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Examination of the effect of the ecPlexin-B1 on MDA-MB-231 cells
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Examination of the effect of the ecPlexin-B1 on MDA-MB-231 cells
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Examination of the effect of the ecPlexin-B1 on MDA-MB-231 cells
by
Yan Ma
A Thesis Presented to the
FACULTY OF THE USC Keck School of Medicine
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
Biochemistry and Molecular Medicine
August 2020
Copyright 2020 Yan Ma
ii
Acknowledgements
I would like to express my overwhelmed gratitude to my research supervisor and thesis
committee chair Dr. Amy S. Lee for her brilliant scientific suggestions and her patient guidance
during past two years. It is all the work that Dr. Lee contributed to this project that made it
concrete and improved: Dr. Lee routinely discussed the experimental results with us team
members and pointed out the direction when we were at our most vulnerable.
Secondly, I would also like to thank the other committee members, Dr. Min Yu and Dr.
David Cobrinik, who both gave me incomparable help and kindly encouragement when I applied
for PhD programs.
Besides, my thanks also go to Dr. Anthony Carlos, for his preliminary work which built a
solid foundation for the project. He also devoted his time and efforts into overcoming technical
issues with me side by side.
Furthermore, I want to give special appreciation to Dat Ha, who gave me invaluable help
in my life. No words can express my gratitude to his kindness. My acknowledgement also goes
to other Lee lab members: Vicky Yamamoto, John Johnson, Han Wan, Richard Van Krieken,
Yuru Wu. The memorable two years in the lab taught me more than science but also rich life
experience, which would be a treasure for my lifetime.
Finally, I would like to give the highest appreciation to my parents, for the love and care
they have incessantly given to me. Their words were like a beacon guiding me in darkness and
help me overcome obstacles on the way.
iii
TABLE OF CONTENTS
Acknowledgements……………………………………………………..……………………...….ii
List of Figures………………………………………………………..……………..…………….iv
Abstract…………………………………………………………...…….…….…………..….……v
Introduction…………………………………………………………………………….………….1
Chapter 1: Materials and Methods…………………………………………..…………..………...9
Plasmids…………………………………………...………………………………......9
Cell culture………………………………………………………………………….....9
Transfection condition………………………………………………………………...9
Protein concentration…………………………………………………………..…….10
Immunoblot analysis………………………………………………………………....10
Chapter 2: Results………………………………………………………………………………..12
EcPlexin-B1 transfection was confirmed by Immunoblot Analysis and Agarose
Gel…………………………………..………………………………………………..12
Extracellular Plexin-B1 was successfully concentrated from media………………...14
Time course experiment showed that 20 minutes is the most suitable treatment time
for AKT and FAK activation………………………………………………………...15
The dose dependent treatment of ecPLXNB1 to measure pFAK and pAkt
activation……………………………………………………………………………..17
Discussion……………………...………………………………………………………………...20
Conclusion……………………………………………………………………………………….23
References………………………………………………………………………………………..24
iv
List of Figures
Figure. 1-1 GRPs and unfolded protein response (UPR)……………………...…………….……6
Figure. 1-2 The role of CS-GRP78………………………………………………………...….….7
Figure. 1-3 Interaction of Sema4D and its receptors……………………………………...….…..8
Figure. 1 Myc tag test and agarose gel confirmation………..…………………………..………13
Figure. 2 Western Blot confirmed the success of ecPleinB1 concentration.…….……….........14
Figure. 3 Time course for serum activation for Akt and FAK…………………....……………16
Figure.4-1 Dose dependent treatment of ecPlexin-B1 upregulates pAkt and pFAK
expression……………………………………………….……………...……………....….18
Figure. 4-2 Repeated dose dependent treatment confirmed that ecPlexin-B1 upregulates
pAkt………………………………………………………....…………………………..…19
v
Abstract
Glucose-Regulated Protein 78 (GRP78) is a chaperone heat shock protein, which plays a vital
role in protein quality control of endoplasmic reticulum (ER). In cancer cells, adaptation to
chronic stress in the tumor microenvironment enhances expression of GRP78 on the cell surface,
which facilitates cell signaling while increasing resistance to apoptosis and tolerance to a wide
variety of therapies. Semaphorins and Plexins are a ligand/receptor pair and comprise a family of
transmembrane and secreted proteins that, together with co-receptors such as the neuropilins,
regulate a variety of developmental and pathological processes. Traditionally, Plexin has long
been studied in its role as a Semaphorin receptor. However, it has recently emerged in the
literature a role for reverse signaling, whereby Semaphorins can act as membrane anchored
receptors to certain cleaved forms of Plexins. We hypothesize that cell surface GRP78 stabilizes
the Plexin-B1/Sema4D complex facilitating downstream signaling pathways. Of particular
interest are focal adhesion kinase pathways, since activation of these pathways could predispose
cancer cells to more readily metastasize. Our project aims to firstly establish the reverse
signaling of Sema4D in triple-negative breast cancer cells, which is to provide a solid foundation
for the next step which entails a GRP78 knockout experiment to investigate the role of csGRP78
in this process of stabilization of signaling pathways downstream of Sema4D/PlexinB1. To
accomplish this goal, we purified and prepared recombinant Plexin-B1 that contains only the
extracellular domains (ecPLXNB1). By using MDA-MB-231 breast cancer cell model, we
successfully showed that treatment with ecPlexinB1 activates pFAK and pAkt levels in these
cells, consistent with the notion that Plexin-B1 can act as a ligand to stimulate Semaphorin-
mediated downstream signaling in human breast cancer cells.
1
Chapter I
Introduction
The glucose regulated proteins are a group of stress inducible chaperones mainly residing in the
endoplasmic reticulum and the mitochondria (Lee 2014). The Grps were firstly discovered by Ira
Pastan in 1977, through the observation that two proteins of molecular size 78 and 94 kDa were
significantly induced in chicken embryo fibroblasts cultured in glucose-free medium. These
proteins were subsequently identified as GRP78 and GRP94 (Lee 2001). The induction of the
expression of mammalian and yeast GRP genes involve a series of stress induction factors: the
expression of misfolded proteins and underglycosylated proteins. Beyond these, other ER stress
induction conditions include ER Ca
2+
depletion, misfolded protein, reductive stress, pathological
state, glycosylation antagonist, micro-environment of solid tumor and mood-altering drug.
The Glucose-Regulated Protein 78,000 (GRP78) or immunoglobulin heavy chain binding
protein (BiP) is an endoplasmic reticulum (ER)-resident chaperone that performs important
functions to regulate protein quality control and degradation. GRP78, encode in humans by
HSPA5, shares 60% amino acid homology with HSP70, including the ATP binding domain
required for their ATPase catalytic activity and is a HSP70 analogue in the endoplasmic
reticulum (Lee 2014). GRP78 is the master regulator of the unfolded protein response (UPR). In
response to ER stress, GRP78 would disassociate from UPR sensors: activating transcription
factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and PKR-like ER kinase (PERK) upon
accumulation of unfolded proteins or ER stress. (Witte 2011) (Figure. 1-1). After the
disassociation from GRP78, PERK dimerizes to promote its autophosphorylation and activation,
activated PERK phosphorylates the eukaryotic translation-initiation factor 2α (Eif2α) to attenuate
the rate of general translation initiation and prevent further protein synthesis; disassociation from
2
GRP78 makes ATF6 to translocate from ER to Golgi, where cleaved ATF9 would migrate to the
nucleus and perform as transcription factor, in order to augment ER folding capacity by
upregulating proteins; activated IRE1 has endoribonuclease activity and splices a 26-base intron
from the mRNA encoding the X-box binding protein 1 (XBP-1), which is a transcriptional factor
with target genes including DnaJ, p58, ERdj4, EDEM, and PDI, all involved in protein folding
and ERAD (Luo and Lee 2012).
While GRP78 is traditionally regarded as an ER luminal protein, studies have emerged
which show that GRP78 can be detected in other cellular compartments including cell surface,
cytosol, nucleus, and mitochondria (Tsai and Lee 2018). Among which, cell surface GRP78
(csGRP78) plays an especially important role in cancer biology. Recently, several csGRP78
ligands have been identified to activate downstream signaling pathways, hence it is believed that
csGRP78 exhibits different functions compared to the role of GRP78 in endoplasmic reticulum.
Evidence is accumulating that GRP78 translocates from the ER to the cell surface under stress
typical of the tumor microenvironment, such as glucose depletion, hypoxia and cancer cells that
have acquired therapeutic resistance express higher csGRP78 levels than the sensitive parental
cells (Zhang et. al, 2010, Zhang and Tseng 2013, Ren et. al., 2013). It has been discovered that
GRP78 is preferentially expressed on the surface of cancer cells rather than normal stromal cells
(Arap et. al., 2004, Yoneda et. al., 2008). So far, multiple ligands of csGRP78 and the
downstream signaling pathway have been identified: csGRP78 binds to MTJ-1, activates PI3K,
then AKT/NF-KB and promotes cell proliferation and apoptosis; csGRP78 binds to β-
integrin/uPAR, activates FAK signaling, and promotes invasion; csGRP78 binds to Cripto,
activates PI3K, SRC, MAPK and promotes growth inhibition, migration and invasion (Figure. 1-
2).
3
Semaphorins are a family of secreted and membrane proteins originally discovered as
axon guidance chemorepellents, and later were discovered as integral signaling components of
the immune response in B and T cells. Semaphorins are a major class of phylogenetically ancient
proteins, which mainly function in immune system signaling and neuronal developmental
signaling. Activated by innate immune mechanisms, Semaphorins are associated with T cell
activation, B cell migration, inhibition and aggregation; they can promote/support humoral
immune response. In neuronal developmental signaling, Semaphorins generally controls axon
guidance and white matter fasciculation; occasionally growth cone motility and stability in some
neural subpopulations. Typically, Semaphorins act as short range through monomeric or dimeric
receptor complexes such as CD72 and Plexins. In cancer, Semaphorins and Plexins are
extremely important to study because: 1) Semaphorins regulate various hallmarks of cancer
through binding to different receptors (Butti et. al., 2018); 2) Semaphorins and plexins are
closely involved in tumorigenesis and metastasis by regulating tumor development and
progression (Worzfeld et. al., 2014); 3) Sema4D employs a potential protective role in benign
tumors since it promotes tumor growth and metastatic inhibition (Gabrovska et. al., 2011); 4)
Semaphorin/plexin signaling either promote or suppress tumor growth by directly controlling
cell migration or cell apoptosis (Tseng et. al., 2011).
The discovery of Sema4D can be dated to 1992, when Boumsell’s team reported a novel
150-kDa glycoprotein dimer; it was expressed on T lymphocytes that had been activated with
CD3 monoclonal antibody or phytohemagglutinin (Zhang et. al., 2013). The SEMA4D gene was
then cloned and identified three years later as the 1
st
Semaphorin with immune system functions.
Unlike the prototypical Semaphorins, which are neuronal chemorepellents, Sema4D, Sema3A,
Sema4A, Sema6D and Sema7A have been described as immune Semaphorins, because they are
4
expressed in T cells, B cells, natural killer cells and dendritic cells. Lately, emerging evidence
has supported that Sema4D and its high affinity receptor PlexinB1 play a vital role in tumor
angiogenesis, tumor invasiveness and regulation of tumor-associated macrophages (Figure1-3).
The identified properties of Sema4D through PlexinB1 include induction of cell migration,
tubulogenesis, angiogenesis, regulation of monocytic lineage cells and tumor-associated
macrophages, controlling of invasive growth (Ch’ng 2010). Canonically, PlexinB1 is commonly
studied as the receptor. However, recent studies indicate that Semaphorins can also function as a
receptor to certain cleaved and solubilized forms of Plexins. For example, Sun et al. found that
there is a reverse signaling between transmembrane Semaphorin 4A (Sema4A) and PlexinB1
(Sun et. al., 2016). Drosophila Semaphorin-1a (Sema1a) can also function as a receptor during
neural development through Semaphorin receptor Plexin A (PlexA) (Yu et.al., 2010). Sema4D is
important to study in breast cancer because studies have shown that it activates Erb-B2/HER2
signaling to promote breast cancer metastasis and its expression has been demonstrated in
primary breast cancers (Yang et. al., 2016); Klotz et. at. found that Sema4D overexpression
promoted cancer circulating cells (CTC) to transmigrate the blood-brain-barrier in vitro (Klotz
et.al, 2018). Clearly, Sema4D serves an important role in tumor cell motility and even
organotrophic metastasis. Whether cell surface GRP78 modulates Sema4D remained unclear yet
and needed to be answered.
The disease we focused on is triple-negative breast cancer (TNBC), which is a cancer
type that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein,
which means traditional hormonal therapy medicines or HER2 receptor-targeted medicines
ineffective. According to National Breast Cancer Foundation, approximately 10%-20% of breast
5
cancers belong to triple-negative breast cancers. Compared to other breast cancers, TNBC is a
more basal-like type which makes it more aggressive, with a poorer prognosis.
Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays an important
role in survival signaling and has multiple roles in breast cancer. According to Zhou et.al, FAK
protects nuclear stabilizing protein (NS) from proteasomal degradation to allow breast cancer
growth (Zhou et.al., 2019). The FAK gene is amplified and overexpressed in a large fraction of
breast cancer specimens (Luo, 2009). Increased FAK expression and activity has been associated
with multiple poor prognostic indicators in breast cancer patients, while inhibition of FAK may
reduce the metastatic potential of breast cancer (Rigiracciolo et. al., 2019). Since FAK has been
confirmed to interact with Sema1A (Cho et. al., 2012) and Sema3A (Acevedo et.al, 2008) to
promote angiogenesis and cell migration, we hypothesize that Sema4D increases FAK
phosphorylation at focal adhesions to modulate triple-negative breast cancer cell motility and
ultimately, metastasis.
This thesis aimed to address whether cell surface GRP78 facilitates Sema4D/PlexinB1
signaling, and whether this leads to temporally enhanced or attenuated signaling such as
increased magnitude of signaling via Focal Adhesions. Lastly, we want to check whether
activation of Focal Adhesion signaling will bring functional changes in the downstream.
6
Figure. 1-1 GRPs and unfolded protein response (UPR). Under ER stress, GRP78 is titrated away from the ER stress
sensors and therefore activates PERK, IRE1 and ATF6 signaling pathways. ATF6 undergoes Golgi processing and is translocate
to nucleus. IRE1 and PERK are polymerized and phosphorylated to induce XBP1 splicing and eIF2a phosphorylation
respectively, which leads to arrest of translation and ER- associated protein degradation (ERAD) (Lee 2014).
7
Figure. 1-2 The role of CS-GRP78. csGRP78 interacts with various surface receptors to
promote cell proliferation and survival, migration, and invasion in cancer. (Gopal, 2018)
8
Figure. 1-3 Interaction of Sema4D and its receptors. Sema4D interacts with its low and high affinity
receptors, CD72 and Plexin-B1, respectively to elicit various physiologic responses as well as
oncogenesis (Ch’ng 2010).
9
Chapter Ⅱ
Materials and Methods
2.1 Plasmids and DNA
The secreted Myc-His-tagged form of Plexin-B1 in the pSecTag2 vector was obtained from our
collaborator, Dr. Worzfeld. For high-quality preparations, DNA was extracted using a GeneJET
Plasmid Maxiprep Kit. To prepare the bacterial culture, three single colonies were picked and
inoculated 5ml of LB medium supplemented with penicillin. The incubation lasted 8 hours at
37℃. The starter culture was then diluted from 1:1000 to 1:10000 in LB medium and incubated
overnight at 37℃. The cells were harvested by centrifugation at 5,000×g for 10 min. Pelleted
cells were resuspended, neutralized and washed according to manufacturer’s protocol. To
increase the concentration of eluted ecPLXNB1 DNA, the volume of the Elution Buffer was
reduced to 0.7ml, plus an additional elution step with 0.5ml Elution Buffer.
2.2 Cell culture
Human cell line 293T, human epithelial breast cancer cell line MDA-MB-231 were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Inc. Manassas, VA) containing
10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA) and 1% penicillin antibiotics.
Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% humidity.
2.3 Transfection condition
Experiments with forced expression of constitutively active ecPlexinB1 were done in 293T cells
in 10 cm dishes with 70%-90% confluence transfected with pSec2 vector provided by Dr.
Worzfeld, using the transfection reagent BioT (Bioland Scientific, Paramount, CA) following the
10
manufacturer’s instructions (Bioland Scientific). After 24 hours of transfection, the medium was
changed to fresh medium. The cells were collected after 72 hours of transfection.
For serum starvation experiments, cells were placed in DMEM (4.5 g/L glucose) not containing
FBS for 16 hours.
2.4 Protein concentration
We utilized the Amicon Ultra 10K devices coupled with swinging bucket rotor to concentrate the
protein. Before use, the ultrafiltration membranes were rinsed twice with buffer to avoid the
interference. To improve the yield of the final concentrated protein, 20ml of total extracellular
protein was added to the Ultra filter device, then span at 4,000 x g for 30 minutes. The
concentrate was then reconstituted to the original sample volume for buffer exchange to remove
salts and solvents. The amount of the final concentrated protein was 200μL after buffer
exchange.
2.5 Immunoblot analysis
Protein lysates from cells were extracted using ice-cold radioimmunoprecipitation assay buffer
(50mmol/L Tris-Cl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS)
with added protease and phosphatase inhibitors (Thermo Scientific), and by centrifugation
(13,000 rpm for 15 min). Proteins were separated by 8% or 10% SDS-PAGE gel and transferred
to nitrocellulose membrane at 4°C at 35V overnight. Target proteins were probed by primary
antibodies as follow: GRP78 (Santa Cruz Biotechnology), phosphorylated AKT (BD
Biosciences), AKT (Cell Signaling Technology), phosphorylated FAK (Cell Signaling
Technology), FAK (Santa Cruz Biotechnology), c-Myc (Tonbo Bioscience), and GAPDH (Santa
11
Cruz Biotechnology). The immunoblot membranes were incubated with primary antibodies at
4°C overnight. Proteins were detected with SuperSignal West Femto stable peroxide buffer after
reacting with HRP-conjugated secondary antibodies. Western blot densitometry was analyzed
using Image Lab.
12
Chapter Ⅲ
Results
3-1 EcPLXNB1 expression was confirmed by Immunoblot Analysis and Agarose Gel.
To obtain pure ecPLXNB1, the original extracellular PlexinB1 plasmid was amplified by an
Escherichia coli expression system and then purified with ThermoFisher GeneJET Plasmid
Maxiprep Kit to get large scale isolation of high-quality plasmid DNA.
A total of three colonies were selected for Maxiprep (ecPLXN1, ecPLXN2 and
ecPLXN3). Following amplification and isolation of pure plasmid, the MaxiPreps from these
colonies were run on agarose gel and demonstrated that super-coiled DNA from all three
colonies showed similar bands near 6,800bp (Figure 1A).
After agarose gel confirmation, we next tested the expression vector in mammalian
systems. DNA from the three clones (ecPLXN1, ecPLXN2 and ecPLXN3) were transfected into
293T cells, along with a control group which was transfected with pcDNA. The cells lysates
were collected after 72 hours following transfection to allow for sufficient time for expression to
occur. As shown in Figure 1B, Myc-tag antibody detection successfully captured bands near 68
kDa in all three clones, consistent with previous reports of ecPLXNB1 electrophoretic mobility.
To further confirm the 68 kDa bands were ecPLXNB1, we used a His-tag antibody
detection, since pSecTag2-ecPLXNB1 contains C-terminal His and Myc tags. As demonstrated
by Figure 1B and 1C, the ecPLXN1 clone showed highest expression efficiency.
13
Figure. 1 Myc tag test and agarose gel confirmation. A. ecPLXN1, ecPLXN2, ecPLXN3 were from the three
different colonies after Maxiprep; pcDNA was set as the negative control. According to the calculation, the size
of extracellular Plexin-B1 should be near 6,800. B. ecPLXNB1 was transfected into 293T cells and tested by Myc
antibody. Target proteins were analyzed by Western blot with GAPDH serving as loading control. C. Three
expression vectors were subjected to Western blot to test their expression efficiency and His-tag antibody.
A B
C
14
3-2 Extracellular Plexin-B1 was successfully concentrated from media and the efficiency
was tested
To obtain a semi-crude fraction of exPLXNB1 to use as treatment in cancer cell lines, we next
expressed and concentrated ecPLXNB1 from the media of transfected 293T. As control, 293T
cells were transfected with pcDNA3.1.
When we compare between whole cell lysates and media-concentrates from cells
transfected with either ecPLXNB1 or pcDNA, we found that media-concentrate had much higher
concentration of ecPLXNB1. Detected by both Plexin-B1 antibody and Myc antibody, media-
concentrate showed darker bands compared to whole cell lysates, which confirmed that
concentration step was a success (Figure 2). We used collected media from 293T cells
transfected with pcDNA as negative control.
Figure. 2 Western Blot confirmed the success of ecPleinB1 concentration. A. Samples were whole cell
lysates from 293T cells transfected with pcDNA control or ecPLXNB1; and culture media collected from 293T
cells transfected with pcDNA or ecPLXNB1. The ecPLXNB1 protein was detected by PlexinB1 antibody. B.
Samples were the same as described above. ecPLXNB1 protein was detected by Myc antibody. Target proteins
were analyzed by Western blot with GAPDH serving as loading control.
A
B
15
3-3 Time course experiment showed that 20 minutes is the most suitable treatment time for
Akt and FAK activation
Considering that future experiments require the measurement of protein kinase B (PKB/Akt) and
focal adhesion kinase, we conducted the time course experiment for serum activation for Akt and
FAK in our MDA-MD-231 culture system. Treated MDA-MB-231 cells were serum starved for
12 hours before serum activation for 10, 20, 30, 60 minutes respectively or remained untreated.
One dish was treated with 20μL extracellular PlexinB1 for 20 minutes. Cell lysates were
collected and tested by western blot to measure Akt and FAK activation, with GAPDH as the
loading control. As shown in Figure 3B, phosphorylated Akt (pAkt) reached the peak at 20 mins
and then decreased at 30 and 60 minutes, while according to Figure 3C, phosphorylated FAK
(pFAK) reached peak after 20 minutes and sustained throughout the 60 min time course.
Based on these results, we concluded that treatment with ecPLXNB1 can activate pAkt
and pFAK in MDA-MB-231 cells and that the most suitable treatment time to observe peak pAkt
and pFAK is 20 minutes.
16
A
B
C
Figure 3 Time course for serum activation for Akt and FAK A. MDA-MB-231 cells were treated with 10ml DMEM
(serum added) for 10, 20, 30, 60 minutes or remained untreated as the negative control. Another group was treated
with 20μL extracellular Plexin-B1 for 20 minutes. Target proteins were analyzed by Western blot with GAPDH
serving as loading control. For normalization, the first lane was set as the standard, based on which the other lanes
were respectively converted. B. Band intensity quantification of pAkt vs. tAkt under different treatments. The third
line was the extracellular Plexin-B1 treatment group. C. Band intensity quantification of pFAK vs. tFAK. under
different treatments. The third line was the extracellular Plexin-B1 treatment group.
pAkt/tAkt pFAK/tFAK
17
3-4 The dose dependent treatment of ecPLXNB1 to measure pFAK and pAkt activation
We next tested the activation of phosphorylated Akt and FAK under different doses of
ecPLXNB1 treatment. Four experimental groups were designed: cells treated with 10μL or 25μL
pcDNA; cells treated with 10μL or 25μL extracellular Plexin-B1; serum starved group as the
negative control and cells treated with DMEM (serum added) as the positive control. All
treatment was given for 20 minutes. The treatment was performed when MDA-MB-231 cells
were around 70%~90% confluent.
As shown in Figure 4-1 A, ecPLXNB1 activates phosphorylated Akt and FAK when
compared to pcDNA. Furthermore, when compared to serum starvation baseline, ecPLXNB1
induces 6-fold activation of phosphorylated FAK and 1.5-fold activation of phosphorylated Akt.
Peak activation for both pFAK and pAkt is achieved by using 20μL of ecPLXNB1 media-
concentrate. Lastly, we found that for phosphorylated Akt, serum re-stimulation induced pAkt to
1.2-fold over baseline conditions.
To confirm our previous results, we used new lysates from the same cell lineage and kept
other conditions unchanged to control variables. As shown by Figure 4-2, serum activation
induced pAkt by 4-fold and the higher dose of ecPLXNB1 treatment induced a 1.5-fold
upregulation of pAkt when compared to pcDNA negative controls. Therefore, we conclude that
ecPLXNB1 treatment upregulates phosphorylated Akt.
18
Figure. 4-1 Dose dependent treatment of ecPLXNB1 upregulates pAkt and pFAK expression. A. MDA-MB-231
cells were serum starved, treated with 10μL/20μL pcDNA for 20 minutes, treated with 10μL/20μL ecPLXNB1 for
20 minutes or serum activated for 20 minutes. Cells were serum starved for 12 hours in advance. Target proteins
were analyzed by Western blot with GAPDH serving as loading control. B. Band intensity quantification of pAkt
vs. tAkt. C. Band intensity quantification of pFAK vs. tFAK.
pAkt/tAkt
pFAK/tFAK
A
B
C
19
B
Figure. 4-2 Repeated dose dependent treatment confirmed that ecPLXNB1 upregulates pAkt.
A. MDA-MB-231 cells were serum starved, treated with 10μL/20μL pcDNA for 20 minutes, treated
with 10μL/20μL ecPLXNB1 for 20 minutes or serum activated for 20 minutes. Cells were serum
starved for 12 hours in advance. Target proteins were analyzed by Western blot with GAPDH
serving as loading control. B. Band intensity quantification of pAkt vs. tAkt.
A
20
Chapter Ⅴ
Discussion
Over-expression of Plexin-B1 has been observed in many types of cancers including breast
cancer and pancreatic cancer. This provides the precondition for the overexpression of
ecPLXNB1. According to a published paper concerning mechanisms of ectodomain shedding,
ectodomain shedding rapidly converts membrane-associated proteins into soluble effectors, and
at the same time, rapidly reduces the level of cell surface expression (Hayashida et. al., 2010).
Increased extracellular Plexin B1 could facilitate its binding with Semaphorins on the cell
surface due to the high affinity, thereby transducing the signals and promoting cancer
progression. Though extracellular Plexin-B1 is truncated, their juxta-membrane region is
preserved and secures the affinity of binding to the Semaphorins class. It is proposed that
expression of plexin-B1 by the tumor cells themselves might sequester the secreted Sema4D and
reduce its proangiogenic effect. From our research’s perspective, this means that overexpression
of Plexin-B1 could be a starter, then comes more expression of extracellular Plexin-B1 and more
expression of intracellular Semaphorins, along with less Plexin-B1 expression and less secreted
Sema4D expression.
Most of the biological functions exerted by semaphorins rely on their forward signaling,
such as signaling downstream of plexins. However, the role of semaphorins as receptors under
certain circumstances have been widely identified. A number of studies have shown that reverse
signaling through other forms of Semaphorins can induce Akt and receptor tyrosine kinase (Alto
and Terman, 2018). Since high expression levels of Sema4D are often observed in breast cancer
tumors, it is important to understand its novel role as a receptor.
21
Cell surface GRP78 has been found to be preferentially overexpressed in metastatic,
aggressive, and chemo-resistant cancers. In breast cancer, the expression level of csGRP78
gradually increases during cancer progression. Yao et al. found that csGRP78 accelerated breast
cancer cell proliferation and migration by activating STAT3. Conner et al. discovered csGRP78
not only promotes cellular functions in both pluripotent and breast cancer cells, also leads to an
induction of a CD24
-
/CD44
+
tumor initiating cell (TIC) population; as a result, csGRP78
+
breast
cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective
findings show that csGRP78 plays a vital role in oncogenesis and significantly promotes the
metastatic potential of breast cancer cells.
Our lab’s unpublished and preliminary results suggested that a binding interaction may
exist between cell surface GRP78 and Sema4D. Besides, flag-GRP78 coprecipitates with HA-
Sema4D (full length) in 293T cells. Further research found that flag-GRP78 co-precipitates with
all HA-Sema4D constructs (full length, △22-500, △22-551, △22-636) in 293T cell lysate, while
recombinant Sema4D-His (6X His-tag) which was missing intracellular and transmembrane
domains does not co-precipitate with GST-GRP78 by in vitro pull down assay. The
colocalization also showed that in MDA-MB-231 cells, GRP78 and Sema4D colocalizes near
nuclease. All these findings laid a solid theoretical foundation for this project, indicating that
GRP78 binds to the intracellular and transmembrane domains of Sema4D, and probably
PlexinB1 is involved in this process.
Since Semaphorins and plexins both play important regulatory roles in cellular motility,
altering downstream signaling pathways mediated by the Sema4D receptor would affect
pathological aspects such as cancer progression, tumor angiogenesis and invasiveness. However,
22
the role of Plexin-B1 in breast cancer remains largely unknown, let alone the mechanism of
reverse signaling between Plexin-B1 and Sema4D.
To address this question, we investigated whether extracellular Plexin-B1 would bind to
transmembrane Semaphorins on cell surface, and whether this reverse signaling (compared to
traditional Sema4D-Plexin-B1 pathway) would activate other downstream signaling pathways.
Our results showed that treatment of extracellular Plexin-B1 through binding to Semaphorins
would activate phosphorylated FAK and Akt in MDA-MB-231 cells. To conclude, we
established potential reverse signaling of Sema4 as a receptor in MDA-MB-231 cells, which
provided a solid foundation for either studying ecPLXNB1/Sema4D binding or csGRP78
regulation.
23
Chapter Ⅴ
Conclusion
The novel finding of this thesis is that PlexinB1, when serving as a ligand in breast cancer cell
line MDA-MB-231, can induce the phosphorylation of Akt and FAK. Based on this, we further
demonstrated the optimal kinetics and doses to induce these responses.
Sema4D-Plexin-B1 interaction has proven to be essential in tumor angiogenesis, tumor
progression and regulation of tumor-associated macrophages and control of invasive growth.
Emerging data have identified Semaphorin 4D (Sema4D) as a product of osteoclasts acting
through its receptor Plexin-B1 on osteoblasts to inhibit their function. Breast cancers and other
epithelial malignancies overexpress Sema4D. According to Yang et al., Semaphorin 4D
promotes skeletal metastasis in breast cancer. Traditionally, Semaphorins were only taken as the
ligand, while Plexins as the receptor. However, more and more studies supported that Plexins
can act the role of ligand and binds to their high-affinity receptors, Semaphorins. While
overexpression of the Plexin-B1 was found in the majority of primary tumors, the biological role
of Plexin-B1 in breast cancer is largely unknown. Rody et al. scanned n=119 breast cancer
specimens by using Affymetrix microarray analysis and concluded that low Plexin-B1
expression levels characterize a more aggressive tumor phenotype (Rody et. al., 2007).
To investigate the effect of extracellular Plexin-B1 in breast cancer, we treated MDA-
MB-231 cells with concentrated ecPlexin-B1 and tested the downstream signaling. The
upregulation of phosphorylated FAK and Akt, coupled with other supporting evidence from
other lab members, we have proven that: in triple negative breast cancer cell lines, ecPLXNB1
acts as the receptor through Semaphorins; as a result, downstream signaling including FAK
24
(Focal Adhesion Kinase) and Akt would be activated and finally, cancer invasion and metastasis
would be promoted.
A limitation in this study is whether ecPLXNB1 is acting through the Sema4D receptor
or through other receptors to activate Akt or FAK. To address this limitation and for future
directions, we will: 1. Check if pAkt and pFAK activation can still show when we knockout
Sema4D gene. 2. Test whether ecPLXNB1 affects migration and invasion through Sema4D
receptor (Functional assay, such as wound healing assay and invasion assay). 3. Using SubAb (A
bacterial toxin, which cleaves GRP78 at a single site) to block GRP78, which eliminates the
interaction between GRP78 and Sema4D; then repeat functional assay to see if there is still a
downstream effect through Sema4D-Plexin-B1 signaling.
25
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Abstract (if available)
Abstract
Glucose-Regulated Protein 78 (GRP78) is a chaperone heat shock protein, which plays a vital role in protein quality control of endoplasmic reticulum (ER). In cancer cells, adaptation to chronic stress in the tumor microenvironment enhances expression of GRP78 on the cell surface, which facilitates cell signaling while increasing resistance to apoptosis and tolerance to a wide variety of therapies. Semaphorins and Plexins are a ligand/receptor pair and comprise a family of transmembrane and secreted proteins that, together with co-receptors such as the neuropilins, regulate a variety of developmental and pathological processes. Traditionally, Plexin has long been studied in its role as a Semaphorin receptor. However, it has recently emerged in the literature a role for reverse signaling, whereby Semaphorins can act as membrane anchored receptors to certain cleaved forms of Plexins. We hypothesize that cell surface GRP78 stabilizes the Plexin-B1/Sema4D complex facilitating downstream signaling pathways. Of particular interest are focal adhesion kinase pathways, since activation of these pathways could predispose cancer cells to more readily metastasize. Our project aims to firstly establish the reverse signaling of Sema4D in triple-negative breast cancer cells, which is to provide a solid foundation for the next step which entails a GRP78 knockout experiment to investigate the role of csGRP78 in this process of stabilization of signaling pathways downstream of Sema4D/PlexinB1. To accomplish this goal, we purified and prepared recombinant Plexin-B1 that contains only the extracellular domains (ecPLXNB1). By using MDA-MB-231 breast cancer cell model, we successfully showed that treatment with ecPlexinB1 activates pFAK and pAkt levels in these cells, consistent with the notion that Plexin-B1 can act as a ligand to stimulate Semaphorin mediated downstream signaling in human breast cancer cells.
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Asset Metadata
Creator
Ma, Yan
(author)
Core Title
Examination of the effect of the ecPlexin-B1 on MDA-MB-231 cells
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Publication Date
06/22/2020
Defense Date
04/15/2020
Publisher
University of Southern California
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extracellular PlexinB1,OAI-PMH Harvest,reverse signaling,Sema4D,triple-negative breast cancer
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Lee, Amy (
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), Cobrinik, David (
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