrix degradation assay. Left: Representative confocal fluorescent
images of MCF7-LR cells seeded on coverslips coated with FITC-conjugated gelatin and incubated with 76-E6
antibody or control IgG for 24 h. The nuclei were stained by Hoechst 33342 in blue. Scale bar, 20 µm. Right:
Quantification of gelatin degradation compared to control. Data represents means ± S.E.M. from 5 independent image
areas. ***P< 0.001 by Student’s t test. (G) MCF7-LR cells were treated with 76-E6 antibody or control IgG for 24 or
48 h and the cell death was measured by the trypan blue dye exclusion assay. Data represents means ± S.E.M from
two biological repeats. Significance was assessed by Student’s t test. n.s., not significant.
Because MDA-MB-231 cells are fast moving and most of them exhibited unipolar morphology,
we next took these advantages and used this cell line to study the functional roles of csGRP78 in
cell motility and polarity. We conducted time-lapse imaging of spontaneous cell migration and
showed that 76-E6 treatment reduced velocity, straightness (directional persistence) and spreading
55
in MDA-MB-231 cells (Fig. 2.12, A and B). Interestingly, MDA-MB-231 cells treated with 76-
E6 showed about 20% increase of cells exhibiting more than 3 protrusion-like cell edges (unipolar
cells were counted as 2 protrusion-like edges) and most cells showed rounder cell periphery (Fig.
2.12 C). Live MDA-MB-231 cells transfected with mCherry-tagged actin-binding peptide (ABP-
mCherry) and then treated with 76-E6 exhibited imbalanced anteroposterior distribution of F-actin
networks (Fig. 2.12 D). These results indicate that csGRP78 can regulate F-actin dynamics and
cell polarity in MDA-MB-231 cells.
A
B
C
MDA-MB-231
IgG 76-E6
D
IgG 76-E6
MDA-MB-231
**
***
Velocity
Straightness
IgG 76-E6 IgG 76-E6
IgG 76-E6
0
40
80
120
(min)
MDA-MB-231
ABP-mcherry
MDA-MB-231
IgG 76-E6
45%
37%
14%
4%
64%
28%
7%
1%
2 3 4 ≥5
IgG 76-E6
56
Figure 2.12. Antibody against GRP78 suppresses MDA-MB-231 breast cancer cell motility, migration and
alters cell polarity. (A) Upper: Superimposed tracks of control IgG (n=32) and 76-E6 antibody (n=28)-treated MDA-
MB-231 cells during 7 h random migration. Lower: Comparisons of velocity (displacement/time) and straightness
(displacement/total path length) of the cells. **P<0.01 ***P<0.001 (B) Representative time-lapse DIC images of
MDA-MB-231 cells during 2 h observation. The solid lines demark the cell borders. The dotted lines indicate the
positions of the cells. min, minute(s). (C) Upper: Representative bright-field micrographs showed the morphology of
MDA-MB-231 cells treated with 76-E6 antibody or control IgG for 61 h. Protrusion-like cell edges were more
frequently observed in 76-E6 antibody-treated cells (double arrowheads). Lower: The percentage of cells displaying
the indicated number of protrusion-like cell edges. Total number of cells analyzed in the study: 2699, IgG; 942, 76-
E6. (D) Confocal micrographs showing F-actin structure of live MDA-MB-231 cells treated with 76-E6 antibody or
control IgG for 24 h. F-actin was visualized through mcherry-tagged actin binding peptide (ABP-mcherry). Arrow
indicates the front-rear cell polarity. Scale bars, 20 µm.
2.3.7 Targeting csGRP78 by antibody suppresses polarized distribution of IQGAP1 and
active CDC42
To further determine how csGRP78 regulates F-actin network and cell polarity, we purified
cell surface proteins from MCF7-LR cells and performed co-immunoprecipitation (co-IP) with
either control IgG or anti-GRP78 antibody (MAb159), followed by liquid chromatography-tandem
mass spectrometry (LC-MS/MS). We identified a prominent band near 175 kD in the anti-GRP78
co-IP lane but not at the corresponding site of control IgG lane by mass spectrometry as the multi-
scaffold protein, IQGAP1 (IQ motif containing GTPase activating protein 1) (Fig. 2.13 A). We
also identified a panel of proteins associated with csGRP78 that bears important functions in
regulation of F-actin dynamics, cell motility, migration, metastasis, matrix degradation and
exocytosis (Table 2.1). IQGAP1 was shown to directly bind to activated GTP-bound Rho kinases,
including CDC42 and RAC1, and act as a downstream effector protein that crosslinks F-actin and
57
controls cell polarity (Kuroda et al. 1996, Fukata et al. 1997, Watanabe, Wang, and Kaibuchi 2015).
In addition, a previous study has showed that hyaluronan-CD44 interaction with IQGAP1
modulates CDC42 activity (Bourguignon et al. 2005). We then investigated the roles of csGRP78
in regulating IQGAP1 and active CDC42 and observed that 76-E6 treatment blocked polarized
accumulation of both IQGAP1 and active CDC42 at the leading edge of MCF7-LR cells utilizing
confocal microscopy and time-lapse fluorescence resonance energy transfer (FRET) imaging (Fig.
2.13, B and C). Collectively, these results suggest IQGAP1 as a downstream signaling node for
csGRP78, which regulates cell polarity through modulation of subcellular localization of polarity-
related proteins.
Figure 2.13. Targeting csGRP78 by antibody suppresses polarized distribution of IQGAP1 and active CDC42.
(A) Biotinylated cell surface proteins from MCF7-LR cells were subjected to monomeric avidin pull-down and co-
immunoprecipitation (co-IP) using control IgG or antibody against endogenous GRP78 protein (MAb159). SDS-
A
B
175
80
58
46
IgG GRP78
Hc
GRP78
IQGAP1
Puri. CSP
(IP)
IgG 76-E6
F-actin IQGAP1 Enlarged
C
Low
High
IgG
30 45 60 75
76-E6
Fp
Active CDC42
(min)
Cell lysis
Co-IP
Monomeric avidin
pull-down
Biotin Labeling
(cell impermeable)
Elution
SDS-PAGE
LC-MS/MS
Excision
58
polyacrylamide gel electrophoresis (SDS-PAGE) was performed, followed by colloidal blue staining. The gel slices
of bands of interest were subjected to LC-MS/MS. IQGAP1 was identified as one of the top candidates forming
complex with csGRP78. Corresponding sites of gel slices in IgG control were excised and served as controls of LC-
MS/MS. Puri. CSP, purified cell surface proteins; Hc, heavy chain. (B) Immunofluorescent staining and confocal
micrographs showing the distribution of F-actin and IQGAP1 in MCF7-LR cells treated with 76-E6 antibody or
control IgG for 24 h. Boxed regions were enlarged in the right panels. Arrows indicate leading edges. Scale bar, 20
µm. (C) Time-lapse ratiometric FRET/CFP images of MCF7-LR cells expressing Raichu-Cdc42 FRET biosensor.
Cells were treated with either 76-E6 antibody or control IgG for 24 h and then stimulated with 100 µg/mL hyaluronan
before imaging. Time-lapse images were taken from 30 min to 75 min after stimulation. Polarized accumulations of
active CDC42 were indicated by arrows. Hollow arrows indicate directions of cell migration. Red indicates high and
blue indicates low CDC42 activation. Fp: filopodia.
59
60
2.3.8 Antibodies targeting csGRP78 reduce CD44 protein level and suppress CD44
downstream signaling
We next investigated the consequences of perturbing csGRP78 on CD44v and its downstream
signaling. Interestingly, we found that treatment of MCF7-LR cells with 76-E6 led to substantial
reduction of CD44v levels as evidenced by flow cytometry, immunofluorescent imaging and
Western blot analysis (Fig. 2.14, A-C). We also observed reduction of CD44v upon 76-E6
treatment in MDA-MB-231 cells (Fig. 2.14 D). The 76-E6 treatment also resulted in partial
reduction of GRP78 in MCF7-LR cells (Fig. 2.14 C). As membrane-associated matrix
metalloproteinases (MMPs) has been shown to cleave CD44 (Okamoto et al. 1999), we asked if
the reduction of CD44v upon the treatment of 76-E6 was due to proteolytic cleavage by MMPs.
To address this issue, we added the broad-spectrum MMP inhibitor, BB-94, into culture media
together with the 76-E6 treatment and found the level of CD44v reduction was similar to the
DMSO control (Fig. 2.14 C). Therefore, the observed reduction of CD44v was not due to MMP
activity.
61
Figure 2.14. Targeting csGRP78 by antibody results in reduction of CD44 protein level. (A) Flow cytometry
histograms representing the levels of CD44v in the non-treated (NT) MCF7-LR cells or the cells treated with 76-E6
antibody or control IgG for 48 h. The cells were cultured in the collagen I (100 µg/ml) coated plates. CD44v was
detected by anti-CD44v3 antibody. % of max: percentage of maximum staining intensity. Cyan, Anti-CD44v; Red,
Isotype control IgG. Cell number analyzed in each group was about 10,000. (B) Immunofluorescence and compressed
z-stack confocal images showing the CD44v (green) levels on the cell surface of non-permeabilized MCF7-LR cells
treated with 76-E6 antibody or control IgG for 24 h. The cells were seeded on the collagen I (100 µg/ml) coated
coverslips. CD44v was detected by anti-CD44v3 antibody. Nuclei were stained by DAPI in blue. Scale bar, 20 µm.
A
B
C
76-E6 IgG
CD44v
DAPI
D
76-E6 NT IgG 76-E6 NT IgG
DMSO BB-94
GRP78
β−actin
CD44v
MCF7-LR
245
80
46
Anti-CD44v
Isotype IgG
NT IgG 76-E6
% of max
CD44v-Alexa 647
1.00 0.88 0.84
1.00 1.00 0.67
CD44v
GAPDH
GRP78
NT IgG 76-E6
MDA-MB-231
245
32
80
62
(C) Western blot analysis of non-treated (NT) MCF7-LR cells or the cells treated with 76-E6 antibody or control IgG
for 24 h. The broad-spectrum metalloproteinase inhibitor (BB-94) or vehicle control (DMSO) was added into the
culture medium together with the antibody groups. CD44v and GRP78 were detected by anti-CD44v3 and MAb159
antibodies, respectively. (D) Western blot analysis of whole cell lysate from non-treated (NT) MDA-MB-231 cells
and the cells treated with 76-E6 antibody or control IgG for 24 h. CD44v and GRP78 were detected by anti-CD44v3
and MAb159 antibodies, respectively. Numbers below the bands represent relative levels of the indicated proteins and
are derived from the ratio of the indicated proteins to GAPDH.
To further understand the underlying mechanism of the reduction of CD44v, we treated MCF7-
LR cells with 76-E6 or control IgG and then co-stained cells with anti-CD44v and anti-Rab5, an
early endosome marker, antibodies. We analyzed the co-localization of CD44v and Rab5 using
confocal microscopy and the Coloc 2 plug-in (Mander’s overlap coefficient) in the FIJI-ImageJ
software and discovered a modest increase of CD44v endocytosis after 76-E6 treatment compared
to the IgG control (Fig. 2.15, A and B). We then investigated if the reduction of CD44v protein
level was due to decrease of transcription using reverse-transcription quantitative PCR (RT-qPCR)
and primers targeting common regions or specific variable exons (Fig. 2.15 C). We found that 76-
E6 treatment resulted in compensatory increase of overall CD44 transcripts, and this is due to
elevation of CD44 transcripts containing variable exon 3 or 6 but not CD44 standard (CD44s)
isoform (Fig. 2.15 C).
63
Figure 2.15. Targeting csGRP78 by antibody leads to modest increase of CD44v endocytosis and up-regulation
of CD44v RNA expression. (A) Immunofluorescence and confocal images showing the co-localization of CD44v
(red) and Rab5 (green, early endosome marker) in the saponin permeabilized MCF7-LR cells. The cells were treated
with 76-E6 antibody or IgG for 24 h before the staining. CD44v was detected by the anti-CD44v3 antibody. Boxed
regions were enlarged on the right panels. Thickness of single immunofluorescent image section: 0.3 µm. Scale bar,
5 µm. (B) Co-localization of CD44v and Rab5 was analyzed by the Coloc 2 plug-in in the FIJI-ImageJ software.
Mander’s overlap coefficient: M1 is the contribution of CD44v to the co-localized area. Number of analyzed
independent image areas (A) and cells (N): A/N=19/13 (IgG); 17/10 (76-E6). Data represents means ± S.E.M. **P<
0.01 by Student’s t test. This research has made use of the visualization and analysis from BioVinCi version 0.9.1, an
application developed by BioTuring Inc. (C) Quantitative reverse transcription PCR (RT-qPCR) was performed to
measure the quantity of CD44 transcripts after 24 h treatment of the 76-E6 antibody or control IgG in MCF7-LR cells.
0.80
0.75
0.70
0.65
0.60
0.55
0.50
IgG 76-E6
Endocytosis Index (M1)
**
A B
CD44v Rab5 Merge Enlarged
IgG 76-E6
MCF7-LR
No anti-CD44v
primary
No anti-Rab5
primary
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Relative RNA level of CD44
isoforms
IgG
76-E6
***
n.s.
**
*
C
TM C Extracellular (EC)
CD44s
CD44v3-10
v3 v4 v5 v6 v7 v8 v9 v10
1 2 3 4 5 7 8 9 11 12 13 14 15 16 10 17 18 19
5’ 3’
5’ 3’
CD44t
CD44s (162 bp)
CD44v3-f
CD44v3-r
CD44v6-f
CD44v6-r
64
Upper: Schematic illustration of primer target sites for each CD44 isoform. TM, transmembrane C, cytosolic t, totals,
standard f, forward r, reverse Lower: Relative RNA levels. Data represents means ± S.E.M. from three repeats. n.s.,
not significant ***P< 0.001 **P< 0.01 *P≤ 0.05 by Student’s t test.
We next explored whether CD44v could act as a signaling transducer of csGRP78. Previous
studies have shown that STAT3 activation, as indicated by STAT3 phosphorylation at tyrosine
705, played important roles in mediating CD44v signaling, drug resistance and invasion of breast
cancer cells (Wang et al. 2012, Xu et al. 2015). Stable knockdown of CD44 in MDA-MB-231 cells
reduced STAT3 activation and the STAT3 downstream Cyclin D1 protein levels (Fig. 2.16 A).
We then found that treatment of MCF7-LR cells with 76-E6 led to substantial reduction of STAT3
activation compared to the IgG and the non-treated (NT) control. This effect was mediated by
CD44v since overexpression of full-length HA-tagged CD44v nearly completely rescued STAT3
activation suppressed by 76-E6 (Fig. 2.16 B). These results suggest CD44v is an important
signaling mediator of csGRP78 and intracellular STAT3 signaling.
A B
pSTAT3
STAT3
Cyclin D1
β-actin
CD44v
MDA-MB-231
245
32
46
80
80
NT IgG 76-E6 NT IgG 76-E6
pcDNA3 CD44v-HA
pSTAT3
STAT3
GAPDH
HA
MCF7-LR
245
100
100
32
65
Figure 2.16. Targeting csGRP78 by antibody suppresses CD44 downstream pSTAT3 signaling. (A) Western blot
analysis of whole cell lysates from stable CD44-knockdown MDA-MB-231 cells. CD44v was detected by anti-
CD44v3 antibody. (B) Western blot analysis of MCF7-LR cells transfected with HA-tagged CD44v (CD44v-HA) or
pcDNA3 backbone plasmids. Twenty-four h post transfection, cells were left non-treated (NT) or treated with 76-E6
antibody or control IgG for additional 24 h. Phospho-STAT3 was detected by the anti-pSTAT3 (Y705) antibody.
Experiments were repeated twice with similar results.
While the 76-E6 antibody was marketed as anti-GRP78 antibody (Abcam, Cambridge, MA),
surprisingly we discovered that this antibody could bind both recombinant GRP78 (HSPA5) and
HSP70 (HSPA1A) in Western blot analysis (Fig. 2.17 A). Cell surface HSP70 in cancer cells
principally exists as an integral protein and only a minimal sequence (a.a. 450-461) close to the C-
terminal substrate domain is exposed outside the plasma membrane and accessible by the
antibodies (Multhoff and Hightower 2011). We sought to determine if the 76-E6 antibody can
recognize the exposed region of HSP70 protein. Therefore, we first mapped the epitope of the 76-
E6 antibody on the GST-tagged recombinant GRP78 proteins harboring small deletions, and the
purity of proteins was verified by SDS-PAGE and colloidal blue staining (Fig. 2.17 B). We focused
on the region of GRP78 between a.a. 497 and 581, as this region is the epitope of the antibody
advertised by the company (Abcam, Cambridge, MA). Our result showed that the epitope of the
76-E6 antibody on GRP78 is localized between a.a. 557 and 581 on the C-terminal half (Fig. 2.17
C). Because an antibody usually recognizes 5 to 8 amino acid-long epitope and the 76-E6 antibody
showed similar affinities to a.a. 557-571 and a.a. 557-581. The epitope of the 76-E6 antibody can
be further narrowed down to the region between a.a. 557 and 571. Then, we performed sequence
comparison with the HSP70 (HSPA1A) protein, and a.a. 557 to 571 of GRP78 is corresponding to
a.a. 534 to 548 of HSP70. These data suggest that 76-E6 does not target integral HSP70 protein
66
and is likely to principally target GRP78 on the cancer cell surface, since in contrast to HSP70,
csGRP78 mainly exists as a peripheral protein with a.a. 557 to 571 exposed outside the cell surface
(Tsai et al. 2015). The identity of recombinant proteins was confirmed by Western blot analysis
using anti-GRP78 antibodies, N-20 and then C-20 from Santa Cruz Biotechnology (Dallas, TX),
following the immunoblotting using the 76-E6 antibody (Fig. 2.17 D).
Figure 2.17. The 76-E6 antibody targets HSP70 family proteins. (A) SDS-PAGE and Western blot analysis
showing the specificity of each antibody (76-E6: ab25192 from Abcam; MAb159 from Dr. Parkash S. Gill at USC;
C92F3A-5: sc-66048 from Santa Cruz Biotechnology) to the recombinant GST-tagged full-length human GRP78
135
80
58
100
30
25
46
(kD)
Colloidal Blue
A
GRP78
HSP70
135
100
58
46
80
32
76-E6 MAb159 C92F3A-5
WB: 76-E6
135
80
58
100
30
25
46
(kD)
C D
(kD)
B
135
80
58
100
30
25
46
(kD)
WB: 76-E6/N-20/C-20
67
(rGST-GRP78) and recombinant full-length human HSP70 (HSPA1A, rHSP70, ab92415, Abcam). Recombinant
proteins were prepared from E. coli. (B) SDS-PAGE analysis followed by colloidal blue staining of recombinant GST
and GST-tagged full-length GRP78 (FL) and deletion mutants (N, C and the segments indicated by the amino acid
numbers). Two µg was loaded for each protein. a.a., amino acids. (C) Western blot analysis of recombinant GST and
GST-tagged GRP78 FL and deletion mutants using antibodies against GRP78 (76-E6). (D) Western blot analysis of
recombinant GST and GST-tagged GRP78 FL and deletion mutants using antibodies against GRP78 (76-E6 and then
N-20 followed by C-20).
To further confirm and investigate csGRP78-specific functions in regulating CD44v
homeostasis, we utilized the antibody specifically against GRP78 (MAb159) to treat MCF7-LR
cells. Similar to the 76-E6 antibody, cells treated with the MAb159 antibody for 72 h exhibited
reduction of CD44v protein levels by 30 to 50% compared to the IgG or non-treated (NT) control
as evidenced by Western blot analysis using the whole cell lysate (Fig. 2.18 A) and
immunofluorescent staining in non-permeabilized cells (Fig. 2.18 B). We then blocked protein
synthesis with cycloheximide and then treated MCF7-LR cells with the MAb159 antibody, and we
showed that targeting csGRP78 with the MAb159 antibody resulted in modest decrease of CD44v
protein stability compared to the IgG control (Fig. 2.18 C). Moreover, MCF7-LR cells treated with
the MAb159 antibody for 72 h reduced the ability of cells to attach to the culture matrix by about
25% compared to the IgG control (Fig. 2.18 D). We further utilized the specificity of a cell-
impermeable bacterial toxin, subtilase cytotoxin A subunit (SubA), that only targets and cleaves
csGRP78 but not HSP70 (Paton and Paton 2010). We treated MCF7-LR cells with SubA and the
enzymatic activity-dead mutant (SubAA272) for 24 h and then re-seeded cells onto the CD44v
ligand, hyaluronan, -coated culture surface for 18 h. We found that SubA treated cells formed more
aggregates and spread less compared to the mutant (SubAA272) or non-treated (NT)control. The
68
difference between hyaluronan stimulation and DPBS control was not evident at 18 h after re-
seeding (Fig. 2.19 A). Taken together, our results suggest csGRP78 and CD44v are novel partner
proteins in aggressive breast cancer cells, and GRP78 is important for CD44v membrane
homeostasis and downstream signalings, including polarization of IQGAP1/CDC42 and activation
of STAT3 (Fig. 2.19 B).
Figure 2.18. Antibody specifically targeting csGRP78 reduces CD44v level and cell attachment. (A) Western
blot analysis of whole cell lysates from MCF7-LR cells. CD44v was detected by anti-CD44v3 antibody. Numbers
below the CD44v bands represent relative levels of CD44v and are derived from the ratio of CD44v to β-actin in WCL.
(B) Immunofluorescence and compressed z-stack confocal images showing the CD44v (red) levels on the cell surface
of non-permeabilized MCF7-LR cells treated with anti-GRP78 monoclonal antibody (MAb159) or control IgG for 72
h. The cells were seeded on the collagen I (100 µg/ml) coated coverslips. CD44v was detected by anti-CD44v3
antibody. Nuclei were stained by DAPI in blue. Scale bar, 20 µm. (C) The graph shows results of immunoblot analysis
CD44v
β-actin
NT IgG MAb159
1.00 0.97 0.69
MCF7-LR
A
B
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 24 48 72
Relative levels of CD44v
Time (h)
IgG
MAB159
IgG
MAb159
C D
0
20
40
60
80
100
120
Cell attachment (OD
595
)
*
IgG MAb159
MCF7-LR
0
0.2
0.4
0.6
0.8
1
1.2
Relative cs CD44v levels
*
CD44v DAPI Merge
IgG MAb159
MCF7-LR
69
of relative CD44v levels in MCF7-LR cells exposed to cycloheximide (40 μg/ml) and anti-GRP78 antibody (MAb159)
or control IgG for the indicated times. Means ± S.E.M. from three independent experiments for the CD44v/β-actin
band intensity ratio relative to the corresponding value for time zero are shown. (D) Cell adhesion assay. Left:
Representative light micrographs showing crystal violet-stained MCF7-LR cells treated with anti-GRP78 monoclonal
antibody (MAb159) or control IgG for 72 h and then re-seeded onto collagen I-coated culture plates for 1 h. Scale bar,
20 µm. Right: Relative number of attached cells were quantified by the amount of crystal violet dissolved from the
stained cells. Data represents means ± S.E.M. from three biological repeats. *P≤0.05.
Stress fiber
F-actin
STAT3
P
CD44
GRP78
Direction of
migration
N
B
NT SubA
A272
SubA
DPBS Hyaluronan
MCF7-LR
A
70
Figure 2.19. The bacterial cytotoxin specifically targeting csGRP78 suppresses cell spreading and induces cell
aggregation. (A) MCF7-LR cells were treated with the cell-impermeable bacterial subtilase cytotoxin A subunit
(SubA) or the inactive control (SubA A272) for 24 h then were re-seeded on hyaluronan (100 µg/ml)-coated or uncoated
(DPBS vehicle-treated) culture plate for 18 h. Scale bar, 20 µm. (B) Schematic illustration of the GRP78 and CD44v
signalings. GRP78 complexes with CD44 at the plasma membrane and regulates F-actin dynamics, directional cell
migration and polarity through IQGAP1/Active CDC42, pSTAT3 cell signalings.
2.4 Discussion
Finely-tuned cytoskeletal dynamics is critical for cell migration, thus, it is important to
understand how aggressive cancer cells rewire their cytoskeleton and adopt invasive properties.
GRP78 overexpression is widely reported in cancer associating with aggressive growth and
invasion (Lee 2014), and CD44 is an important regulator of cytoskeleton and cancer progression
(Senbanjo and Chellaiah 2017). Here, we report that csGRP78 complexes with the variant isoform
of CD44 in breast cancer cells resistant to hormonal therapy and those with high metastatic
potential, and in such capacity regulates CD44 homeostasis, F-actin integrity and cell signaling,
impacting cell polarity, adhesion and migration. Our study uncovers new pathways that could have
important therapeutic implications for blocking CD44v activity by targeting its partner protein in
aggressive and resistant breast cancers.
The co-localization and punctate staining of csGRP78 and CD44v indicate these two proteins
may collaboratively regulate cytoskeletal dynamics. Interestingly, we discovered that IQGAP1
formed complex with csGRP78 using LC-MS/MS, and targeting csGRP78 by the 76-E6 antibody
inhibited persistent localization of IQGAP1 and active CDC42 at leading edge of MCF7-LR cells.
IQGAP1, a multi-scaffold protein which has emerged as an important regulator of cell polarity,
71
proliferation and cancer invasion, is a binding partner of CD44 (Bourguignon et al. 2005, Skandalis
et al. 2010). It forms complex with active CDC42 and actin cytoskeleton at leading edge of
migrating cells and promotes breast cancer invasion (Watanabe et al. 2004, Jadeski et al. 2008).
The MDA-MB-231 cells treated with the 76-E6 antibody exhibited decreased directional
persistence and aberrant protrusive behaviors. Thus, while the detailed mechanisms remain to be
deciphered, it is possible that csGRP78 modulates anterior-posterior polarity of breast cancer cells
through the CD44/IQGAP1/CDC42 signaling cascade.
The ability to study protein diffusion on a subdiffraction limit scale provides opportunities to
highlight interactions between GRP78 and CD44v at the cell surface. Using dual-color sptPALM,
we uncovered the correlated lateral diffusion and co-confinement of GRP78 and CD44v in plasma
membrane nanodomains, indicating that they dynamically interact with each other in cancer cells.
Our studies revealed that GRP78 knockdown differentially impacts the diffusive behaviors of fast
and slow subpopulations of CD44v. This suggests that GRP78 and CD44v form diverse complexes
at the cell surface and membrane nanodomains. Previous studies have shown that CD44 localized
to lipid raft and non-raft membrane microdomains. For example, the association of CD44 with
lipid rafts activates SRC family protein kinase and Annexin II signalings thereby regulating
cytoskeletal dynamics (Ilangumaran, Briol, and Hoessli 1998, Oliferenko et al. 1999, Lee et al.
2008). Induction of cell migration in MDA-MB-231 breast cancer cells led to reduced affiliation
of CD44 with lipid raft and this was accompanied by increased association of CD44 with active
ezrin, a membrane-cytoskeleton linker, in the non-raft fraction (Donatello et al. 2012). VEGF-
induced cell migration of mesenchymal stem cells rapidly modified the nanodomain size of CD44
which resulted in FAK activation and rearrangement of cytoskeleton (Ke et al. 2015). It is tempting
to speculate that GRP78 facilitates differential CD44v signaling and actin remodeling by
72
associating with subpopulations of CD44v at the cell surface and future studies will be required to
elucidate these intriguing observations.
Targeting CD44 using peptides and antibodies has drawn great attention in cancer therapeutics
but challenges remain due to abundant expression of CD44 in normal tissues including bone
marrow, liver, spleen and skin (Jin et al. 2006, Marangoni et al. 2009, Masuko et al. 2012, Li et al.
2014, Jordan et al. 2015). Therefore, it is critical to identify alternative approaches to target CD44,
possibly through indirect means. Here we discover that the molecular chaperone GRP78 binds to
the transmembrane protein CD44v, which could serve to anchor GRP78, which is a peripheral
protein, on the cell surface of breast cancer cells. Importantly, GRP78 is up-regulated and
preferentially expressed on the cell surface of tumor cells and minimally in normal cells making it
an attractive target for cancer-specific therapy, including aggressive breast cancer (Arap et al. 2004,
Sato et al. 2010, Liu et al. 2013, Lee 2014, Dobroff et al. 2016, D'Angelo et al. 2018, Bakewell et
al. 2018). Here, we discovered that agents against csGRP78, including antibodies and SubA
cytotoxin, resulted in reduction of CD44v protein level and downstream STAT3 activation, and it
also altered polarized localization of IQGAP1/active CDC42. These results suggest that
perturbation of csGRP78 could represent a previously un-identified strategy for anti-CD44 therapy,
which warrants vigorous future investigation.
73
Chapter 3
The C-terminal proline-rich region of GRP78 is a key regulator of its cell surface
expression and viability of tamoxifen-resistant breast cancer cells
3.1 Introduction
Breast cancer is among the leading causes of cancer deaths in women (Siegel, Miller, and
Jemal 2018). Estrogen receptor-positive breast cancer accounts for about 70% of breast cancer
patients (Harvey et al. 1999), thus the estrogen receptor antagonist, tamoxifen, is the most widely
used adjuvant hormonal therapy. However, tamoxifen-resistance is responsible for the relapse of
about one-third of these patients (Davies et al. 2011). MCF7 is one of the best studied estrogen
receptor-positive breast cancer cell lines. We recently discovered that 78 kD glucose-regulated
protein (GRP78, also referred to as BiP or HSPA5) was upregulated intracellularly and on the cell
surface of tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) compared to the parental
non-resistant MCF7-L cells (Zhang et al. 2013). Thus, these cells provide a clinically relevant
model for the investigation of how GRP78 relocalizes from the endoplasmic reticulum (ER) to the
cell surface.
GRP78 belongs to heat-shock protein-70 (HSP70) family, and it is a major chaperone protein
that facilitates protein folding and quality control in the ER (Ni and Lee 2007). It also regulates
ER stress by its interaction with the unfolded protein response (UPR) stress sensors in the ER (Luo
and Lee 2013, Ni and Lee 2007, Lee 2014). GRP78 consists of a N-terminal ER signal sequence,
an ATPase domain, a substrate binding domain (SBD) and a C-terminal KDEL motif, where it
facilitates the retrieval of GRP78 by the KDEL receptor from Golgi back to ER (Munro and
74
Pelham 1986). GRP78 is also observed on the cell surface of various cancer cells, and its cell
surface expression could be further enhanced by stress conditions (Ni, Zhang, and Lee 2011, Zhang
et al. 2013, Tsai et al. 2015). Cell surface GRP78 (csGRP78) regulates novel cell signaling
pathways beyond its traditional protein foldase activity in the ER (Ni, Zhang, and Lee 2011, Zhang
et al. 2013, Tsai et al. 2018). Importantly, csGRP78 is preferentially expressed in cancer cells and
minimally in normal cells making it an attractive target for cancer therapeutics, including breast
cancer (Arap et al. 2004, Sato et al. 2010, Liu et al. 2013, Dobroff et al. 2016, Bakewell et al. 2018,
D'Angelo et al. 2018, Lee 2014). Evidence is emerging on how GRP78 relocalizes from the ER to
the cell surface. First, recent studies revealed that csGRP78 largely exists as a peripheral protein
at the plasma membrane, where it associates with transmembrane or GPI-anchored proteins for its
anchor on the cell surface (Tsai et al. 2015). Second, GRP78 harboring a mutation in the T453D
substrate binding domain exhibited about 80% reduction of its cell surface expression in HeLa
cervical cancer cells, suggesting substrate binding function of GRP78 was required for its cell
surface expression (Tsai et al. 2015). Third, it has been reported that the GRP78 co-chaperone
MTJ-1, a transmembrane protein and also referred to as DnaJC1 (DnaJ homolog subfamily C
member 1), was required for cell surface expression of GRP78 in mouse macrophages (Misra et
al. 2005). However, GRP78 harboring R197H DnaJ binding mutation (Awad et al. 2008) showed
similar level of cell surface expression as wild-type GRP78 in HeLa cells (Tsai et al. 2015). These
results suggest that the requirement of MTJ-1 or the human homolog, HTJ-1, for csGRP78
expression is cell context-dependent.
Since current studies support the notion that cell surface expression of GRP78 is dependent on
its capacity to form complex with transmembrane proteins, GPI-anchored proteins, co-chaperones
and likely folding substrates, we speculate that CD44, a transmembrane cell surface protein
75
abundantly expressed in tamoxifen-resistant MCF7 cells (Hiscox et al. 2012, Bellerby et al. 2016)
may be a folding substrate of GRP78, as in such capacity, contribute to the cell surface expression
of GRP78 in the context of these resistant cells. CD44 is a highly heterogeneous single-pass type
I transmembrane glycoprotein, and it is regulated by post-translational modifications and
alternative splicing (Zoller 2011, Yae et al. 2012). It is a major cell surface receptor of hyaluronan
(Ghatak et al. 2010) and serves as a co-receptor of FGF2, HGF, VEGF and osteopontin (Zoller
2011). The expression of CD44 variant isoforms enhanced the lung colonization of breast cancer
stem-like cells (Yae et al. 2012), and CD44v3-10 but not CD44v8-10 or CD44s was correlated
with poor prognosis of breast cancer patients (Hu et al. 2017). In this study, we determined that
GRP78 forms complex with CD44v3-10 through the region containing PPP polyproline sequence
localized close to the C-terminus of GRP78, and further discovered that mutation of this PPP
polyproline sequence impedes GRP78 expression on the cell surface in the MCF7-LR cells.
Enforced expression of an expression plasmid containing secretory signal peptide and 20 amino
acids spanning the PRR reduced CD44v protein level and led to increase in apoptotic markers. Our
studies uncovered critical functions of the C-terminal proline-rich region of GRP78 in its cell
surface expression and may have clinical implications in lowering the viability of tamoxifen-
resistant breast cancer.
3.2 Materials and methods
Cell culture
Tamoxifen-resistant MCF7 breast cancer cells were a kind gift from Dr. Rachel Schiff (Baylor
College of Medicine, TX) and cultured as previously described (Hiscox et al. 2012) in phenol-red
76
free RPMI 1640 medium containing 5% charcoal-stripped fetal bovine serum, 2.5 µg/ml fungizone,
200 mM glutamine, 10 IU/ml penicillin and 10 µg/ml streptomycin supplemented with 100 nM 4-
hydroxy tamoxifen. The cells were authenticated by STR DNA profiling analysis at the reagent
core facility in the USC Norris Comprehensive Cancer Center. Only mycoplasma-negative cells
were used.
Antibodies
We used the following primary antibodies for the proteins listed: GRP78 from Dr. Parkash S.
Gill at USC (MAb159, gift from Dr. Parkash Gill at USC), CD44v3 from Thermo Fisher Scientific
(BMS144, Waltham, MA), GST tag from Santa Cruz Biotechnology, Inc. (sc-138, Dallas, TX),
HA tag from Santa Cruz Biotechnology, Inc. (sc-805, Dallas, TX), FLAG M2 from Sigma-Aldrich
(F1804, St. Louis, MO), Annexin II from BD Biosciences (610068, San Jose, CA), β-actin from
Sigma-Aldrich (A5316, St. Louis, MO), Phospho-STAT3(Y705) from Cell Signaling Technology
(9145, Danvers, MA), STAT3 from BD Biosciences (610190, San Jose, CA), Cyclin D1 from
Santa Cruz Biotechnology, Inc. (sc-753, Dallas, TX), cleaved Caspase-3 from Cell Signaling
Technology (9661, Danvers, MA), cleaved PARP from Cell Signaling Technology (5625, Danvers,
MA). Secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX),
LI-COR Biosciences (Lincoln, NE) and Enzo Life Sciences (Farmingdale, NY).
Plasmids and cloning
The GST (glutathione S-transferase)-tagged full-length human GRP78 (FL, a.a. 19-654) and
truncated mutants including N-terminal half (N, a.a. 19-407), C-terminal half (C, a.a. 413-654),
ΔKDEL (a.a. 19-650), ΔC11 (a.a. 19-643), ΔC17 (a.a. 19-637), ΔC73 (a.a. 19-581) and C73 (a.a.
582-654) were inserted in-frame into pGEX-4T-1 expression plasmid (GE Healthcare, Chicago,
77
IL) at BamHI and XhoI sites. The FLAG-tagged full-length human GRP78 bearing a C-terminal
PPP to AAA (a.a. 640-642) mutation was generated by PCR amplification of the GRP78 coding
sequencing from FLAG-tagged human GRP78 (wild-type) expression plasmid using the reverse
primer containing modified sequence. The PCR product was inserted in-frame into pcDNA3
expression vector (Thermo Scientific, Waltham, MA) at BamHI and XhoI sites. The hemagglutinin
(HA)-tagged CD44v3-10 (CD44v-HA) expression plasmid was generated by PCR amplification
from cDNA library of MCF7-LR cells using the reverse primer containing HA tag sequence, and
the PCR product was inserted in-frame into pcDNA3 expression vector (Thermo Scientific,
Waltham, MA) at KpnI and ECoRI sites. All constructs were verified by sequencing. We used the
following primers: CD44v3-10-HA 5’-
CGGGGTACCATGGACAAGTTTTGGTGGCACGCAGCCT-3’ and 5’-CCGGAATTCTT
AAGCGTAATCTGGAACATCGTATGGGTACACCCCAATCTTCATGTCCACATTCT-3’;
GST-GRP78 (FL) 5’-CGCGGATCCATGGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTACAACTCATCTTTTTCTGCT-3’; GST-GRP78 (N) 5’-
CGCGGATCCATGGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTAACCAGAGAGCACACCAGC-3’; GST-GRP78 (C) 5’-
CGCGGATCCATGGACCTGGTACTGCTTGATGTA-3’ and 5’-CCGCTCGAGCTACAAC
TCATCTTTTTCTGCT-3’; GST-GRP78 (ΔKDEL) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTATTCTGCTGTATCCTCTTCACCAG-3’; GST-GRP78 (ΔC11) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTAAGTTGGGGGAGGGCCTGCA-3’; GST-GRP78 (ΔC17) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
78
CCGCTCGAGCTAACTTCCATAGAGTTTGCTGATAATTGG-3’; GST-GRP78 (ΔC73) 5’-
CGCGGATCCGAGGAGGAGGACAAGAAGGAGGA-3’ and 5’-
CCGCTCGAGCTACTTTTCTTTATCTCCAATCTGATTC-3’; GST-GRP78 (C73) 5’-
CGCGGATCCCTGGGAGGTAAACTTTCCTCTG -3’ and 5’-
CCGCTCGAGCTACAACTCATCTTTTTCTGCTG -3’; FLAG-GRP78 (AAA mutant) 5’-
CGCGGATCCATGAAGCTCTCCCTGGTGGC-3’ and 5’-
CCGCTCGAGCTACAACTCATCTTTTTCTGCTGTATCCTCTTCACCAGTTGCGGCAGCG
CCTGCACTTCCATAGAGTTTGCTGA-3’.
The expression plasmids for small peptides containing a previously described (Yin et al. 2003)
secretory and sorting sequence of α-melanocyte-stimulating hormone at N-terminus were
generated by insertion of the annealed synthetic DNA oligonucleotides into pcDNA3 expression
plasmid at BamHI and ECoRI sites. All constructs were verified by sequencing. For the plasmid
containing wild-type sequence (P), we used the following oligonucleotides: 5’-
GATCCATGCCGAGATCGTGCTGCAGCCGCTCGGGGGCCCTGTTGCTGGCCTTGCTGC
TTCAGGCCTCCATGGAAGTGCGTGGCTGGTGCCTGGAGAGCAGCCAGTGTCAGGAC
CTCACCACGGAAAGCAACCTGCTGGAGTGCATCCGGGCCTGCAAGCCCCGCGAGGG
CAAGCGCATCAGCAAACTCTATGGAAGTGCAGGCCCTCCCCCAACTGGTGAAGAGG
ATACAGCAGAATAAG-3’ and 5’-
AATTCTTATTCTGCTGTATCCTCTTCACCAGTTGGGGGAGGGCCTGCACTTCCATAGA
GTTTGCTGATGCGCTTGCCCTCGCGGGGCTTGCAGGCCCGGATGCACTCCAGCAGGT
TGCTTTCCGTGGTGAGGTCCTGACACTGGCTGCTCTCCAGGCACCAGCCACGCACTT
CCATGGAGGCCTGAAGCAGCAAGGCCAGCAACAGGGCCCCCGAGCGGCTGCAGCA
CGATCTCGGCATG -3’; For the plasmid containing mutant sequence (mP), we used the
79
following oligonucleotides: 5’-
GATCCATGCCGAGATCGTGCTGCAGCCGCTCGGGGGCCCTGTTGCTGGCCTTGCTGC
TTCAGGCCTCCATGGAAGTGCGTGGCTGGTGCCTGGAGAGCAGCCAGTGTCAGGAC
CTCACCACGGAAAGCAACCTGCTGGAGTGCATCCGGGCCTGCAAGCCCCGCGAGGG
CAAGCGCATCAGCAAACTCTATGGAAGTGCAGGCGCTGCCGCAACTGGTGAAGAGG
ATACAGCAGAATAAG-3’ and 5’-
AATTCTTATTCTGCTGTATCCTCTTCACCAGTTGCGGCAGCGCCTGCACTTCCATAGA
GTTTGCTGATGCGCTTGCCCTCGCGGGGCTTGCAGGCCCGGATGCACTCCAGCAGGT
TGCTTTCCGTGGTGAGGTCCTGACACTGGCTGCTCTCCAGGCACCAGCCACGCACTT
CCATGGAGGCCTGAAGCAGCAAGGCCAGCAACAGGGCCCCCGAGCGGCTGCAGCA
CGATCTCGGCATG-3’.
siRNA knockdown and GRP78 overexpression
Cells were transfected with Lipofectamine™ RNAiMAX Reagent (Thermo Fisher Scientific,
Waltham, MA) containing siRNA (Dharmacon Inc., Lafayette, CO) to the final concentration of
60 pM by reverse transfection. Twenty-four hours post siRNA transfection, plasmids containing
FLAG-tagged wild-type or mutant GRP78 were transfected into cells using BioT reagent (Bioland
Scientific, Paramount, CA). Culture media were replaced 5 h post- BioT transfection with fresh
media containing 60 pM siRNA. We used the following siRNAs: siGrp78_3’UTR 5’-
CUUAAGUCUCGAAUGUAAUdTdT-3'; sictrl: 5’-GAGAUCGUAUAGCAACGGUdTdT-3’.
Plasmid transfection
Plasmids were transfected into subconfluent cells with BioT reagent (Bioland Scientific,
Paramount, CA) according to the manufacturer’s instruction. Culture media were replaced 5 h
80
post-transfection, and cells were harvested 48 h post-transfection for further analyses.
Western blotting
Cells were lysed by radioimmunoprecipitation (RIPA) lysis buffer containing 50 mM Tris-HCl
pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and a protease and
phosphatase inhibitor cocktail (Roche, Indianapolis, IN). Whole cell lysates were cleared by
centrifugation at 4°C and 13,000 rpm for 15 min. Proteins were analyzed by 10% SDS-PAGE and
then transferred at 4°C overnight on nitrocellulose membranes (Bio-Rad Laboratories, Hercules,
CA). Membranes were blocked by Tris-buffered saline containing 0.05% Tween-20 (TBST) and
5% non-fat dry milk at room temperature for 1 h followed by incubation with primary antibody at
4°C overnight. Membranes were washed three times with TBST and then incubated with
fluorescent IRDye-labeled antibodies or HRP-conjugated secondary antibodies. Fluorescent
IRDye signal was detected by Odyssey (LI-COR Biosciences, Lincoln, NE). HRP signal was
visualized by an ECL chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) and
then quantified with Image Lab software (Bio-Rad Laboratories, Hercules, CA).
Immunofluorescence and confocal microscopy
For detection of endogenous GRP78 and CD44 containing v3 exon intracellularly in MCF7-
LR cells, cells were grown for 48 h to subconfluence on sterile coverslips. Coverslips were
sequentially coated with 50 μg/ml poly-L-lysine in ultrapure water (Sigma-Aldrich, St. Louis, MO)
at RT for 1 h and then 100 µg/mL collagen I from rat tail (Corning Inc., Corning, NY) in 0.02%
acetic acid at RT for 2 h. The cells were fixed in 4% paraformaldehyde (Electron Microscopy
Sciences, Hatfield, PA) in Dulbecco's Phosphate-Buffered Saline (DPBS) at RT for 10 min and
then permeabilized by 0.3% saponin in PBS at RT for 15 min. Then the cells were incubated with
81
blocking buffer containing 4% BSA and 0.01% saponin in PBS at RT for 3 h. The primary antibody
against GRP78 (MAb159, a kind gift from Dr. Parkash S. Gill at USC) was incubated with the
cells at 4°C overnight in blocking buffer, followed by staining with the AlexaFluor-568 secondary
antibody (Thermo Scientific, Waltham, MA) at 37°C for 40 min. Then the cells were treated with
M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories, Inc., Burlingame, CA) at 37°C for 3
h to block mouse immunoglobulin from the primary antibody against GRP78. The cells were then
incubated with the primary antibody against CD44 variable exon 3 at 4°C overnight in blocking
buffer, followed by staining with the AlexaFluor-647 secondary antibody (Thermo Scientific,
Waltham, MA) at 37°C for 40 min. Each step was followed by four washes in PBS containing
0.01% saponin. Coverslips were rinsed once with ultrapure water (Sigma-Aldrich, St. Louis, MO)
before mounting with Vectashield anti-fade medium containing DAPI (Vector Laboratories, Inc.,
Burlingame, CA). Z-stack images were obtained on a Leica TCS SP8 confocal microscope
equipped with a 63x/1.4 NA oil DIC objective lens and Leica Application Suite 10 Software (Leica
Microsystems, Wetzlar, Germany).
For detection of endogenous GRP78 and CD44 containing v3 exon on the cell surface, MCF7-
LR cells were grown for 48 h to subconfluence on sterile coverslips. Coverslips were coated as
described above. The cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences,
Hatfield, PA) in DPBS at RT for 10 min and then incubated with blocking buffer containing 4%
BSA in PBS at RT for 1 h. The primary antibody against GRP78 (MAb159) was incubated with
the cells at 4°C overnight in blocking buffer, followed by staining with AlexaFluor-594 secondary
antibody (Thermo Scientific, Waltham, MA) at RT for 1 h. Then, the cells were treated with
M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories, Inc., Burlingame, CA) at RT for 2
h. Then the cells were incubated with the primary antibody against CD44 variable exon 3 (Thermo
82
Scientific, Waltham, MA) at 4°C overnight in blocking buffer, followed by staining with
AlexaFluor-488 secondary antibody (Thermo Scientific, Waltham, MA) at RT for 1 h. Coverslips
were washed four times following each step and rinsed once with ultrapure water (Sigma-Aldrich,
St. Louis, MO) before mounting with Vectashield anti-fade medium containing DAPI (Vector
Laboratories, Inc., Burlingame, CA). Z-stack images were obtained on a Zeiss LSM 510 confocal
microscope equipped with a Plan-Apochromat 100x/1.4 NA oil DIC objective lens and LSM 510
version 4.2 SP1 acquisition software (Carl Zeiss, Oberkochen, Germany).
Purification of GST-tagged recombinant proteins
Plasmids containing GST-tagged full-length GRP78 or deletion mutants was transformed into
E. coli (BL21). The expression of the GST-fusion proteins was induced with 4 mM isopropyl-β-
D-thiogalactoside (IPTG) when the optical density (OD600) of bacterial broth culture reached 0.5.
Bacteria were then incubated at 37°C and 200 rpm for 4 h to allow the expression of recombinant
proteins. Cells were then lysed in TBS containing 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mg/ml
lysozyme, 1% Triton X-100 and protease and phosphatase inhibitor cocktails (Thermo Scientific,
Waltham, MA). Bacterial cells were then sonicated for 4 min with 20 sec on and 20 sec off,
followed by centrifugation at 4°C and 11500 rpm for 1 h. Supernatant was collected and incubated
with Glutathione-Sepharose 4B beads (GE Healthcare, Chicago, IL) at 4°C for 12 h. Recombinant
GST-tagged protein was eluted with freshly prepared reduced glutathione (10 mM, Sigma-Aldrich,
St. Louis, MO) at 4°C for 12 h. The solution containing recombinant proteins was then buffer-
exchanged to TBS using protein concentrators (Pall Corporation, Port Washington, NY).
Recombinant proteins in TBS containing 15% glycerol were snap-frozen in liquid nitrogen and
then stored at -80°C.
83
In vitro GST pull-down assay
Recombinant GST-tagged proteins were coupled to Glutathione-Sepharose 4B beads (GE
Healthcare, Chicago, IL) at 4°C for 4 h. Then, the beads were incubated with 1 mg whole cell
lysate collected from 293T cells transiently expressing HA-tagged CD44v3-10 at 4°C overnight
in IP lysis buffer (Thermo Fisher Scientific, Waltham, MA; 25 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1 mM EDTA, 5% glycerol, 1% NP-40). The beads were then washed 6 times with IP lysis
buffer, and the bound proteins were eluted from the beads with equal volume of 2x SDS sample
buffer.
Purification of cell surface proteins
Experiments were performed according to previously described protocol (Tsai et al. 2015).
Briefly, cell surface proteins were biotinylated with 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin
(Thermo Fisher Scientific, Waltham, MA) at 4°C for 30 min, and excessive biotin was quenched
by four washes with glycine (100 mM) in PBS at 4°C. Cells were then lysed with RIPA lysis buffer
(50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and
a protease and phosphatase inhibitor cocktail). The biotinylated cell surface proteins were captured
on high capacity NeutrAvidin agarose resin (Thermo Fisher Scientific, Waltham, MA).
WST-1 viability assay
Cell viability was assessed with the WST-1 reagent (Roche, Indianapolis, IN). Briefly, 24 h
post transfection in 6-well culture plate, 3,000 cells per well were re-seeded into 96-well culture
plates with 100 µL culture medium per well. Then, in another 24 h, the cell viability was measured
by incubating each plate with 10 μL per well of WST-1 substrate for 3 h and then the plates were
read at a wavelength of 450 nm with a reference wavelength of 655 nm.
84
Statistical analysis
Data are presented as means ± S.E.M. from three biological repeats. P values were calculated
via two-tailed unpaired Student’s t test. Statistical significance was represented as *P≤0.05,
**P<0.01 and ***P<0.001.
3.3 Results
3.3.1 GRP78 co-localizes with CD44v both in the ER compartment and on the cell surface
In permeabilized MCF7-LR cells, immunofluorescent staining and confocal microscopy
showed that endogenous GRP78 was expressed abundantly in the perinuclear as expected for its
ER protein folding function (Fig. 3.1 A). As a transmembrane protein, CD44 is synthesized in the
ER and traffics to the cell surface. CD44 containing variable exon 3 to 10, CD44v, that is
abundantly expressed in the MCF7-LR cells, was detected in the cytosolic and perinuclear region
as well as inside the nucleus. Co-localization of GRP78 and CD44v was detected in the perinuclear
region (Fig. 3.1 A). Since GRP78 is a major chaperone protein in the ER, it is likely that CD44v
is a folding substrate of GRP78. In non-permeabilized cells, GRP78 and CD44v were detected on
the cell surface and showed substantial co-localization (Fig. 3.1 B). These results suggest that
GRP78 and CD44v co-localize both in the ER compartment and on the cell surface.
85
A
B
GRP78 No CD44v primary Merge
MOM control
No primary control
GRP78 CD44v Merge DIC
MCF7-LR (Non-permeabilized)
MCF7-LR (Permeabilized by
saponin)
CD44v DAPI Merge GRP78
GRP78 No CD44v primary Merge
MOM control
No primary control
86
Figure 3.1. GRP78 co-localizes with CD44v in MCF7-LR breast cancer cells. (A) Immunofluorescence and
confocal images showing the distribution and co-localization of GRP78 (red) and CD44v (green) in the saponin-
permeabilized MCF7-LR breast cancer cells. GRP78 and CD44v were detected by MAb159 and anti-CD44v3
antibodies, respectively. The mouse-on-mouse (M.O.M.) control staining was performed with the same protocol as
double-stained cells but lacking the primary antibody targeting CD44v. No primary control staining was conducted
without the primary antibodies but with secondary antibodies. The nuclei were stained by DAPI in blue. Thickness of
single immunofluorescent image section: 0.3 µm. Signals were visualized with 63x/1.4 NA objective on a Leica TCS
SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany). Scale bars, 20 µm. (B) Immunofluorescence and
confocal images showing the distribution and co-localization of GRP78 (red) and CD44v (green) on the cell surface
of non-permeabilized MCF7-LR breast cancer cells. GRP78 and CD44v were detected by MAb159 and anti-CD44v3
antibodies, respectively. The M.O.M. control staining was performed with the same protocol as double-stained cells
but lacking primary antibody targeting CD44v. No primary control staining was conducted without the primary
antibodies but with secondary antibodies. The nuclei were stained by DAPI in blue. DIC: differential interference
contrast. Thickness of single immunofluorescent image section: 0.8 µm. Cell peripheries were outlined with black
lines. Signals were visualized with 100x/1.4 NA objective on a LSM 510 confocal microscope. Scale bars, 20 µm.
3.3.2 The C-terminal PRR of GRP78 is essential for forming complex with CD44v in vitro
We next characterized the region of GRP78 binding to CD44v. A schematic drawing of the
identified ER signal, ATPase domain, substrate binding domain and the KDEL Golgi-to-ER
retrieval motif of GRP78 is shown in Figure 2A. A panel of GST-tagged GRP78 deletion mutants
was created (Fig. 3.2 A). We purified the recombinant GST-tagged GRP78 full-length (FL) and
deletion mutants from E. coli (BL21) and then incubated them with whole cell lysates containing
transiently expressed HA-tagged CD44v (vHA, Fig. 3.2 B). The immunoblots of these GST-pull
down assays revealed that GRP78 binds CD44v via the regions localized in its C-terminal half
region (Fig. 3.2 C). The binding of FL, C (a.a. 413-654) and ΔKDEL mutants to vHA were
87
comparable when the amount of binding was normalized to the level of input GST-tagged proteins.
Interestingly, an increased complex formation was observed upon deletion of last 11 amino acids
of GRP78 (ΔC11, a.a. 19-643), suggesting this deletion likely exposed this region for more
effective binding and/or forming a more stable complex. The deletion of last 17 amino acids of
GRP78 (ΔC17, a.a. 19-637) reduced its capacity to form complex with vHA. The deletion of last
73 amino acids of GRP78 (ΔC73, a.a. 19-581) abolished binding to vHA. These results suggest
that the amino acids between 638 and 643 of GRP78 are essential for forming complex with vHA.
We then explored if the C-terminal region of GRP78 devoid of the substrate binding domain was
sufficient for complex formation with vHA. Surprisingly, the last 73 amino acids of GRP78 alone
(C73) could not form complex with vHA (Fig. 3.2 D). Collectively, these results indicate that a.a.
638 to 643 of GRP78 is essential but not sufficient for GRP78 to form complex with CD44v. A
closer look at the amino acid sequence between a.a. 638 and 643 of GRP78 revealed a PRR
containing three consecutive prolines between a.a. 640 and 642 localized outside the substrate
binding domain (SBD). A computational 3-dimentional protein structure of human GRP78
modeled by I-TASSER (Roy, Kucukural, and Zhang 2010, Yang et al. 2015, Zhang 2008) showed
that these three consecutive prolines created a turn in a less structured region of last 27 amino acids
of GRP78 (C27) as depicted in Fig. 3.2 E.
88
Figure 3.2. The C-terminal proline-rich region of GRP78 is essential for forming complex with CD44v in vitro.
(A) Schematic representation of the human GST-tagged GRP78 wild-type and deletion mutants cloned into pGEX-
4T-1 backbone vector. a.a., amino acids. FL, a.a. 19-654; N, a.a. 19-407; C, a.a. 413-654; ΔKDEL, a.a. 19-650; ΔC11,
a.a. 19-643; ΔC17, a.a. 19-637; ΔC73, a.a. 19-581; C73, a.a. 582-654. The locations of the ER signal, ATPase domain,
substrate binding domain, proline-rich region and KDEL motif of GRP78 are depicted on top. (B) Schematic
representation of the expression construct of HA-tagged human CD44 containing variable exon 3 to 10. EC,
extracellular; TM, transmembrane; IC, intracellular. (C-D) Western blot analysis of samples from in vitro GST pull-
down assay. GST or GST-tagged GRP78 wild-type and mutant proteins purified from E. coli (BL21) were incubated
with 293T whole cell lysate containing overexpressed CD44v-HA (vHA). (E) Upper panel: I-TASSER model of full-
length human GRP78 protein. ATPase domain is in light blue. Substrate binding domain (SBD) is in orange. The last
27 amino acids close to the carboxyl-terminus (C27) is lighted in rainbow color and by dashed circle. Arrow indicates
vHA
GST
vHA
GST
A
C D
E
700 1
CD44v3-10-HA
ER signal TM
EC IC
21 607 629 (a.a.)
HA
B
ATPase
domain
SBD
P640
P641
P642
C27
Proline-rich region
ATPase DQDT
G
Substrate binding QPIISKLYGSAGPPPTGEEDTAEKD
EL
628 654 19 407 413 627
638 643
ER signal
1
(N’) (C’)
GST
GST
GST
GST
GST
GST ΔC73
ΔC17
FL
N
C
ΔC11
(a.a.)
pGEX4T1
GST C73
GRP78
GST
ΔKDEL
89
the PPP polyproline amino acids (red). Lower panel: The enlarged C27 area shows the polyproline amino acids (a.a.
640-642; red) and the adjacent protein structure.
3.3.3 The C-terminal PRR of GRP78 is critical for its cell surface expression and can
modulate STAT3 signaling
Proline-rich region plays important roles in mediating protein-protein interactions and the
specificity of interactions can be finely-tuned by the proline-rich sequence (Kay, Williamson, and
Sudol 2000). On the cell surface, GRP78 largely exists as a peripheral protein (Tsai et al. 2015)
and associates with GPI-anchored (Kouznetsova et al. 2018, Philippova et al. 2008, Tsai et al.
2018) or transmembrane partner proteins such as MTJ-1, MHC class I, and tissue factor (Misra et
al. 2005, Triantafilou, Fradelizi, and Triantafilou 2001, Bhattacharjee et al. 2005). Since the PRR
is essential for GRP78 to form complex with CD44v, we hypothesized that the C-terminal PRR of
GRP78 may be critical for its cell surface expression through its ability to form complex with
partner proteins such as CD44v. To test this hypothesis, we created expression plasmids in
pcDNA3 backbone vector (v) for FLAG-tagged human GRP78 (F-GRP78) wild-type (WT) and
mutant harboring a PPP to AAA mutation (AAA) at a.a. 640-642 (Fig. 3.3 A). We then purified
cell surface proteins of MCF7-LR cells transfected with v, WT or AAA plasmids by cell surface
biotinylation and high-capacity avidin pull-down, and we found that the AAA mutant showed
about 65% decrease of cell surface expression compared to the WT (Fig. 3.3, B and C). These
results indicate that the C-terminal polyproline sequence of GRP78 plays a previously unidentified
role in mediating its cell surface expression in tamoxifen-resistant breast cancer cells.
We next explored the signaling function of this polyproline sequence by transfecting the v, WT
and AAA mutant into MCF7-LR cells. As a proof-of-principle, we focused on the activation of
90
signal transducer and activator of transcription 3 (STAT3) through phosphorylation at Tyr705
(pY705) because it is a common signaling pathway of CD44 (Wang et al. 2012, Xu et al. 2015)
and GRP78 (Yao et al. 2015) and plays important roles in cell proliferation and migration in breast
cancer. GRP78 is abundantly expressed in MCF7-LR cells, thus we co-transfected the plasmids
for transient expression with the siRNA targeting the 3’ untranslated region (UTR) of endogenous
Grp78 mRNA (Fig. 3.3 D). We observed about 30% reduction of STAT3 activation upon
knockdown of GRP78 and an increase of STAT3 activation when WT GRP78 was transiently
expressed, and this stimulation was blunted when AAA was introduced (Fig. 3.3, E and F). This
result suggests that the polyproline sequence close to the C-terminus of GRP78 is a novel regulator
of STAT3 activation in tamoxifen-resistant breast cancer cells.
Figure 3.3. The C-terminal polyproline sequence of GRP78 is critical for its cell surface expression and
modulates STAT3 signaling in MCF7-LR breast cancer cells. (A) Schematic representation of FLAG-tagged
0
0.2
0.4
0.6
0.8
1
1.2
Relative cs F-GRP78 levels
**
FLAG
v WT AAA v WT AAA
Annexin II
β-actin
WCL Cell surface
MCF7-LR
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1 2 3 4
pSTAT3/STAT3
pSTAT3
v v WT AAA
STAT3
siGrp78, 3’UTR sictrl
GRP78
β-actin
MCF7-LR
1 2 3 4
A B C
E
AAA
654
WT
FLAG
1 19
ER signal KDEL
407 627 413
ATPase SBD PPP
FLAG
ATPase SBD AAA
AAA
WT
Gr p 7 8 open reading frame
3’ UTR
5’ UTR
siRNA target
site within
3’ UTR
FLAG
PPP
FLAG
AAA
D F
*
P=0.06
*
91
human GRP78 (F-GRP78) wild-type (WT) and mutant harboring a PPP to AAA mutation (AAA). a.a., amino acids.
SBD, substrate binding domain. (B) Western blot analysis of MCF7-LR cells transfected with FLAG-tagged GRP78
WT, AAA or pcDNA3 backbone vector (v). Annexin II and β-actin are loading controls for cell surface fraction and
whole cell lysate (WCL), respectively. (C) Quantitative analysis of cell surface (cs) F-GRP78 levels. Data represents
means ± S.E.M. from three biological repeats. **P< 0.01 by Student’s t test. (D) Schematic illustration of siRNA
designed to target 3’ untranslated region (UTR) of Grp78. WT and AAA expression plasmids do not contain 3’ UTR.
(E) Western blot analysis of MCF7-LR cells co-transfected with v, WT or AAA and control (sictrl) or Grp78 siRNA
targeting 3’ UTR (siGrp78, 3’ UTR). Numbers below the β−actin bands represent individual experimental conditions.
(F) Quantification of the ratio of pSTAT3 (Y705) and STAT3 of each experimental condition as shown in panel (E).
Data represents means ± S.E.M. from three biological repeats. *P≤ 0.05 by Student’s t test.
3.3.4 The C-terminal polyproline sequence of GRP78 is evolutionally conserved in higher
eukaryotic organisms
A closer examination at the amino acid sequence of human GRP78 revealed that another
consensus proline-rich region (a.a. 487-496) containing a classical xPPxP (IPPAP, a.a. 487-491)
motif and a PQ sequence (a.a. 495 and 496) preceded the PPP polyproline motif (a.a. 640-642).
The 3-dimensional localizations of these two proline-rich regions were mapped to the
corresponding sites on the computational GRP78 protein structure (a.a. 413-654) modeled by I-
TASSER, and the result showed that these two sequences were both localized to more unstructured
regions of GRP78 protein (Fig. 3.4 A). The proline residue, P
495
, localized between a.a. 487 and
496, was reported to facilitate the substrate binding function of GRP78 (Shen et al. 2005), however,
the function of the PPP sequence close to the C-terminus of GRP78 is largely unknown.
Multiple sequence alignment provides insights into the evolutionary, structural and functional
relationships among the amino acid sequences. We applied Clustal analysis using MEGA-X
92
software (Kumar et al. 2018) to compare the proline-rich sequences of GRP78 homologs from a
diverse set of species (Fig. 3.4, B and C). Interestingly, we found that the GRP78 C-terminal PPP
polyproline sequence was conserved among Homo sapiens, Rattus norvegicus, Gallus gallus,
Xenopus laevis and Stylophora pistillata. Although Latimeria chalumnae, Priapulus caudatus and
Octopus bimaculoides did not show tri-proline sequence, they exhibited two consecutive proline
residues. However, Saccharomyces cerevisiae and GRP78 homolog, DnaK, in Escherichia coli
did not show proline residues in their C-termini (Fig. 3.4 B). These results suggest that the presence
of GRP78 C-terminal polyproline sequence is likely a unique feature in higher eukaryotic
organisms. On the other hand, the IPPAP/PQ proline-rich region was highly conserved among all
species, with Octopus bimaculoides and GRP78 homolog, DnaK, in Escherichia coli exhibited
single amino acid variances (Fig. 3.4 C). We then asked whether the PPP polyproline sequence is
unique to GRP78 or common to heat shock family proteins. Therefore, we examined the C-
terminal amino acid sequences of human heat shock 70 (HSP70) and 90 (HSP90) family
chaperones (Fig. 3.4 D). We did not observe PPP polyproline sequence in these heat shock family
proteins. Of note, HSC70, HSP90α and HSP90β showed two consecutive prolines. This result
indicates that the C-terminal PPP polyproline sequence is unique in GRP78 among the listed heat-
shock proteins in human.
93
H. sapiens
TEQDEDEEMDVGTDEEEETAKESTAEKDEL GRP94:
VDKLAELKNLGQPIKIRFQESEERPNYLKN HSP70:
PTADDTSAAVTEEMPPLEGDDDTSRMEEVD HSP90α:
VAAEEPNAAVPDEIPPLEGDEDASRMEEVD HSP90β:
MAYKKMASEREGSGSSGTGEQKEDQKEEKQ GRP75:
EKVCNPIITKLYQSAGGMPGGMPGGFPGGGAPPSGGASSGPTIEEVD HSC70:
B
D
A
C
IPPAP
PPP
a.a. 413-654
PQ
94
Figure 3.4. Comparisons of the proline-rich regions of GRP78. (A) I-TASSER model of human GRP78 (a.a. 413-
654) demonstrating the 3-dimentional localizations of PPP and IPPAP/PQ proline-rich sequences. The classical IPPAP
motif were highlighted in magenta, the PQ amino acids in blue and the C-terminal PPP polyproline amino acids in
red. (B) Results of multiple sequence alignment of GRP78 C-terminal proline-rich regions from a broad range of
species using MEGA-X software with polyproline residues in red. DnaK is a bacterial homolog of GRP78. Asterisk
indicates conserved residue among all species. Dots show conserved residues with human (Homo sapiens) GRP78.
Dashes indicate gaps. (C) Results of multiple sequence alignment of GRP78 IPPAP/PQ proline-rich regions from a
broad range of species using MEGA-X software with the proline residues localized at the classical IPPAP motif in
magenta and the proline residue localized at the PQ amino acids in blue. DnaK is a bacterial homolog of GRP78.
Asterisks indicate conserved residues among all species. Dots show conserved residues with human GRP78. (D) The
C-terminal amino acid sequences of other human (H. sapiens) heat shock proteins with polyproline residues in red.
3.3.5 Enforced expression of a short peptide bearing the PRR region of GRP78 reduced cell
viability and promoted apoptosis in tamoxifen-resistant breast cancer
Overexpression of GRP78 in tumor lesions from breast cancer patients has been associated
with the development of therapeutic resistance, and knockdown of GRP78 can resensitize
tamoxifen-resistant breast cancer cells to tamoxifen (Cook and Clarke 2015). Thus, we
investigated the feasibility of utilization of small peptide encoding the C-terminal PRR of GRP78
to target potential protein-protein interactions mediated by the PRR of GRP78 in tamoxifen-
resistant breast cancer cells. We first constructed expression plasmids in a pcDNA3 backbone
vector (v) containing wild-type (P) or mutant (mP) sequence encoding 20 amino acids spanning
the C-terminal polyproline sequence (a.a. 631-650) of GRP78. These sequences were preceded by
a previously described secretory and sorting peptide of α-melanocyte-stimulating hormone (SSP)
(Yin et al. 2003) (Fig. 3.5 A). We selected this region because it is localized in between SBD and
KDEL motif, and it preserves some conformational and sequence information in addition to the
95
PPP sequence, as previous studies have shown that the flanking region of core proline-rich
sequence also substantially contributed to the specificity and affinity of PRR-mediated protein-
protein interactions (Holtzman et al. 2007, Ball et al. 2000). The inclusion of the sorting and
secretory sequences predicted that the peptides would be localized to the ER as well as secreted
into the medium. We then analyzed the MCF7-LR cells transfected with the plasmids as well as
the cells applied with the conditional media (cM) obtained from the culture media of transfected
cells as depicted in Fig. 3.5 B. Notably, more cells became rounded and detached from the culture
surface when they were transfected with the plasmid containing wild-type PRR sequence (P)
compared to the cells transfected with v and mP plasmids (Fig. 3.5 C). Further analyses of the cell
lysates from transfected cells by SDS-PAGE and immunoblotting revealed that the cells
transfected with the plasmid containing wild-type PRR sequence (P) exhibited a significant
reduction of CD44v level as well as a reduction of proliferation marker, Cyclin D1. Additionally,
they demonstrated an increase in apoptotic markers, cleaved Caspase-3 and PARP, compared to
the v and mP controls (Fig. 3.5 D). In agreement with this result, cells transfected with the plasmid
containing wild-type PRR sequence (P) displayed a decrease of cell viability as shown by WST-1
assay (Fig. 3.5 E). These results further demonstrate the functional importance of the C-terminal
PRR of GRP78.
To test the functionality of the secreted peptides, we treated MCF7-LR cells with the
conditional media (cM) harvested from the culture media of transfected cells (Fig. 3.5 F). Similar
to cells transfected with the plasmids, cells treated with the cM collected from the cells transfected
with the plasmid containing wild-type PRR sequence (P), but not the mP sequence, showed a
significant reduction of CD44v level and a decrease in proliferation marker, Cyclin D1 (Fig. 3.5
F). However, these reductions did not lead to increase in cell apoptotic markers (Fig. 3.5 F). This
96
was likely because the forced expression would continuously supply the small peptides while the
amount in the collected conditional media is limiting.
Figure 3.5. Enforced expression of short peptide encoding the proline-rich region of GRP78 reduced cell
viability and promoted apoptosis in MCF7-LR breast cancer cells. (A) Schematic illustration of expression
plasmids containing wild-type or mutant sequences encoding 20 amino acids spanning the proline-rich region of
GRP78 (a.a. 640-642). The sequences were preceded by a secretory and sorting peptide sequence of α-Melanocyte-
stimulating hormone (SSP). The plasmids were constructed in pcDNA3 backbone vector (v). P, the plasmid containing
wild-type proline-rich sequence. mP, the plasmid containing PPP to AAA mutation. (B) Schematic representation of
experimental procedures. cM, conditional media. h, hours. (C) Bright-field images of MCF7-LR cells with the
enforced expression of v, P or mP. Images were photographed 48 h post transfection. Scale bar, 50 µm. (D) Left:
Western blot analysis of whole cell lysates from MCF7-LR cells transfected with v, P or mP. Cells lysates were
collected 48 h post transfection. CD44v and GRP78 were detected by anti-CD44v3 and MAb159 antibodies,
respectively. β-actin is loading control. Right: Quantification of CD44v levels compared to the control. Data represents
means ± S.E.M. from three biological repeats. *P≤ 0.05 by Student’s t test. (E) Quantitative analysis of cell viability
0
0.2
0.4
0.6
0.8
1
1.2
1.4
v
P
mP
Relative levels of CD44v
*
0
20
40
60
80
100
120
v
P
mP
Cell viability (% of control)
***
**
***
ISKLYGSAGPPPTGEEDTAE
ISKLYGSAGAAATGEEDTAE
SSP
SSP
P
mP
pcDNA3
48h
cM
Transfection
(v, P, or mP)
48h
cM-treated
(v, P, or mP)
0
0.2
0.4
0.6
0.8
1
1.2
v
P
mP
Relative levels of CD44v
*
A
B
C
F D E
v P mP
Transfection
mP
CD44v
v P
GRP78
MCF7-LR
CyclinD1
β-actin
Transfection
cCaspase3
cPARP
CyclinD1
β-actin
CD44v
v P mP
cM-treated
GRP78
MCF7-LR
cCaspase3
cPARP
MCF7-LR
97
by WST-1 assay in MCF7-LR cells overexpressing v, P or mP. Cells were analyzed 48 h post transfection. Data
represents means ± S.E.M. from four experimental repeats. **P< 0.01 ***P<0.001 by Student’s t test. (F) Left:
Western blot analysis of whole cell lysates from MCF7-LR cells treated with conditional media (cM) collected from
MCF7-LR cells transfected with v, P or mP. Conditional media were collected 48 h post transfection and then applied
to new cells for another 48 h. CD44v and GRP78 were detected by anti-CD44v3 and MAb159 antibodies, respectively.
β-actin is loading control. Right: Quantification of CD44v levels compared to the control. Data represents means ±
S.E.M. from three biological repeats. *P≤ 0.05 by Student’s t test.
3.4 Discussion
Tamoxifen-resistance presents a major challenge for the treatment of estrogen-receptor
positive breast cancer as that is associated with disease relapse and a poor prognosis. Therefore, it
is important to identify molecular pathways that can be used to target tamoxifen-resistant breast
cancer cells. Our previous studies revealed that in a human tamoxifen-resistant breast cancer cell
line MCF7-LR, the molecular chaperone GRP78 was elevated both intracellularly and on the cell
surface (Zhang et al. 2010). This same cell line also expressed a very high level of CD44v.
Interestingly, both GRP78 and CD44 have been documented to play important roles in mediating
endocrine resistance in breast cancer (Fu, Li, and Lee 2007, Cook et al. 2012, Bellerby et al. 2016,
Hiscox et al. 2012). In this study, while we explored the physical and functional relationship
between GRP78 and CD44v in the MCF7-LR cells, we uncovered several novel findings that
advance our understanding on the requirement of GRP78 to express on the cell surface and how
CD44v level and signaling are modulated in these cells.
Through confocal microscopy, we observed substantial co-localizations of GRP78 and CD44v
in the intracellular compartments and on the cell surface of MCF7-LR cells. Their cytosolic co-
localizations are presumably in the ER since GRP78 is a major ER resident protein, and this
98
suggests that CD44v is a folding substrate of GRP78. Interestingly, we found most of the cultured
cells showed nuclear CD44v. Because we used the antibody against the extracellular variable exon
3 of CD44v for the immunofluorescent staining, it is likely that it is the full-length CD44v in the
nuclei. All CD44 isoforms contain a bipartite nuclear localization signal (NLS) at their cytosolic
C-terminal region (Janiszewska et al. 2010), and the full-length CD44 was reported internalized
upon ligand binding. The internalized full-length CD44 formed complex with STAT3 in the
cytosol and facilitated the acetylation and activation of STAT3, and then the complex traveled
together into the nucleus and binding to the promoter of Cyclin D1, leading to elevated expression
of Cyclin D1 (Lee, Wang, and Chen 2009). The same study (Lee, Wang, and Chen 2009) also
showed that the nuclear CD44 bound to the 5’ sequence of the Grp78 allele and it remains to be
determined whether nuclear CD44 can directly facilitate the expression of Grp78 at the
transcriptional level.
GRP78 has well-established roles as a chaperone protein that facilitate substrate folding
through its SBD domain (a.a. 413-627) and the subsequent releasing of the substrate through the
ATP hydrolysis by the ATPase domain (a.a. 19-407). Despite these advances, the function of the
C-terminal sequence between a.a. 628-650 preceding the KDEL Golgi-to-ER retrieval motif is
largely unknown, although antibodies against the C-terminal region of GRP78 have shown
efficacy in suppressing cancer growth and metastasis (Liu et al. 2013, Ray et al. 2012). Here, we
uncovered that the GRP78 C-terminal proline-rich region containing three consecutive prolines
(a.a. 640-642) is essential for complex formation with CD44v as demonstrated by the in vitro GST-
binding assay. However, such capacity is not sufficient and likely also dependent on the substrate
binding function of GRP78 as we have found that the last 73 amino acids alone is unable to form
complex with CD44v, whereas the subdomain spanning the C-terminal half from a.a. 413 to 654
99
is sufficient. Importantly, we identified that this PPP polyproline sequence is critical for GRP78
expression on the cell surface of MCF7-LR cells. Since GRP78 co-localized with CD44v both
intracellularly and on the cell surface, it is tempting to speculate that the cell surface
transmembrane protein CD44v forms complex with GRP78 in the ER and facilitate GRP78 cell
surface translocation.
Proline-rich sequence is an important mediator of protein-protein interactions (Kay,
Williamson, and Sudol 2000). A previous genome-wide study (Mandal, Mandal, and Park 2014)
showed that the frequencies of PPP and PPG motifs were low in prokaryotes and drastically
increased with the complexity of eukaryotic organisms. Here, we found the C-terminal PPP
polyproline sequence is uniquely presented in the GRP78 of higher eukaryotic organisms but not
the yeast (S. cerevisiae) and E. coli as shown from the result of multiple sequence alignment. This
implies that the PPP polyproline sequence of GRP78 may have functions specific to higher
eukaryotes. Interestingly, the result of multiple sequence alignment suggests that the PPP sequence
is evolved from a D/E rich region. Proline is non-polar and is the only cyclic amino acid. It is often
found in turns or loops or at the end of α-helix. Its amino acid property is very different from the
acidic amino acid aspartic acid (D) and glutamic acid (E). This further supports the idea of
functional divergence of the PPP polyproline sequence during evolution. Additionally, the PPP
polyproline sequence is localized in a region with a lot more sequence variance compared to the
region bearing IPPAP/PQ sequences within the substrate binding domain among the analyzed
species. In future studies, it will be interesting to determine whether the PPP sequence/region may
bear functions independent of the traditional view of GRP78 as a foldase.
GRP78 and CD44v have both been reported to mediate STAT3 signaling in breast cancer cells
(Chung, Aroh, and Vadgama 2013, Yao et al. 2015), and STAT3 signaling is also important in
100
mediating tamoxifen-resistance in breast cancer cells (Wang et al. 2012). We found this
polyproline sequence is essential for STAT3 activation mediated by enforced expression of GRP78,
providing the first evidence that the C-terminal polyproline sequence is a previously unidentified
modulator of STAT3 activation. In testing the functional consequence of enforced expression of
the C-terminal peptide of GRP78 (aa 631-650) in MCF7L-R cells, we discovered that enforced
expression of the wild-type peptide but not the mutant bearing AAA mutation to the PPP motif,
caused dramatic rounding of the cells leading to detachment, associating with substantial decrease
in CD44v level, increase of apoptotic markers and reduction in cell viability. This raises the
interesting possibility that enforced expression of this GRP78 C-terminal peptide may disrupt the
interaction of GRP78 with CD44 as well as other unidentified binding/folding candidate proteins
critical for promoting tamoxifen resistance, thus representing a novel approach to combat
endocrine resistance in breast cancer. Taken together, we identified that the C-terminal polyproline
sequence of GRP78 is a new evolutionary feature gained by higher eukaryotic organisms, and our
biochemical and cellular studies supported the perspective that this sequence harbors previously
unidentified signaling functions in regulating cell surface expression of GRP78 and STAT3
activation in tamoxifen-resistant breast cancer cells. The basic and clinical implications of these
findings warrant vigorous investigation study in the future.
101
Chapter 4
Conclusions and Perspectives
Endoplasmic reticulum (ER) is often the largest organelle in eukaryotic cells, and it hosts major
machineries controlling the lipid synthesis, balance of calcium ions and maturation of proteins
(Schwarz and Blower 2016). GRP78 is a major chaperone protein with calcium binding capacity
in the ER, and it facilitates protein folding and regulates unfolded protein response under ER stress
(Ni and Lee 2007, Pfaffenbach and Lee 2011). Therefore, GRP78 is a key regulator of ER and
cellular homeostasis. Recent studies have revealed the unconventional localizations of GRP78 on
the cell surface, mitochondria, cytosol and nucleus (Ni, Zhang, and Lee 2011, Zhang et al. 2010).
Cell surface GRP78 (csGRP78) has drawn great attention to scientists because it mediates novel
cell signalings and largely presents on the tumor cells and minimally on the normal cells. Thus,
csGRP78 is vividly under investigation for cancer therapeutics (Lee 2014, Sato et al. 2010).
Emerging evidences have shown that GRP78 can anchor on cell surface through GPI-anchored
proteins such as Cripto, T-cadherin, and CD109 (Kelber et al. 2009, Kouznetsova et al. 2018, Tsai
et al. 2018) or transmembrane proteins such as MTJ-1, MHC class I, and tissue factor (Misra et al.
2005, Triantafilou, Fradelizi, and Triantafilou 2001, Bhattacharjee et al. 2005). However, the
underlying mechanisms mediating the signaling function of csGRP78 and its cell surface
translocation are still largely unknown. In this dissertation, we set forth to explore novel binding
partners of csGRP78 and the mechanisms responsible for its localization on the cell surface.
In Chapter 2, we discovered that type-I transmembrane protein CD44 is a previously un-
identified partner protein of csGRP78 in aggressive breast cancer cells. CD44 itself lacks kinase
activity but it can activate Src kinase and downstream focal adhesion dynamics (Bourguignon et
al. 2001, Lee et al. 2008). It can also regulate cytoskeleton rearrangement through direct binding
102
to ezrin/radixin/moesin (ERM) and ankyrin proteins (Goodison, Urquidi, and Tarin 1999).
Additionally, CD44 can associate multi-scaffold protein IQGAP1 (Skandalis et al. 2010,
Bourguignon et al. 2005), which has exhibited capacity to regulate F-actin dynamics, ERK1/2
activation, E-cadherin, β-catenin and microtubules (Noritake et al. 2005). CD44 is a major receptor
of hyaluronan with ability to bind to other extracellular matrix components, and the variant
isoforms of CD44 can function as a co-receptor for HGF, osteopontin, FGF2 and VEGF (Ponta,
Sherman, and Herrlich 2003, Zoller 2011). CD44 isoforms have been associated with cancer cell
adhesion, migration, stemness, multi-drug resistance, recurrence and metastasis (Ponta, Sherman,
and Herrlich 2003, Zoller 2011). The bipartite nuclear localization signal at the C-terminus allows
the internalized carboxyl tail or the full-length CD44 to activate downstream cell signaling, such
as STAT3 activation, and initiate gene transcription (Lee, Wang, and Chen 2009). Thus, CD44
serves as a signaling hub, and targeting CD44 is of great potential to block multi-faceted molecular
pathways critical for cancer progression. We identified two anti-GRP78 antibodies, 76-E6 and
MAb159, that can reduce CD44v protein levels and block cell adhesion/migration in breast cancer
cells. Therefore, targeting the partner proteins of CD44 such as GRP78 can serve as a new cancer
therapeutic approach for anti-CD44 therapy, particularly in breast cancer.
Cell surface GRP78 acts as a “co-receptor or receptor” on the cell surface for soluble ligands
such as oxidized phospholipids (OxPAPC) in human endothelial cells (Birukova et al. 2014), α2-
macroglobulin (α2M) in mouse macrophages (Misra, Deedwania, and Pizzo 2005), and vaspin in
liver cells and tissues (Nakatsuka et al. 2012) through complexing with transmembrane protein
MTJ-1/HTJ-1. Upon ligand binding, the GRP78/MTJ-1(HTJ-1) complex can activate AKT/PI3K
pro-survival pathway to alleviate ER stress and Src/Fyn kinase leading to cortical F-actin
remodeling. GRP78 also directly bind to tissue factor (TF), a transmembrane protein on the surface
103
of endothelium, and negatively regulates TF-mediated procoagulant activity (Bhattacharjee et al.
2005). The transmembrane protein β1–integrin was reported as a binding partner of csGRP78 and
regulates colorectal cancer cell migration and invasion through activation of downstream focal
adhesion kinase (FAK) pathway (Li et al. 2013), however, the resolution and quality of this work
should be improved to meet the authors’ claim. The GPI-anchored protein, Cripto, can form
complex with csGRP78 in fetal and adult mammary stem cells ex vivo and promote the self-
renewal of stem cells (Spike et al. 2014). The Cripto/csGRP78 complex can also sustain dormant
hematopoietic stem cells in hypoxic environment (Miharada et al. 2011) and this have great
implications in cancer stem cells, which often reside in hypoxic niches. GRP78 also forms complex
with GPI-anchored protein, T-cadherin, in endothelial cells and facilitates T-cadherin-mediated
survival signaling via Akt (Philippova et al. 2008). Additionally, GPI-anchored protein, CD109,
was reported to form complex with csGRP78 in HeLa cervical cancer cells and , in this capacity,
inhibit TGF–β-mediated Smad2 signaling (Tsai et al. 2018). However, the biological significance
of this interaction requires thorough investigation. Cell surface GRP78 also binds to soluble tumor
differentiation factor (TDF) in human steroid-resistant BT-549 and steroid-responsive MCF7
breast cancer cells (Sokolowska et al. 2012) to stimulate cell differentiation and α2M in rat and
human sperms (Lobo and Parte 2019) to activate Rac/Cdc42 signaling resulting in F-actin
reorganization during epididymal maturation with unknown partner proteins. Despite these
advances, limited number of partner proteins of csGRP78 in cancer is known, and the partner
proteins of csGRP78 in some of the known signaling functions of csGRP78 mentioned above are
not yet identified. In chapter 2, we established the functional importance of csGRP78 in cell
adhesion, motility, migration, matrix degradation and polarity. We further showed that
CD44v/csGRP78 can regulate STAT3 activation and transduce plasma membrane signals to the
104
cytoskeleton likely through intracellular multi-scaffold protein, IQGAP1. The STAT3 and
IQGAP1 proteins are important mediators of cancer cell proliferation, metastasis and chemo-
resistance (Wang et al. 2012, Yao et al. 2015, Bourguignon et al. 2005, Jadeski et al. 2008, Pan et
al. 2017, Zeng et al. 2018, White et al. 2011). Although our work does not provide solid
mechanistic explanation of CD44v/GRP78 and its connections to STAT3 or IQGAP1signaling, it
provides opportunities to further study the functions of csGRP78 in cytoskeletal dynamics and
cancer progression through partnership with CD44v.
CD44 is abundantly expressed in many tissue types and stem cells (Zoller 2011), and isoform
switch of CD44 through alternative splicing promotes malignant transformation of cells under
stress conditions (Yae et al. 2012). CD44 isoforms can be differentially post-translationally
modified and exert diverse signaling and cellular functions, including maintenance of cancer
initiating/stem cells (Zoller 2011, Wang et al. 2018, Senbanjo and Chellaiah 2017, Chen et al.
2018). The stemness of cancer cells is often correlated with capacity of metastasis, therapeutic
resistance and relapse, and CD44 is an important marker for breast cancer stem cells (Palomeras,
Ruiz-Martí nez, and Puig 2018, Sin and Lim 2017). It is of interest to study if perturbation of
csGRP78 can reverse stemness of cancer cells, particularly through the interference of the CD44
level and/or functions. A recent report from the American Association for Cancer Research
(AACR) meeting (Lager et al. 2018) disclosed that cell surface GRP78 is enriched in iPSC and
subset of breast cancer cells exhibiting CD44
+
/CD24
-
stemness characteristics, and csGRP78+
cells gains enhanced capability for distant metastasis. This report also revealed that csGRP78
interacts with a naturally secreted peptide, Dermcidin, on the cell surface of cancer cells and iPSCs,
and this complex plays important roles in regulating cancer and stem cell migration, proliferation
and survival. This study highlights the importance of csGRP78 in breast cancer stem cells and
105
supports our observation that the expression of csGRP78 is higher in subset of CD44+ breast
cancer cells. We observed that unipolar MDA-MB-231 cells expressed higher levels of csGRP78
than non-polar cells, and csGRP78 is usually localized in the cell spreading area, likely the
lamellipodia and filopodia. This phenomenon is not as clear in MCF7-LR cells. Although we did
not characterize and report these observations in detail, these observations pointed out that
csGRP78 may exert specific cell functions depending on the cell types and/or cellular localizations.
These observations also provide preliminary explanation to support the reported higher metastatic
capacity of csGRP78+ breast cancer cells (Lager et al. 2018) since MDA-MB-231 cells are highly
metastatic. Notably, while CD44v is abundantly and more ubiquitously expressed on the cell
surface of MDA-MB-231 cells, GRP78 shows localized expression pattern and largely co-
localizes with subset of CD44v in the restricted area. This indicates a polarized/directed
transportation system of the CD44v/GRP78 complex and likely involves additional proteins to the
assembly of the complex(es). It was reported that IQGAP1 is a key regulator of cell polarity and
migration, and it also coordinates secretory vesicles through interacting with exocyst complex
(Noritake et al. 2005, Hedman, Smith, and Sacks 2015). Since we have identified that IQGAP1
forms complex with csGRP78 by LC-MS/MS, further studies will be required to elucidate if
IQGAP1 is required for csGRP78 expression and its role in polarized expression of csGRP78.
Additionally, we uncovered specific CD44v3-10 isoform that forms complex with csGRP78 in
breast cancer cells. We also showed that CD44s isoform can form complex with GRP78 in the in
vitro systems. Previous study has shown that CD44v3-10 but not CD44s or CD44v8-10 is critical
for lung metastasis of breast cancer cells (Hu et al. 2017). It is of interest to further investigate if
GRP78 can facilitate CD44 isoform-specific signaling and cellular function in vivo.
106
The anti-GRP78 antibody (clone:76-E6) that we used to target GRP78 shows broader
specificity to HSP70 family proteins. It is interesting to consider the possibility that targeting the
csHSP70 family proteins may be an effective approach to suppress cancer progression than
targeting csGRP78 alone since both csGRP78 and the other major csHSP70 family protein, HSP70
(HSPA1A), are expressed at higher levels in cancer cells compared to normal cells. However, the
intrinsic behaviors of csGRP78 and HSP70 are very different thus the 76-E6 antibody is unlikely
to target both csGRP78 and csHSP70 proteins in live cancer cells. Cell surface GRP78 exists
largely as a peripheral protein and is associated with membrane proteins (Tsai et al. 2015). There
is no clear evidence, to the best of my knowledge, to point out a direct association of GRP78
protein to the lipids. On the other hand, csHSP70 is a membrane-embedded integral protein, and
only a small portion of the protein is exposed outside the plasma membrane. According to our
epitope mapping and sequencing comparison results, the 76-E6 antibody does not target the
portion of HSP70 protein exposed outside the plasma membrane. Therefore, the 76-E6 antibody
is unlikely to target membrane-embedded HSP70 protein. However, HSP70 protein shows high
affinity to phosphatidylserine (PS) presented in artificial unilamellar lipid vesicles (Vega et al.
2008, Schilling et al. 2009). PS is exclusively localized on the inner leaflet of plasma membrane
and the presence of PS on the outer leaflet is a maker of early cell apoptosis (Tesniere et al. 2008).
Therefore, it is possible that PS can facilitate translocation of HSP70 from the inner leaflet to the
outside of plasma membrane in the early apoptotic cells, and the 76-E6 antibody may target this
population of csHSP70. The presence of HSP70 protein on the outer leaflet of plasma promotes
killing of tumor cells (Schilling et al. 2009). How would the 76-E6 antibody affect the interaction
of HSP70, PS and immune cells and then collectively influence the viability and behaviors of
tumor cells require further investigations. Additionally, the density of csHSP70 is not affected by
107
the alteration of extracellular pH and salt concentration, therefore it is not likely that HSP70
interacts with membrane proteins (Gehrmann et al. 2008). Thus, our observation that the 76-E6
antibody treatment reduced the CD44v protein level is likely a result of specific targeting of
csGRP78 instead of csHSP70. This observation is also supported by the specific targeting of
csGRP78 by the MAb159 antibody.
In Chapter 3, we identified previously unknown functions of the C-terminal PPP polyproline
sequence of GRP78 protein in regulating its cell surface expression and modulating STAT3
activation. We focused our research on the functional significance of this polyproline region and
did not elucidate the underlying mechanisms for our observations, thus the major contribution of
this study is to open new possibilities to further investigate GRP78 and chaperone biology. In this
work, we identified the PPP polyproline region by characterizing the interaction between CD44v
and GRP78. However, we did not show how the PPP to AAA mutation (a.a. 640-642) of GRP78
would affect its affinity to CD44v. Therefore, the observed reduction of the mutant GRP78 on the
cell surface lacks mechanistic explanation, although the co-localization of CD44v and the wild-
type GRP78 in both the ER and cell surface suggests that CD44v may facilitate the translocation
of GRP78 from the ER to the plasma membrane. Additionally, although the PPP sequence is
localized outside the SBD domain and is not immediately adjacent to the KDEL Golgi-to-ER
retrieval motif, we cannot exclude the possibility that the mutation of the PPP sequence will affect
the ability of GRP78 to bind to its substrate and to interact with the KDEL receptor thus affecting
the cell surface expression of GRP78 protein. Therefore, future studies will be required to decipher
the roles of PPP polyproline sequence in the interaction of GRP78 with known partner proteins,
such as CD44v, MTJ-1/HTJ-1, and Cripto, the folding substrates and the KDEL receptor.
108
The discovery of the role of PPP polyproline sequence in STAT3 activation indicates that this
region has a novel signaling function. Future studies will be required to further explore if there are
interacting proteins specific to this polyproline motif and together with GRP78 play unidentified
roles in cell signaling and functions independent of the traditional chaperone function of GRP78.
The overexpression of the small peptide encoding the C-terminal proline-rich region of GRP78
reduced the viability and increased apoptotic markers in MCF7-LR cells, supporting the idea of
the functional importance of the newly identified C-terminal region containing the polyproline
sequence. However, the clinical importance is far from sufficient and is not the focus of this
research. The potential applications in the cancer therapeutics warrant rigorous future investigation.
Furthermore, we found that the C-terminal polyproline sequence of GRP78 is specific to higher
eukaryotic organisms. Therefore, it is intriguing to speculate that this sequence facilitates the
orchestration of complex tissue structures and signaling pathways. It will require future studies to
elucidate its roles in normal development and malignant transformation.
Taken together, in this dissertation, we unveiled previously unidentified partner protein,
CD44v, of csGRP78 in breast cancer cells. We also showed that targeting csGRP78 by the
antibodies, 76-E6 and MAb159, suppressed CD44v expression at the protein level and inhibited
cell adhesion. Additionally, the 76-E6 antibody can potently suppress cell migration, spreading,
motility, polarity and matrix degradation. Therefore, targeting csGRP78 open new possibilities to
suppress CD44+ cancer cells, particularly the breast cancer cells with the stemness characteristics
that are responsible for the relapse of the disease. We also identified an important intracellular
multi-scaffold protein, IQGAP1, as a potential downstream signaling node of csGRP78 by LC-
MS/MS. This provides a strong base to further study the function of csGRP78 in regulation of
cytoskeletal dynamics. The nature of the observed plasma membrane dynamics of GRP78 and
109
CD44v in the nanodomains are not clear, however, our results point out new directions to further
study the heterogenous protein behaviors and functional importance of these two proteins. The
discovery of the functional importance of the C-terminal PPP polyproline sequence of GRP78
sheds light on the potential mechanism of csGRP78 expression and facilitates our understanding
of GRP78/chaperone biology. Our work may have broader therapeutic implications in other cancer
types that express CD44 and csGRP78.
110
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Abstract (if available)
Abstract
The 78 kD glucose-regulated protein (GRP78) is a major chaperone protein in the endoplasmic reticulum (ER). It conducts protein folding and quality control and regulates unfolded protein response (UPR) under ER stress. The expression and cell surface translocation of GRP78 was elevated in aggressive tumors. Cell surface GRP78 (csGRP78) majorly exists as a peripheral protein and exerts novel signaling functions beyond its traditional role as a chaperone protein in the ER. However, the underlying mechanisms enabling GRP78 to be expressed on the cell surface and conduct novel signaling functions at the plasma membrane are just emerging. ❧ In this dissertation, we discovered that the type-I transmembrane protein CD44 is a novel partner protein of GRP78 on the cell surface and in the cytosol of breast cancer cells. Studies have shown that CD44 is an important regulator of breast cancer progression. Using conventional confocal microscopy and super-resolution dual-color single particle tracking, we found that GRP78 interacts with CD44 variant isoform (CD44v) in plasma membrane nanodomains of breast cancer cells. We further showed that this interaction regulates CD44v membrane homeostasis and multifaceted cell signaling impacting cell adhesion, spreading, migration and polarity. Thus, this work provides new mechanistic insights on how the GRP78/CD44v axis regulates cell migration in aggressive breast cancer. ❧ Furthermore, we mapped the region of GRP78 that is important for forming complex with CD44v by the in vitro GST pull-down assay and discovered that the C-terminal proline-rich region (PRR) containing the PPP polyproline sequence is essential for the complex formation. Mutation of the PPP polyproline sequence of GRP78 suppresses its cell surface expression in the tamoxifen-resistant breast cancer cells and inhibits STAT3 signaling, suggesting novel functions of PPP polyproline sequence in regulation of csGRP78 expression and cell signaling. We further designed an expression plasmid encoding a secretory signal peptide and 20 amino acids spanning the PPP polyproline sequence of GRP78, and we showed that overexpression of this plasmid reduced cell viability and led to an increase in apoptotic markers in tamoxifen-resistant breast cancer cells. Our study uncovered a previously unidentified interaction between two major players in cell and cancer biology, GRP78 and CD44, and discovered novel functions of the C-terminal proline-rich region of GRP78 in breast cancer cells.
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Creator
Tseng, Chun-Chih
(author)
Core Title
The role of endoplasmic reticulum chaperone protein GRP78 in breast cancer
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
03/19/2020
Defense Date
04/29/2019
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BiP,breast cancer,CD44,GRP78,OAI-PMH Harvest,Palm,single molecular imaging,single particle tracking,super-resolution,tamoxifen-resistant
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), Lee, Amy S. (
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), Pinaud, Fabien (
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), Yu, Min (
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breast cancer
CD44
GRP78
single molecular imaging
single particle tracking
super-resolution
tamoxifen-resistant