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Utilizing novel small molecules to modulate GRP78 expression and activity in cancer
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Utilizing novel small molecules to modulate GRP78 expression and activity in cancer
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Content
Utilizing Novel Small Molecules to Modulate GRP78 Expression and Activity in Cancer
by
Bintao Wang
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 2022
Copyright 2022 Bintao Wang
Acknowledgements
First and foremost, I would like to express my sincere gratitude toward my mentor and thesis
committee chair, Dr. Amy Lee, for her generous support and guidance throughout the past two
years. Researching has not been easy during the past two years because of all the turbulences and
hindrances brought on by the pandemic; however, Dr. Lee’s perseverance and enthusiasm toward
conducting research and publishing high quality papers served as a beacon which guided me
toward achieving my goals in this chaotic period of time. Dr. Lee also tried her best to provide
me with opportunities to present new data and learn new techniques to help me become more
familiar with the field of biological research.
I would also like to express my appreciation toward the members my research committee, Dr.
Min Yu and Dr. Baruch Frenkel, for their insights and suggestions toward my research. They
have also kindly supported me in my decision to apply for PhD programs and provided me with
recommendation letters which helped me immensely.
Furthermore, I would like to thank my mentor Dr. Vicky Yamamoto, and all the rest of Lee lab’s
member, for their generous support and help during my training here. Dr. Vicky Yamamoto had
always been encouraging and patient to me. She provided me with thorough and detailed
instruction when teaching me new techniques, and she had always been very patient when I have
questions. Moreover, Dr. Ze Liu and Dr. Dat Ha had also taught me many new experimental
techniques and proper ways to analyze data, which I am grateful for. I would also like to thank
Dr. Bo Huang and Mr. Guanlin Liu for all their support, help, and all the good times we had
together. My research experience would not be as interesting and memorable without them.
ii
Table of Contents
Acknowledgements ii
List of Figures v
Abstract vi
Chapter I Introduction 1
Chapter II Using dCas9 activation system to induce overexpression of 6
endogenous GRP78
2.1 Introduction 6
2.2 Material and Methods 9
2.3 Results 12
2.3.1 Amplifying Piggybac plasmid and confirming plasmid 12
identity
2.4 Discussion 15
2.4.1 Construct of dCas9 activation system to induce 15
endogenous GRP78 overexpression
2.4.2 Proposed follow-up experiments utilizing dCas9 16
activation system
Chapter III Small molecule AA147 upregulates GRP78 and confers 19
resistance against Cisplatin in head and neck cancer cell
3.1 Introduction 19
3.2 Material and Methods 23
3.3 Results 25
3.3.1 AA147 upregulates GRP78 by preferentially activating 25
ATF6 branch of UPR
3.3.2 AA147 upregulates GRP78 in head and neck cancer cell 26
lines and lung cancer cell lines without activating global UPR
3.3.3 AA147 upregulation of GRP78 confer resistance against 26
cisplatin in SCC15
3.4 Discussion 33
Chapter IV Requirement of GRP78 in head and neck cancer cell 35
survival and resistance against cisplatin treatment
4.1 Introduction 35
iii
4.2 Material and Methods 39
4.3 Results 41
4.3.1 Knockdown of GRP78 induces apoptosis in SCC15 cell line 41
4.3.2 Inhibition of GRP78 activity induces apoptosis in HNSCC 41
cell lines
4.3.3 YUM70 can resensitize cisplatin-resistant SCC15 to 42
cisplatin treatment.
4.4 Discussion 48
Chapter V Conclusion 50
References 52
iv
List of Figures
Figure 1-1. Role of GRP78 in regulating ER stress signaling and UPR activation. 5
Figure 2-1. SunTag dCas9 activation system and proposed scheme to induce endogenous 8
GRP78 overexpression.
Figure 2-2. Construct of the PB-TetON-dual-SunTag-Hygro vector. 13
Figure 2-3. Xbal restriction enzyme digestion and sequencing result. 14
Figure 3-1. ATF6 activator AA147 screening and mechanism of action. 22
Figure 3-2. AA147’s effect on UPR markers in lung cancer cell line A549. 28
Figure 3-3. Effect of AA147 treatment on induction of apoptosis determined by 29
Western Blot.
Figure 3-4. AA147’s ability to upregulate GRP78 without evoking ER stress in 31
HNSCC and lung cancer cell lines.
Figure 3-5. AA147 confers resistance against cisplatin in SCC15 cell. 32
Figure 4-1. GRP78 expression and survival outcome of HNSCC patients. 37
Figure 4-2. YUM70’s structure and cytotoxicity. 38
Figure 4-3. Knockdown of GRP78 by siGRP78 induces apoptosis in SCC15 cell. 43
Figure 4-4. YUM70 effect on HNSCC cell lines. 45
Figure 4-5. Cell image of SCC15 cell after YUM70 treatment. 46
Figure 4-6. YUM70 can resensitize cisplatin resistant SCC15 cell to cisplatin treatment. 47
v
Abstract
The unfolded protein response (UPR) is an adaptive reaction in response to endoplasmic
reticulum (ER) stress that is highly conserved in eukaryotes. Activation of the UPR will
stimulate the expression of chaperone proteins, which can ameliorate ER stress and restore ER
homeostatsis; however, prolonged or irreversible ER stress will trigger UPR-mediated apoptosis.
As a major regulator of the UPR, the glucose regulated protein GRP78 is a ER chaperone protein
that can directly influence the outcome of UPR.
GRP78 is often upregulated in cancer cell to alleviate ER stress and maintain cell
viability. It plays a critical role in the survival of cancer cell, and elevated GRP78 can confer
drug resistance. In this study, we utilized novel small-molecule ATF6 activator AA147, which
can elevate the expression of GRP78, to investigate GRP78’s role in the development of drug
resistance. On the other hand, we used small-molecule GRP78 inhibitor YUM70 to demonstrate
the requirement of GRP78 in the survival of head and neck cancer cells and their resistance to
cisplatin. Overall, this study has identified small-molecule GRP78 inhibitors as promising cancer
therapeutics, especially when administered in combination with traditional chemotherapeutics.
vi
Chapter I
Introduction
GRP78 in endoplasmic reticulum stress response
GRP78, a 78kDa glucose-regulated protein, is a molecular chaperone belonging to the
heat shock protein(HSP) family. HSPs play an important role in the survival of stressed cells due
to their ability to prevent intracellular proteins from aggregating and misfolding and restore
cellular homeostasis [1]. While most HSPs are found in the cytosol, GRP78 are mostly found in
the endoplasmic reticulum(ER), an important organelle for regulating protein quality control and
trafficking [2]. GRP78 has been established to be an indicator for endoplasmic reticulum (ER)
stress; although GRP78 is a ubiquitous protein that is constitutively expressed at basal level, its
level is significantly increased by the onset of ER stress [2,3].
As a regulator of ER stress signaling pathway, GRP78 binds and inhibits ER stress
transducers PERK, IRE1, and ATF6 in the ER lumen[4]. Accumulation of misfolded proteins in
the ER will cause GRP78 to bind to the misfolded proteins and release ER stress transducers,
leading to the activation of unfolded protein response (UPR) (figure 1-1) [2,4]. During UPR,
activated ER stress transducer PERK phosphorylates eukaryotic translation initiation factor 2α
(eIF2α), which blocks most protein translation and reduces influx of nascent proteins into the ER
[2,4]. The release of IRE1 from GRP78 lead to the activation of IRE1’s endonuclease activity,
which splice the mRNA coding for transcriptional factor X-box-binding protein 1 (Xbp-1) [2,4].
Activated ATF6 translocates from the ER to the Golgi complex and get cleaved by proteases
S1P/S2P into activated transcriptional factor [2,4]. Overall, during UPR, transcriptional factors
Xbp-1 and ATF6 upregulate the expression of ER chaperon proteins and protein degradation
1
molecules, along with the transient inhibition of most protein translation, can prevent misfolded
protein accumulation and the induction of UPR-mediated apoptosis [4,5].
GRP78 role in diseases
One important aspect of cancer progression is the ability of the cancer cells to proliferate
rapidly, which requires increased protein synthesis [6]. ER activity is enhanced during cancer
proliferation to facilitate the folding and transportation of membrane and secreted proteins [6].
GRP78 is one of the ER chaperones which maintain ER homeostasis, while preventing apoptosis
caused by stressful environment such as hypoxia and glucose deprivation from rapid growth and
inadequate angiogenesis during cancer proliferation [6]. Moreover, GRP78 can be transported to
the cell surface, where it can act as a cell surface receptor; cell surface GRP78 is also involved in
tumor survival, proliferation, and drug resistance [7]. Cell surface GRP78 acts as a receptor for
activated plasma protease inhibitor α2-macroglobulin, leading to ERK and AKT activation [2].
Activated AKT induces autophagy, which can serve as a mechanism to restore ER homeostasis
and prevent apoptosis [2,5,6]. Expression of GRPs can act as an adaptive response to the stress
induced by cancer treatment, which impede therapeutic efficacy and confer therapeutic resistance
[8].
Furthermore, cell surface GRP78 can act as a receptor for multiple families of viruses,
including Borna disease, Japanese encephalitis, dengue virus serotype 2, and even corona virus
spike protein [7]. Recent studies had shown that compared to Covid-19 negative pneumonia
patients, GRP78 expression is significantly higher in Covid-19 positive pneumonia patients, thus
suggesting that cell surface GRP78 can be a potential therapeutic target for the development of
anti-SARS-CoV-2 drugs [9].
2
While elevated GRP78 is often linked with more rapid tumor progression in cancer
patients, GRP78 plays a protective role for neuronal cells in neurodegenerative diseases [4,10].
Previous studies had demonstrated that induced overexpression of GRP78 can ameliorate ER
stress and promote neuronal cell survival undergoing degeneration associated with activated
UPR [11].
GRP78 as a therapeutic target
Due to the critical role of GRP78 in maintaining ER homeostasis and preventing
apoptosis in cancer proliferation and progression, many studies had proposed GRP78 as a
therapeutic target [2,5,6]. One of the main challenges in cancer treatment is the development of
drug resistance, and the expression of GRP78 is a major contributor to drug tolerance and
resistance development [2]. Experiments had shown that knockdown of GRP78 will sensitize
tumor cells to a wide range of cancer therapies including chemotoxic, anti-hormonal, DNA
damaging and anti-angiogenesis agents in various cancers [5,6].
Moreover, the presence of GRP78 on cell surface, which is primarily found in malignant
tumor cells but not in normal healthy cells, has also been identified as a target for cancer therapy
and drug delivery with high specificity [34]. Cell surface GRP78 binding with Cripto can inhibit
apoptotic pathway induced by TGF-β signaling and activate proliferating MAP kinase pathway
[35]. Also, studies utilizing various heterozygous knockout mice have shown that a 50%
decrease of GRP78 expression has no negative effect on normal organs, but significantly reduced
tumor proliferation and angiogenesis [36], suggesting that GRP78 inhibition has great
therapeutic potential for cancer patients.
Targeting GRP78
3
GRP78 inhibition can be achieved by inhibiting GRP78’s transcription, translation,
stability, or activity. Various drugs have been identified to inhibit GRP78. Natural products such
as the soy phytoestrogen genistein can block the transcription of GRP78, and the green tea
flavonoid epigallocatechin gallate can inhibit the ATPase of GRP78, thereby inhibiting its
activity. Moreover, bacterial product subtilase cytotoxin (SubAB), derived from Shiga toxigenic
Escherichia coli strain cleaves the ATPase domain of GRP78 from substrate binding domain,
leading to increased ER stress and corresponding UPR-mediated apoptosis [37,38,39]. Screening
for small molecules inhibitor of GRP78 can provide promising drugs which can be used in
combination with traditional cancer therapies to prevent the development of resistance and
increase efficacy [5,6,8].
4
Figure 1-1. Role of GRP78 in regulating ER stress signaling and UPR activation. When
misfolded proteins accumulate in ER, GRP78 is released from binding with ATF6, IRE1, and
PERK, allowing the activation of these ER stress sensors, which results in the onset of UPR.
GRP78 can also form complex with procaspases located on ER membrane, thus preventing
apoptosis mediated by procaspases such as caspase-7 and caspase-12 [2].
5
Chapter II
Using dCas9 activation system to induce overexpression of
endogenous GRP78
2.1 Introduction
Due to the pivotal role of GRP78 in tumor progression and development of drug
resistance [2,4], our lab is interested in exploring new strategies to inhibit GRP78’s expression or
activity, including testing novel small molecules. While silencing GRP78’s expression by siRNA
can be readily performed and can greatly diminish the expression of GRP78, inhibiting GRP78
with small molecule treatment holds more clinical relevance. Nonetheless, one of the major
downsides of small molecule treatment is the lack of specificity and potential off-target effects.
Therefore, we constructed an inducible overexpression of endogenous GRP78 system, which
allow us to perform small molecule treatment and rescue experiment on the same cells, to
confirm the small molecule’s mechanism of action is through inhibiting GRP78. Overexpression
of endogenous GRP78 can be achieved by activating GRP78 promotor region with dCas9
activation system. The dCas9 system consists of two parts, a dCas9 with a SunTag tail which can
recruit transcription factor VP64, and a gRNA that guides the dCas9 to the promotor region of
desired gene. When combined with gRNA complementing to the proximal upstream region of
GRP78 promotor region, dCas9 can bring transcription factor VP64 to the region and activate
expression of GRP78 with high precision and minimal off-target effect [12]. If a potential small
molecule induces apoptosis in cancer cells through inhibiting GRP78 as its mechanism of action,
6
overexpression of GRP78 by dCas9 activation system should mitigate the small molecule’s effect
on the induction of apoptosis in cancer cells.
7
Figure 2-1. SunTag dCas9 activation system and proposed scheme to induce endogenous GRP78
overexpression. The SunTag system involves an enzymatically inert dCas9 linked with GCN4
epitope(the tail). scFv diffusible antibody linked with VP64 is also expressed by the same vector,
and the scFv antibody has a high affinity for the GCN4 epitope tail. Upon expression of gRNA
targeting sequences upstream of the endogenous GRP78 promotor region, the gRNA will guide
the dCas9, along with the VP64 to proximal region of the endogenous GRP78 promotor region.
The VP64 will then induce the expression of GRP78.
8
2.2 Material and Methods
2.2.1 gRNA design
CHOPCHOP was used to identify suitable gRNA sequences complementary to the
promotor region of GRP78. Sequences of 20 nucleotides preceding “NGG” protospacer adjacent
motif (PAM) of Cas9 on both strands were selected (5”-AATCGGCGGCCTCCACGAC
GGGG-3’; 5’- AGGGGGCCGCTTCGAATCGGCGG-3’). Other factors that were taken into
consideration when designing the gRNA sequence were: having a GC content between
40%-60%, off-target with 0, 1, and 2 mismatches were excluded to maximize efficiency.
2.2.2 Selecting appropriate dCas9a and gRNA expression vector
The dCas9a vector expressing a dCas9 SunTag system was named PB-TetON-dual-
SunTag-Hygro and obtained from Addgene (#121119). The PiggyBac vector for expression of
gRNA and puromycin resistance was obtained from Addgene (#121121). Selected sgRNA
preceded by 5′-CACC and 5′TTTG overhang can be cloned into the PB-gRNA-Puro vector.
2.2.3 Bacterial transformation and harvesting plasmid
Competent and Super Competent E.Coli was transformed by PB-TetON-dual-SunTag-
Hygro vector with heat shock technique. The transformed E.Coli was incubated in 500µL of
Luria Broth containing ampicillin for 1 hour, then 200µL of L.B. containing transformed bacteria
was planted on selective agar containing ampicillin and incubated at 37°C overnight. After
individual colonies were picked and expanded in L.B., the PB-TetON-dual-SunTag-Hygro vector
9
was harvested with GeneJET Plasmid Miniprep Kit (ThermoFisher, Waltham, MA) and the
concentration of harvested plasmid was 504.2 ng/µL.
2.2.4 Confirming plasmid identity with restriction enzyme digestion
The PB-TetON-dual-SunTag-Hygro plasmid was digested with Xbal restriction enzyme
by mixing 8µL of plasmid with 1µL CutSmart buffer (NEB #6004, Ipswich, MA) and 1µL of
Xbal. The digested plasmid was then run through a 1% agarose gel in TAE buffer with RedSafe
(Bulldog Bio, Portsmouth, NH) added to separate the DNA fragments based on size.
Proposed follow-up experiments
2.2.5 Transfecting HEK293 cells with GRP78-SunTag and gRNA expression vector
Lipofectamine 2000 from ThermoFisher (Waltham, MA) can be used to transfect
HEK293T cell with PB-TetON-dual-SunTag-Hygro plasmid and gRNA plasmid. After treatment
with doxycycline, which induces the expression of GRP78-SunTag plasmid containing HygR,
successfully transfected cells are resistant to Hygromycin and will be selected with the treatment
of Hygromycin.
2.2.6 Microscopy
Live cell immunofluorescent microscopy should be performed to check the expression of
dCas9 system. Cells that express dCas9 should be green and blue due to the expression of GFP
on svFc antibody and BFP on dCas9 plasmid.
10
2.2.7 Immunoblot analysis
After the GRP78-dCas9 tumor cell line has been established, treat one culture with
doxycycline, which will induce the activation of dCas9 system. Perform a western blot using the
cell lysis of cell culture with doxycycline and compare with control to confirm that the
expression level of GRP78 is increased in dox(+) culture. SDS-PAGE gel will be used to
separate proteins based on their sizes, primary antibody against GRP78 (BiP, 1:2000, BD
Transduction Laboratories #610979) is used to detect GRP78.
2.2.7 RNA-extraction and reverse transcription RNA can be extracted with TRIzol from ThermoFisher (Waltham, MA). Reverse
transcription will be carried out by mixing extracted RNA with random hexamers or specific
primers of GRP78 mRNA for strand-specific RT-qPCR to confirm that the transcription of
GRP78 mRNA level has increased.
11
2.3 Results
2.3.1 Amplifying Piggybac plasmid and confirming plasmid identity
After we received the PB-TetON-dual-SunTag-Hygro vector from Pablo Navarro’s lab in
France (Figure 2-2), the plasmid was successfully amplified and harvested. The concentration of
plasmid after it was harvested was determined with a spectrophotometer by measuring
absorbance at 260nm, which reached 504.2 ng/µL. With an ample amount of plasmid, we
proceeded to verify the identity of the plasmid before transfecting HEK293 cell with it. The
plasmid contains a Xbal restriction site, thus we digested the 1µL of the plasmid with Xbal
restriction enzyme. Since there was only one Xbal restriction site, we expected to see a band that
traveled less distance than control group (6kb) after separating and staining the cleaved DNA
fragments; however, there was an unidentified band at 2kb, indicated that there may be more
than one Xbal restriction sites, which was cleaved into a small fragment of DNA (Figure 2-3).
Due to the failed verification of plasmid identity, we then sent the plasmid to sequencing service
provided by GeneWiz, and sequencing of the plasmid confirmed that the plasmid identity could
not be verified (Figure 2-3).
12
Figure 2-2 Construct of the PB-TetON-dual-SunTag-Hygro vector. The vector contains a tet-on
promotor, dCas9 fused with a SunTag tail, a BFP tag, transcription factor VP64, and Hygromycin
resistance gene. The Xbal restriction site on the plasmid is indicated by the red arrow.
13
(A)
(B)
Figure 2-3. Xbal restriction enzyme digestion and sequencing result. (A) PB-TetON-dual-
SunTag-Hygro plasmid was harvested from transformed super competent E.Coli and transformed
competent E.Coli. After restriction enzyme digestion, the cleaved DNA contains a band that
traveled more distance than the control group. (B) The sequencing result from GeneWiz showed
failed verification of plasmid.
14
2.5kb
10kb
6kb
2.4 Discussion
2.4.1 Construct of dCas9 activation system to induce endogenous GRP78 overexpression
The critical role of GRP78 in maintaining cancer cell proliferation and development of
cancer therapy resistance makes GRP78 a promising target for cancer therapy. Generating a
system to achieve inducible overexpression of endogenous GRP78 can be a useful tool to
confirm the mechanism of action of potential anti-GRP78 small molecules. If the small
molecule’s mechanism of action is targeting GRP78’s transcription, translation, stability, or
activity, induced overexpression of GRP78 should mitigate the effect of the small molecule.
In order to achieve specific overexpression of the gene coding for GRP78, a dCas9
activation system is constructed. Designing the gRNA by selecting sequences of 20 nucleotides
preceding “NGG” protospacer adjacent motif (PAM) of Cas9 with minimal mismatches can
provide high specificity and low off-target effect. The SunTag system vector expresses both
diffusible antibody scFv, and an enzymatically inert Cas9 linked with a GCN4 epitope. The scFv
antibodies are fused with GFP and transcription factor VP64, and have high affinity for the
GCN4 epitope; this system allows dCas9 to bring VP64 to the region targeted by gRNA with
high specificity and efficiency [12].
The PB-TetON-dual-SunTag-Hygro and gRNA plasmid are cloned into PiggyBac
transposon plasmids in between two terminal repeat (TRs) sequences. when co-transfected with
PBase helper plasmid, which encodes for the transposase, the transposase will insert the
sequence between the two TRs into the host genome [12]. To achieve inducible expression, the
expression of dCas9 system is under the control of Tetracycline Response Element (TRE). Also,
15
BFP is linked to the dCas9 through P2A sequence; when the cells are treated with Doxycycline
(Dox), successfully transfected cells will become blue.
We did not proceed with this project because we did not receive the correct PB-TetON-
dual-SunTag-Hygro vector. It took over a month for us to receive the vector from France, and
due to the limited time of master research, we had to abort this project. However, this system
should theoretically be able to induce the overexpression of endogenous GRP78. In the future,
the proposed follow-up experiments should be conducted to validate the specificity and
efficiency of this system to induce overexpression of endogenous GRP78.
2.4.2 Proposed follow-up experiments utilizing dCas9 activation system
HEK293T cell line and A549 lung cancer cell line were proposed to be transfected due to
their high proliferation rate and ease of transfection. After selecting cells that have been
successfully transfected by PB-TetON-dual-SunTag-Hygro vector, we can then introduce a
vector expressing gRNA that complements the upstream PAM sequence of GRP78 promoter
region into the cells and test the expression level of GRP78.
To confirm that the expression of GRP78 is elevated in the transfected cells, induce the
expression of the dCas9-SunTag by treatment of Doxycycline. Introduce vector expressing
gRNA targeting GRP78 promoter region to one group of dCas9-SunTag cells (test) and introduce
vector expressing random gRNA sequence that will not target any gene’s promoter region to
another group of GRP78-SunTag cells (control). Compare the level of GRP78 of both groups by
performing a western blot using the cell lysates of the two groups of cells. dCas9-SunTag cells
with gRNA targeting the GRP78 promoter region should have a higher level of GRP78 compared
to the control group.
16
If the level of GRP78 is not significantly different between the two groups, perform a
reverse transcription followed by qPCR to compare the mRNA level of Grp78 between the test
group and the control group. If GRP78 mRNA level is elevated but the protein level remains
unchanged, then the translation of GRP78 may be effected by other factors, or the GRP78 protein
stability might be affected.
After confirming the effectiveness of GRP78-SunTag system in elevating the GRP78
level in transfected cells, the cells will be treated with known GRP78 inhibitors to investigate the
system ability to mitigate apoptotic effect of GRP78 inhibitors. Natural products such as
epigallocatechin gallate and honokiol, which are known to inhibit GRP78 by binding to its
ATPase domain, along with drugs that can induce ER stress and corresponding apoptosis, can be
used to inhibit GRP78[6]. After GRP78 inhibitor treatment, Doxycycline and gRNA vector
targeting promotor region of GRP78 will be provided to the dCas9-SunTag cells to induce
overexpression of endogenous GRP78. The apoptosis rate will be compared with the control
group, which did not receive Dox, or received a non-specific gRNA vector. If the induced
overexpression of GRP78 can alleviate ER stress and decrease apoptosis caused by known anti-
GRP78 small molecule, the system can be used as a valuable tool to confirm the mechanism of
action of potential anti-GRP78 small molecules. However, whether the SunTag system will be
able to mitigate the effect of anti-GRP78 small molecules that inhibit the transcription of GRP78
remains questionable and require further testing.
After the effectiveness of inducible overexpression of GRP78 has been proven to
alleviate apoptosis caused by known anti-GRP78 drugs, we can use the system to examine the
newly developed anti-GRP78 drug’s mechanism of action. Small molecules will be screened to
17
target the structure of the ATPase region and substrate binding region of GRP78. When the
selected small molecule induces ER stress and apoptosis in the dCas9-SunTag cell, Dox will be
provided to the dCas9-SunTag cells to induce overexpression of GRP78. If induced
overexpression of GRP78 can ameliorate the ER stress caused by the selected small molecule,
we can conclude that one of the mechanism of action of selected small molecule is through
inhibiting GRP78. The selected small molecule can then be used in conjunction with traditional
cancer therapy to increase efficacy and impede resistance development. On the other hand, if the
induced overexpression of GRP78 cannot rescue the cells from the proposed anti-GRP78 small
molecule, we can conclude that the selected small molecule does not target GRP78 as expected,
and the mechanism of action will require further testing.
18
Chapter III
Small molecule AA147 upregulates GRP78 and confers resistance
against Cisplatin in head and neck cancer cell
3.1 Introduction
During cancer proliferation, stressful tumor microenvironment and increased protein
synthesis can lead to the onset of ER stress and the disruption of intracellular homeostasis [4,6].
To alleviate ER stress and restore ER homeostasis, the Unfolded Protein Response (UPR) will be
activated [13]. GRP78 plays a major regulatory role in the activation of UPR by binding with
three ER stress transducer proteins that reside in the ER, namely activating transcription factor 6
(ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase R-like ER kinase (PERK) [3].
Upon detection of unfolded proteins, GRP78 will bind to the unfolded protein and release itself
from binding with ATF6, IRE1, and PERK [3]. Once released, these three transmembrane
proteins can trigger a signaling cascade and initiate UPR-related transcriptional and translational
modulations, such as increased chaperone proteins expression and halted overall protein
translation in response to ER stress [13]. Through these mechanisms, UPR can help ER stressed
cells survive and restore ER homeostasis, including cancer cells [3,13].
ATF6 is a type II transmembrane protein in the ER with a luminal domain that can be
post-translationally modified by N-linked glycosylation or intramolecular disulfide bridge
formation [14]. After post-translational modification, ATF6 will form monomeric or oligomeric
ATF6, or heteromeric complexes with other ER proteins [15]. Upon the onset of ER stress, ATF6
will be reduced into monomeric ATF6, which allows ATF6 to translocate to the Golgi apparatus
19
from ER through coat protein complex II (COPII) [15]. Once ATF6 relocates to the Golgi
apparatus, site 1 and site 2 proteases will cleave ATF6 into a leucine zipper transcriptional
activator, which can then enter the nucleus and upregulate the expression of chaperone proteins,
including GRP78 [14,15].
Since ATF6 plays an important role in the UPR response, ATF6 activator can potentially
restore ER homeostasis and reduce unfolded proteins by upregulating the expression of GRPs.
Plate et al. utilized cell-based high throughput screening to screen over 60,000 small molecules,
and identified small molecule AA147 as a non-toxic ATF6 activator through transcriptional
profiling [16]. One major challenge in identifying novel small molecule activator is to eliminate
compounds functioning through off-target effect. Transcriptional profiling was used by Dr.
Wiseman’s lab to confirm that small molecules AA147 preferentially activates ATF6 branch of
UPR instead of activating global UPR activation (figure 3-1) [16]. Wiseman’s lab had also
demonstrated that ATF6 activation by AA147 can preferentially decrease synthesis and
aggregation of unfolded and amyloidogenic proteins, while leaving functional, non-
amyloidogenic proteins unaffected [16]. Based on Plate et al. study, AA147 is a low-cytotoxic
small molecule that preferentially activates that ATF6 pathway of the UPR without causing
global UPR activation. Since GRP78 is a major downstream target of ATF6, AA147 should be a
suitable inducer of GRP78 transcription without evoking global UPR.
Here in this study, we utilized AA147 to upregulate GRP78 in several cancer cell lines.
The mechanism of AA147 activation of ATF6 involves compound metabolic activation and
covalent modification of ER-localized protein disulfide isomerases (PDIs), which increases the
reduced monomeric ATF6 for trafficking to the Golgi and proteolytic activation [17]. Cancer
20
cells often have elevated GRP78 expression level due to stressful cancer microenvironment, such
as hypoxia, glucose starvation, and increased protein synthesis [2,5]. In many types of cancer,
elevated GRP78 is commonly associated with therapeutic resistance [5,6]. To mimic the elevated
level of GRP78 in cancer cells, lung cancer cell line A549, H1975, H1993, and head and neck
cancer cell lines SCC15, SCC25 were treated with AA147. After confirming AA147’s ability in
elevating GRP78 without causing global ER stress in cancer cells, head and neck cancer cell line
SCC15 was treated by the combination of AA147 and cisplatin, a traditional chemotherapeutic
drug, to demonstrate the role of elevated GRP78 in promoting chemotherapy resistance in head
and neck cancer.
21
Figure 3-1 ATF6 activator AA147 screening and mechanism of action. (A) The small molecules
were first screened with luciferase reporter system to select those that can upregulate the
transcription of Endoplasmic Reticulum Stress Element (ERSE). Renilla Luciferase reporter
system was then used to counter-screen any small molecule that can activate IRE1 branch of
UPR, which is an indicator for global UPR activation. Then, screened molecules went through
multiplex transcriptional profiling to confirm the selected small molecule upregulate GRP78 by
preferentially activating the ATF6 branch of UPR. (B) AA147 stimulates the proteolytically
activation of ATF6, monomeric activated ATF6(N) can enter the nucleus and elevate GRP78
expression.
22
(A) (B)
3.2 Material and Method
3.2.1 Compound
Small molecule AA147 was provided to our lab by Dr. Wiseman’s lab. The compound was
dissolved in sterile dimethyl sulfoxide (DMSO) to reach a concentration of 10mM. The dissolved
aliquots were stored in -80°C. Thapsigargin (Tg) (MilliporeSigma, Burlington, MA) was
dissolved in DMSO at a concentration of 0.75mM.
3.2.2 Cell culture
HEK293AD, SCC15, and SCC25 (kindly provided by Dr. V . Yamamoto, University of Southern
California, Los Angeles) were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium
(DMEM) (ThermoFisher, Waltham, MA) supplemented with 10% fetal bovine serum (FBS)
(Corning) and 1% penicillin/streptomycin. A549, H1993, and H1975 (kindly provided by Dr. Z.
Liu) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with
10% fetal bovine serum (FBS) (Corning, Corning, NY) and 1% penicillin/streptomycin. All cell
lines were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
3.2.3 Small molecule treatment
Cells were rescued in T25 flask from being frozen at -80°C, and seeded into 6-cm dishes. Cells
were grown until they reached 60-80% confluency before AA147 treatment. Cells were treated
with 10µM of AA147 for 48 hours by adding 5µL of 10mM AA147 aliquot into 5mL of growth
medium in 6-cm dishes. 5µL of DMSO was also administered to the control groups for 48 hours.
23
In the induced ER stress positive control groups, ER stress was induced by adding 300nM of Tg
for 24 hours before harvesting.
3.2.4 Immunoblot analysis
Cell samples were rinsed twice with 5mL of cold Dulbecco's phosphate-buffered saline (DPBS)
(Fisher, Waltham, MA) before lysed with RIPA buffer (50mM Tris-HCl, 150mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with Protease and
Phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA) on ice. Protein concentration was
measured with Bio-Rad Protein assay and a spectrometer (Bio-rad, Hercules, CA). Cell lysate
were subjected to 8% or 12% SDS/PAGE and separated based on size. The proteins were then
transferred onto nitrocellulose membrane (Bio-rad laboratory, Hercules, CA) at 30V overnight.
Primary antibodies that were used to detect the proteins of interest were as follows: GRP78 (BiP,
1:2000, BD Transduction Laboratories #610979), GAPDH ( (6C5), 1:2000, Santa Cruz
#sc-32233), CHOP ( (L63F7), 1:1000, Cell Signaling #2895), and ATF-4 ( (D4B8), 1:1000, Cell
Signaling #11815). Secondary antibodies used were as follows: HRP-conjugated goat anti-mouse
(1:5000, Santa Cruz #sc-2005), HRP-conjugated goat anti-rabbit(1:3000, Santa Cruz #sc-2005).
3.2.5 Cell viability assay
Cell were seeded in 96-well plate and cell viability was measured by Cell Proliferation Reagent
WST-1 (SigmaAldrich, St. Louis, MO) assay. The experiment was performed according to the
assay protocol provided by SigmaAldrich.
24
3.3 Results
3.3.1 AA147 upregulates GRP78 by preferentially activating ATF6 branch of UPR
Lung cancer cell line A549 was treated with 10μM of AA147 for 48 hours to investigate
the robustness of AA147 in upregulating GRP78 and the cytotoxicity of AA147. Thapsigargin
(Tg) treatment, which induces ER stress and activates UPR, was also administered either alone
or in combination with AA147 to generate positive control samples for ER stressed cells. The
expression level of GRP78 was measured by Western Blot, and the result confirms that AA147
can elevate GRP78 level by 2.59 folds in A549 cell after normalization to loading control
GAPDH (Figure 3-2).
The level of activating transcription factor 4 (ATF4) and C/EBP homologous protein
(CHOP), which are downstream of the PERK branch of UPR, were also measured by Western
Blot to validate the mechanism of AA147 upregulation of GRP78 is through preferentially
activating ATF6 without causing global ER stress and UPR activation. As expected, the ATF4
and CHOP level of AA147 treated samples remained unchanged compared to the control group,
whereas the Tg treated A549 samples displayed a strong increase in ATF4 and CHOP level
(Figure 3-2).
Other apoptotic markers, namely cleaved poly-ADP-ribose polymerase (c-PARP) and
cleaved caspase-3 (c-Caspase 3), were also probed to investigate whether AA147 can induce
apoptosis (Figure 3-3). As expected, AA147 treatment did not elevate the level of c-PARP or c-
Caspase3 in A549 cell compared to the control group, while Tg treated samples displayed a large
increase in the level of these apoptotic markers (Figure 3-3).
25
3.3.2 AA147 upregulates GRP78 in head and neck cancer cell lines and lung cancer cell
lines without activating global UPR
Head and neck cancer cell lines SCC15 and SCC25, and Lung cancer cell lines H1975
and H1993 underwent 48 hours of 10μM AA147 treatment to decipher AA147’s ability to elevate
GRP78 in these cancer cell lines. AA147 treatment was also performed on HEK293AD cell line
to investigate AA147’s ability to upregulate GRP78 in non-cancerous cell line. The AA147
treatments were done in duplicates.
AA147 induces a strong elevation of GRP78 in HEK293AD cells by 9.28 folds, reaching
a level almost as strong as the elevation of GRP78 induced by ER stress caused by Tg (11.4
folds) (Figure 3-4). In head and neck cancer cell lines, SCC15 is more responsive to the AA147
upregulation of GRP78, which increased GRP78 level by 3.67 folds, comparing to SCC25, in
which GRP78 level was increased by 1.17 folds (Figure 3-4). In lung cancer cell line H1975 and
H1993, AA147 upregulated the expression level of GRP78 by 1.31 folds and 1.87 folds
respectively (Figure 3-4).
It is worth noting that in non-cancerous cell HEK293AD, AA147 was able to further
elevate GRP78 level when used in combination with Tg, comparing to Tg treatment alone
(Figure 3-4). Moreover, Tg induced ER stress was ameliorated in SCC25 cell, as denoted by the
decreased ATF4 level (Figure 3-4).
3.3.3 AA147 upregulation of GRP78 confer resistance against cisplatin in SCC15
A WST-1 cell viability assay was performed by my research mentor Dr. Vicky Yamamoto
using head and neck cancer SCC15 cell line. SCC15 cell line was selected as our model cell line
26
due to its stronger response toward AA147. SCC15 cell were treated by 12µM of traditional
chemotherapeutic drug cisplatin, with or without AA147, for 48 hours. 48 hours of 12µM of
cisplatin treatment can significantly reduce the viability of SCC15 to below 30% of the control
group’s viability (Figure 3-5). Interestingly, the addition of AA147 was able to counteract the
cytotoxic effect of cisplatin and restore the cell viability back to around 60% of the original level,
which is about a two-fold increase compared to the cell viability of cisplatin treated groups
(Figure 3-5).
27
Figure 3-2. AA147’s effect on UPR markers in lung cancer cell line A549. (A) Right: A549 was
treated with AA147, Tg, or the combination of AA147 and Tg for 48 hours. Left: Tg treated
MDA-MB-231 sample provided by Dr. Dat Ha was used as positive control samples for clearer
identification of correct protein bands. Whole cell lysates were subjected to immunoblot analysis
for protein levels of GRP78, ATF4, and CHOP, with GAPDH serving as a loading control. (B)
Quantification of GRP78 protein level after normalization is shown. (C) Quantification of ATF4
and CHOP protein level.
28
(A)
(B) (C)
Figure 3-3. Effect of AA147 treatment on induction of apoptosis determined by Western Blot.
(A) The level of apoptosis markers c-PARP and c-Caspase 3 of AA147/Tg treated A549 cell were
measured by Western Blot. (B) Quantification of apoptosis markers c-PARP and c-Caspase3 after
AA147/Tg treatment is shown.
29
(A)
(B)
30
(A)
(B)
(C)
Figure 3-4. AA147’s ability to upregulate GRP78 without evoking ER stress in HNSCC and
lung cancer cell lines. The selected cell lines were treated with DMSO(control), AA147, Tg, or
the combination of AA147 and Tg for 48 hours. GRP78, ER stress sensor ATF4, and loading
control GAPDH were probed by western blot. The quantification of the proteins after
normalization to GAPDH is shown.
31
(D)
(E)
Figure 3-5. AA147 confers resistance against cisplatin in SCC15 cell. SCC15 cell were treated
with 10μM AA147, 12μM cisplatin, or the combination of AA147 and cisplatin for 48 hours
before cell viability was measured by WST-1 cell viability assay. Data obtained from Dr. Vicky
Yamamoto.
32
3.4 Discussion
Plate et al. constructed a sophisticated three-tiered screening strategy utilizing cell-based
high throughput screening, counterscreening by transcriptional reporter system, and
transcriptional profiling to identify ATF6 activating small molecules. AA147 was selected due to
its ability to upregulate GRP78 by preferentially activating the ATF6 branch of the UPR. In this
study, we investigated the effect of AA147 on several different cell lines including non-cancerous
cell lines (HEK293AD), lung cancer cell line (A549, H1975, H1993), and head and neck cancer
cell lines (SCC15, SCC25). Although the degree of GRP78 elevation induced by AA147 varied
among different cell lines, from as strong as 9.28 folds in HEK293AD (Figure 3-3A) to 1.18 fold
in SCC25 (Figure 3-3C), AA147 did not cause elevation of ATF4, which was consistent across
different cell lines (Figure 3-2, 3-3). ATF4, a transcriptional activator under the PERK branch of
UPR independent from the ATF6 branch [40], was measured as an indicator for global ER stress
and UPR activation. Moreover, CHOP, which is a precursor for ER-stress induced apoptosis [2],
was also measured to confirm that AA147 does not induce ER stress-mediated apoptosis (Figure
3-2). The results suggested AA147 can induce elevated expression level of GRP78 without
causing global ER stress or activating UPR.
To ensure my experiments were performed properly, I included several positive controls
were included in this project because this was the first project that I executed by myself. All the
cell lines were also treated with Thapsigargin (Tg) to generate positive control of ER-stressed
samples. Tg is a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor that can
induce ER stress by disturbing ER homeostasis and causing the accumulation of misfolded
protein [18]. The addition of Tg treated positive control samples can provide easier identification
33
of the correct protein bands and display the level of elevation of ER stress markers when ER
stress is induced. These positive control samples can also help us rule out the possibility that the
lack of increase in ER stress markers is due to the failed exposure or wrong identification of the
correct protein bands. Furthermore, Dr. Dat Ha had also kindly provided Tg treated MBA-
MB-231 samples as ER-stressed positive control at the beginning of this project for the
identification of the correct protein bands.
After we had successfully confirmed the ability of AA147 to upregulate GRP78 without
causing global UPR activation, we utilized AA147 to induce the overexpression of endogenous
GRP78 to mimic the elevated GRP78 level commonly seen in cancer cells. To demonstrate the
critical role of GRP78 in cancer cell survival and the development of drug resistance, head and
neck cancer cell line SCC15 was treated with a traditional chemotherapy drug cisplatin in
combination with AA147. WST-1 cell viability assay was carried out by my research mentor Dr.
Vicky Yamamoto to measure and compare the viability of the treated cells. The result indicated
that SCC15 cells are quite sensitive to cisplatin treatment, as the cell viability dropped to below
30% of the control group’s level (Figure 3-5). Meanwhile, AA147 treatment alone did not
significantly increase cell viability, suggesting that AA147 treatment does not promote cancer
cell proliferation (Figure 3-5). However, when SCC15 cell was treated with AA147 in addition to
cisplatin, the cell viability restored to around 60% of the control group’s level (Figure 3-5). This
result had demonstrated the protective role of GRP78, and supported the notion that GRP78 is
required for cancer cell’s survival and acquisition of therapeutic resistance. This observation was
further investigated in the following chapter that either silenced the expression of GRP78 or
inhibited the activity of GRP78.
34
Chapter IV
Requirement of GRP78 in head and neck cancer cell survival and
resistance against cisplatin treatment
4.1 Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer
worldwide, affecting around 600,000 patients annually, and approximately half of the cases
resulted in death [19,20]. The major reasons for the high incidence of HNSCC-related mortality
include the lack of effective therapies and the recurrence of HNSCC [20]. One of the FDA
approved therapies for HNSCC treatment is cetuximab, a monoclonal antibody that targets the
epidermal growth factor receptor (EGFR); however, cetuximab treatment only has a response
rate of 13% in HNSCC patients when used alone [21]. Thus, cetuximab is often used in
conjunction with cisplatin, which induces apoptosis by damaging DNA and prevents cell division
by inhibiting DNA synthesis and mitosis [22]. The combination of cetuximab and cisplatin
treatment can raise the therapy response rate to 36% in HNSCC patients [23]. However,
recurrence of HNSCC often happens after initially responding to the treatment due to the
acquisition of drug resistance [20,23]. On the other hand, GRP78 had been identified to play a
cytoprotective role and confer drug resistance against chemotherapeutics in many types of cancer
[24,25]. We had demonstrated in the previous experiment that elevated GRP78, induced by
AA147, can increase SCC15 cell viability after cisplatin treatment (Figure 3-5). In addition,
elevated GRP78 expression has been correlated with poorer overall survival in HNSCC patients
(Figure 4-1) [26]. Therefore, we started to question if we can increase the sensitivity of HNSCC
35
cell to traditional chemotherapeutic cisplatin by the combination treatment of cisplatin with small
molecules GRP78 inhibitors.
YUM70, a hydroxyquinoline analog, was designed and synthesized by Neamati’s lab to
target GRP78 [27]. Samanta et al. tested the efficacy of YUM70 in inhibiting GRP78 and
impeding tumor progression in pancreatic cancer xenograft model and concluded that YUM70
can upregulate ER stress-related gene and induce apoptosis with no detectable cytotoxicity to
normal tissue (Figure 4-2) [27]. They have also identified that YUM70 can directly bind with
GRP78 and inhibit GRP78 ATPase domain activity [27]. Therefore, we selected YUM70 to
inhibit GRP78 in head and neck cancer cell lines SCC15, SCC25, and SCC351. We proposed the
inhibition of GRP78 by YUM70 will reduce HNSCC proliferation and enhance the sensitivity of
HNSCC toward cisplatin treatment.
36
Figure 4-1. GRP78 expression and survival outcome of HNSCC patients (Schneider M, 2022).
HNSCC patients with higher GRP78 expression have a significantly lowered (p-value < 0.05)
survival probability.
37
Figure 4-2. YUM70’s structure and cytotoxicity (Samanta S, 2021). (A) The structure of
YUM70, a synthesized hydroxyquinoline analog. (B) The IC50 of YUM70 for pancreatic cancer
cell lines and normal pancreatic tissue derived cells (HPNE) is shown.
38
(A) (B)
4.2 Material and Methods
4.2.1 Compound
YUM70 was kindly gifted to us by Dr. Nouri Neamati from University of Michigan. YUM70
was dissolved in DMSO to a concentration of 10mM and stored in -80°C.
4.2.2 Cell culture
SCC15, SCC25, and SCC351 (kindly provided by Dr. V . Yamamoto, University of Southern
California, Los Angeles) were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium
(DMEM) (ThermoFisher, Waltham, MA) supplemented with 10% fetal bovine serum (FBS)
(Corning, Corning, NY) and 1% penicillin/streptomycin. All cell lines were maintained at 37°C
in a humidified atmosphere of 5% CO2 and 95% air.
4.2.3 Transfection of siRNA
Custom siRNA were purchased from GE Healthcare Dharmacon, Inc. (Chicago, IL).
Transfection was performed using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) in
accordance with the protocol provided by the company. Cell lysate were harvested 48 hours after
transcfection of siRNA.
siRNA Sequence
siControl 5’-GAGAUCGUAUAGCAACGGU-3’
siGRP78 5’-GGAGCGCAUUGAUACUAGA-3’
39
4.2.4 Immunoblot analysis
Cell samples were rinsed twice with 5mL of cold Dulbecco's phosphate-buffered saline (DPBS)
(Fisher, Hampton, NH) before lysed with RIPA buffer on ice. Protein concentration was
measured with Bio-Rad Protein assay and a spectrometer (Bio-Rad, Hercules, CA). Cell lysate
were subjected to 8% or 12% SDS/PAGE and separated based on size. The proteins were then
transferred onto nitrocellulose membrane (Bio-Rad laboratory, Hercules, CA) at 30V overnight.
Primary antibodies that were used to detect the proteins of interest were as follows: GRP78 (BiP,
1:2000, BD Transduction Laboratories #610979), Cleaved-PARP ( (Asp214) (D64E10), 1:1000,
Cell Signaling #5625), GAPDH ( (6C5), 1:2000, Santa Cruz #sc-32233), CHOP ( (L63F7),
1:1000, Cell Signaling #2895), Cleaved-Caspase 7 ( (Asp198) (D6H1), 1:1000, Cell Signaling
#8438). Secondary antibodies used were as follows: HRP-conjugated goat anti-mouse (1:5000,
Santa Cruz #sc-2005), HRP-conjugated goat anti-rabbit(1:3000, Santa Cruz #sc-2005).
4.2.5 Cell viability assay
Cells were seeded in 96-well plate and cell viability was measured by Cell Proliferation Reagent
WST-1 (SigmaAldrich, St. Louis, MO) assay. The experiment was performed according to the
assay protocol provided by SigmaAldrich.
40
4.3 Result
4.3.1 Knockdown of GRP78 induces apoptosis in SCC15 cell line
GRP78 knockdown by siRNA targeting the coding region of GRP78 was performed to
demonstrate the lack of GRP78 can trigger the onset of apoptosis in SCC15 cells. After the
transfection of siGRP78 in SCC15 cell line, the expression of GRP78 decreased significantly, to
below 20% of the control group’s level (Figure 4-3). Meanwhile, the level of apoptotic markers
cleaved-PARP and CHOP elevated after the transfection of siGRP78 (Figure 4-3).
4.3.2 Inhibition of GRP78 activity induces apoptosis in HNSCC cell lines
HNSCC cell lines SCC15, SCC25, and SCC351 were treated with YUM70 for 48 hours.
The response of the HNSCC cell lines to YUM70 treatment was investigated by treating the cells
with increasing concentration of YUM70 ranging from 1.25µM to 20µM. In general, GRP78
expression was elevated as the concentration of YUM70 increased, which is an indicator for
onset of ER stress and UPR activation (Figure 4-4). As expected, the level of apoptotic markers
cleaved-PARP, cleaved-Caspase 7, and CHOP also increased as the concentration of YUM70
increased, suggesting inhibition of GRP78 by YUM70 can induce UPR-mediated apoptosis in
HNSCC cell lines (Figure 4-4). The Western Blot result also showed that SCC351 and SCC25
were more resistant to YUM70 treatment than SCC15 (Figure 4-4). Live cell images were taken
right before harvesting the cell lysate, and these images also indicated that YUM70 treatment
caused morphology change and reduced the cell density of SCC15 (Figure 4-5).
41
4.3.3 YUM70 can resensitize cisplatin-resistant SCC15 to cisplatin treatment
Cisplatin resistant SCC15 cell line (cisR-SCC15) was cultured by Dr. Vicky Yamamoto
by treating parental SCC15 with low dose of cisplatin and passage the survived cell after
allowing the cells to recover. By this selection, cisR-SCC15 became not responsive to 25µM
cisplatin (Figure 4-6), which served as a model cell line for cisplatin resistant HNSCC. However,
when YUM70 was used in combination with cisplatin, the cell viability of cisR-SCC15 cell
decreased as the concentration of cisplatin increased, suggesting cisR-SCC15 became sensitive
to cisplatin treatment again (Figure 4-6).
42
Figure 4-3. Knockdown of GRP78 by siGRP78 induces apoptosis in SCC15 cell. siControl and
siGRP78 were transfected into SCC15 cell line. The levels of GRP78 and apoptosis markers
cleaved-PARP and CHOP were probed with western blot. The quantifications of the relative level
of the proteins are shown.
43
44
Figure 4-4. YUM70 effect on HNSCC cell lines. HNSCC cell lines SCC15, SCC25 and SCC351
were treated with increasing concentrations of YUM70 ranging from 1.25µM to 20µM for 48
hours before cell lysates were harvested. The levels of GRP78, apoptosis markers cleaved-PARP
and CHOP, and loading control GAPDH were probed by western blot. Quantifications of the
protein are shown below each western blot.
45
Figure 4-5. Cell image of SCC15 cell after YUM70 treatment. Pictures were taken after SCC15
were treated with DMSO or YUM70 for 48 hours. The scale bars represent 10µm.
46
DMSO 5µM YUM70
10µM YUM70 20µM YUM70
Figure 4-6. YUM70 can resensitize cisplatin resistant SCC15 cell to cisplatin treatment. Cell
viability of CisR-SCC15’s after cisplatin and YUM70 combination treatment were measured by
WST-1 cell viability assay. Data was obtained from Dr. Vicky Yamamoto.
47
4.4 Discussion
The requirement of GRP78 for proliferation and survival in HNSCC cell lines was
demonstrated by both the knockdown of GRP78 and the inhibition of GRP78 activity.
Knockdown of GRP78 by custom siRNA targeting the coding region of GRP78 was quite
efficient, as the protein level of GRP78 decreased to less than 20% of the control group’s GRP78
level (Figure 4-3). Meanwhile, the level of apoptotic markers cleaved-PARP and UPR-mediated
apoptotic marker CHOP both elevated significantly when the expression of GRP78 decreased
(Figure 4-3). On the other hand, although YUM70 treatment generally elevates the expression of
GRP78 in HNSCC cell lines, the inhibition of GRP78 ATPase domain by YUM70 leads to UPR-
mediated apoptosis. In combination, the results indicated that GRP78 is required for the survival
of HNSCC, and the inhibition of GRP78 can effectively induce apoptosis in HNSCC.
One problem with many small molecules is the unintended interaction with unknown
targets, and the off-target effect might contribute to the cytotoxicity of the small molecule [27].
Therefore, we performed knockdown of GRP78 experiment to show the response of HNSCC
when GRP78 expression is hindered. As expected, HNSCC responded in similar fashion after the
transfection of siGRP78 and YUM70 treatment. From the similar response, we concluded that
the induction of apoptosis by YUM70 is likely contributed by YUM70’s ability to inhibit GRP78
activity.
siGRP78 can efficiently reduce the expression of GRP78 and induce UPR-mediated
apoptosis; however, siGRP78 held less clinical relevance due to the difficulties and limitations in
siRNA-based cancer therapy [28]. Therefore, we chose to inhibit GRP78 through small-molecule
YUM70 to decipher the role of GRP78 in the resistance against cisplatin in cisplatin resistant
48
SCC15 cell line. The cisplatin resistance of cisR-SCC15, cultured by Dr. Vicky Yamamoto, was
confirmed, as the viability of cisR-SCC15 only showed a minimal decrease after 25µM cisplatin
treatment (Figure 4-6). However, when YUM70 was administered in combination with cisplatin,
the viability of cisR-SCC15 showed a much higher level of decrease with increasing cisplatin
concentration (Figure 4-6). Based on the result, we concluded that GRP78’s activity is required
for the survival and the development of cisplatin resistance in HNSCC. Due to the critical role of
GRP78 in cisplatin resistance, we proposed that GRP78 inhibitors such as YUM70 have great
therapeutic potential in HNSCC therapy. Furthermore, GRP78 inhibitors should be considered as
a novel option for combination treatment with cisplatin to increase the efficacy of chemotherapy.
49
Chapter V
Conclusion
As a major regulator of the UPR, GRP78 can influence the outcome of UPR, resulting in
either the alleviation of ER stress or UPR-mediated apoptosis [29]. In this study, we demonstrate
the requirement of GRP78 for the survival and the cisplatin resistance of HNSCC through using
small molecules to induce the upregulation of GRP78 or inhibit the activity of GRP78. As
expected, HNSCC cell lines became less responsive to cisplatin treatment after GRP78
upregulation by ATF6 activating small molecule AA147. On the other hand, the inhibition of
GRP78 activity by small-molecule YUM70 in HNSCC can efficiently induce UPR-mediated
apoptosis and augment the efficacy of cisplatin.
While the cytoprotective role of GRP78 in cancer survival and development of drug
resistance had been thoroughly studied and established [30], the novelty of this study is the
utilization of novel small molecules to modulate GRP78 expression and activity. GRP78
inhibitor YUM70’s ability to impede HNSCC proliferation and reduce cisplatin resistance
reveals the therapeutic potential of GRP78 inhibitors in HNSCC therapy, especially when used in
combination with other traditional chemotherapeutics. Furthermore, this study has also illustrated
AA147’s ability to upregulate GRP78 without inducing global ER stress. Although AA147 is
used to show that elevated GRP78 in HNSCC can confer resistance against cisplatin in this
study, AA147’s ability to upregulate GRP78 makes it a promising candidate for treating
degenerative diseases caused by aggregation of misfolded protein [31].
Small molecule targeted drugs offer many advantages in efficacy and safety compared to
traditional chemotherapeutics; however, small molecule targeted therapy still faces many
50
obstacles, such as low response rate or unintended off-target effects [32]. One limitation of this
study is the possibility of unknown off-target effects which may contribute to YUM70’s ability to
induce apoptosis. We utilize knockdown of GRP78 through siRNA, which specifically targets
GRP78, to illustrate the apoptotic response of HNSCC when the expression of GRP78 is
hindered. Through the similar response of HNSCC after transfection of siGRP78 or YUM70
treatment, we conclude that YUM70’s ability to inhibit GRP78 is the major contributor to its
induction of apoptosis. However, we cannot rule out the possibility of unintended cytotoxic off-
target effects which may contribute to the induction of apoptosis by YUM70 without further
transcriptomic analysis.
51
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55
Abstract (if available)
Abstract
The unfolded protein response (UPR) is an adaptive reaction in response to endoplasmic reticulum (ER) stress that is highly conserved in eukaryotes. Activation of the UPR will stimulate the expression of chaperone proteins, which can ameliorate ER stress and restore ER homeostatsis; however, prolonged or irreversible ER stress will trigger UPR-mediated apoptosis. As a major regulator of the UPR, the glucose regulated protein GRP78 is a ER chaperone protein that can directly influence the outcome of UPR.
GRP78 is often upregulated in cancer cell to alleviate ER stress and maintain cell viability. GRP78 plays a critical role in the survival of cancer cell, and elevated GRP78 can confer drug resistance. In this study, we utilized novel small-molecule ATF6 activator AA147, which can elevate the expression of GRP78, to investigate GRP78’s role in the development of drug resistance. On the other hand, we used small-molecule GRP78 inhibitor YUM70 to demonstrate the requirement of GRP78 in the survival of head and neck cancer cells and their resistance to cisplatin. Overall, this study has identified small-molecule GRP78 inhibitors as promising cancer therapeutics, especially when administered in combination with traditional chemotherapeutics.
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Creator
Wang, Bintao
(author)
Core Title
Utilizing novel small molecules to modulate GRP78 expression and activity in cancer
School
Keck School of Medicine
Degree
Master of Science
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Biochemistry and Molecular Medicine
Degree Conferral Date
2022-08
Publication Date
07/08/2023
Defense Date
05/17/2022
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cancer apoptosis,chemotherapeutic resistance,GRP78,head and neck cancer,lung cancer,OAI-PMH Harvest
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Lee, Amy (
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), Frenkel, Baruch (
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), Yu, Min (
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barrytowang7@gmail.com,bintaowa@usc.edu
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Tags
cancer apoptosis
chemotherapeutic resistance
GRP78
head and neck cancer
lung cancer