The Pennsylvania State University

The Graduate School

College of Medicine

INVESTIGATING THE FUNCTION AND REGULATION OF PROSTATE

APOPTOSIS RESPONSE-4 IN CANCER

A Dissertation in

Molecular Medicine

by

Jeffrey Nguyen

© 2016 Jeffrey Nguyen

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2016

ii

This dissertation was reviewed and approved* by the following:

Rosalyn Irby

Associate Professor of Medicine

Department of Medicine

Dissertation Advisor

Chair of Committee

Jennifer Baccon

Associate Professor of Pathology

Arun Sharma

Associate Professor of Pharmacology

Robert Levenson

Distinguished Professor of Pharmacology

Charles Lang

Distinguished Professor of Cellular and Molecular Physiology and Surgery

Chair of Molecular Medicine PhD Program

*Signatures are on file in the Graduate School

iii

Abstract

Cancer is a disease where normal cells proliferate uncontrollably, which can

ultimately lead to significant morbidity and death. The aggressiveness and mortality of

cancers vary by type: certain cancers respond well to treatment, such as pediatric

leukemias, whereas pancreatic cancer and glioblastoma have a high mortality and low

five-year survival. In an effort to improve current cancer therapies, I focus on elucidating

the function and regulation of prostate apoptosis response-4 (Par-4) in various cancers.

Par-4 is a tumor-suppressor that has been shown to induce cancer cell selective

apoptosis and to sensitize cancer cells to apoptotic stimuli, such as chemotherapeutics

and radiation, and therefore has therapeutic potential. In the first part of this work, I

focused on studying the effect of Par-4 on cell migration, invasion, and the epithelial-

mesenchymal transition in colon cancer cells. I found that ectopic expression of Par-4

inhibited both cell migration and cell invasion, while knocking down Par-4 promoted cell

migration in SW480 and SW620 colon cancer cells. In addition, I found that Par-4

overexpression appeared to induce a mesenchymal-epithelial transition in SW620 cells.

In the second part of this work, I sought to identify novel regulators of Par-4 and

elucidate the mechanism of regulation. I identified Trim21 as a novel binding partner in

colon cancer cells, and show that Trim21 overexpression in the presence of cisplatin

downregulates Par-4 in colon and pancreatic cancer cell lines, and show that

modulating levels of Trim21 and Par-4 affects the sensitivity of cancer cells to cisplatin.

Finally, I demonstrate that Trim21 mRNA levels correlate with survival in pancreatic

cancer patients, with lower Trim21 levels correlating with increased overall survival and

disease-free survival and high Trim21 levels correlating with reduced disease-free

iv

survival. In the third part of this work, I sought to determine whether Par-4 could

enhance the effectiveness of chemotherapeutics and small molecule drugs. I chose to

focus on glioma due to the lack of effective therapeutics. I show that ectopic Par-4

expression alone is sufficient to reduce cell viability and to induce apoptosis in glioma

cell lines, A172 and SNB19. Furthermore, I demonstrate that Par-4 transfected glioma

cells are sensitized to 5-fluorouracil and ISC-4. Taken together, the results presented in

this dissertation suggest novel roles and regulatory mechanisms of Par-4 in cancer, and

provide rationale for its use in cancer treatment; as well as suggesting a novel

prognostic marker for pancreatic cancer.

v

Table of Contents List of Figures ............................................................................................................................................. viii

Abbreviations ............................................................................................................................................... ix

Acknowledgements ...................................................................................................................................... xi

1. Chapter 1 ............................................................................................................................................... 1

1.1. Prostate apoptosis response-4 ..................................................................................................... 1

1.1.1. Domains ...................................................................................................................................... 2

1.1.2. Intracellular functions of Par-4 ................................................................................................... 3

1.1.3. Extracellular functions of Par-4 ................................................................................................... 4

1.1.4. Mechanisms of regulation .......................................................................................................... 5

1.1.5. Non-apoptosis related functions ................................................................................................ 7

1.2. Colon cancer ................................................................................................................................. 9

1.2.1. Par-4 and Colon Cancer ............................................................................................................. 13

1.3. Pancreatic cancer ........................................................................................................................ 13

1.3.1. Par-4 and Pancreatic Cancer ..................................................................................................... 16

1.4. Glioblastoma ............................................................................................................................... 17

1.4.1. Classification ............................................................................................................................. 18

1.4.2. Prognosis and Treatment .......................................................................................................... 19

1.4.3. Molecular Genetics ................................................................................................................... 20

1.5. Par-4 and glioma ......................................................................................................................... 21

1.6. Apoptosis .................................................................................................................................... 22

1.6.1. Mechanism of apoptosis ........................................................................................................... 22

1.7. Conclusion ................................................................................................................................... 24

2. Chapter 2 ............................................................................................................................................. 26

2.1. Introduction ................................................................................................................................ 26

2.2. Materials and Methods ............................................................................................................... 27

2.2.1. Cell culture and transfection ..................................................................................................... 27

2.2.2. Western blot analyses ............................................................................................................... 27

2.2.3. MTT assay .................................................................................................................................. 28

2.2.4. Scratch assay ............................................................................................................................. 29

2.2.5. Boyden Chamber assays ........................................................................................................... 29

2.2.6. RT-PCR analyses ........................................................................................................................ 30

vi

2.2.7. Cell proliferation assay .............................................................................................................. 30

2.2.8. Statistical analyses .................................................................................................................... 30

2.3. Results ......................................................................................................................................... 30

2.3.1. Par-4 increases susceptibility of metastatic SW620 cells to 5-FU ............................................ 30

2.3.2. Par-4 inhibits cell migration and invasion in SW480 and SW620 cells ..................................... 34

2.3.3. Par-4 induces a mesenchymal-epithelial transition in SW620 cells ......................................... 40

2.3.4. Par-4 regulates tight-junction protein expression in SW620 cells ............................................ 45

2.4. Discussion .................................................................................................................................... 48

2.5. Acknowledgements ..................................................................................................................... 51

2.6. Conflict of Interest ...................................................................................................................... 51

2.7. Publication Note ......................................................................................................................... 52

3. Chapter 3 ............................................................................................................................................. 53

3.1. Introduction ................................................................................................................................ 53

3.2. Results ......................................................................................................................................... 55

3.2.1. Trim21 is a novel interacting partner of Par-4 .......................................................................... 55

3.2.2. Trim21 interacts with Par-4 through its PRYSPRY domain ....................................................... 59

3.2.3. Trim21 is not sufficient to downregulate Par-4 levels .............................................................. 62

3.2.4. Ectopic expression of Trim21 downregulates Par-4 in the presence of cisplatin ..................... 65

3.2.5. Cisplatin downregulates Par-4 in a dose- and proteaseome-dependent manner ................... 68

3.2.6. Cisplatin downregulates Par-4 in both the cytoplasmic and nuclear compartments .............. 71

3.2.7. Cisplatin downregulates Par-4 in pancreatic cancer cells ......................................................... 73

3.2.8. Trim21 is a potential therapeutic target in colon and pancreatic cancer................................. 76

3.3. Discussion .................................................................................................................................... 80

3.4. Materials/Methods ..................................................................................................................... 82

3.4.1. Cell culture, transfection, plasmids, reagents, and antibodies ................................................. 82

3.4.2. Western blot analyses ............................................................................................................... 83

3.4.3. Co-IP/Mass-Spec ....................................................................................................................... 84

3.4.4. MTT assay .................................................................................................................................. 85

3.4.5. Immunofluoresence .................................................................................................................. 85

3.4.6. Nuclear-Cytoplasmic Fractionation ........................................................................................... 86

3.4.7. Statistical analyses .................................................................................................................... 86

3.5. Acknowledgements ..................................................................................................................... 87

vii

4. Chapter 4 ............................................................................................................................................. 88

4.1. Introduction ................................................................................................................................ 88

4.2. Materials and Methods ............................................................................................................... 89

4.2.1. Cell culture and transfection ..................................................................................................... 89

4.2.2. Western blot analyses ............................................................................................................... 89

4.2.3. MTT viability assay .................................................................................................................... 90

4.2.4. PE Annexin V apoptosis assay ................................................................................................... 91

4.2.5. Statistical analysis ..................................................................................................................... 91

4.3. Results ......................................................................................................................................... 91

4.3.1. Akt inhibitors reduce cell viability in glioblastoma cells ........................................................... 91

4.3.2. Par-4 is sufficient to reduce cell viability in GBM cells ............................................................. 95

4.3.3. Par-4 sensitizes GBM cells to ISC-4 and 5-FU ............................................................................ 98

4.3.4. Par-4, ISC-4, and their combinations induce apoptosis in GBM cells ..................................... 101

4.4. Discussion .................................................................................................................................. 104

5. Chapter 5 ........................................................................................................................................... 106

References ................................................................................................................................................ 112

viii

List of Figures

Figure 2-1. Overexpression of Par-4 increases susceptibility of metastatic SW620 cells to 5-FU. ............. 33

Figure 2-2. Par-4 overexpression inhibits metastatic processes in SW620 cells. ....................................... 37

Figure 2-3. Par-4 inhibits migration in SW480 cells. ................................................................................... 39

Figure 2-4. Par-4 induces a mesenchymal-epithelial transition in SW620 cells. ........................................ 43

Figure 2-5. Par-4 induces E-cadherin transcription and tight-junction protein upregulation. ................... 47

Figure 3-1. Trim21 is a novel interacting partner of Par-4. ......................................................................... 58

Figure 3-2. Trim21 interacts with Par-4 via its PRY-SPRY domain. ............................................................. 60

Figure 3-3. Trim21 is not sufficient to downregulate Par-4 protein levels. ................................................ 63

Figure 3-4. Ectopic expression of Trim21 downregulates Par-4 in the presence of cisplatin in colon cancer

cells. ............................................................................................................................................................ 66

Figure 3-5. Cisplatin downregulates Par-4 in a dose- and proteasome-dependent manner. .................... 69

Figure 3-6. Cisplatin downregulates Par-4 in both the cytoplasmic and nuclear compartments. ............. 72

Figure 3-7. Cisplatin downregulates Par-4 in pancreatic cancer cells. ....................................................... 75

Figure 3-8. Trim21 is a potential therapeutic target in colon and pancreatic cancer. ............................... 79

Figure 4-1. Akt inhibitors decrease cell viability in glioblastoma cells. ....................................................... 94

Figure 4-2. Par-4 is sufficient to reduce cell viability in GBM cells. ............................................................ 96

Figure 4-3. Par-4 sensitizes GBM cells to 5-FU and ISC-4. ........................................................................ 100

Figure 4-4. Par-4, ISC-4, and their combinations induce apoptosis in GBM cells. .................................... 102

ix

Abbreviations

Par-4 – Prostate apoptosis response-4; Trim21-Tripartite motif-containing protein 21;

EMT-Epithelial-mesenchymal transition; MET-Mesenchymal-epithelial transition; CDDP-

cis-diamminedichloridoplatinum (II); MTT-3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide; 5-FU-5-fluorouracil; ISC-4-Isoselenocyanate; DNA-

Deoxyribonucleic acid; cDNA-complementary DNA; RNA-Ribonucleic acid; shRNA-

short hairpin RNA; NF-κB-Nuclear factor kappa-light-chain-enhancer of activated B

cells; PARP-Poly (ADP-ribose) polymerase; VEGF-Vascular endothelial growth factor;

PDGF-Platelet-derived growth factor; EGFR-Epidermal growth factor receptor; Bcl-2-B-

cell lymphoma 2; DR5-Death receptor 5; IRF-Interferon regulatory factor; c-FLIP-FLICE-

like inhibitory protein; PI3K-Phosphatidylinositol-4,5-bisphosphate 3-kinase; NLS-

Nuclear localization signal; SAC-Selective for apoptosis in cancer; FADD-Fas-

associated protein with death domain; TRADD-Tumor necrosis factor receptor type 1-

associated death domain; PKC-Protein kinase C; WT1-Wilm’s tumor 1; TOP1-DNA

topoisomerase 1; ER-Endoplasmic reticulum; GRP78-78 kDa glucose-regulated protein;

PKA-Protein kinase A; Akt1-RAC-alpha serine/threonine-protein kinase; FBXO45-F-box

only protein 45; IDH1-Isocitrate dehydrogenase 1;Foxo3a-Forkhead box O3a; miRNA-

microRNA; UTR-Untranslated region; TGF-β-Transforming growth factor-β; ROS-

Reactive oxygen species; APC-Adenomatous polyposis coli; k-RAS-V-Ki-ras2 Kirsten

rat sarcoma viral oncogene homolog; TP53-Tumor protein p53; MSH-MutS protein

homolog; MLH-MutL homolog; PDAC-Pancreatic ductal adenocarcinoma; BRCA-Breast

cancer susceptibility gene; PanIN-Pancreatic intraepithelial neoplasia; RB-

Retinoblastoma gene; Apaf-1-Apoptotic protease activating factor 1; ATP-Adenosine

x

triphosphate; TNF-Tumor necrosis factor; RING-Really interesting new gene finger

domain; RT-PCR-Reverse transcription polymerase chain reaction; TRAMP-Transgenic

adenocarcinoma of the mouse prostate; RPMI-Roswell park memorial institute medium;

FBS-Fetal bovine serum; PBS-Phosphate buffered saline; BCA-bicinchoninic acid;

SDS-Sodium dodecyl sulfate; TBS-Tris buffered saline; HRP-Horseradish peroxidase;

ECl-Enhanced Chemiluminescence; MT1-MMP-Membrane-type 1 matrix

metalloproteinase; bHLH-Basic helix-loop-helix; ZO-1-Zonula occludens 1; DDX41-

DEAD-Box helicase 41; dsDNA-double stranded DNA; TCGA-The cancer genome

atlas; DMSO-Dimethyl sulfoxide; FITC-Fluorescein isothiocyanate; Ro-Rhodamine;

DAPI-4',6-diamidino-2-phenylindole; GBM-Glioblastoma; TMZ-Temozolomide; PrPc-

Cellular prion protein; IL-2-Interleukin 2; CD-28-Cluster of differentiation 28

xi

Acknowledgements

I would like to thank Dr. Rosalyn Irby for her guidance in helping me develop as a

scientist. After suddenly finding myself in the position of being forced to switch labs after

a year into my PhD, she graciously accepted me as a student. She has always been

very patient with me, and has allowed me to pursue my own ideas. I am very grateful for

that. I also would like to thank my committee for their helpful guidance throughout the

process. In addition, I would like to thank my family, especially my parents, for their

support and encouragement throughout my life. They instilled in me the values which

have shaped me into the person that I am and have led me to this point. Finally, I would

like to thank my wife, Felicia, for her patience, encouragement, and love throughout this

process. It is because of her that I have been able to find the resolve to get through the

more difficult moments during my PhD.

1

1. Chapter 1

Introduction

1.1. Prostate apoptosis response-4

Prostate apoptosis response-4 (Par-4) is a gene that was originally discovered in

rat prostate cancer cells that were induced to undergo apoptosis.1 Par-4 was one of a

group of early-upregulated genes in these prostate cancer cells, and was the only one

that had not yet been described. The initial characterization of Par-4 demonstrated that it

is highly expressed in a wide variety of normal tissues.2 Initial studies showed that Par-4

expression was induced in response to physiologic stimuli, such as androgen withdrawal.2

Studies in cancer cells revealed that Par-4 could sensitize cells toward apoptosis-inducing

agents, such as chemotherapeutics and radiation.3-6 In certain types of cancer cells, such

as androgen-independent cancer cell lines, ectopic Par-4 expression is sufficient to

induce apoptosis.7 Interestingly, Par-4 protein has also been shown to be secreted, and

this extracellular Par-4 protein is sufficient to induce apoptosis.8 An attractive property of

the apoptosis-inducing ability of Par-4 is that it is selective for cancer cells. In other words,

it leaves normal cells unaffected (for mechanisms, see sections 1.1.3 and 1.1.4). This

selectivity for cancer cells is ideal therapeutically, since several of the toxic side-effects

of commonly used chemotherapies is due to their non-selective mechanism.9 More recent

studies on Par-4 have begun to examine its role in regulating cellular processes beyond

apoptosis, including migration and autophagy.10-13 Taken together, the vast majority of

2

studies on Par-4 function suggest that it functions as a tumor suppressor. This is

supported by expression data from clinical specimens. Par-4 is shown to be

downregulated in renal cancer,14 leukemia, lung cancer,15 and endometrial cancer.16 In

addition, oncogenes have been shown to downregulate Par-4.17, 18 The tumor-

suppressive function of Par-4 in also suggested from work in animal models: Par-4

knockout mice (KO) are prone to tumor development in a variety of tissues, especially

hormone-dependent tissues, such as the endometrium and prostate; Par-4 KO mice are

also more sensitive to developing carcinogen-induced tumors.19

1.1.1. Domains

Par-4 is a 343 amino acid protein with several conserved domains. At the C-

terminus, there is a leucine-zipper domain, which, like other leucine zippers, is an alpha-

helix that contains a series of heptad repeats, where every fourth position is a leucine.20

These regularly spaced leucines give the alpha-helix a hydrophobic face that allows for

dimerization – with itself and other proteins. This leucine zipper domain of Par-4 is

responsible for mediating almost all of the protein-protein interactions of Par-4.21 Also at

the C-terminus is a nuclear export sequence, though the function of this sequence has

not been studied. Near the N-terminus, there are two localization sequences, NLS1 and

NLS2: NLS1 is not required for the nuclear localization of Par-4, whereas NLS2 is

required.21 There is also a domain in the middle of Par-4 termed the selective-for-

apoptosis-in-cancer domain (SAC). This domain was discovered by serially deleting

fragments from either termini in order to discover the minimal sequence that was sufficient

for Par-4 apoptotic activity.21 This 59 amino acid SAC domain has demonstrated apoptotic

activity against cancer cells when overexpressed intracellularly and when cells are treated

3

with recombinant SAC protein, which is comparable to the apoptotic activity of full-length

Par-4.8 Additionally, ectopic expression of the SAC domain has been shown to induce

apoptosis in cells that were resistant to full-length Par-4.22 Par-4 also contains many other

putative domains and sites for post-translational modification, such as phophorylation

sites and glycosylation sites; however, their function has yet to experimentally validated.23

1.1.2. Intracellular functions of Par-4

The vast majority of studies on Par-4 function have focused on the intracellular

role of Par-4 in apoptosis. Par-4 affects apoptosis by regulating both the intrinsic and

extrinsic pathways of apoptosis.

Par-4 modulates the extrinsic pathway by facilitating the trafficking of both Fas and

FasL to the cell membrane.24 Fas and FasL, in turn, recruit FADD and activate the

extrinsic pathway of apoptosis. Another mechanism by which Par-4 regulates apoptosis

is by inhibiting NF-κB.25 NF-κB is a transcription factor that upregulates pro-survival and

pro-inflammatory genes in response to cytokines.26 Given its pro-survival function, NF-κB

is also, not surprisingly, found to be upregulated in a wide variety of cancers. Par-4 inhibits

NF-κB function in multiple ways. For example, Par-4 can inhibit NF-κB transcriptional

activity in the nucleus,27 though the exact mechanism for this is currently unknown. It

could act via direct DNA binding and inhibition of NF-κB activity, or it could act by

recruiting co-repressors, which in turn inhibit NF-κB activity. In addition, Par-4 has been

shown to inhibit NF-κB activation in the cytoplasm.25 Furthermore, Par-4 can influence

NF-κB activity indirectly by binding to and inhibiting protein kinase C zeta (PKCζ).25, 28

4

PKCζ is an atypical isoform of the PKC family of kinases that activates NF-κB by

phophorylating IκB.

Though its ability to affect Fas/FasL translocation and its ability to inhibit NF-κB

are its most well-known functions, Par-4 also has been described to act on other

intracellular proteins. For example, Par-4 enhances the repressor activity of Wilm’s Tumor

1 (WT1).29, 30 WT1 is a transcription factor that acts as a tumor suppressor by repressing

the transcription of genes and thereby inhibiting proliferation. Par-4 has also been shown

to interact with topoisomerase 1 (TOP1).31 TOP1 functions to relax supercoiled DNA,

which is essential for DNA replication to proceed. By interacting with TOP1, Par-4 inhibits

its ability to relax DNA supercoils.

1.1.3. Extracellular functions of Par-4

The function of extracellular Par-4 is a relatively recent finding. Studies have

shown that Par-4 is secreted by both normal cells and cancer cells.8 Like other secreted

proteins, Par-4 is synthesized and secreted from the cell via the classical ER-Golgi

pathway. It was also found that ER stress inducers increase Par-4 secretion. Importantly,

it was found that secreted Par-4 can induce cancer-cell selective apoptosis by binding to

glucose-regulated protein 78 (GRP78), which in turn activates the extrinsic apoptosis

pathway.8, 32 GRP78 is normally found in the endoplasmic reticulum and normally acts as

a sensor for ER stress and activator of the unfolded protein response, but for unknown

reasons, a significant fraction is found at the cell surface in cancer cells.33, 34 It is this

cancer-cell specific membrane distribution of GRP78 that is thought to confer the ability

of extracellular Par-4 to induce cancer-cell selective apoptosis.9

5

1.1.4. Mechanisms of regulation

Though most studies of Par-4 have focused on elucidating its function, there has

also been some work done examining how Par-4 expression and activity is regulated.

Gene regulation can be categorized as transcriptional, post-transcriptional, or post-

translational, depending on at what point in the process of gene expression that a

regulatory mechanism acts.

Most studies of Par-4 regulation have focused on the post-translational

mechanisms that control Par-4 activity. One of the most common post-translational

mechanisms to control protein activity is phosphorylation. The phosphorylation of Par-4

by protein kinase A (PKA) was one of the first regulatory mechanisms discovered.35 PKA

phosphorylates Par-4 at a threonine residue located at position 155. This position is

located within the SAC domain, and was found to be essential for the nuclear

translocation of Par-4, essential for the inhibition of NF-κB, essential for translocation of

Fas/FasL to the cell membrane, and essential for its pro-apoptotic activity.35 This

modification is significant, because ability of ectopically expressed Par-4 to induce

apoptosis selectively in cancer cells is attributed to the differential expression of PKA

between cancer cells and normal cells: cancer cells, in general, have higher PKA levels

than normal cells, and it is thought that this activates the apoptotic functionality of Par-4.

In contrast, phosphorylation of Par-4 at a serine residue at position 249 by another kinase,

AKT1,36 is responsible for inhibiting Par-4 activity. In this case, once Par-4 is

phosphorylated by AKT1, it is sequestered by 14-3-3δ protein, and it is this sequestration

in the cytosol that prevents Par-4 from carrying out its apoptotic activity. Other

mechanisms of post-translational regulation of Par-4 include ubiquitination37 and

6

proteolytic cleavage.38, 39 Ubiquitination of target proteins by E3 ligases leads to their

proteolytic cleavage by the proteasome.40 Par-4 was shown to a substrate of the E3

ligase, FBXO45, and the ubiquitination and subsequent downregulation of Par-4 by

FBXO45 was shown to regulate cancer cell apoptosis, survival, and colony formation.37

Multiple reports have shown the ability of Par-4 to be cleaved by caspases in response

to various apoptotic stimuli.38, 39 Interestingly, cleaved Par-4 appears to have some

interesting characteristics: it contains the essential SAC domain, it localizes to the

nucleus, and appears to have greater apoptotic activity than full-length Par-4.41, 42

While most reports on Par-4 regulation have focused on its post-translational

regulation, a few reports have highlighted transcriptional mechanisms of Par-4 regulation.

As an example of the transcriptional regulation of Par-4, in endometrial epithelial cells

during a normal menstrual cycle, Par-4 is very highly expressed and this was correlated

with NF-κB activity.43 Furthermore, Par-4 levels in endometrial carcinoma cells were also

correlated with NF-κB activity despite overall downregulation of Par-4 levels relative to

normal endometrium. Further, in vitro studies showed that Par-4 is a direct target of NF-

κB. As another example, it was found that gliomas with mutant isocitrate dehydrogenase

1 (IDH1) had significantly lower levels of Par-4 relative to gliomas without mutant IDH1.44

In vitro studies showed that the product of mutant IDH1 protein, D-2-hydroxyglutarate,

suppresses Par-4 transcription through inhibition of promoter activity. In addition, it

enhances Par-4 mRNA degradation. In another study, Par-4 was shown to be a

transcriptional target of FOXO3a.45 In this study, treatment with Withaferin A, a small

herbal molecule that inhibits AKT1, promotes FOXO3a translocation to the nucleus, which

7

in turn induces Par-4 transcription. Finally, one group reported on the activation of Par-4

transcription by targeting the Par-4 promoter with small-activating RNAs.46

No studies have reported on the post-transcriptional regulation of Par-4, such as

regulation by microRNAs (miRNAs). miRNAs are endogenous short 20-22 nucleotide

sequences of RNA that negatively regulate target genes by binding to the 3’- UTRs of

target mRNAs and negatively regulate gene expression by leading to mRNA cleavage or

by inhibiting mRNA translation.47 Like protein-coding genes, the spectrum of activity of

individual miRNAs can be classified as tumor-suppressive or oncogenic, and miRNAs

have been shown to play a role in cancer progression.48 Undoubtedly, Par-4 is the target

of some miRNAs. It would be interesting to see what role miRNAs play in regulating Par-

4 expression during cancer development.

1.1.5. Non-apoptosis related functions

Recent work on Par-4 has attempted to look beyond its role in regulating apoptosis

and has begun to examine its role in other cellular process, such as migration, and

autophagy.10-13, 49

Cell migration is an important phenotype, especially in the context of cancer. As

cancer cells accumulate mutations, they also gain the ability to escape from their primary

site and to metastasize and colonize distant sites.50 The epithelial-mesenchymal

transition (EMT) is thought to play in important role in the process of cancer cells

developing a metastatic phenotype.51 EMT, which was originally discovered as a

developmental process, is a process by which epithelial cells lose their characteristic

epithelial features, such as cell-cell contacts, and acquire mesenchymal features, such

8

as spindle-shaped cells with minimal cell-cell contacts. Par-4 was first shown to regulate

EMT in a study that was examining the mechanisms of cisplatin resistance in pancreatic

cancer.10 In that study, a cisplatin resistant pancreatic cell line was created, and the

resistant cells acquired a mesenchymal phenotype, which correlated with Par-4

downregulation. Further in vitro studies showed that ectopic Par-4 expression in these

resistant cells reversed EMT and cisplatin resistance, and they demonstrated that Par-4

regulated EMT in a PI3K/Akt-dependent manner. Finally, Par-4 knock-down in cisplatin

sensitive cells induced EMT and cisplatin resistance. In endometrial and breast cancer

cell lines, the opposite effect of Par-4 on EMT was found. In that study, Par-4 mediated

TGF-β induced EMT.11 In this study, Par-4 transcription was induced by SMAD, and Par-

4 in turn upregulated Vimentin and other mesenchymal markers. In chapter 2 of this

dissertation, I examine the role of Par-4 in regulating migration and EMT in colon cancer.

Autophagy is the process by which the cell recycles damaged or senescent

components.52 During this regulated process, old components are surrounded by a

double membrane, resulting in an autophagosome. Later, the autophagosome fuses with

the lysosome, and the contents are broken down. In the context of disease, autophagy

can be pro-survival or anti-survival. In some instances, autophagy is a cellular response

to stress, such as in times of starvation; however, in some cases it can promote cell-

death.52 The first study to link Par-4 to autophagy was a study that examined the

mechanisms by which Par-4 enhanced response to chemoradiotherapy in

hypopharyngeal carcinoma cells.13 The results from that study showed that Par-4 induced

both apoptosis and autophagy and sensitized cells toward both chemotherapeutics and

x-ray irradiation. Another study showed that Par-4 induced autophagic cell death in glioma

9

cells in response to curcumin.12 In this study, Par-4 expression was induced in a reactive

oxygen species-dependent (ROS) manner in response to curcumin, which led to

autophagic cell death. In this case, Par-4 overexpression sensitized glioma cells toward

curcumin, whereas antioxidants blunted the ROS-dependent Par-4 induction and

autophagic cell death.12

1.2. Colon cancer

Colon cancer is a type of cancer that originates from the epithelial cells lining the

colon. The most distal portion of the gastrointestinal tract, called the rectum, is also a

site of malignancy. Collectively, colon and rectal cancers are referred to as colorectal

cancer. Combined, colorectal cancer is one of the leading causes of mortality among all

types of cancer, second only to lung cancer.53 The median age at diagnosis is 68 years.

The incidence of colorectal cancer is about 41 per 100,000 per year. In the year 2014,

136,830 people are expected to be diagnosed with colon cancer, while 50,310 people

are estimated to die from the disease. Overall, the five-year survival rate is at 64.7%

with the survival rate inversely proportional to disease stage at diagnosis – localized

disease, where cancer cells are confined to the primary site of origin, has a survival rate

of 90.1%, while metastatic disease, where cancer cells have spread to distant organs,

has a survival rate of 13.5%.54

Colon cancer arises from a series of mutations in key genes.55, 56 Over time, this

leads to a series of morphological changes that convert normal colonic epithelium into a

carcinoma, which is a general term that refers to a cancer that arises from epithelial

tissue. Although the exact genetic changes and the order in which they occur may vary

from individual to individual, the transition from epithelium to carcinoma is thought to

10

occur through one of two distinct pathways: the adenoma-carcinoma sequence or the

mismatch-repair pathway.55 The pathways differ by the genes that are mutated and the

mutational mechanisms that give rise to them. The adenoma-carcinoma sequence is

characterized by chromosomal instability, which gives rise to disease-causing mutations

in genes, such as APC, K-RAS, TP53, and CTNNB157. These accumulated genetic

changes, in turn, result in a series of morphological changes at the cellular and tissue

level. Initially, there is focal epithelial proliferation. With time this epithelial proliferation

creates a physical protrusion, called a polyp, consisting of dysplastic cells. As mutations

continue to accumulate, the protrusion grows and breaks through adjacent tissue layers,

thereby becoming an invasive cancer. In contrast, the mismatch-repair pathway of colon

carcinogenesis is characterized by mutations in DNA mismatch repair genes. Mutations

in mismatch repair genes, such as MSH2, MSH6, or MLH1, are most likely the initiating

genetic lesions that give rise to this pathway of colon carcinogenesis.56 Macroscopically,

the progression from normal tissue to cancerous tissue does not occur in a stepwise

progression, unlike the adenoma-carcinoma pathway. Carcinomas arising through the

mismatch-repair pathway can arise with no detectable precursor lesion in some

instances; in other instances, cancer can arise from a precursor lesion, called a sessile

serrated adenoma, a term that describes a polyp with a flat morphology without a

detectable stalk.

A large component of the risk of developing colon cancer is related to

environmental factors, such as diet.57 Specifically, consuming excess amounts of

calories, red meat, and fat puts one at higher risk of developing colon cancer. Though

many hypotheses have been proposed to explain the link, the mechanistic basis for how

11

these risk factors enhance the development of colon cancer is still unclear. Other risk

factors include heritable cancer predisposition syndromes, such as Lynch syndrome,

where germline mutations in DNA mismatch repair genes lead to a higher risk of

developing colorectal cancer and cancers in other sites.58 In addition, people with a

history of inflammatory bowel disease are at high risk of developing colorectal cancer,59

highlighting the role that chronic inflammation of the intestines plays in colon cancer

development.60

Patients with colon cancer can present with a variety of symptoms based on the

location of the tumor. For example, patients with tumors in the ascending colon can

become sizable since stool is relatively liquid and little water has been absorbed by the

intestines at that point. Likewise, patients with such tumors will be unlikely to have

experienced any changes in bowel movements. Instead, due to the ability of a tumor at

that location to grow large, these tumors tend to ulcerate, leading to chronic blood loss.

Thus, patients can experience fatigue, palpitations, and anemia secondary to this blood

loss. In contrast to tumors arising in the ascending colon, tumors arising in the

transverse or descending colon tend to cause a different set of symptoms. For example,

since stool has less water and is more solid at these portions of the colon, patients with

tumors arising at these locations tend to experience abdominal pain, cramping, and

obstructive symptoms, such as changes in bowel habits and changes in the size and

caliber of stool.

Once the diagnosis of colorectal cancer is made, the prognosis and ultimate

treatment depend on the stage of the tumor. Staging, in turn, is determined by the depth

of penetration of the tumor into normal tissue. Normal colonic tissue consists of multiple

12

layers. The layer that is in contact with the lumen of the colon is the epithelium. Just

underneath the epithelial layer is a thin layer of muscle, called the muscularis mucosa.

Underneath the muscularis mucosa layer lie the submucosa, muscularis propria, and

serosa, respectively. Finally, the serosa is in contact with a connective tissue layer

consisting of fat, blood vessels, and lymph nodes.61 Stage I colon cancer can be divided

into two subgroups, T1 or T2: T1 disease is defined as a tumor that has not penetrated

completely through the submucosa; T2 disease is defined as a tumor that has not

penetrated completely through the muscularis propria. Stage II colon cancer is defined

as a tumor that has completed breached the muscularis propria, but has not yet spread

to the lymph nodes. Like stage I, stage III colon cancer can also be divided into two

subgroups, N1 or N2: N1 disease is defined as a tumor that has spread to 1-3 lymph

nodes; N2 disease is defined as a tumor that has spread to greater than 4 lymph nodes.

Finally, a patient has stage IV colon cancer if there is evidence of metastatic spread to

distant sites, such as the liver.61

Like prognosis, treatment also depends on the stage of the tumor at diagnosis. In all

cases, surgical resection of the tumor is indicated. Depending on the stage of disease,

further treatment may be warranted. With stage I disease, where there is no evidence of

any local or distant spread, no chemotherapy is indicated.61 In patients with stage II

disease, additional chemotherapy is controversial. In patients with stage III and IV

disease, chemotherapy is an essential part of treatment - 5-FU, which is the backbone

of treatment, in combination with other drugs, such as oxaliplatin or irinotecan, can

reduce the rate of the recurrence and prolong survival in such settings.61 Chemotherapy

drugs used in the treatment of colon cancer act through a variety of mechanisms. For

13

example, 5-fluorouracil inhibits thymidylate synthase, whereas oxaliplatin and irinotecan

are a DNA cross-linking agent and topoisomerase inhibitor, respectively.61

1.2.1. Par-4 and Colon Cancer

Much work has been done on the role of Par-4 in colon cancer. The first study to

report on the role of Par-4 in colon cancer demonstrated that treatment of HCA-7 colon

cancer cells with cyclooxygenase inhibitors upregulated Par-4 expression.62 Future

studies examined the ability of Par-4 to sensitize colon cancer cells to

chemotherapeutics. Specifically, Par-4 plasmid delivered with nanoliposomes sensitized

HT-29 colon tumor cells to 5-FU in a nude mouse model.63 A microarray study showed

that Par-4 sensitizes to 5-FU by inhibiting NF-κB and regulating a network of miRNAs.64

Later studies showed that both ISC-465 and a combination of Src inhibitor and 5-FU66

can activate Par-4 resulting in reduced tumor growth and cell death, respectively.

1.3. Pancreatic cancer

Pancreatic cancer is an umbrella term for a family of different cancers arising from

cells of the pancreas. The various types are categorized by histology and cell-of-origin.

Infiltrating ductal adenocarincoma (PDAC) is the most common and constitutes greater

than ninety-percent of all pancreatic cancer cases.67 In this dissertation, PDAC will be a

focus.

PDAC is a type of cancer that originates from the ductal epithelial cells of the

exocrine pancreas.67 The median age at diagnosis is 70.54 Pancreatic cancer is one of

the most aggressive forms of cancer and the fourth leading cause of cancer death with

an incidence 12.4 per 100,000 per year. This leads to an estimated 53,070 new cases

14

in 2016. In 2016, about 41,780 people are expected to die from pancreatic cancer.54

Thus, virtually all patients will die from their disease. The five-year overall survival for

pancreatic cancer is 7.7%. Stratified by stage, the five-year survival is: 29.3% for

localized disease, 11.1% for regional disease, and 2.6% for metastatic disease.54

Like colon cancer, pancreatic cancer also arises as a consequence of the

accumulation of genetic mutations.68, 69 As mutations accumulate, the pancreatic ductal

cell acquires the ability to proliferate uncontrollably and to infiltrate adjacent structures.

The most common mutations involved in pancreatic cancer are KRAS, P16, P53,

SMAD4, and BRCA2.70, 71 Also, like colon cancer, as the mutations accumulate, the

pancreatic ductal cell progresses through a series of histologically defined stages on its

way to becoming an invasive cancer. These stages are called pancreatic intraepithelial

neoplasisas (PanINs), and together the sequence of pancreatic carcinogenesis is

termed the PanIN-carcinoma sequence.67 The stages represent a series of increasingly

dysplastic pancreatic epithelium, PanIN 1 through 3, finally terminating in invasive

pancreatic adenocarcinoma. In this progression, activating mutations in KRAS are

thought to occur relatively early, whereas the inactivating mutations of tumor-suppressor

genes, such as P16, P53, SMAD4, and BRCA2 occur relatively late in the sequence.72-

75

Risk factors for pancreatic cancer include smoking, diabetes, obesity, chronic

pancreatitis, and a diet high in fats.76 Smoking is the biggest risk factor and accounts for

roughly 25% of pancreatic cancer patients, and is thought to double the risk of

developing pancreatic cancer. Though diabetes and chronic pancreatitis increase one’s

15

risk, it is unclear whether the disease processes have a causal role in pancreatic

carcinogenesis, since they both can also occur secondary to pancreatic cancer.77 Age,

socioeconomic status, and race also play a role in increasing one’s risk for pancreatic

cancer. Roughly 80% of cases occur in patients between the ages of 60 and 80.54

Pancreatic cancer is more common in blacks compared to whites, and also is more

common in Ashkenazi jews. Finally, various cancer predisposition syndromes can

increase one’s risk for developing pancreatic cancer - examples of such syndromes

include: familial pancreatic cancer syndrome, Peutz-Jager’s syndrome, familial

pancreatitis, Li-Fraumeni syndrome, and Lynch syndrome.78, 79

Depending on the location of the tumor, patients can experience a variety of

symptoms. When the tumor originates in the head of the pancreas, for example, the bile

duct can be obstructed, which can lead to abdominal pain, itchiness, lethargy, and

weight loss. Obstruction of the pancreatic duct from a pancreatic tumor can lead to

symptoms, such as epigastric pain, new-onset diabetes mellitus, and acute pancreatitis.

Finally, constriction of the junction between the distal stomach and duodenum can result

in nausea and vomiting.

Imaging is used to diagnose pancreatic cancer, usually a spiral CT scan. Most

patients are diagnosed with pancreatic cancer at a late stage: only 9% have localized,

resectable disease, whereas the remainder have either metastatic or locally advanced

disease.54

As with colon cancer, the prognosis depends on the stage at diagnosis. Staging of

pancreatic cancer utilizes the TNM staging system: stage I disease is defined as a

16

tumor without lymph node involvement, and such tumors are usually around 2 cm; once

the tumor extends beyond the pancreas and/or involves the lymph nodes, it is stage II,

by definition;61 if the celiac axis or superior mesenteric artery is involved, then the tumor

is stage III; if there is evidence of distant metastatic lesions, then the tumor is stage IV.61

The five-year survival rate by stage is inversely proportional to the stage of disease at

diagnosis: if a patient has localized, resectable, stage I disease, the five-year survival

rate is 16%; if a patient has stage II or III disease, the five-year survival rate is 8%; if the

patient has stage IV disease, the five-year survival rate is 2%.61

Like prognosis, treatment of pancreatic cancer also depends on stage. Stage I

disease is, by definition, resectable. Therefore, surgical resection is warranted, followed

by adjuvant chemotherapy, since adjuvant chemotherapy in this setting has been shown

to improve survival. Stage II disease warrants neoadjuvant chemotherapy, followed by

surgical resection and adjuvant chemotherapy. Finally, stage III and IV disease call

purely for chemotherapy, since surgical resection is not curative in this setting. In all

cases, chemotherapy used in the treatment of pancreatic cancer consists of a

combination of gemcitabine, 5-FU, and radiation.61

1.3.1. Par-4 and Pancreatic Cancer

A significant body of work has been done on the role of Par-4 in pancreatic cancer.

For example, when oncogenic KRAS was expressed in pancreatic cancer cell lines,

Par-4 expression was downregulated.18 In addition, in an analysis of Par-4 and KRAS

expression in clinical specimens, KRAS mutational status was shown to correlate with

Par-4 expression.18 Additionally, Par-4 expression had prognostic significance: Par-4

17

expression was correlated with prolonged survival.18 Finally, small molecule inducers of

Par-4 expression sensitized pancreatic cancer cells to chemotherapeutics.80

1.4. Glioblastoma

The brain is a complex tissue consisting of many different cell types, which can be

classified into two types: neurons and glial cells. Neurons are cells that communicate

with one another and are responsible for our cognition, and through nerves, coordinate

and control all of our bodily functions. Glial cells are supportive cells that serve a variety

of functions, such as structural support for neuronal cells. In addition, they help to

regulate blood flow by constricting local vasculature and function as insulation for

neuronal cells. Finally, certain glial cells act as a defense against foreign pathogens.

Some examples of glial cells are astrocytes, Schwann cells, oligodendrocytes,

ependymal cells, and satellite cells.

Brain tumors can arise from any cell type, neurons or glia. The annual incidence

of primary brain tumors is about 10-17 per 100,000 per year.81 About half of patients

that are diagnosed with brain cancer have a primary tumor, while half have metastatic

disease from another primary site.82 Primary brain tumors have an uneven distribution

among age groups: while they comprise only 1-2% of all cancers, they make up about

20% of all childhood cancers.82

Brain cancers have several features that distinguish them from most other

cancers. Unlike colon cancer, and similar to pancreatic cancer, the distinction between

benign and malignant tumors is obscure. All grades of brain cancer have a relatively

poor prognosis.82 In addition, due to its sensitive location and the infiltrating nature of

18

brain cancers, the ability to resect a given tumor is limited. Finally, primary brain tumors

rarely metastasize outside of the central nervous system, even grade 4 brain cancers.82

Patients that develop primary brain tumors can present with a variety of

symptoms based on the location of the tumor. Usually patients present with a focal

neurologic deficit, such as paralysis.61, 82 These focal neurologic deficits are the result of

the compression of adjacent neurons and nerves. Inflammation and edema around the

tumor can also contribute to this compression. Patients can also present with seizures.

Seizures result from a growth of a tumor that disrupts the neuronal connections leading

to an imbalance between excitatory and inhibitory circuits, shifting the balance in favor

of over-excitation.61 Likewise, patients can present with non-focal neurologic symptoms

such as headache, altered mental status, or a change in personality. These non-focal

symptoms are the result of increased intracranial pressure secondary to the tumor. The

tumor can lead to increased intracranial pressure by a variety of mechanisms: tumor

growth compressing adjacent brain tissue, inflammation and edema, tumor hemorrhage,

or obstruction of cerebrospinal fluid pathways.61, 82

1.4.1. Classification

Brain tumors are classified according to the histology and cell type.83 Of the glial

tumors, the most common types arise from astrocytes, oligodendrocytes, and

ependymal cells.82 Astrocytic, specifically glioblastoma (discussed later), tumors will be

a focus of this dissertation. Tumors arising from other types of glial cells or tumors

arising from neuronal cells are outside the scope of this dissertation and will not be

discussed further.

19

Astrocytic tumors are the most common of the primary glial tumors and are

divided into four grades based on histology. A grade I astrocytoma is called a pilocytic

astrocytoma and is well circumscribed on histology and imaging. A pilocytic astrocytoma

is considered benign. A grade II astrocytoma is called a diffuse astrocytoma. Instead of

being well circumscribed, the borders of a diffuse astrocytoma are infiltrative on

histology and imaging. A grade III astrocytoma is called an anaplastic astrocytoma. On

histology an anaplastic astrocytoma has infiltrative borders and the presence of

numerous mitotic figures. A grade IV astrocytoma is called a glioblastoma. On histology,

a glioblastoma has an infiltrative border, numerous mitotic figures, and the presence of

vascular proliferation and/or necrosis. Grades III & IV astrocytomas are considered

malignant.82

1.4.2. Prognosis and Treatment

As with colon cancer and pancreatic cancer, the prognosis and treatment of

primary astrocytic tumors depend on how advanced the cancer is.

Pilocytic astrocytomas are the most common type of glial neoplasms in children.

They tend to affect the cerebellum and optic nerves. They are often well circumscribed

and are slow-growing with an excellent prognosis with total excision.61

Diffuse astrocytomas make up about 10-15% of all glial neoplasms and have a

peak incidence in the fourth decade. The edges of the tumor are not well defined due to

the infiltrative nature of the cancer cells. The mean survival of patients with diffuse

astrocytomas is 6-8 years. Most patients ultimately progress to anaplastic astrocytoma

or glioblastoma as mutations in their tumor accumulate.61

20

Anaplastic astrocytomas have a peak incidence in the fifth and sixth decades. As

with diffuse astrocytomas, the edges of the tumor are also ill-defined and infiltrative. In

addition, the presence of mitotic figures without evidence of vascular proliferation or

necrosis by histology characterizes anaplastic astrocytomas. The mean survival of

patients with anaplastic astrocytoma is 3 years.82

Glioblastomas have a peak incidence in the sixth and seventh decades, and

make up the majority of gliomas (50-60%). As with diffuse and anaplastic astrocytomas,

the borders of glioblastomas are irregular and on histology; however, there is also

evidence of dedifferentiation, mitotic figures, and either vascular proliferation, necrosis,

or both. The mean survival of patients with glioblastoma is 8-10 months.82

Treatment for pilocytic astrocyomas is surgical resection, which though not

necessarily curative, allows for a very favorable prognosis, with a five-year survival in

some cohorts over 90%.61 Treatment for diffuse astrocytomas, anaplastic astrocytomas,

and glioblastomas, consists of combinations of surgical resection and

chemoradiotherapy.84 Unfortunately, due to the infiltrative nature of the more advanced

grades of astrocytomas, treatment is rarely curative and the tumors almost always

recur.61

1.4.3. Molecular Genetics

The progression from low grade to high grade astrocytomas correlate with a series

of mutations in the tumor.85 Low-grade astrocytomas are associated with inactivating

mutations in P53 and activating mutations in PDGF and its receptor. In contrast, high-

21

grade astrocytomas are additionally associated with inactivating mutations in RB and

P16.86

There are two clinically distinct subsets of gliobastomas: new-onset glioblastomas,

also called primary glioblastomas; and secondary glioblastomas, which are

glioblastomas that present in patients with a prior history of a lower grade astrocytoma.

Primary glioblastomas tend to occur in older patients, whereas secondary glioblastomas

tend to occur in younger patients and have a better prognosis.61 Though primary and

secondary glioblastomas have different clinical courses and prognoses, they share

some common genetic abnormalities, such as mutations in P53. In addition, they have

unique genetic abnormalities. For example, whereas activating mutations in platelet

derived growth factor receptor more often are seen in secondary glioblastomas,

activating mutations in epidermal growth factor receptor are found more often in primary

glioblastoma.82

1.5. Par-4 and glioma

Very little work has been done on the role of Par-4 in glioma. Temozolomide, the

drug used in the treatment of glioblastoma, has been shown to induce PrPc expression

in glioma cell lines, which in turn inhibits Par-4 activation.87 In this study, knocking down

Par-4 enhances cell death in response to Temozolomide. Par-4 has also been shown to

mediate glioma cell death in response to curcumin12 and tamoxifen.88 Interestingly,

IDH1 mutation gliomas, which have a favorable prognosis, have downregulated Par-4

expression.44

22

1.6. Apoptosis

Apoptosis is an evolutionarily conserved cell-death process that was originally

discovered in 1842 that is characterized by a series of morphological changes.89 Such

classic morphological features of apoptosis include: membrane blebbing, cell shrinkage,

nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and

global mRNA decay. These morphological changes are due to a fundamental series of

biochemical processes that occur in the cell.89, 90

Apoptosis is a process that plays an important role in human development and in

health and disease. The purpose of apoptosis is to eliminate aged, damaged, harmful, or

unwanted cells. For example, during development, apoptosis plays a role in

organogenesis, developmental involution, and implantation.91 In the adult organism,

apoptosis is responsible for maintaining homeostasis of cell number in tissues that consist

of proliferating cells, for involution of hormone-dependent tissues during hormone

withdrawal, and for eliminating self-reactive lymphocytes. In disease settings, apoptosis

serves to eliminate cells that are damaged beyond repair - for example, in response to

DNA damage by chemotherapeutics, in response to the immune response against a

virally-infected cell, or in response to an accumulation of unfolded proteins.82

1.6.1. Mechanism of apoptosis

Apoptosis can be broadly divided into two pathways: the extrinsic pathway and intrinsic

pathway.92 This classification is based on the nature of the apoptosis-inducing stimulus

and the sensor/effector components involved. A family of cysteine proteases, called

caspases, are the main effectors of apoptosis regardless of the pathway.90, 93

23

In the intrinsic pathway, a cell stimulus leads to an increase in mitochondrial

permeability, which in turn leads to leakage of initiators,94, 95 such as cytochrome C, from

the mitochondria into the cytosol. Upon release, cytochrome C binds to apoptotic

protease activating factor-1 (APAF1) and ATP, which then binds to procaspase 9, itself

an initiator capsase, forming the apoptosome.96 Formation of the apoptosome leads to

the cleavage of pro-caspase 9 into its active form. Active caspase 9 then cleaves pro-

caspase 3 into active caspase 3. Caspase 3 is an effector caspase, which leads to

cleavage of multiple intracellular components, including DNA, resulting in cell death.

Thus, regulating mitochondrial permeability is important for regulating the intrinsic

pathway, since it is the initial step in the intrinsic pathway. The BCL family of proteins is

responsible for regulating mitochondrial permeability, which consists of groups that are

pro-apoptotic and anti-apoptotic.97 Certain conditions, such as presence of growth

factors, induce the production of anti-apoptotic molecules over pro-apoptotic molecules;

however, under certain stressors - such as heat, radiation, or nutrient deprivation – the

balance of BCL family proteins shifts toward pro-apoptotic effectors.

The extrinsic pathway is mainly initiated by receptor-ligand interactions, where

death receptors at the cell surface bind to their respective ligands and initiate apoptosis.98-

100 The cell-surface death receptors are members of the TNF receptor family. There are

two main ligands that have been demonstrated to initiate apoptosis: Fas ligand and TNF.

When a ligand binds its death receptor, the receptor trimerizes, allowing the recruitment

of adaptor proteins, such as FADD or TRADD, to the cytoplasmic domain of the receptors.

These adaptor proteins then bind to multiple inactive forms of caspase 8, an initiator

caspase.101 The aggregation of caspase 8 molecules leads to their activation, by

24

proteolytic cleavage, to yield active caspase 8. Active caspase 8 then activates various

effector caspases and cell death results from caspase cleavage of intracellular

components. The end result of apoptosis is the formation of apoptotic bodies, which are

phagocytosed by immune cells, leaving no inflammation or damage to surrounding cells

and tissues.102

1.7. Conclusion

From the body of work presented above, it is clear that advanced colon cancer,

pancreatic cancer, and glioblastoma have very poor prognoses, and there is a need for

novel and effective therapies. In addition, Par-4 is a positive regulator of apoptosis with

cancer cell selective properties, and Par-4 plays a role in colon cancer, pancreatic

cancer, and glioblastoma. Given its tumor-suppressive properties and therapeutic

potential, further study of Par-4 in the context of those cancers is warranted. To study

Par-4, three different approaches will be taken:

1. Elucidating novel Par-4 function.

2. Identifying and characterizing new regulators of Par-4.

3. Demonstrating the usefulness of Par-4 in increasing the sensitivity to

chemotherapeutics.

My central hypothesis is that Par-4 acts as a general tumor-suppressor and inhibits

cancer progression and can be used to increase the effectiveness of

chemotherapeutics. Toward this end, I chose to pursue three specific aims:

25

1. Determine the impact of Par-4 on colon cancer cell migration, invasion, and

EMT.

2. Determine novel regulators of Par-4 in colon and pancreatic cancer.

3. Determine whether Par-4 can enhance the efficacy of therapeutics and

radiation toward glioma cell death.

Specific aims 1, 2, and 3 will be covered in chapters 2, 3, and 4 of this dissertation,

respectively. Chapter 1 has been previously published.82

26

2. Chapter 2

Overexpression of the pro-apoptotic protein Prostate Apoptosis

Response-4 (Par-4) in colon cancer cells can inhibit metastasis by

upregulating E-cadherin expression

2.1. Introduction

The poor prognosis of advanced colorectal cancer previously discussed

underscores the need for novel strategies to inhibit colorectal cancer metastases.

As also previously mentioned, Par-4 plays a role in apoptosis in a cell-type-

specific manner. Par-4 overexpression is sufficient to induce apoptosis in vitro and in

vivo in a myriad of cancer cell types: breast cancer22, 35, androgen-independent and

androgen-dependent prostate cancer cell line, TRAMP, lung cancer, cervical cancer,

nasopharyngeal cancer, and melanoma7. In other cell types - Jurkat T lymphocytes4,

androgen-dependent prostate cancer cell line, LNCaP24, 36, melanoma cells103, and

renal carcinoma - Par-4 increases the susceptibility of cancer cells to pro-apoptotic

stimuli, including UV irradiation, serum-withdrawal, ionizing radiation, doxorubicin, and

camptothecin. In colon cancer cells, Par-4 overexpression increases apoptosis in

response to the chemotherapeutic agent 5-fluorouracil63.

Par-4 not only induces cell death in cancer cells, but it may also inhibit their

metastasis. This was suggested in a previous study, where mRNA and microRNA

microarray analyses on Par-4 overexpressing HT-29 colorectal cancer cells showed that

Par-4 altered the expression of genes involved in cell movement, including cell

27

migration and invasion64. In addition, Par-4 induced the upregulation of 13 and

downregulation of 9 microRNA’s. Among the predicted target mRNAs of these

dysregulated microRNAs, a significant number are involved in the WNT/β-catenin

pathway, a pathway that has been strongly implicated in colon cancer metastasis. In

vivo, recombinant Par-4 protein inhibits the formation of lung nodules by mouse Lewis

lung carcinoma cells in a tail vein metastasis model32. The goal of this study is to

uncover the mechanisms by which Par-4 inhibits metastasis.

2.2. Materials and Methods

2.2.1. Cell culture and transfection

SW480 and SW620 colorectal cancer cells were maintained in RPMI + 10% fetal

bovine serum (FBS) + 1% penicillin-streptomycin. The cells were transiently transfected

with empty vector (mock), a plasmid vector encoding for human Par-4 (OriGene

Technologies, Rockville, MD), or a plasmid vector encoding for anti-Par-4 shRNA

(Thermo Scientific, Waltham, MA) using either Lipofectamine 2000 Transfection

Reagent (Life Technologies, Grand Island, NY) or PolyJet DNA Transfection Reagent

(SignaGen Laboratories, Rockville, MD), according to the manufacturer’s instructions.

Stable transfectants were isolated using geneticin selection 24 hrs post-transfection.

2.2.2. Western blot analyses

Cells were washed twice with PBS and were lysed into lysis buffer (50mm

HEPES, 100 mm NaCl, 10 mm EDTA, 0.5 % NP40, 10% glycerol, supplemented with

0.0001% Tween 20, 0.1 mM PMSF, 0.1 mM NaVO4, 0.5 mM NaF, 5 μg/ml leupeptin,

0.1 mm DTT). The proteins were quantified according to the BCA Assay (Thermo

28

Scientific Inc., Rockford, IL) and loaded equally onto 10% SDS-polyacrylamide gels.

Proteins were electrophoresed at 150 V and transferred to nitrocellulose membranes

using a semi-dry blotter (BioRad, Hercules, CA). Membranes were blocked with 5%

non-fat dry milk or 5% BSA for 1 hr and incubated with primary antibody overnight. The

blots were washed 3X in TBS with 0.1% Tween20 and incubated for 1 hr in appropriate

HRP-conjugated secondary antibodies (Amersham, Piscataway, NJ). Blots were

washed 3X and chemiluminescent detection was performed using Amersham ECL

Prime Western Blotting Detection Reagent (Thermo Scientific Inc., Rockford, IL). The

blots were either exposed to autoradiography film (GE Healthcare Life Sciences,

Pittsburgh, PA) and scanned or imaged using the Molecular Imager Gel Doc XR System

(Bio-Rad). Densitometric analyses were performed with NIH Image J software104.

Primary antibodies used were: E-Cadherin, Claudin-1, Occludin, ZO-1, Vimentin (Cell

Signaling, Danvers, MA), Par-4 (Santa Cruz Biotechnology, Santa Cruz, CA), and Actin

(Sigma-Aldrich, Saint Louis, MO).

2.2.3. MTT assay

SW620 cells were seeded at a density of 6 x 103 cells/well in a 96-well culture

plate. After 24 hrs, the medium was replaced with medium containing different

concentrations of 5-fluorouracil (5-FU). Forty-eight hours later, MTT reagent was added

(Calbiochem) and the cells were incubated for 3.5 hrs at 37◦ C. After incubation, the

media was aspirated off, crystals were dissolved in MTT solvent (4 mM HCl, 0.1%

Nonidet P-40, in isopropanol), and viability was assessed by measuring the absorbance

at 570 nm with 630 nm absorbance as the reference.

29

2.2.4. Scratch assay

Mock, Par-4, and anti-Par-4 shRNA transfected cells were plated in either 6-well

or 12-well plates and grown to confluence. A scratch was made with sterile pipette tip.

The wells were washed with PBS and photomicrographs were collected under 200x

magnification. The cells were incubated for 24 hrs and the scratch areas were again

photographed.

2.2.5. Boyden Chamber assays

Matrigel-coated inserts (BD Biosciences) were prepared according to the

manufacturer’s instructions. The inserts were aseptically-transferred into 12 well culture

plates containing medium with 10% FBS. Mock and Par-4 transfected SW620 cells

were suspended in culture medium with 0.1% FBS and seeded into each insert. The

cells were allowed to invade for 72 hrs. After removing the culture medium and

scrubbing the cells off of the inside of the insert, the cells on the underside of the insert

were stained with 1% crystal violet in 50% methanol and counted under a microscope.

Cell culture inserts without matrigel were aseptically transferred into 24 well

culture plates containing 10% FBS-RPMI. Mock, Par-4 and shRNA transfected SW480

cells (1x105) were suspended in 1% FBS-RPMI and seeded into the inserts. The cells

were allowed to migrate for 24 hrs. After removing the culture medium, cells were fixed

with 4% paraformaldehyde, permeabilized with 100% methanol, and stained with 0.1%

crystal violet in 50% methanol. After scrubbing off non-migrated cells from the inside of

the insert with a cotton swab, the number of migrated cells on the outside of the insert

was quantified at 100X magnification.

30

2.2.6. RT-PCR analyses

RNA was isolated from mock and Par-4-transfected SW620 cells using RNeasy

kit (Qiagen). cDNA was synthesized using a High Capacity cRNA reverse transcription

kit (Applied Biosystems). Real-time quantitative PCR was performed using ABI Gene

Expression Assay primers on the ABI7900 HT Sequence Detection System. Using the

2ΔΔCt analysis method with the ABI SDS2.2.2 software, relative amounts of target

mRNA were quantitated using actin as an internal control.

2.2.7. Cell proliferation assay

SW480 and SW620 cells were seeded into a 12-well culture dish (1 x 105

cells/well). At the appropriate timepoints, cells were trypsinized, and the number of

viable cells was quantitated using a hemacytometer and trypan blue.

2.2.8. Statistical analyses

The statistical analyses were carried out using GraphPad Prism software, version

6.04 (GraphPad Software, Inc., San Diego, CA, USA). Unpaired two-tailed t-tests are

carried out in order to determine statistically significant differences between control and

transfectants, unless otherwise noted. The threshold for significance is a P < 0.05.

*P<0.05; **P<0.01; ***P<0.001.

2.3. Results

2.3.1. Par-4 increases susceptibility of metastatic SW620 cells to 5-FU

Stable Par-4 transfectants and control transfectants were created by transfecting

SW620 colon cancer cells with Par-4 plasmid or control plasmid, respectively, followed

by geneticin selection. First, Par-4 expression in the stable clones was validated by

31

measuring mRNA and protein levels by qRT-PCR and Western blotting, respectively.

The Par-4 clones (Pcl5 and Pcl7) had increased Par-4 mRNA levels (Fig. 2-1a) and

protein levels (Fig. 2-1b) relative to the control transfectants (Mock). The susceptibility

of mock and Par-4 transfected SW620 cells to the chemotherapeutic agent 5-

fluorouracil (5-FU) was assessed by performing MTT viability assays on treated cells.

As can be seen in Fig. 2-1c, both Par-4-overexpressing clones tested had lower viability

in comparison to a mock-transfected clone when treated with 385 μM 5-FU for 48 hours.

32

33

Figure 2-1. Overexpression of Par-4 increases susceptibility of metastatic SW620 cells to 5-FU.

SW620 colorectal cancer cells were transfected either with a plasmid vector containing a

human Par-4 construct or an empty vector (mock). A) Par-4 mRNA expression was assessed in

mock-(M) and Par-4-transfected (Pcl5 and Pcl7) cells by RT-PCR analysis. The bars in the graph

reflect the fold upregulation of Par-4 mRNA expression in Par-4-transfected cells over the

expression in mock-transfected cells. Data shown are means + SE of three biological replicates.

B) Western blot analyses were performed to assess Par-4 protein expression in mock- and Par-

4-transfected cells. The intensities of the Par-4 bands were normalized against the respective

intensities of the bands for the loading control, actin, to calculate the -fold increase in

expression. C) The susceptibility of mock- and Par-4 transfected SW620 cells to the

chemotherapeutic agent 5-FU was assayed by MTT. Cells were treated with either the vehicle

control (DMSO) or with the indicated concentrations of 5-FU for 48 hours. The bars in the graph

reflect the absorbance readings of the 5-FU-treated cells normalized against those of the

vehicle-treated cells. Data shown are means + SE of at least three biological replicates. The data

presented in this figure is the work of Christina Leah Kline.

34

2.3.2. Par-4 inhibits cell migration and invasion in SW480 and SW620 cells

Next, the effects of Par-4 overexpression on two key steps of metastasis,

migration and invasion, were examined. To examine migration, scratch assays on

SW620 cells were performed and the number of cells that migrated into the area of the

scratch after 24 hours were quantified. Fewer Par-4 overexpressing cells than mock-

transfected cells migrated into the scratch area (Fig. 2-2a and b). The scratch assay

showed that Par-4 overexpression inhibited the migratory ability of SW620 cells. Next,

the invasive ability of mock- and Par-4-transfected SW620 cells was assessed in a

Matrigel assay. As can be seen in Fig. 2-2c, fewer Par-4 overexpressing cells were able

to invade through the Matrigel than mock transfected cells. It is possible that more of the

mock-transfected cells appeared to invade through the Matrigel, because increased

Par-4 expression reduced cell proliferation. To assess this, the cell growth of mock- and

Par-4 transfected SW620 cells (Fig. 2-2d) was monitored. No significant differences in

cell proliferation were observed as a result of increased Par-4 expression.

To extend the previous observations beyond a single cell line, SW480 cells were

transiently transfected with either control or anti-Par-4 shRNA, and scratch assays were

performed to assess migratory ability. Consistent with the inhibitory effect of Par-4 on

migration in SW620 cells, Par-4 knock-down in SW480 cells enhanced migration as

evidenced by complete closure of the scratch relative to the control transfected cells

(Fig. 2-3a). To validate this observation, SW480 cells were transiently transfected with

control plasmid, Par-4 expression plasmid, or anti-Par-4 shRNA, then seeded into a

Boyden chamber, and the number of cells that migrated across the Boyden chamber

after 24 hrs was quantified (Fig. 2-3b). As can be seen in Fig. 2-3b, Par-4

35

overexpression inhibited migration, whereas knock-down of Par-4 enhanced migration

across the Boyden chamber. Western blots confirming overexpression and knock-down

of Par-4 are also shown in Fig. 2-3b. Cell proliferation assays were performed to

compare the growth of Par-4 and control transfected SW480 cells. Although there is a

statistically significant difference in cell number by day 3 post-transfection, the 24-hr

time point of the Boyden chamber assay corresponds to day 1 post-transfection;

therefore, differences in proliferation cannot account for the inhibition of migration seen

in Par-4 transfected SW480 cells in Fig. 2-3b.

36

37

Figure 2-2. Par-4 overexpression inhibits metastatic processes in SW620 cells.

Par-4- and mock-transfected SW620 cells were seeded into six-well or twenty-four-well plates,

and migration and invasion assays were performed to measure the effect of Par-4 on these

metastatic processes. A) Scratch assays were performed to compare the ability of mock- and

Par-4 transfected cells to migrate in 24 hours. The lines are drawn to indicate the boundaries of

the scratch made at the 0 hour timepoint. B) The number of cells that migrated into the scratch

area after 24 hours were graphed. Data shown are means + SE of at least two biological

replicates and a total of six scratch areas were imaged. C) Mock- and Par-4 transfected cells

were cultured in low-serum (0.1%) media in Matrigel-coated inserts to assess invasive

capability. The bars in the graphs reflect the number of cells that invaded through the insert

after 72 hours. Data shown are means + SE of at least 5 biological replicates. D) Cell

proliferation was assessed by counting viable cells in trypan blue assays. Data shown are means

± SE of at least two replicates. The data presented in this figure is the work of Christina Leah

Kline.

38

39

Figure 2-3. Par-4 inhibits migration in SW480 cells.

A) Scratch assays were performed to compare the ability of mock- and anti-Par-4 shRNA-

transfected cells to migrate in 24 hours. A representative image of the scratch at the 0 and 24

hour timepoint is shown. B) SW480 cells that were transiently transfected with either Par-4

expression plasmid, anti-Par-4 shRNA (A11, A12, B1, and B2), or empty vector were cultured in

media with 1% FBS in the inner chamber of a Boyden chamber, while media with 10% FBS in the

lower chamber acted as a chemoattractant. Cells are allowed to migrate for 24 hrs, then the

number of migrated cells were quantitated (# cells/high-powered field). Data shown are the

means ± SE of five random fields. Western blots confirming the overexpression and knock-down

of Par-4 levels are also shown. C) Cell proliferation was assessed by counting viable cells in trypan

blue assays. Data shown are means ± SE of at least two replicates.

40

2.3.3. Par-4 induces a mesenchymal-epithelial transition in SW620 cells

Given the novel observations that Par-4 inhibits migration and invasion of colon

cancer cells, the mechanisms behind these effects were investigated. Par-4

overexpression altered the morphology of SW620 cells (Fig. 2-4a). This is significant,

since the SW620 cell line is one that was derived from a metastatic site, a lymph node,

from a patient with colorectal cancer. Both mock-transfected clones exhibited a mixture

of round cells and elongated cells with minimal cell-to-cell contacts. However, with Par-4

overexpression, the cells demonstrate more cell-to-cell contacts. It is proposed that a

cancer cell that has gained the ability to metastasize undergoes a phenotypic change in

a process referred to as epithelial-mesenchymal transition (EMT). One characteristic of

this change is a morphologic change where the epithelial cell loses cell-to-cell contacts

and becomes more elongated. Thus, the morphologic changes observed in the SW620

cell line with Par-4 overexpressed are suggestive of an EMT reversal, namely a

mesenchymal-epithelial transition (MET).

Another hallmark of EMT is the loss of the epithelial protein, E-cadherin,

concomitant with the gain of the mesenchymal marker, vimentin. Thus, the levels of E-

cadherin and vimentin were examined. Western blot analyses showed that E-cadherin

protein expression was increased in the Par-4 overexpressing cells, while vimentin

levels were reduced (Fig. 2-4b). Par-4 overexpression in SW480 cells also increased

the expression of E-cadherin (Fig. 2-4c). Immunofluorescence studies on the

localization of E-cadherin in the mock and Par-4 transfected SW620 cells shows that

more E-cadherin can be detected in the membranes of Par-4 overexpressing cells than

in mock-transfected cells (Fig. 2-4d). E-cadherin associates with β-catenin at the cell

41

membrane. This association inhibits the activation of the WNT/β-catenin pathway, a

pathway that is involved in colon cancer progression. Immunofluorescence results show

that there is increased localization of β-catenin at the membrane of Par-4

overexpressing cells (Fig. 2-4e). Taken together, these data further support the

observation of an MET phenotypic change.

42

43

Figure 2-4. Par-4 induces a mesenchymal-epithelial transition in SW620 cells.

A) Phase-contrast microscope images of mock and Par-4-transfected SW620 cells were

collected, and B) lysates were processed for Western blot analyses of E-cadherin, the

mesenchymal marker, vimentin, and actin. C) SW480 cells were transfected with Par-4 and

Western blot analyses were performed for Par-4, E-cadherin, and actin. D) Par-4 overexpression

increases E-cadherin localization in the membrane. Immunofluorescence for the epithelial

44

marker E-cadherin (green) was performed with mock and Par-4-transfected cells. Nuclei were

stained with DAPI. E) Par-4 overexpression increases β-catenin localization in the membrane.

Immunofluorescence for the E-cadherin-interacting protein β-catenin was performed. The

percentage of cells expressing β-catenin in their surface was quantitated. The data shown

indicate mean + SE of at least 3 sections.

45

2.3.4. Par-4 regulates tight-junction protein expression in SW620 cells

E-cadherin protein expression can be altered by different mechanisms, including

transcriptional, post-transcriptional, and epigenetic. To determine whether Par-4

increases E-cadherin mRNA expression, qRT-PCR analyses were performed on mock

and Par-4 transfected SW620 cells. As can be seen in Fig. 2-5a, E-cadherin mRNA

levels were increased in the Par-4 overexpressing SW620 clones. Western blot

analyses were performed to identify the transcriptional repressor(s) that may be altered

in response to Par-4 overexpression. Snail is a transcription factor that promotes the

repression of E-cadherin, thus regulating EMT during embryonic development. Snail

upregulation and nuclear localization have been shown to downregulate E-cadherin and

upregulate vimentin. Western blot analyses indicated that Snail expression may be

slightly decreased (Fig. 2-5b). Like Snail, Slug is also a transcriptional repressor of E-

cadherin during embryonic development. Western blot analyses of Par-4

overexpressing cells show that Slug expression was not changed (Fig. 2-5b). Even if

Snail expression was not markedly altered, I assessed the transcriptional activity of

Snail by examining the expression of its other targets -- claudin, occludin, and ZO-1.

Par-4 overexpression caused the upregulation of these proteins, especially occludin and

ZO-1 (Fig. 2-5c).

46

47

Figure 2-5. Par-4 induces E-cadherin transcription and tight-junction protein upregulation.

A) RT-PCR analyses for E-cadherin mRNA expression in mock and Par-4-transfected cells were

performed. The bars in the graph reflect the fold upregulation of E-cadherin mRNA expression

in Par-4-transfected cells over the expression in mock-transfected cells. Data shown are means

+ SE of three biological replicates. B) Par-4 overexpression does not alter the expression of a

subset of E-cadherin transcriptional repressors. Western blot analyses for E-cadherin

transcriptional repressors Snail, Slug, and Zeb1 were performed. C) Western blot analyses for

tight junction proteins, claudin 1, occludin, ZO-1, and actin were performed.

48

2.4. Discussion

The potential of Par-4 in cancer therapy has been increasingly appreciated given

its ability to induce cell death by itself or in combination with chemotherapeutics and

radiation 105. In this study, we have shown that SW620 cells are sensitized to 5-FU upon

ectopic Par-4 expression. This sensitization corroborates our prior findings in HT-29

cells in response to 5-FU and ISC-4 treatment, respectively63-66. Although it has been

shown that recombinant Par-4 can inhibit metastasis32, the mechanisms behind this

effect have been scarcely elucidated. Furthermore, ectopic Par-4 expression in HT-29

cells was shown to deregulate mRNA’s and microRNA’s involved in cell migration and

motility64. We have shown in this study that Par-4 can inhibit the ability of colon cancer

cell lines, SW480 and SW620, to migrate and invade.

The ability of SW620 cells to migrate and invade was reduced with increased

expression of Par-4. Moreover, the ability to SW480 cells to migrate was also inhibited

by Par-4 overexpression, while downregulation of Par-4 resulted in increased migration.

One of the mechanisms behind this reduction in migration and invasion may be the Par-

4-induced upregulation of E-cadherin. Increased expression of E-cadherin in mammary

and prostate epithelial carcinoma cells has been shown to inhibit migration and invasion

106, 107. Small molecules that can restore E-cadherin expression in SW620 cells have

been shown to reduce invasion 108. The ability of E-cadherin to inhibit cell migration and

invasion has been shown to be independent of its role in mediating cell-cell adhesions,

and instead is due to its role in downregulating the β-catenin/TCF pathway 107. E-

cadherin sequesters β- catenin, keeping β- catenin from interacting with actinin-4. On

the other hand, in the absence of E-cadherin or when E-cadherin is downregulated, β-

49

catenin has been found to colocalize with actinin-4 in bleb-like membrane protrusions in

colorectal cancer cells 109. Overexpression of actinin-4 has been shown to increase

motility of colorectal cancer cells 110. The mechanism underlying the β-catenin/actinin-4

complex-induced migration remains to be elucidated. Another mechanism by which E-

cadherin can inhibit migration and invasion is due to downregulation of hNanos1.

hNanos1 is a zinc finger protein that acts a translational repressor by binding to the 3’-

UTR of mRNA targets. Overexpression of hNanos1 is sufficient to induce invasion in

collagen type I gels and increase migration 111. This may be due in part to the hNanos1-

induced expression of the matrix metalloproteinase MT1-MMP (membrane type1-matrix

metalloproteinase) 112.

The loss or heterogenous expression of E-cadherin in colorectal cancer tissue

samples has been correlated to an advanced clinical stage and liver metastasis 113. This

makes the upregulation of E-cadherin induced by Par-4 overexpression in SW480 and

SW620 cells a significant finding. E-cadherin expression is regulated via different

mechanisms 114. Although the CpG island on the E-cadherin promoter is methylated in a

number of cancers 115, including colorectal cancer 116, 117, this may not be the dominant

mechanism of E-cadherin downregulation in colorectal cancer 118. E-cadherin

expression is also inhibited by a number of transcriptional repressors, including

members of the ZEB and basic helix-loop-helix (bHLH) families 119. In this study, we

reported the transcriptional upregulation of E-cadherin as a result of Par-4

overexpression. In addition, the expression levels of other tight junction proteins –

claudin-1, occludin, and ZO-1 – were upregulated. The mechanisms by which Par-4

regulates the expression of these genes is an area for further study.

50

Increasing Par-4 levels in SW620 cells was sufficient to alter the cell morphology

from a rounded phenotype with minimal cell-to-cell contacts to one with many cell-to-cell

contacts, which is characteristic of epithelial cells. Par-4-overexpressing cells had a

decreased expression of mesenchymal markers, such as vimentin, and upregulation of

the epithelial cell protein, E-cadherin. This is significant, because it suggests an MET

transition, the opposite of the epithelial-mesenchymal transition (EMT) that primary

tumor cells must undergo in order to metastasize. In colorectal cancer, EMT has been

observed in cells at the invasive front of primary tumors and lymph node metastases 120.

Characteristics of cells undergoing EMT include the decreased expression of epithelial

markers, in particular E-cadherin, and upregulation of mesenchymal markers, like

vimentin. Another characteristic of EMT is the increased nuclear localization of β-

catenin. In this study, we report that by increasing the levels of Par-4 in the metastatic

cell line, SW620, MET was induced, which is significant in that it helps to resolve a

discrepancy in the literature regarding the regulation of EMT by Par-4. In a study by Tan

et. al., Par-4 downregulation was shown to induce EMT in the cell line, BxPc-3,

concomitant with the acquisition of cisplatin resistance10; however, in a more recent

study, Par-4 was shown to mediate TGF-β-induced EMT11. In that study, TGF-β induced

EMT resulted in a Smad-dependent Par-4 upregulation, which in turn downregulated

epithelial markers and upregulated mesenchymal markers with a concomitant increase

in cell motility in several different cell lines. Subsequent overexpression and knockdown

of Par-4 showed that these phenotypic changes were mediated by Par-411. Thus, our

results corroborate the data by Tan et. al. and contradict the data by Chaudhry et. al. A

potential explanation for this discrepancy is that Par-4 regulates EMT in a cell-type

51

dependent manner. In the study by Tan et. al., the studies were carried out in a

pancreatic cancer cell line10, whereas in the study by Chaudhry et. al., endometrial,

cervical, ovarian, and breast cancer cell lines were used11. Our study was carried out in

colon cancer cell lines. The exact, cell-specific mechanisms by which Par-4 regulates

EMT remain to be elucidated. Taken together, in this study, the increased cell-to-cell

contacts due to the increased expression of tight junction proteins along with the EMT

reversal help to explain the observed inhibitory effect of Par-4 on the migration and

invasion of colon cancer cells.

In summary, our data show that Par-4 inhibits the migration and invasion of

SW480 and SW620 colon cancer cells, and the upregulation of epithelial markers and

downregulation of mesenchymal markers concomitant with a mesenchymal-epithelial

transition can partially explain the observed phenotype. These findings provide an

additional impetus to explore the use of Par-4 in colon cancer therapy. A potential

limitation of this study is that the migration and invasion experiments were entirely in

vitro. Future experiments in in vivo models, such as a tail-vein injection model, will help

to address this limitation. Future experiments should also examine the effect of Par-4 on

migration and invasion in other types of cancer and further elucidate the cell-specific

mechanisms by which Par-4 regulates migration.

2.5. Acknowledgements

This work was supported by Penn State Hershey start-up funds of Rosalyn B. Irby.

2.6. Conflict of Interest

The authors declare no conflicts of interest.

52

2.7. Publication Note

The work contained within this chapter was published in the Journal of Colon and Rectal

Cancer.121

53

3. Chapter 3

Trim21 is a novel regulator of Par-4 in colon and pancreatic cancer

cells

3.1. Introduction

As discussed above, metastatic colon cancer and pancreatic cancer both have a

very grim prognosis. The poor prognoses are due to a variety of factors. In colon

cancer, inherent chemoresistance and acquired resistance to treatment both play a

role.122 The inherent difficulty in treating pancreatic cancer is related to late diagnosis

and the resistance of pancreatic cancer cells to chemotherapy.123 This underscores an

important need for more effective therapeutics and for identifying novel therapeutic

targets.

Cancer care is shifting from the use of traditional chemotherapy with a broad-

based mechanism of action to targeted therapy, which is designed to interfere

selectively with cancer cells thereby limiting side-effects. Some examples of targeted

therapies used in the treatment of colorectal cancer and pancreatic cancer include

Avastin and Erlotinib. Avastin is a monoclonal antibody directed against vascular

endothelial growth factor (VEGF), thereby inhibiting angiogenesis, and is used in the

treatment of colorectal cancer.122 Erlotinib is a small-molecule inhibitor of epidermal

growth factor receptor (EGFR), thereby inhibiting cell survival and proliferation, and is

used in the treatment of pancreatic cancer.123 While the combination of standard

54

chemotherapy with targeted therapy improves survival compared with standard therapy

alone, resistance often develops.

Par-4 is a tumor suppressor with cancer-cell selective properties. In colon cancer

cells, Par-4 overexpression sensitizes cells to apoptosis in response to the

chemotherapeutic agent, 5-fluorouracil63, and AKT inhibitor, ISC-465. In pancreatic

cancer cells, treatment with inhibitors of NF-κB and BCL-2 induce Par-4 expression,

which in turn sensitizes cells to chemotherapeutic-induced apoptosis.80, 124

While most Par-4 studies have focused on characterizing the function of Par-4,

its regulation is a relatively understudied area. Par-4 is upregulated in response to

treatment with various natural products and small-molecules.12, 62, 125 Recently, Par-4

has also been shown to be transcriptionally upregulated by FOXO3a in response to

treatment with Withaferin A.45 Ubiquitination of target proteins by E3 ligases followed by

proteasomal degradation is a common mechanism for downregulating protein levels

and activity. The ubiquitination of Par-4 by FBXO45 and the subsequent proteasomal

degradation of Par-4 regulates cancer cell survival.37

Trim21, as an E3 ligase, also functions by ubiquitination of target substrates. For

example, Trim21 regulates innate immune signaling through ubiquitination of DDX41,

an intracellular DNA sensor, thereby inhibiting the innate immune response to

intracellular dsDNA.126 In addition, multiple members of the IRF family of proteins, which

is a family of transcription factors that are activated and act downstream of toll-like

receptors, are substrates of Trim21.127, 128 Thus, Trim21 negatively regulates the innate

immune response to foreign pathogens.

55

While most studies have examined the role of Trim21 in regulating innate

immune signaling, some studies have implicated Trim21 in the regulation of other

cellular processes. For example, Trim21 has been shown to positively regulate

apoptosis via ubiquitination of apoptosis inhibitors, such as BCL-2 and c-FLIP, leading

to their degradation.129, 130 Furthermore, Trim21 is a negative regulator of B-cell

proliferation.131 The above findings suggest a possible tumor-suppressive role of

Trim21. In line with this, two recent studies have demonstrated that reduced Trim21

expression is correlated with poor prognosis in hepatocellular carcinoma and diffuse

large B-cell lymphoma.132, 133

Identifying novel regulators of Par-4 represents a potential avenue for identifying

new drug targets for colon and pancreatic cancer. In this study, I identify Trim21 as a

novel interaction partner of Par-4 in colon cancer cells. Furthermore, I show that Trim21

can regulate Par-4 levels in response to cisplatin in both pancreatic cancer and colon

cancer cell lines. Finally, I show that Trim21 may represent a potentially novel

therapeutic target and biomarker.

3.2. Results

3.2.1. Trim21 is a novel interacting partner of Par-4

In order to discover new regulators of Par-4, I sought to identify novel binding

partners of Par-4. To identify novel binding partners of Par-4, I performed an

immunoprecipitation of Par-4 from HCT-116, HT-29, and KM12C colon cancer whole

cell lysates that had been transiently transfected with Par-4 plasmid. The purpose of the

transfection was to increase the signal. Proteins that co-precipitated with Par-4 were

analyzed by mass spectrometry for identification. Trim21 was identified as an interacting

56

protein in all three cell lines tested. A representative list of the top identified proteins in

the pull-down and the negative control pull-down is shown in Fig. 3-1A. To validate the

mass-spectrometry result and to confirm that the interaction between Par-4 and Trim21

was not an artifact of the ectopic Par-4 expression, reciprocal co-immunoprecipitations

were performed with endogenous Par-4 in HCT-116, HT-29, and KM12C whole cell

lysates. As can be seen in Fig. 3-1B through Fig. 3-1D, Par-4 and Trim21 interact

endogenously in colorectal cancer cells. To corroborate the interaction data, an

immunofluoresence analysis was performed in order to examine the co-localization of

Trim21 and Par-4 proteins intracellularly. The co-localization of Par-4 and Trim21 further

suggests that Trim21 and Par-4 proteins interact endogenously (Fig. 3-1E).

57

58

Figure 3-1. Trim21 is a novel interacting partner of Par-4.

HCT-116, HT-29, and KM12C cells are grown and either transfected with Par-4 expression

plasmid or control plasmid for 48 hrs. Par-4 was immunoprecipitated from whole cell lysates

using anti-Par-4 antibody and protein G magnetic beads. An in-solution trypsin digestion was

performed on the eluted proteins, and the digests were sent for mass spectrometry analysis. A)

A representative list of the most abundant proteins identified in the digest reveals Trim21 as a

potential novel interacting partner of Par-4. Co-immunoprecipitations are performed in HT-29,

HCT-116, and KM12C cells (B, C, D, respectively) in order to validate the Trim21/Par-4

interaction. KM12C cells were grown on coverslips, fixed, permeabilized, and probed for Par-4

and Trim21. E) A representative image showing co-localization of Par-4 and Trim21 in KM12C

cells.

59

3.2.2. Trim21 interacts with Par-4 through its PRYSPRY domain

Next, I sought to characterize the Par-4/Trim21 interaction. Specifically, I wanted

to identify which domain of Trim21 is responsible for mediating its interaction with Par-4.

Trim21 is an E3 ligase that is part of the Tripartite motif (TRIM) family. TRIM family

members contain a RING finger domain, a B-box domain, and a coiled-coil domain.

Trim21 is unique in that it also contains a C-terminal PRYSPRY domain, which is a

protein-protein interaction domain.40 A schematic of Trim21 can be seen in Fig. 3-2A.

To determine which domain of Trim21 mediates its interaction with Par-4, I

obtained three Trim21 constructs: full-length, exon 1 deletion (Trim21ΔEx1), and exon 6

(Trim21ΔEx6) deletion. The exon 1 deletion does not contain the RING domain nor the

B-box domain. These domains are responsible for the E3 ligase activity of Trim21. The

exon 6 deletion does not contain the PRYSPRY domain. A schematic of these

constructs can also be seen in Fig. 3-2A. To test which domain of Trim21 is responsible

for its interaction with Par-4, I co-transfected HCT-116 cells with combinations of the

Par-4 plasmid and each of the three Trim21 constructs. Then, I performed a co-

immunoprecipitation against Par-4, and probed for the presence of Trim21 by Western

blotting. The co-immunoprecipitation performed in cells with the Trim21ΔEx6 mutant

shows decreased binding of Trim21 to Par-4. This suggests that the PRYSPRY domain

of Trim21 is required for Trim21/Par-4 binding (Fig. 3-2B).

60

Figure 3-2. Trim21 interacts with Par-4 via its PRY-SPRY domain.

HCT-116 cells are co-transfected with Par-4 plasmid and different Trim21 constructs for 48 hrs.

Par-4 is immunoprecipitated from whole cell lysates using anti-Par-4 antibody and protein G

magnetic beads. The proteins were eluted from the beads and a Western blot analysis is

performed using anti-Trim21 antibody. A) A diagram representing the various Trim21

constructs that are used B) Western blots demonstrating that when co-transfecting with the

Trim21 construct that does not contain the PRY-SPRY domain, Trim21 does not interact with

61

Par-4; in contrast, when co-transfecting with the Trim21 constructs that contain the PRY-SPRY

domain, Trim21 does interact with Par-4.

62

3.2.3. Trim21 is not sufficient to downregulate Par-4 levels

Given that Trim21 is an E3 ligase and interacts with Par-4, I hypothesized that

Trim21 may downregulate Par-4 protein levels via ubiquitination and subsequent

proteasomal degradation. The endogenous expression patterns of Par-4 and Trim21

across a panel of colorectal cancer cell lines appears to show an inverse relationship

(Fig. 3-3A). This inverse relationship suggests that Trim21 could downregulate Par-4

levels. To test this hypothesis, I ectopically expressed the three Trim21 constructs in

SW480 cells by transient transfection, and probed for the expression of Par-4 by

Western blotting. The Par-4 protein expression was not significantly changed in the

Trim21 transfectants compared to the control, which demonstrate that Trim21

overexpression is not sufficient to downregulate Par-4 levels (Fig. 3-3B). These results

were corroborated by repeating the experiment using HCT-116 cells (data not shown).

This conclusion is further supported by the observation that ectopic Trim21 expression

and Trim21 knock-down do not affect the half-life of Par-4.37

63

Figure 3-3. Trim21 is not sufficient to downregulate Par-4 protein levels.

A) Western blots of endogenous expression levels of Par-4 and Trim21 in colon cancer cell lines

show an inverse correlation in expression. B) SW480 cells are transfected with either a control

64

plasmid or various Trim21 constructs for 48 hours. Whole cell lysates are collected and Par-4

protein levels are analyzed by Western blot with actin as a loading control.

65

3.2.4. Ectopic expression of Trim21 downregulates Par-4 in the presence of

cisplatin

Given that Trim21 was not sufficient to downregulate Par-4 levels, I hypothesized

that Trim21 may regulate Par-4 levels in response to a stimulus. Cisplatin (CDDP) is

one such stimulus that has been shown to affect Par-4 activity via promoting its

cleavage by caspase 3.41 Furthermore, Par-4 downregulation induces cisplatin

resistance in pancreatic cancer cells via a PI3K/AKT-dependent EMT pathway.10

To test whether Trim21 modulates Par-4 levels in response to cisplatin treatment,

I transfected HCT-116 and HT-29 cells with Trim21 full-length plasmid or control

plasmid, and treated the transfected cells with increasing concentrations of cisplatin for

24 hrs. I probed for changes in Par-4 levels by Western blotting. As can be seen in Fig.

3-4A and Fig. 3-4B, in the presence of Trim21 overexpression, cisplatin downregulates

Par-4 levels relative to control transfected cells. In HT-29 cells, this downregulation is

evident even at the lowest cisplatin dose tested, and this downregulation occurs at all

doses up to 15 μg/ml (Fig. 3-4A). In HCT-116 cells, this downregulation occurs at all

doses at or above 3.75 μg/ml with the exception of the 7.5 μg/ml dose (Fig. 3-4B).

66

Figure 3-4. Ectopic expression of Trim21 downregulates Par-4 in the presence of cisplatin in colon cancer cells.

Colon cancer cell lines, transfected with or without Trim21 expression plasmid for 48 hrs, were

treated with increasing doses of cisplatin for 24 hrs. The cisplatin concentrations are in units of

67

μg/ml. Western blots of Par-4 and Trim21 levels are shown with actin shown as a loading

control. A) HT-29 B) HCT-116.

68

3.2.5. Cisplatin downregulates Par-4 in a dose- and proteaseome-

dependent manner

Given the previous observations that cisplatin downregulates Par-4 at multiple

doses, I asked whether cisplatin downregulates Par-4 in a dose-dependent manner. To

determine this, I treated Trim21-transfected HCT-116 cells with increasing doses of

cisplatin and performed a time-course. Par-4 levels from whole cell lysates were then

examined via Western blotting. The data show that cisplatin-induced Par-4

downregulation occurs in a dose-dependent manner: Par-4 levels are reduced more

quickly at higher doses (Fig. 3-5A).

Given that Trim21 is an E3 ligase, I asked whether the downregulation of Par-4

occurs through proteasomal degradation. To test this hypothesis, I transfected HCT-116

cells with control plasmid or Trim21 plasmid, treated cells with two different doses of

cisplatin, and treated cells either with or without 10 μM MG132, a proteasome inhibitor.

The effects on Par-4 levels were determined by Western blotting. The data show that

the Par-4 downregulation in reponse to Trim21/cisplatin is abrogated by co-treatment

with MG132 (Fig. 3-5B). This suggests that the downregulation of Par-4 in response to

cisplatin occurs at least partially through the proteasome pathway.

69

Figure 3-5. Cisplatin downregulates Par-4 in a dose- and proteasome-dependent manner.

A) Western blots showing Par-4 expression levels in Trim21-transfected HCT-116 cells over time

at different doses of cisplatin. Cisplatin doses are in units of μg/ml. Time is in units of hours.

Actin is shown as a loading control. B) HCT-116 cells were transfected with or without Trim21

expression plasmid for 48 hrs, then treated with the indicated doses of cisplatin in μg/ml for 24

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hrs, and with or without 10 μM MG132. Blot shows Par-4 expression levels, and actin is shown

as a loading control. A vertical line demarcates the boundary between separate blots.

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3.2.6. Cisplatin downregulates Par-4 in both the cytoplasmic and nuclear

compartments

To determine an intracellular location of the Par-4 downregulation in response to

cisplatin, I transfected HCT-116 cells with or without Trim21 and treated with 3.75 μg/ml

of cisplatin or carrier, then performed a nuclear-cytoplasmic fractionation to determine

whether the Par-4 downregulation was localized to a specific subcellular compartment.

Par-4 protein levels were examined by Western blotting. As can be seen in Fig. 3-6, the

Par-4 downregulation occurs in both the cytoplasmic and nuclear compartments, with

the downregulation being slightly more pronounced in the nuclear compartment. Lamin

A/B and β-tubulin were probed to confirm the integrity of the subcellular fractionation

and to ensure that there was no cross-contamination between compartments during the

fractionation.

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Figure 3-6. Cisplatin downregulates Par-4 in both the cytoplasmic and nuclear compartments.

HCT-116 cells were either transfected with plasmid encoding Trim21 or control plasmid for 48

hrs and treated with or without 3.75 μg/ml of cisplatin. Then, nuclear-cytoplasmic fractionation

was performed and Par-4 and Trim21 levels were examined by Western blotting, with Lamin

A/B and β-tubulin serving as markers validating the integrity of the fractionation.

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3.2.7. Cisplatin downregulates Par-4 in pancreatic cancer cells

Given that Par-4 was downregulated in response to cisplatin in the presence of

overexpressed Trim21 in colon cancer cells, I wanted to examine whether I could

extend this observation to a different type of cancer. To test that hypothesis, I

transfected AsPc-1, BxPc-3, and MiaPaca-2 cells, which are all pancreatic cancer cell

lines, with or without Trim21 for 48 hrs, and treated the cells with increasing doses of

cisplatin for 24 hrs, and probed whole cell lystates for Par-4 expression via Western

blotting. I observed an enhanced dose-dependent downregulation of Par-4 in response

to cisplatin treatment in Trim21-transfected cells compared to control cells (Fig. 3-7A-C).

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75

Figure 3-7. Cisplatin downregulates Par-4 in pancreatic cancer cells.

Pancreatic cancer cell lines were either transfected with Trim21 plasmid or control plasmid for

48 hrs, and then treated with increasing concentrations of cisplatin for 24 hrs. Cisplatin doses

are in units of μg/ml. Western blots showing Par-4 expression are shown with actin as a loading

control. Western blots validating Trim21 overexpression are also included. A) BxPc-3 B)

MiaPaca-2 C) AsPc-1.

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3.2.8. Trim21 is a potential therapeutic target in colon and pancreatic

cancer

Given the effect of Trim21 on Par-4 in response to cisplatin, I hypothesized that

Trim21 could contribute to cisplatin resistance. To address this question, I performed an

MTT viability assay on HCT-116 cells that had either been transfected with Trim21

plasmid or control plasmid, and then treated with increasing concentrations of cisplatin.

The data show that Trim21 decreases the sensitivity of HCT-116 cells to cisplatin (Fig.

3-8A). Specifically, HCT-116 cells had statistically significant increases in viability at

cisplatin doses between 0.5-10 μg/ml, with a right shift in IC50 from approximately 5

μg/ml to 10 μg/ml between the control and Trim21 transfected cells, respectively.

Likewise, I performed an analogous experiment in Panc-1 cells, a pancreatic cancer cell

line. To my surprise, ectopic Trim21 expression in Panc-1 cells had no effect on Panc-1

cell viability in response to cisplatin (data not shown). Despite that finding, I show in Fig.

3-8B that ectopic Par-4 expression sensitizes Panc-1 cells to cisplatin-induced

apoptosis as indicated by PARP cleavage, specifically at the 15 and 30 μg/ml doses.

PARP cleavage was examined by Western blotting and the percentage of the cleavage

product was determined using densitometry. Densitometric analysis revealed that Par-4

overexpression increased the levels of apoptosis in Panc-1 cells by two- to three-fold

relative to the control transfected cells at the 15 and 30 μg/ml doses of cisplatin (Fig. 3-

8B).

Given the effects of Trim21 on Par-4, and in light of the recent literature

suggesting that Trim21 is a favorable prognostic marker in cohorts of hepatocellular

carcinoma and diffuse large B-cell lymphoma patients,132, 133 I asked whether Trim21

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has prognostic significance in either colorectal cancer or pancreatic cancer. To answer

this question, I downloaded data from the TCGA database, stratified by Trim21

expression: Trim21 high (Z-score > 2), Trim21 low (Z-score < -2), and Trim21

intermediate (-2 < Z-score < 2). Then, I plotted Kaplain Meier survival curves of these

three Trim21 expression groups in the colorectal and pancreatic cancer cohorts. There

was no statistically significant difference in overall survival or disease-free survival in the

colorectal cancer cohort between the three Trim21 expression groups (data not shown).

In contrast, in pancreatic cancer there was a dramatic increase in overall survival in the

Trim21 low cohort relative to the intermediate and high cohorts (p = 0.0043), as can be

seen in Fig. 3-8C. Though the difference in overall survival was not significantly different

between the Trim21 high and intermediate groups (p = 0.2121), the Trim21 high group

seems to be trending toward reduced overall survival relative to the intermediate group

(Fig. 3-8C). Likewise, the trends in disease-free survival follow the same patterns as the

trends in overall survival in the pancreatic cancer cohort. Specifically, low Trim21

expression correlates with increased disease-free survival, whereas high Trim21

expression correlates with reduced disease-free survival relative to the Trim21

intermediate cohort (Fig. 3-8D). In the disease-free survival curve, the Trim21 high,

intermediate, and low expression groups were all significantly different from each other

(p < 0.01).

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Figure 3-8. Trim21 is a potential therapeutic target in colon and pancreatic cancer.

A) HCT-116 cells were either transfected with a plasmid encoding Trim21 or control plasmid for

48 hrs, and then treated with increasing doses of cisplatin for 24 hrs, and viability was assessed

by MTT assay. Viability is plotted as a percentage of control samples. Asterisks indicate

statistically significant differences in viability between Trim21 and control transfected cells. B)

Panc-1 cells were either transfected with a plasmid encoding Par-4 or control plasmid for 48

hrs, and then treated with increasing doses of cisplatin. Cisplatin doses are shown in units of

μg/ml. Levels of PARP and Par-4 levels were examined by Western blotting. Percentage of

cleaved PARP, as determined by densitometric analysis, is shown below the blots. Kaplan-Meier

survival curves showing overall survival, Fig. 3-8C and progression-free survival, Fig. 3-8D, of a

cohort of pancreatic cancer patients stratified by Trim21 mRNA expression levels. Data was

obtained from TCGA database.

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3.3. Discussion

In this study, I identified Trim21 as a novel interaction partner of Par-4, and

demonstrate that they interact endogenously. I show that the binding occurs through the

PRYSPRY domain of Trim21. Though Trim21 is not sufficient to downregulate Par-4

levels, I show that in response to cisplatin Trim21 downregulates Par-4 in a dose- and

proteasome-dependent manner in both the nucleus and cytoplasm. Furthermore, I show

that Trim21 can increase the resistance of cancer cells to cisplatin and that by

overexpressing Par-4, cancer cells can be sensitized to cisplatin-induced apoptosis.

Finally, I demonstrate that Trim21 expression can predict overall- and disease-free

survival in a cohort of pancreatic cancer patients.

Par-4 is an important tumor-suppressor whose expression is downregulated in

several cancers.14-16 In addition, its ability to selectively induce and sensitize cancer

cells toward apoptosis underscores the tremendous therapeutic potential of Par-4.134-136

Therefore, understanding the mechanisms of Par-4 regulation is an important, yet

understudied, area of research, and this work represents an advance in that field. Chen

et. al. showed that Par-4 levels could be post-translationally regulated by the

proteasome through targeting by FBXO45.37 Brasseur et. al. showed that cleaved Par-4

is also regulated by the proteasome.42 I now report a stimulus-dependent regulation of

Par-4 by the proteasome. That stimulus is cisplatin. This observation is significant, since

cisplatin and its analogues are used in the treatment of colon and pancreatic cancer.

Additionally, the subcellular localization of Par-4 is important, because its nuclear

localization is correlated with its apoptotic function.21 Thus, the finding that its

downregulation is present in the nucleus, along with in the cytoplasm, is also significant.

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By identifying a novel mechanism of regulation of Par-4, I in turn discovered a

potentially new therapeutic target: Trim21. This is especially important for pancreatic

cancer, where most patients ultimately succumb to their disease,54 and are therefore in

need of novel, effective therapies. Here, I show that low Trim21 levels correlate with

prolonged overall and disease-free survival in pancreatic cancer patients. Conversely, I

showed that high Trim21 levels correlate with lower disease-free survival. This intriguing

finding is the opposite of the protective effects of Trim21 in hepatcellular carcinoma and

diffuse large B-cell lymphoma.132, 133 This illustrates the complexity of disease biology,

and the prognostic differences of Trim21 may reflect differences in downstream Trim21

targets, such as Par-4, in different types of cancer. Further study of the role of Trim21 in

pancreatic cancer is needed.

Other work has been done on the relationship between cisplatin and Par-4. For

example, cisplatin has been shown to activate Par-4 via cleavage by caspase 3.41 In

this instance, the cleaved Par-4 had more apoptotic activity and a greater nuclear

localization than full-length Par-4. In another study, Par-4 downregulation was shown to

confer resistance to cisplatin through the PI3K/AKT pathway.10 In this example, Par-4

overexpression reversed the resistance phenotype. This work adds to the complex

relationship between cisplatin and Par-4, and gives rise to interesting questions. For

example, understanding the mechanisms immediately downstream of cisplatin-induced

DNA damage that determine whether Par-4 is cleaved to a more active form versus

downregulated would be an important area of study. Finding ways to switch from

cisplatin-induced Par4 downregulation to cisplatin-induced Par-4 cleavage could be

beneficial. In addition, it would be interesting to examine whether Par-4 downregulation

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occurs solely in response to the DNA-crosslinking that occurs during cisplatin

treatment,137 or whether this response extends to other types of DNA damage, such as

double-strand breaks.

In conclusion, our data show that Trim21 is a novel interaction partner of Par-4

and a novel regulator of Par-4 in response to cisplatin treatment. Our data suggests that

Trim21 can influence sensitivity of cancer cells to cisplatin. Targeting Trim21 may

potentially be a way to enhance the effectiveness of cisplatin treatment, and may

represent an important therapeutic target, especially in pancreatic cancer.

3.4. Materials/Methods

3.4.1. Cell culture, transfection, plasmids, reagents, and antibodies

HCT-116, SW480, and HT-29 colorectal cancer cells were obtained from ATCC

and maintained in RPMI (Cellgro, Manassas, VA) + 10% fetal bovine serum (FBS).

Panc-1 cells were a kind gift from Dr. Arun Sharma and were maintained in DMEM

(Cellgro, Manassas, VA) + 10% FBS. FBS was obtained from Atlanta Biologicals

(Norcross, GA). Cell lines were grown at 37◦C and 5% CO2. For experiments, cells were

seeded in six-well plates at a seeding density of 300,000 cells/well. Transient

transfections were performed 24 hrs post-seeding. Drug treatments were performed 48

hrs post-seeding. The cells were transiently transfected using PolyJet DNA Transfection

Reagent (SignaGen Laboratories, Rockville, MD), according to the manufacturer’s

instructions. The Par-4 plasmid and anti-Trim21 shRNA plasmids were obtained from

Origene Technologies (Rockville, MD). The plasmids encoding for full-length Trim21

and deletion mutant Trim21 were kind gifts from Dr. Caroline Jefferies at the Royal

College of Surgeons in Ireland. The anti-Trim21, anti-Lamin A/C, and anti-Par-4

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antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-

actin and anti-β-tubulin antibodies were obtained from Sigma-Aldrich (Saint Louis, MO).

The anti-PARP antibody was obtained from Cell-Signaling (Danvers, MA). Cisplatin was

obtained from Acros Organics (Thermo Fisher, Waltham, MA). MG132 was obtained

from Fisher (Thermo Fisher, Waltham, MA).

3.4.2. Western blot analyses

To examine changes in protein expression, cells were washed twice with PBS

and were lysed into lysis buffer (50mm HEPES, 100 mm NaCl, 10 mm EDTA, 0.5 %

NP40, 10% glycerol, supplemented with 0.0001% Tween20, 0.1 mM PMSF, 0.1 mM

NaVO4, 0.5 mM NaF, 5 μg/ml leupeptin, 0.1 mm DTT). The proteins were quantified

according to the BCA Assay (Thermo Scientific Inc., Rockford, IL) and loaded equally

onto 10% SDS-polyacrylamide gels. Proteins were electrophoresed at 150 V and

transferred to nitrocellulose membranes using a Trans-Blot SD Semi-Dry Transfer Cell

(BioRad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk for 1 hr and

incubated with primary antibody overnight. The blots were washed 3X in TBS with 0.1%

Tween20 and incubated for 1 hr in appropriate HRP-conjugated secondary antibodies

(Amersham, Piscataway, NJ). Blots were washed 3X and chemiluminescent detection

was performed using Amersham ECL Prime Western Blotting Detection Reagent

(Thermo Scientific Inc., Rockford, IL). The blots were either exposed to autoradiography

film (GE Healthcare Life Sciences, Pittsburgh, PA) and scanned or imaged using the

Molecular Imager Gel Doc XR System (Bio-Rad, Hercules, CA).

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3.4.3. Co-IP/Mass-Spec

To identify novel binding partners of Par-4, HCT-116, HT-29, and SW480 cells

were cultured in two 10-cm dishes and transfected with 5 μg of Par-4 plasmid per dish

using SignaGen Polyjet transfection reagent, according to the manufacturer’s

instructions. After 48 hours of transfection, cells were lysed in lysis buffer (50mm

HEPES, 100 mm NaCl, 10 mm EDTA, 0.5 % NP40, 10% glycerol, supplemented with

0.0001% Tween20, 0.1 mM PMSF, 0.1 mM NaVO4, 0.5 mM NaF, 5 μg/ml leupeptin, 0.1

mm DTT), and the total protein concentration was determined as above.

To perform the co-immunoprecipitation, approximately 3,000 μg of total protein

from the lysate was mixed with 2 μg of anti-Par-4 antibody. As a negative control, the

same amount of total protein from the lysate was mixed with anti-HA antibody. The

antibody-lysate mixtures were allowed to mix overnight at 4◦ C. The next day the

antibody-antigen complexes were pulled down using Dynabeads Protein G (Thermo

Fisher Scientific, Leesport, PA) and washed with lysis buffer, according to the

manufacturer’s instructions. Proteins were eluted from the beads by boiling at 100◦C for

10 mins in digestion buffer (6 M urea, 50 mM Tris-HCl, pH = 8.0). Samples were then

cooled on ice and eluate removed from beads.

To perform the in-solution digestion, 5 μl of 200 mM DTT/50 mM Tris-HCl/pH =

8.0 was added to solution, mixed, and allowed to incubate for 1 hr at room temperature.

After 1 hr, 20 μl of 200 mM Iodoacetamide/50 mM Tris-HCl/pH = 8.0 was added, mixed,

and allowed to incubate at room temperature in the dark for 1 hr. After 1 hr, 775 μl of 50

mM Tris-HCl/1 mM CaCl2/pH = 7.6. 2 microliters of trypsin (0.2 μg/ml) was added,

mixed, and allowed to incubate at 37◦C overnight. Formic acid was added to bring pH

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down to 3-4. Solutions were snap frozen in liquid nitrogen and spun to dryness under

vacuum. Residue was resuspended in milliQ water (EMD Millipore, Billerica, MA), snap

frozen in liquid nitrogen, and dried using a speed vac. This process was repeated two

more times. The final residue was used for protein identification via MALDI-TOF mass

spectrometry. Proteins were identified from mass spectra using the Paragon Algorithm.

3.4.4. MTT assay

To examine changes in cell viability, cells were seeded at a density of 6 x 103

cells/well in a 96-well culture plate. After 24 hours, the cells were transfected with the

appropriate plasmid. Twenty- four hours post-transfection, the medium was replaced

with medium containing different concentrations of cisplatin. Twenty-four hours post-

cisplatin treatment, MTT reagent was added (Calbiochem) and the cells were incubated

for 3.5 hrs at 37◦ C. After incubation, the media was aspirated off, crystals were

dissolved in MTT solvent (4 mM HCl, 0.1% Nonidet P-40, in isopropanol), and viability

was assessed by measuring the absorbance at 570 nm with 630 nm absorbance as the

reference. Percent viability is expressed as the absorbance normalized to the DMSO

control.

3.4.5. Immunofluoresence

Cells were grown to approximately 50% confluence on 22 x 22 mm cover slips

(#1.5). Media was removed and cells were washed with PBS, followed by fixation with

4% paraformaldehyde in PBS for 15 mins at room temperature. Cells were washed in

PBS, then permeabilized 3 x 5 mins with 0.1% Triton X-100 in PBS. Cells were washed

with PBS, then blocked with 10% BSA in PBS for 1 hr at room temperature. After one

86

hour, the cells were washed with PBS, and incubated with a mixture of anti-Par-4

antibody (1:50) and anti-Trim21 antibody (1:50) in 4% BSA/PBS for two hours at room

temperature. After the incubation in primary antibody, the cells were washed 4X with

PBS for 10 mins each at room temperature with gentle shaking. After washing, the cells

were then incubated in a mixture of secondary antibody in 4% BSA/PBS: anti-rabbit

FITC (1:100) and anti-mouse Ro (1:100). The secondary antibody incubation was

carried out in the dark for two hours at room temperature. After incubation in secondary

antibody, the cells were washed 4X with PBS for 10 mins each at room temperature

with gentle shaking. After washing, the cells were incubated with DAPI (0.5 μg/ml in

PBS) for 5 mins at room temperature in the dark. After incubation with DAPI, the cells

were washed 4X with PBS for 10 mins each at room temperature with gentle shaking.

Finally, the coverslips were mounted on glass slides using ProLong Diamond Antifade

mountant (Thermo Fisher, Leesport, PA). Slides were imaged using DeltaVision Elite

Inverted Microscope. Six random fields are imaged to ensure representativeness.

3.4.6. Nuclear-Cytoplasmic Fractionation

To perform the nuclear-cytoplasmic fractionation, the NE-PER Nuclear and

Cytoplasmic Extraction Kit (Thermo Fisher, Leesport, PA) was used, and the

manufacturer’s instructions were followed. The resultant extracts were quantified and

loaded equally onto a 10% acrylamide gel, and proteins probed by Western blotting.

3.4.7. Statistical analyses

The statistical analyses were carried out using GraphPad Prism software, version

6.04 (GraphPad Software, Inc., San Diego, CA, USA). All plots were created using

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GraphPad Prism. All experiments were repeated three times with representative

experiments shown in the results. For the MTT assay, two-way ANOVA was used to

determine differences between transfectants and controls, using the Sidak correction to

correct for multiple comparisons. For the Kaplan-Meier survival curves, the log-rank test

was used to determine differences between groups. The pancreatic cancer TCGA data

was accessed and downloaded from www.cbioportal.org. Trim21 high expression was

considered to be those that had a Z-score greater than 2. Trim21 low expression was

considered to be those than had a Z-score lower than -2. Patients with intermediate

Trim21 expression had Z-scores between +/- 2. Densitometric analysis was performed

using ImageJ.104 The threshold for statistical significance is P < 0.05.

3.5. Acknowledgements

The Penn State Hershey Imaging Core is acknowledged for their help in obtaining

images. This work was funded by start-up funds granted to Rosalyn B. Irby.

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4. Chapter 4

Prostate apoptosis response-4 sensitizes glioma cells to

chemotherapeutics

4.1. Introduction

Despite the current standard of care - which consists of surgical resection,

chemoradiotherapy, and adjuvant temozolomide (TMZ) - the median survival for GBM

14.6 months.81 This highlights the need for novel monotherapies and combination

therapies.84

Prostate apoptosis response-4 is a tumor suppressor that can induce apoptosis

in cancer cells.23 While the role of Par-4 has been studied in a variety of cancers, its role

in glioma is relatively understudied. One of the earliest studies of Par-4 in glioma

showed that ectopic expression of Par-4 induced apoptosis in glioma and other types of

brain tumors.138 Another study showed that upon TMZ treatment, cellular prion protein

was upregulated, which bound to and inhibited Par-4 activation; knock-down of cellular

prion protein dramatically enhanced TMZ induced apoptosis, which was mediated by

Par-4.87 Other studies have demonstrated a role of Par-4 in drug-induced apoptosis of

tamoxifen and drug-induced autophagic cell death of curcumin.12, 88 These studies

underscore the importance of Par-4 in mediating cell death in glioma cells alone and in

response to treatment.

AKT/PKB is a serine/threonine protein kinase that acts downstream of various

growth factor receptors to mediate signaling that leads to cell growth, proliferation,

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migration, and apoptosis-inhibition.139 Multiple studies have demonstrated that AKT is

activated in the majority of gliomas through mutations in upstream signaling proteins.85

Despite this, there are few AKT inhibitors with demonstrated efficacy in vitro and in

vivo.140

In this work, I demonstrate that the novel AKT inhibitor, ISC-4, and its analogs

show activity in reducing glioma cell viability, and Par-4 sensitizes glioma cells to ISC-4.

Furthermore, I show that Par-4 is sufficient to reduce cell viability, induce apoptosis, and

sensitize glioblastoma cells toward 5-fluorouracil.

4.2. Materials and Methods

4.2.1. Cell culture and transfection

A172 and SNB19 glioblastoma cells were obtained from ATCC and maintained in

RPMI/10% FBS in a 37°C incubator with 5% CO2 and a humidified atmosphere. The

cells were transfected with either empty vector or plasmid vector encoding for human

Par-4 (OriGene Technologies, Rockville, MD) using Signagen Polyjet DNA transfection

reagent (SignaGen Laboratories, Rockville, MD) according to the manufacturer’s

instructions.

4.2.2. Western blot analyses

To measure changes in protein expression, cells were washed twice with PBS and were

lysed into lysis buffer (50mm HEPES, 100 mm NaCl, 10 mm EDTA, 0.5 % NP40, 10%

glycerol, supplemented with 0.0001% Tween 20, 0.1 mM PMSF, 0.1 mM NaVO4, 0.5

mM NaF, 5 μg/ml leupeptin, 0.1 mm DTT). The proteins were quantified according to

the BCA Assay (Thermo Scientific Inc., Rockford, IL) and loaded equally onto 10%

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SDS-polyacrylamide gels. Proteins were electrophoresed at 150 V and transferred to

nitrocellulose membranes using a semi-dry blotter (BioRad, Hercules, CA). Membranes

were blocked with 5% non-fat dry milk or 5% BSA for 1 hr and incubated with primary

antibody overnight. The blots were washed 3X in TBS with 0.1% Tween20 and

incubated for 1 hr in appropriate HRP-conjugated secondary antibodies (Amersham,

Piscataway, NJ). Blots were washed 3X and chemiluminescent detection was

performed using Amersham ECL Prime Western Blotting Detection Reagent (Thermo

Scientific Inc., Rockford, IL). The blots were either exposed to autoradiography film (GE

Healthcare Life Sciences, Pittsburgh, PA) and scanned or imaged using the Molecular

Imager Gel Doc XR System (Bio-Rad). Densitometric analyses were performed with

NIH Image J software104. Primary antibodies used were: phospho-Akt, phospho-Par-4,

and PARP (Cell Signaling, Danvers, MA), GRP78 (Santa Cruz Biotechnology, Santa

Cruz, CA), and Actin (Sigma-Aldrich, Saint Louis, MO).

4.2.3. MTT viability assay

To measure in vitro cytotoxic efficacy, we used 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide to measure cell viability. A172 and SNB19 cells were

seeded at a density of 7 x 103 cells/well in a 96-well culture plate. After 24 hours, the

cells were transfected with Par-4 plasmid or control plasmid. Twenty-four hours later,

cells were treated with the indicated concentrations of drugs. Forty-eight hours later,

MTT reagent was added (Calbiochem) and the cells were incubated for 3.5 hrs at 37◦ C.

After incubation, the media was aspirated off, crystals were dissolved in MTT solvent (4

mM HCl, 0.1% Nonidet P-40, in isopropanol), and viability was assessed by measuring

the absorbance at 570 nm with 630 nm absorbance as the reference.

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4.2.4. PE Annexin V apoptosis assay

To measure apoptosis, the PE Annexin V flow cytometry assay was used

according to the manufacturer’s protocol. Briefly, after cells seeded in six-well plates

were transfected and treated with drugs for the appropriate time period, cells were

trypsinized, washed twice with cold PBS, and resuspended in binding buffer at a

concentration of 1 x 106 cells/ml. One-hundred microliters of the cell suspension were

transferred to a 5 ml culture tube, and 5 microlitres of PE Annexin V and 5 microliters of

7-AAD were added. The mixture was vortexed gently and allowed to incubate for 15

mins at room temperature in the dark. Four-hundred microliters of binding buffer were

added, mixed, and analyzed by flow cytometry.

4.2.5. Statistical analysis

All data analysis was conducted using a student’s T-test.

4.3. Results

4.3.1. Akt inhibitors reduce cell viability in glioblastoma cells

A172 cells were seeded into 96-well plates and treated with varying

concentrations of the AKT inhibitors, NISC-6, ISC-4, NNITC-2, NNISC-2 for 48 hours.

Viability was determined using the MTT viability assay. As can be seen in Fig. 4-1A, the

AKT inhibitors all decreased cell viability with IC50 values ranging from 2 to 8 μM. I then

wanted to examine the effectiveness of AKT inhibition on a wider panel of GBM cell

lines, and ISC-4 was chosen given that it has been well-characterized.65 As can be seen

in Fig. 4-1B and Fig. 4-1C, ISC-4 is effective at decreasing cell viability across a wide

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panel of GBM cell lines at 24 and 72 hours, though some cell lines, such as CRL1690,

seem to be resistant to ISC-4 even at 72 hours.

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Figure 4-1. Akt inhibitors decrease cell viability in glioblastoma cells.

A) A172 cells were seeded into 96-well plates, then treated with various concentrations of

NISC-6, ISC-4, NNITC-2, and NNISC-2 for 48 hrs. MTT assay was used to determine cell viability.

Various GBM cell lines were seeded into 96-well plates and treated with different

concentrations of ISC-4 for 24 (B) and 72 (C) hrs. MTT assay was used to determine cell viability.

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4.3.2. Par-4 is sufficient to reduce cell viability in GBM cells

A172 and SNB19 cells were seeded into 96-well plates and transfected with

various concentrations of Par-4 for 24, 48, and 72 hours. Viability was determined by

the MTT viability assay. As can be seen from Fig. 4-2A and Fig. 4-2B, Par-4 is sufficient

to reduce cell viability in a dose-dependent manner. A172 cells are particularly sensitive

to ectopic Par-4.

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Figure 4-2. Par-4 is sufficient to reduce cell viability in GBM cells.

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A172 (A) and SNB19 (B) cells were seeded into 96-well plates then transfected with various

concentrations of Par-4 plasmid for 24, 48, and 72 hours and viability was assessed by MTT

assay.

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4.3.3. Par-4 sensitizes GBM cells to ISC-4 and 5-FU

After determining that ISC-4 and Par-4 were both sufficient at reducing cell

viability in GBM cell lines, I sought to determine whether Par-4 could sensitize GBM

cells to ISC-4 treatment. In addition, I sought to determine whether Par-4 could sensitize

GBM cells toward the commonly used therapies: TMZ and 5-FU. As can be seen in Fig.

4-3A and Fig. 4-3B, Par-4 sensitizes A172 cells toward 5-FU and SNB19 cells toward

ISC-4 treatment, respectively. Par-4 did not sensitize GBM cells toward TMZ treatment

(data not shown).

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100

Figure 4-3. Par-4 sensitizes GBM cells to 5-FU and ISC-4.

A) A172 cells were either untreated, treated with 5-FU (700 μM), transfected with Par-4 (0.08

μg/ml), or treated with the combination for 48 hrs B) SNB19 cells were transfected with either

empty vector or Par-4 plasmid and treated with increasing amounts of ISC-4 for 48 hours. MTT

assays were used to determine cell viability

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4.3.4. Par-4, ISC-4, and their combinations induce apoptosis in GBM cells

To gain insight into the mechanisms of the decrease in cell viability seen with

Par-4 transfection, drug treatments, or their combination, I sought to determine whether

cells were dying by apoptosis. A172 and SNB19 cells were seeded into six-well plates,

transfected with Par-4 plasmid or control, and then treated with DMSO control, ISC-4 (5

μM) or TMZ (200 μM) for 24 hrs. Then, the PE Annexin V flow cytometry assay was

used to determine the percentage of apoptotic cells under these conditions. As can be

seen in Fig. 4-4, Par-4, ISC-4, and their combinations led to increased levels of

apoptosis in A172 and SNB19 GBM cells. TMZ treatment alone did not lead to

significant levels of apoptosis in A172 and SNB19 cells.

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Figure 4-4. Par-4, ISC-4, and their combinations induce apoptosis in GBM cells.

103

A172 cells (A) and SNB19 cells (B) were either transfected with Par-4 plasmid or control plasmid

and then treated with ISC-4 (5 μM) or TMZ (200 μM). The percentage of apoptotic cells was

determined by PE Annexin V flow cytometry assay.

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4.4. Discussion

In this study, I show that treating glioblastoma cells with selenocyanate AKT

inhibitors potently reduced cell viability. In addition, I showed that these types of

inhibitors are active across multiple GBM cell lines. I also found that ectopic Par-4

expression is sufficient to reduce cell viability in GBM and can sensitize cells toward

ISC-4 and 5-FU treatment. Finally, I show that the reduction in viability induced by Par-4

or its treatment combinations at least partially occurs through apoptosis.

These findings are significant, because little is known about the role and potential

therapeutic significance of Par-4 in gliomas, and much of that work is focused on glioma

stem cells. The first study on Par-4 in gliomas demonstrated that ectopic Par-4

expression induced apoptosis in a variety of different brain cancers, including

neuroblastoma, medulloblastoma, and gliomas.138 These findings corroborate the ability

of Par-4 to induce apoptosis in gliomas. Experiments are ongoing in our lab that are

attempting to elucidate the mechanisms by which Par-4 induces apoptosis. The study

by Vetterkind et. al. suggests that the mechanism is independent of BCL-2

downregulation and PKCζ inhibition, while being partially dependent on the Fas death

receptor pathway.138 This suggests that a novel mechanism of Par-4 induced apoptosis

is at play. Given the role of NF-κB activity in glioma141 and the essential role of NF-κB

inhibition in Par-4 activity,105 one possibility is enhanced inhibition of NF-κB

transcriptional activity in the nucleus.

Given that the PI3K/AKT pathway is activated in a majority of gliomas and given

that AKT inhibition has not been explored extensively, the finding that ISC-4 and its

105

derivatives reduce cell viability and is efficacious across a variety of glioma cell lines is

important. Furthermore, the ability of Par-4 to sensitize glioma cells to ISC-4 is

significant, because it underscores the therapeutic potential of Par-4 in glioma

treatment. Further experiments could expand on this by studying whether extracellular

Par-4 can sensitize to ISC-4 treatment. In addition, studies examining the efficacy of

ISC-4 alone and in combination with Par-4 in an in vivo model of glioma would be a

fruitful area of research.

In summary, our data show that Par-4 is sufficient to reduce cell viability alone

and in combination with ISC-4 and 5-FU. Given the lack of effective therapies for

glioblastoma patients, this work demonstrates the potential of using a combination of

AKT inhibitor, ISC-4, along with Par-4 in the treatment of glioma.

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5. Chapter 5

Summary and Future Directions

Par-4 has been shown to have therapeutic potential in a number of cancers due

to its ability to induce cancer-cell selective apoptosis and its ability to sensitize cancer

cells to apoptosis-inducing agents, including chemotherapy and radiation.23 The long-

term goal of our lab is to utilize Par-4 to enhance cancer therapy, and at the start of this

dissertation the focus of the lab has been on studying Par-4 in colon cancer. At the time,

our lab had shown: 1) Delivery of Par-4 plasmid via nanoliposomes can sensitize colon

tumor cells to 5-fluorouracil.63 2) Par-4 overexpression leads to gene expression

changes and to changes in microRNA levels, which seemed to be mediated by NF-κB

inhibition, that could account for its sensitization to 5-fluorouracil.64 3) Endogenous Par-

4 can be activated by treatment with a combination of 5-fluorouracil and c-Src inhibitor

or by ISC-4, which can lead to cell death in vitro and in vivo.65, 66 The goal of this

dissertation was to expand on this foundation, and to elucidate novel mechanisms of

Par-4 activity, regulation, and potential applicability to cancer treatment. In the first part

of this work, I examined the effect of Par-4 on cell migration in colon cancer. Through

this work, I demonstrated that Par-4 inhibited migration, invasion, and induced a

mesenchymal-epithelial transition in colon cancer cells. In the second part of this work, I

sought to identify novel regulators of Par-4. In the work described here, I showed that

Trim21 is a novel interactor with Par-4, and that in response to cisplatin, Trim21

overexpression results in enhanced downregulation of Par-4. In addition, Trim21

expression can lead to cisplatin resistance. I also found that Trim21 levels correlated

107

with survival in a cohort of pancreatic cancer patients, with low Trim21 levels correlating

with prolonged overall and disease-free survival and high Trim21 levels correlating with

reduced disease-free survival. The third part of this work examined whether Par-4 could

enhance the effects of chemotherapy against glioblastoma cells. I found that Par-4 is

sufficient to reduce cell viability in GBM cells and that it also sensitizes GBM cells to the

AKT inhibitor, ISC-4, and chemotherapeutic, 5-FU. Taken together, these results

highlight novel tumor-suppressive mechanisms of Par-4, outside of its ability to regulate

apoptosis, which provides further rationale for its development as a cancer therapy.

Additionally, these results reveal a novel mechanism of Par-4 regulation, and in the

process uncovered a potentially novel biomarker for pancreatic cancer. Finally, the

results in glioma cells demonstrate the potential of Par-4 as gene therapy alone and in

combination with chemotherapeutics in the treatment of glioblastoma.

There is currently an unmet clinical need for novel, effective therapies for

advanced colon cancer, along with pancreatic cancer and glioblastoma.54, 81 With this

dissertation work, I illustrate the potential of Par-4 in these different contexts.

Understanding the role of Par-4 in regulating cell migration is a relatively new area of

research on Par-4 function. The vast majority of studies on Par-4 function have focused

on its ability to regulate apoptosis, and only recently have studies began to look at the

role that Par-4 plays in other processes such as migration and autophagy. Only two

other studies have reported on the role of Par-4 on migration. In one, a cisplatin

resistant pancreatic cancer cell line, BxPc-3, was created, and it was found that

phenotypic changes consistent with EMT resulted.10 In addition, Par-4 was

downregulated in the resistant cells relative to sensitive cells, and Par-4 overexpression

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in resistant cells reversed these changes, while Par-4 knock-down in sensitive cells

conferred a similar cisplatin-resistant phenotype. In contrast, in the second study, TGF-

β mediated EMT in endometrial, breast, ovarian, and cervical cancer cell lines was

found to be dependent on Par-4.11 Overexpression of Par-4 induced the expression of

EMT markers and led to an increase in motility, whereas Par-4 knock-down decreased

the expression of EMT markers and prevented TGF-β induced EMT. These contrasting

results suggest that the ability of Par-4 to regulate EMT and motility depends on the

intrinsic cell-specific mutations and genetic background of different groups of cancers.

Future work is warranted to elucidate these cell-specific mechanisms and targets of

Par-4 that can account for these differences in Par-4 activity.

In my work on Trim21 as a regulator of Par-4, the finding that Trim21 correlates

with survival in pancreatic cancer patients is perhaps the most significant finding. This is

especially true given the current standard-of-care for pancreatic cancer. Despite the

current chemotherapy cocktail used for pancreatic cancer treatment, almost all

pancreatic cancer patients will die from their disease.54 Though this high mortality is due

partially to late diagnosis, it is also largely due to ineffective treatment.142 Knowing that

patients with tumors that have low Trim21 expression will survive significantly longer,

informing patients of their Trim21 status can be source of hope for patients and allow

them to cope with the psychological burden of a cancer diagnosis. Indeed, the survival

is prolonged such that the survival curve is essentially horizontal for the overall and

disease-free survival. Interesting basic science questions also arise from the data. For

example, most of the studies on Trim21 regulation of processes that are critical to

cancer development, such as apoptosis and proliferation, suggest that, overall, Trim21

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acts as a tumor suppressor. This has been corroborated by studies that show that high

Trim21 expression predicts longer survival in cohorts of diffuse large B-cell lymphoma133

and hepatocellular carcinoma;132 however, my data show the opposite trend in

pancreatic cancer. One explanation for this difference in prognostic value of Trim21

across different cohorts could lie in the differences in expression of Trim21 targets

between pancreatic cancer compared to hepatocellular carcinoma and diffuse large B-

cell lymphoma. For example, while Trim21 overexpression inhibited proliferation in a B-

cell line,131, 133 other studies have shown that Trim21 can positively regulate cell

proliferation and cell cycle progression. In one study, Trim21 knock-down inhibited

progression through the cell-cycle via p27 accumulation.143 In another study, Trim21

overexpression enhanced IL-2 expression in response to CD28 treatment.144 Thus,

future studies could examine the effect of overexpression or knock-down of Trim21 on

pancreatic cell migration, proliferation, and resistance to cell death. Based on the data

in this dissertation, I hypothesize that Trim21 will be a positive regulator of cell migration

and proliferation and increase resistance to cell death. If, indeed, Trim21 appears to be

pro-oncogenic in the context of pancreatic cancer, developing small molecule inhibitors

of Trim21 would be warranted.

In the work of Par-4 and glioma, I demonstrated the utility of Par-4 in the

treatment of glioma. This work can be extended by moving to in vivo models of glioma.

In addition, elucidating the mechanism of Par-4-induced apoptosis and sensitization to

chemotherapeutics in glioma is warranted. Future studies could examine the role of NF-

ĸB inhibition and AKT inhibition, given the known roles of those two pathways in glioma

development and Par-4 activity.

110

Beyond the scope of the work in this dissertation, there are outstanding

questions in the Par-4 field that deserve further study. For example, given its recent

discovery, relatively little work has been done on the extracellular role of Par-4.8,22,32

Extracellular Par-4 inhibited lung carcinoma metastasis in vivo32, yet the mechanism of

inhibition was not elucidated. It could act via its reported receptor, GRP78, or through

some unknown receptor. Pursuing this avenue of research could be promising, since

extracellular Par-4 seems to have similar activity to intracellular Par-4: it both induces

apoptosis and inhibits metastasis of cancer cells. In addition, it has certain advantages

over delivering Par-4 plasmid as gene therapy, especially in the context of glioma –

given that Par-4 mediates neuronal cell death in models of neurodegeneration23, there

is a possibility of off-target neuronal cell death when using Par-4 as gene therapy in the

context of glioma; with extracellular Par-4 this side effect would not be a concern.

Another interesting research question would be clarifying the mechanisms of the

cancer-cell selectivity of Par-4. The prevailing paradigm is that the selectivity of

intracellular Par-4-induced apoptosis is due to the greater PKA activity in cancer cells

relative to normal cells.35 The explanation for the cancer-cell selectivity of extracellular

Par-4 is due to the selective membrane localization of GRP78 in cancer cells.8,32

However, this explanation is incomplete. Intracellular Par-4 does not induce apoptosis in

all cancer cells – only a subset. Furthermore, Par-4 nuclear localization correlates with

the sensitivity of cancer cells to Par-4-induced apoptosis. This suggests that there is an

intranuclear activity of Par-4 that is essential for its activity, yet little is known about its

roles in the nucleus besides interacting with TOP131, NF-ĸB25, and WT129,30. A co-

immunoprecipitation screen with Par-4 protein as bait and using nuclear lysates as

111

potential prey could begin to address this question with subsequent reporter assays to

explore the functional significance of the discovered interactions.

In conclusion, while more work remains before the goal of using Par-4 in cancer

treatment is realized, the work in this dissertation provides further rationale for

continuing to study the role of Par-4 in cancer and to develop it as a cancer treatment.

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CURRICULUM VITAE

JEFFREY NGUYEN

19 Rosedale Apartments, Hershey, PA 17033 | (309)706-0376 | jnguyen2@hmc.psu.edu

EDUCATION

Pennsylvania State University College of Medicine, Hershey, PA

MD/PhD candidate 2009 - Present

Saint Louis University, Saint Louis, MO

B.S. Biochemistry 2007

TEACHING EXPERIENCE

Penn State College of Medicine, Hershey, PA

Undergraduate Summer Student Mentor 2012 - Present

ABSTRACTS AND PRESENTATIONS

Nguyen J, Irby R. Trim21: a novel regulator of Par-4 and oncogenic signaling. AACR Annual Meeting, Philadelphia, PA: April 18-22, 2015.

Nguyen J. Elucidating the role of prostate apoptosis response protein-4 in colon cancer. Penn State College of Medicine, MD/PhD seminar,

Hershey, PA: August 13, 2015.

Nguyen J. The regulation of EMT and colon cancer metastasis b y Par-4. Penn State College of Medicine, MD/PhD seminar, Hershey, PA:

March 13, 2014.

Nguyen J, Kline L, Irby R. The pro-apoptotic protein, Par-4, induces mesenchymal-epithelial transition and inhibits migration/invasion.

Medicine/Biochemistry Research Day, Penn State College of Medicine, Hershey, PA: May 2013.

PUBLICATIONS AND PAPERS

Nguyen J, Irby R. Trim21 is a novel regulator of Par-4 in colon and pancreatic cancer cells. Cancer Biology and Therapy. 2016. (Accepted/In

Press).

Nguyen, J, Kline, CL, Caballero, N, Irby R. Overexpression of prostate apoptosis response protein-4 in colon cancer cells can inhibit

metastasis by upregulating E-cadherin expression. Journal of Colon and Rectal Cancer. 2015; 1(1): 20-34.

EXTRACURRICULARS

APAMSA treasurer, Penn State College of Medicine, Hershey, PA: 2010-2011

PULSE, Penn State College of Medicine, Hershey, PA: 2010-2011

MEMBERSHIPS

American Association for Cancer Research