DYSREGULATION OF THE HOST IMMUNE RESPONSE BY THE HUMAN

PAPILLOMAVIRUS ONCOPROTEIN E7

by

JOSEPH WESTRICH

B.S., Colorado State University, 2009

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Program in Microbiology

2018

This thesis for the Doctor of Philosophy degree by

Joseph A Westrich

has been approved for the

Microbiology Program

by

Linda van Dyk, Chair

Dohun Pyeon, Advisor

Eric T. Clambey

Thomas E. Morrison

Jill Slansky

Date: August 17, 2018

ii Westrich, Joseph A. (PhD, Microbiology)

Dysregulation of the Host Immune Response by the Human Papillomavirus Oncoprotein E7

Thesis directed by Associate Professor Dohun Pyeon

ABSTRACT

High-risk human papillomaviruses (HPVs) are causally associated with multiple cancers. HPV persistence is necessary for cancer progression, but factors that drive cancer progression and how HPV evades the host immune response during persistence remain under defined. The HPV oncoprotein E7 enacts numerous functions within the infected cell, including disrupting immune mechanisms. I have identified two novel mechanisms by which the HPV oncoprotein E7 promotes immune evasion and cancer progression.

APOBEC3 (A3) proteins are cytidine deaminases that play a role in immunity and possibly cancer development. A3 mutational signatures have been observed in many human cancer genomes, including HPV-associated cancers. However, factors that promote off-target A3 activity remain unclear. Here, I describe that E7 from high-risk HPVs, but not low-risk HPVs, prolongs the cellular half-life of A3A protein. I have revealed that the HPV oncoprotein E7 mediates A3A stabilization through the interaction with cullin 2 (CUL2), a core component of the ubiquitin ligase complex. My findings provide a novel mechanistic insight into cellular triggers of A3 mutations during HPV-driven cancer progression.

Our group found that CXCL14 is significantly downregulated in HPV-positive cancers by E7-mediated hypermethylation of the CXCL14 promoter. Interestingly, restoration of

CXCL14 expression in HPV-positive head and neck cancer cells prevents tumor development in immunocompetent syngeneic mice, but not in immunodeficient mice. I have further discovered that CXCL14 expression significantly increases natural killer and T cell chemotaxis in vitro and their infiltration into the tumor-draining lymph nodes in vivo.

Furthermore, my studies have revealed that the CXCL14-mediated tumor suppression is reliant on antigen-specific CD8+ T cells. CD8+ T cells are capable of responding to and

iii eliminating tumor cells in vitro. Interestingly, CXCL14 expression in tumor cells restores

MHC-I expression on tumor cell surface. Finally, I show that CXCL14-mediated tumor suppression disappears by depletion of MHC-I expression. Taken together, these results suggest that the HPV oncoprotein E7 suppresses the antigen-specific CD8+ T cell response by downregulating CXCL14 expression in HPV-infected cells. My findings provide new mechanistic insights into HPV-mediated immune evasion that contribute to cancer progression.

The form and content of this abstract are approved. I recommend its publication.

Approved: Dohun Pyeon

iv

I dedicate this work to my beautiful wife Erin, she is the light of my life. Her continual love and support has provided untold strength throughout this journey.

I would also like to dedicate this work to my parents, Cos and Jane, and my siblings,

Jason, Julia, Lisa, Katie, and Laura. Their patience and support through the years have helped me achieve my dreams and I feel entirely blessed to have such a great family.

v ACKNOWLEDGMENTS

I would like to thank my mentor, Dohun Pyeon, for his amazing guidance over the years. He has given me the confidence to tackle big questions and important opportunities to develop as a researcher.

I would also like to thank my committee members, Linda van Dyk, Eric Clambey,

Tem Morrison, and Jill Slansky for all of their support and for letting me drop in for a chat at a moment’s notice. Their feedback, direction, and brainstorming during committee meetings was invaluable.

Finally, I would like to thank all the Pyeon lab members past and present. Between shenanigans and thoughtful discussion, they made every day an adventure.

.

vi TABLE OF CONTENTS

CHAPTER Page

I. INTRODUCTION ...... 1

Papillomaviruses Genome Organization and Lifecycle ...... 1

HPV Associated Disease ...... 4

HPV Prevalence ...... 4

HPV Related Diseases and Rates ...... 5

HPV Oncoproteins ...... 6

HPV E5 ...... 6

HPV E6 ...... 7

HPV E7 ...... 8

Mechanisms of Immune Defense Against HPV ...... 9

Innate Defense Mechanisms ...... 9

Extracellular Defense Mechanisms ...... 11

Mechanisms of HPV Immune Evasion ...... 12

Deregulation of DNA Methylation by HPV ...... 12

Immune Evasion by Dysregulating Protein Functions ...... 13

Intracellular Sequestration of MHC Molecules by HPV ...... 15

APOBEC3 and HPV ...... 16

Rationale for Research ...... 17

Figures ...... 18

II. MATERIALS AND METHODS ...... 21

Cell Lines and Reagents ...... 21

Plasmid Constructs ...... 22

Lentivirus Production and Transduction ...... 22

Drug Treatment and Cell Viability Assays ...... 23

vii

Western Blotting ...... 23

Co-immunoprecipitation ...... 23

Cytidine Deaminase Assay ...... 24

Reverse Transcriptase-Quantitative PCR ...... 24

Bisulfite Modification, Methylation-Specific PCR (MSP), and Bisulfate Sequencing ...... 24

Cell Migration Assays ...... 25

Mice and Tumor Growth ...... 25

Antibodies and Flow Cytometry ...... 26

In Vivo Immune Cell Depletion ...... 27

Immunofluorescence ...... 27

T-Cell Purification and Cultures ...... 28

ELISA and In Vitro CTL Lactate Dehydrogenase Assay ...... 28

Statistical Analysis ...... 28

III. HUMAN PAPILLOMAVIRUS 16 E7 STABILIZES APOBEC3A PROTEIN BY INHIBITING CULLIN-2 DEPENDENT PROTIEN DEGRADATION ...... 30

Importance ...... 30

Introduction ...... 30

Results ...... 32

HPV16 E7 Increases A3A Protein Levels ...... 32

HPV16 E7 Prevents A3A Protein Degradation ...... 33

High-risk HPV E7, but not Low-risk HPV E7, Prevents A3A Protein Degradation ...... 33

A3A Protein is Stabilized in HPV-positive CxCa and HNC Cell Lines ..... 34

The CUL2 Binding Site in E7 is Important for A3A Protein Stabilization ...... 35

viii CUL2 is Necessary for A3A Degradation ...... 36

CUL2 Interacts with A3A and HPV16 E7 ...... 37

HPV16 E7-Stabilizes A3A Maintains Deaminase Activity ...... 38

A3A Protein Stabilization by HPV16 E7 is Independent of TRIB3 ...... 38

Discussion ...... 39

Figures ...... 44

IV. SUPRESSION OF ANTITUMOR IMMUNE RESPONSES BY HUMAN PAPILLOMAVIRUS THROUGH EPIGENETIC DOWN REGULATION OF CXCL14 ...... 53

Importance ...... 53

Introduction ...... 53

Results ...... 55 Proinflammatory Chemokines are Upregulated during CxCa Progression ...... 55

CXCL14 Expression is Downregulated in HPV-Associated Cancer Progression ...... 56

CXCL14 Down Regulation in HPV-Positive Keratinocytes is Associated with Promoter Hypermethylation ...... 58

CXCL14 Expression Hinders Cell Migration in vitro ...... 60

Restoration of Cxcl14 Expression Clears HPV-Positive Tumors in Immunocompetent Mice, but not in Rag1-Deficient Mice ...... 61

Restored Expression of CXCL14 Increases Natural Killer (NK), CD4+ T, and CD8+ T cells in the Tumor-Draining Lymph Nodes in vivo ...... 62

Expression of Cxcl14 Induces Chemotaxis of NK, CD4+ T, and CD8+ T in vitro ...... 63

Discussion ...... 64

Figures ...... 67

V. ANTIGEN-SPECIFIC CD8+ T CELL RESPONSE IS REQUIRED FOR CXCL14-MEDIATED TUMOR SUPPRESION IN HPV-POSITIVE HEAND AND NECK CANCER ...... 81

Importance ...... 81

ix

Introduction ...... 81

Results ...... 83

CXCL14-Mediated Tumor Suppression Requires CD8+ T Cells ...... 83

CXCL14-Mediated Tumor Suppression Requires Antigen Specific CD8+ T Cells ...... 85

CXCL14 Expression Restores MHC-I Expression on Tumor Cell Surface ...... 86

CXCL14-Mediated Tumor Suppression is Abrogated by Knockdown Of MHC-I Expression in Tumor Cells ...... 86

CXCL14 Expression in Tumor Cells Induces CD8+ T Cell Activation ...... 87

Discussion ...... 89

Figures ...... 94

VI. DISCUSSION AND CONCLUDING REMARKS ...... 104

Discussion ...... 104

HPV-E7 Stabilization of A3A ...... 105

Viral Stabilization of Host Proteins ...... 105

A3A Stabilization by High-Risk E7 ...... 105

Mechanisms of HPV E7-mediated A3A Protein Stabilization ...... 106

The contribution of A3A Stabilization in HPV-associated Cancer ...... 108

HPV-E7 Suppression of CXCL14 ...... 109

Chemokines in HPV-associated Cancer Progression ...... 109

CXCL14 and Cancer ...... 110

CD8+ T cells in HPV-Positive Cancer ...... 111

Tumor Associated antigens recognized by CD8+T Cell Response ...... 112

Mechanism of MHC-I Expression Restored by CXCL14 ...... 113

The Immunosuppressive Tumor Microenvironment in HPV-associated Cancers ...... 114

x

Concluding Remarks ...... 116

Figures ...... 117

REFERENCES ...... 121

APPENDIX ...... 146

A. Primers used in this study ...... 159

B. Gene Expression Microarray Details and Methodology ...... 160

xi CHAPTER 1

INTRODUCTION1

Papillomaviruses Genome Organization and Lifecycle

Papillomaviruses are an ancient family of non-enveloped DNA viruses that have co- evolved with their host species and infect a wide distribution of vertebrates (1). Studies suggest that papillomaviruses arose roughly 330 million years ago making them one of the oldest and largest virus families (1, 2). Currently, more than 300 different types of papillomaviruses have been described with full-length genomes, infecting species spanning from mammals, to birds and reptiles (3, 4). Over the ages, papillomaviruses have developed a viral lifecycle where the majority of infections facilitate virus production and exhibit minimal apparent disease. Although the majority of infections are asymptomatic, severe diseases can occur due to these infections occasionally. Papillomaviruses that infect humans are known collectively as human papillomaviruses (HPVs). An excess of 200 genotypes of HPVs have been identified to date. The HPVs are divided into 5 genera based on sequence similarity: Alpha, Beta, Gamma, Mu, and Nu. Members of the Alpha genus can be divided based on tropism to cutaneous or mucosal epithelial layers. The mucosal tropic viruses are further subdivided into high-risk and low-risk genotypes based on their capacity to promote cancer progression.

HPVs typically have an approximately 8-kilobase circular double-stranded DNA genome, generally encoding eight to nine open reading frames (ORFs) on one DNA strand

(5). The HPV genome can be divided in three regions: early genes (denoted with E), late genes (denoted with L), and the long control region (LCR) (Figure 1-1). The proteins encoded in the E region are expressed early in infection and include replication proteins (E1,

1 Portions of this chapter were published with permission from Westrich et al 2017 Evasion of Host Immune Defenses by Human Papillomavirus. Virus research (231) 21-23 doi.org/10.1016/j.virusres.2016.11.023

1 E2, and E4) and oncoproteins (E5, E6 and E7), with some exceptions (6). The L region encodes the major and minor viral capsid proteins, L1 and L2, respectively, and is expressed late in the viral lifecycle. The LCR is a regulatory region of the viral genome and has the origin of replication (ori) (4). Early in infection, the HPV early promoter (PE) is activated, and the early proteins are expressed from a single polycistronic primary RNA that utilizes active RNA splicing to generate multiple isoforms of mRNAs (7). In the later stage of infection in differentiated keratinocytes, the major viral late promoter (PL) becomes active, expressing the polycistronic transcript for the L proteins. As with the E transcript, the late polycistronic transcript utilizes alternative RNA splicing for L1 and L2 expression.

E1 and E2 are viral proteins that play key roles in viral genome replication and maintenance (6). E1 is a viral helicase that binds to the viral ori in the long control region of the viral genome and prepares the viral genome for replication by recruiting cellular DNA replication factors and unwinding the viral genome (8). E1 is the only viral protein to have inherent enzymatic activity. The E2 protein serves several functions within the infected cell

(9). E2 assists in viral replication by facilitating the binding of E1 to the viral ori. E2 is responsible for the maintenance of genome copy number and faithful distribution of viral genomes to the daughter cells after cell replication by tethering of the HPV genomes to the host genome (10). Acting as a transcriptional regulator of viral promoters, E2 binds sequence motifs in the LCR to activate or repress transcription, depending on the associated cellular factors and the nature of the binding site (11). Importantly, E2 negatively regulates the expression of the oncogenes E6 and E7 in HPV-infected keratinocytes. E2 expression is often disrupted during the inadvertent integration of the HPV genome into the host genome (12). The loss of E2 suppression of E6 and E7 expression results in aberrant expression of the oncoproteins, and subsequent increase of the oncoproteins’ activity. E5,

E6 and E7 are small viral proteins that function to promote a pro-viral environment within the infected cells (5). The pathways E5, E6 and E7 dysregulate are similar to pathways

2 disrupted in cancer, such as inactivation of p53 and pRb (5). E5, E6 and E7 from high risk

HPVs exhibit potent activity against their respective pathways and contribute heavily to HPV associated cancer progression, thus they are considered oncoproteins. Further details on the function of these proteins will be explored in the following section. The E4, L1, and L2 proteins are involved in viral encapsidation and release of virions from the cell. Although E4 is denoted with the early expression signifier “E”, only a relatively low level of E4 protein is expressed during the early stages of viral infection (13). A dramatic increase of E4 protein is seen in the differentiated layers of the papilloma, with estimates of up to 30% of protein production devoted to E4 (13). E4 protein is thought to facilitate virion release into the environment by disrupting intermediate filaments of the keratinocyte cytoskeleton (13).

Upon translation, the L1 capsid protein spontaneously self-assembles into pentamers that are stabilized by disulfide bonds between neighboring L1 proteins. The minor capsid protein

L2, in addition to cooperating with L1 to package the viral DNA into the virion, has been shown to play a major role in chaperoning the HPV genome to the nucleus during infectious entry process (14).

HPV replication is closely linked to epithelial differentiation (Figure 1-2) (5). The natural host cells infected by papillomaviruses are the basal keratinocytes at the base of epithelial layers. These basal keratinocytes are typically protected by the layers of later stages of differentiated cells but are made accessible to the virus through micro-abrasions in the epithelium (5). The infecting HPV virions appear to attach to the basal keratinocyte via keratinocyte-specific heparan sulfate proteoglycans (15, 16) After the initial binding of the virion to the cell, L2 must be cleaved by the cellular protease furin to facilitate virion uptake

(17). Virion internalization into the cell is based on an actin-dependent micropinocytosis (18).

Endosome acidification facilitates L1 uncoating (19) and facilitates endosomal escape of L2 with the HPV genome (20). After endosome escape, L2 with the HPV genome, retrograde traffics through the trans-Golgi network, whereupon during cell division and nuclear

3 envelope breakdown, the HPV genome enters into the nucleus (21, 22) and traffics to nuclear domain 10 (ND10) bodies, sub-nuclear domains rich in transcription factors (23).

The HPV genome undergoes rapid but tightly regulated expansion of between 10-200 genomes with the assistance of E1 and E2 (6). When the infected basal epithelial keratinocyte undergoes cell division, E2 ensures equal distribution of the HPV genomes between the parental and daughter keratinocytes (9). As the daughter cells are pressed upward through the stratified layers of the epithelium, different HPV proteins are expressed.

In the suprabasal layer of the epithelium, in addition to E1 and E2 promoting genome maintenance, E5, E6 and E7 proteins are expressed to promote immune evasion and enhance cell proliferation (5). Genome amplification and capsid protein (L1 and L2) expression occur in the late suprabasal/early granular layers (6). In the final stages of epithelial differentiation, the capsid proteins self-assemble with the viral genome, generating the infectious particle. With the assistance of the E4 protein, the nascent virion escapes the cell to go on to infect the next host cell.

HPV Associated Disease

HPV Prevalence

HPV is the most prevalent sexually transmitted infection in the United States (24). It is estimated that 79 million Americans are currently infected with HPV, with 14 million new cases occurring annually (25). It is estimated that 80 percent of sexually active men and women will be infected at some point in their lifetime. The majority of HPV infections, including those of high-risk HPV genotypes, typically spontaneously resolve within one to two years and often without knowledge of the infection (26, 27). In infections that are not cleared (about 10%), a persistent infection can last for decades. A persistent infection with a high-risk HPV genotype increases the likelihood for the HPV disease progression, but only a small percentage ultimately progress to cancer (28).

4 HPV Related Diseases and Rates

Although most HPV infections have minimal outward presentation, several diseases are associated with a subset of HPV infections. Infection with low-risk HPVs is associated with several non-cancer pathologies including genital and non-genital warts, recurrent respiratory papillomatosis, and the very rare inherited disease epidermdysplasia verruciformis (29). With the exception of epidermdysplasia verruciformis, these pathologies are often not highly associated with patient mortality. High-risk HPVs are causally associated with several malignancies and are estimated to account for roughly 5% of total cancer incidences (30). The association between HPV infection and cancer was first described by

Harold zur Hausen in early 1980s (31). Since that observation HPV has been determined to be the cause of almost all cervical cancers (CxCa) incidences (32). The high-risk genotypes

HPV16 and HPV18 account for approximately 70% of CxCa cases (33, 34). HPV is also causally associated with several other anogential cancers including vulvar (51%), vaginal

(64%), anal (93%) and penile (36%) (Reviewed in (32)). HPV is also associated with several non-anogential cancers; it is estimated that HPV is associated with 63% of oropharyngeal cancers (OPSCC) (32). HPV-negative OPSCCs are primarily associated with alcohol and tobacco use and often and occur in an older population. In contrast, HPV-positive OPSCCs occur in a younger population and are associated with sexual risk factors (35, 36). In the

United States, the incidence of HPV-positive OPSCCs has been rising at an alarming rate while the incidence of HPV-negative OPSCCs has been declining (36, 37). The incidence of

HPV-positive OPSCCs now exceeds that of HPV-negative and, if current trends continue,

HPV-positive OPSCCs will surpass the number of cervical cancer cases by the year 2020

(36).

HPV Oncoproteins

While HPV-positive cancer progression is reliant on expression of the high-risk HPV oncoproteins, it is important to understand the oncogenic activity of these proteins represent

5 functions related to promoting the viral life cycle and promoting long term persistent infection within the cell. Severe disease and cancer development arises as an inadvertent consequence of specific viral replication strategies. Despite this, the HPV proteins E5, E6 and E7 of high-risk genotypes are considered to be potent oncoproteins. The HPV oncoproteins have an enormous repertoire of function within the infected cell but only the most potent and relevant mechanisms will be highlighted in the following sections.

HPV E5

The HPV E5 oncoprotein is maintained as a homodimer with a highly hydrophobic region that serves as a transmembrane domain (38). The E5 dimer is localized to membranes of the endoplasmic reticulum (ER) and Golgi apparatus (39). E5 plays a role in immune evasion by down-regulating the cell-surface expression of major histocompatibility complexes (MHCs) described in detail in subsequent sections. E5 also performs additional functions to enhance viral survival and cellular proliferation. E5 enhances proliferation through stimulation of epithelial growth factor receptor (EGFR) (40), suppresses apoptosis

(41), and promotes activation of the Akt pathway and cell cycle deregulation (Reviewed in

(42)).

Although E5 expression plays several enhancing roles within the infected cell, its expression is not critical for productive infection as shown by not all HPV genera encode E5

(6). In cultured cells, expression of E5 alone exhibits cellular toxicity that is nullified by the expression of the other oncoproteins (43). In the absence of the other HPV oncoproteins, high-risk E5 displays weak transforming activity (38, 42, 44). Unlike E6 and E7, E5 expression is expendable for tumor maintenance as E5 expression is often lost upon integration of the viral genome into the host genome (45). While E5 is dispensable for viral infections and cellular transformation, its activity further contributes to these functions.

6 HPV E6

E6 plays many roles within the infected cell mediated by protein-protein interactions.

The best-defined function of high-risk E6 is targeting the tumor suppressor protein p53 for proteasomal degradation, thus reducing the cell's ability to respond to DNA damage (46).

Under normal conditions, p53 protein is regulated by the E3-ubiquitin ligase, MDM2. When the cell undergoes cellular stress (UVR, DNA damage, e.g.), MDM2 activity is halted and p53 is stabilized (47). The functions of p53 is to facilitate the DNA damage response, arrest the cell cycle to allow for DNA repair, and initiate apoptosis in the event of crisis (48). As such, functional p53 expression represents a threat to viral replication (49). In the context of

HPV infected cells, MDM2 is inactive and p53 turnover is regulated by a trimeric complex composed of E6, E6-associated protein (E6AP) ubiquitin ligase and p53 which facilitates p53 proteasomal degradation (46, 50, 51). The E6 mediated proteasomal degradation of p53 is found only in high-risk genotypes (52), although low risk genotypes maintain the ability to bind and sequester p53, abolishing its transcriptional activity (53).

Through several mechanisms independent of p53, E6 further suppresses the cell’s ability to respond to cellular stress by disrupting pathways involved in apoptosis (54-56) and with the DNA damage repair response (57). Furthermore, E6 promotes continued cellular proliferation though induction of telomerase activity by transcriptional transactivation of hTERT, thus preventing shortened telomeres and the subsequent senescence (58, 59).

E6 has enormous oncogenic potential and is sufficient to immortalize human mammary epithelial cells (60, 61). However, in terms of full transformation, E6 alone has weakly transforming capacity in rodent cells (62). This transforming activity is made more efficient when combined with activated ras oncogene expression (63-65). In a transgenic mouse model of cervical cancer, E6 neutralized p53 and elevated centrosome copy numbers, but did not exhibit neoplasia or progress to cancer (66). This study and others have demonstrated that the combined activity of E6 and E7 are necessary for HPVs full

7 oncogenic potential (66-69)

HPV E7

As with the other HPV oncoproteins, E7 lacks intrinsic enzymatic and specific DNA binding activity but facilitates a spectrum of activities through protein-protein interactions

(70). Structurally, HPV16 E7 can be roughly broken down into 3 domains: conserved region

(CR) 1, CR2, and the somewhat unfolded C terminus. The CR1 and CR2 sequences have high homology with adenovirus (AdV) and the simian vacuolating virus 40 (SV40), E1A and large tumor antigen (T) respectively (71, 72). As with AdV E1A and SV40 T, these two conserved regions significantly contribute to the transforming capacity of the high-risk HPV

E7 (73-75). The C terminus of E7 contains a zinc-binding domain that facilitates dimerization of E7 (76, 77) as well as binding domains for numerous cellular proteins (78).

E7 has a wide array of functions within the infected cell, most notably activation of the E2F-dependent transcription through mediating proteasomal degradation of retinoblastoma (pRB) protein (Figure 1-3) (78). Under normal conditions, the pRB/E2F complex acts as a transcriptional repressor, maintaining the cell in G1 phase.

Phosphorylation of pRb by cyclin dependent kinases leads to the disruption of the pRB/E2F complex, allowing the dissociated E2F to transcriptionally activate genes necessary for S- phase entry and progression. A LXCXE domain in the CR2 domain is necessary and sufficient for the association of E7 with pRB and of other related pocket proteins (p107 and p130) (79, 80). In addition to the LXCXE motif in CR2, carboxyl terminal E7 sequences are necessary for disruption of pRB (81, 82) via a mechanism of interaction with the cullin 2

(CUL2) ubiquitin ligase complex (78). Low-risk genotypes are able to bind to pRB, albeit at a lower affinity, but only high-risk E7s are capable of mediating pRb degradation (80, 83). In addition to promoting protein degradation, HPV has been shown to promote the stability of other cellular proteins. E7 has been shown to promote the stability of p53, although the mechanism of stabilization remains unknown (84, 85). Interestingly, despite increased levels

8 of p53, it was shown to have no functional activity within the cell (86). p21cip1 is an additional cellular protein stabilized by E7 (87). p21cip1 is a potent cyclin-dependent kinase inhibitor (CKI) and inhibits the activity of cyclin complexes that promote cell cycle progression. Thus, in addition to influencing other pathways, E7 is a key driver of cell cycle progression in the infected cells.

Early investigations to determine the transforming capacity of E7 utilized rodent cells and showed that it was the “major driver of transforming activity” in high-risk HPV (72, 88,

89). In human cells, E7 is sufficient to cause life span extension, inhibit keratinocyte differentiation, and facilitate immortalization (90-93). In evaluating these cells for tumor growth in mice, it was shown that the cells remained non-tumorigenic, unless cultured over extended periods of time or when additional oncogenes such as ras or fos oncogenes were co-expressed (66, 94-97).

Although only a portion of the activities of the HPV oncoproteins were described, they play a significant role in altering cellular pathways to promote cellular survival and persistent infection.

Mechanisms of Immune Defense Against HPV

Innate Defense Mechanisms

The unique lifecycle and strict tropism of HPV generates significant physical barriers for virus entry into basal keratinocytes, the native host cells of HPV. To initiate infection, HPV first needs to translocate across skin and mucous membranes, a process that is facilitated by tissue damage. The mucous membrane poses a major physical barrier to virus infection due to the secretion of a viscous protective fluid and antimicrobial peptides found therein.

Once HPV reaches the extracellular matrix, extracellular proteases trigger conformational changes in the virus capsid that facilitates virus internalization. Following uptake of virus particles by macropinocytosis, viruses travel along the endocytic pathway to acidic late endosomes/lysosomes, and retrograde traffics through the Golgi to reach the nucleus (21,

9 98-100). During intracellular trafficking, a vast majority of virus particles are degraded and eliminated by host autophagy (101). The nuclear envelope poses another physical barrier to

HPV DNA entry into the nucleus. Nuclear envelope breakdown during prometaphase is required for the successful establishment of HPV infection (21, 100). Beyond these physical barriers, human α-defensins, particularly α-defensin 5, were found as potent antagonists of

HPV infection through inhibition of furin cleavage of the HPV minor capsid protein L2 at the cell surface (102, 103).

Once HPV enters a host cell, HPV DNA can be recognized by innate pathogen sensors. Absent in melanoma 2 (AIM2), interferon-γ (IFN-γ) inducible protein 16 (IFI16), and cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) are cytosolic

DNA sensors, while IFI16 also detects foreign DNA in the nucleus (104-107). Triggering of the AIM2 inflammasome by viral DNA leads to the maturation of caspase-1 and interleukin-

1β (IL-1β), which are commonly found activated in HPV16-infected lesions and keratinocytes (108). IFI16 restricts HPV genome replication and gene transcription by enhancing heterochromatin association with the early and late promoters (109). Further, there is a significant correlation in women between clearance of initial HPV infection and higher expression of nucleic acid-sensing toll-like receptors (TLR3, TLR7, TLR8, and TLR9) as well as TLR2 (110). One of the downstream effects of pathogen recognition by pattern recognition receptors (PRRs) is the production of type I interferons (IFN-α and -β). IFN-β treatment hinders HPV entry and promotes clearance of latent HPV episomes in persistently infected cells (111-113). HPV genomes in HPV-positive cervical lesions and IFN-β treated cervical keratinocytes are edited by several IFN-inducible cytidine deaminase APOBEC3 family members (114, 115). We and other groups have demonstrated that one of these family members, APOBEC3A, significantly restricts HPV infection (116, 117)

10 Extracellular Defense Mechanisms

Innate immune cells are also involved in early host responses against HPV infection.

HPV infection recruits dendritic (DC), Langerhans (LC), natural killer (NK), and natural killer

T (NKT) cells to the HPV-infected microenvironment (118). In addition, plasmacytoid dendritic cells (pDC) have been shown to respond to the presence of HPV16 virus-like particles (VLPs) in CxCa tissue and secrete various cytokines, including IFN-α, IL-6, tumor necrosis factor α (TNF-α), and IL-8 following activation of MyD88-dependent signaling (119-

121). Additionally, increased HPV infection and HPV-associated cancer incidence has been observed in individuals with various functional NK cell deficiencies (122). Taken together, these findings suggest that the early inflammatory response might be critical for initiating a robust host defense against HPV infection.

The HPV lifecycle is strictly intraepithelial and virions are produced only from the fully differentiated upper layer of skin. Thus, there is no virus-induced cytolysis or viremia, which limits the exposure of HPV to systemic immune responses. Nevertheless, results from many studies have agreed that host T cell responses are required to eliminate HPV-infected cells.

The regression rate of cervical precancerous lesions strongly correlates with the presence of intraepithelial granzyme B-positive cytotoxic T cells (123). A recent study has shown that

LCs isolated from women with persistent HPV16 infection can present HPV antigens and activate HPV16-specific CD8+ T cells (124). Additionally, using the recently established mouse papillomavirus (Mus musculus papillomavirus 1 or MmuPV1) model, Handisurya et al. revealed that productive MmuPV1 infection and papilloma formation require both CD4+ and CD8+ T cell functions; while CD4- or CD8-knockout C57BL/6 mice were resistant to productive MmuPV infection, depletion of CD4+ and CD8+ T cells from immunocompetent mice led to infection and papilloma formation (125). In addition, UVB irradiation-induced systemic immune suppression causes mice to become highly susceptible to MmuPV1 infection, which ultimately results in the development of squamous cell carcinoma (126).

11 Taken together, these findings exemplify the pivotal roles of T cell-mediated immune responses in host clearance of HPV infection. The immunogenicity and efficacy of current

HPV vaccines further confirmed that HPV infection can also be prevented by antibody- driven immunological memory (127). However, antibody titers from natural HPV infection are usually too low to show a protective effect, suggesting that HPV efficiently evades the host antibody response during natural infection (128),

Mechanisms of HPV Immune Evasion

In order to evade host immune defenses and establish persistence, HPV modulates host gene expression by deregulating host DNA methylation, histone modification, and transcription factors.

Deregulation of DNA Methylation by HPV

Epigenetic regulation by DNA methylation provides distinct gene expression patterns in different developmental stages, organ tissue types, and disease states (129-131). DNA methylation also functions as a host defense mechanism. By methylating CpG residues of foreign DNA in eukaryotic cells, pathogen activity can be shut down due to alterations in pathogen transcriptional profiles (132, 133). HPV DNA is also frequently methylated and viral gene transcription is repressed following DNA methylation (134, 135). Previous studies have shown that several pathogens hijack the host DNA methylation machinery to manipulate host transcription to their benefit (136-138). Interestingly, the HPV oncoprotein

E7 interacts with the DNA methyltransferase DNMT1, and stimulates its methyltransferase activity (139). This may partially explain the global changes in the host methylome that we have observed in HPV-positive keratinocytes (Cicchini et al., unpublished results). We have recently shown that expression of the chemokine CXCL14 is downregulated during HPV- associated cancer progression by promoter hypermethylation in an E7-dependent manner

(140). Since restoration of CXCL14 expression in HPV-positive HNC cells significantly suppresses tumor growth in vivo, HPV E7-mediated CXCL14 promoter methylation may

12 represent an immune evasion strategy during HPV persistence. Our and other studies have shown that CXCL14 induces direct chemotaxis of various immune cells including DCs, LCs,

NK, and T cells (140-142). Additionally, using transgenic mouse models, it has been demonstrated that HPV16 E7 expression in the epidermis creates a strong immunosuppressive area where LC and CD8+ T cell functions are significantly suppressed

(143-145). This immune suppression appears to be mediated by an influx of HPV16 E7- induced regulatory T (Treg) cells and tolerization of cytotoxic T cells (144, 146). In addition to HPV E7, HPV E6 is also involved in deregulation of host DNA methylation. High-risk HPV

E6 represses IFN-κ, which is constitutively expressed in keratinocytes and serves to reinforce IFN-stimulated gene expression. E6-mediated repression of IFN-κ can be reversed by treatment with a DNA methyltransferase inhibitor (147, 148). These findings suggest that

HPV manipulates host DNA methylation to evade host immune defenses.

Immune Evasion by Dysregulating Protein Functions

HPV E6 and E7 frequently inhibit host protein functions by direct binding. The HPV18

E6 protein directly interacts with non-receptor tyrosine-protein kinase 2 (TYK2), resulting in a reduction of IFN-α-induced phosphorylation in TYK2 and Signal Transducer and Activator of

Transcription 2 (STAT2) proteins (149). TYK2 is a member of the JAK family and is important for signal transduction following receptor binding by various cytokines, including

IL-6, IL-12, and type I IFNs. The IFN regulatory transcription factor IRF3 is selectively bound and inhibited by HPV16 E6 protein, while HPV6, HPV11, and HPV18 E6 proteins bind poorly to IRF3 (150). IRF3 plays a central role in inducing innate immune response against viral infections (151). The interaction of HPV16 E6 to IRF3 does not lead to ubiquitination or degradation of IRF3, but significantly represses its activity on target gene transcription and

IFN-β production. This suggests that HPV16 E6 binding is sufficient for inhibition of IRF3 functions. Cytosolic DNA can be detected by cGAS, which initiates innate immune signaling through the adapter proteins STING and IRF3 (152, 153). Interestingly, HPV18 E7

13 antagonizes the activation of the cGAS-STING pathway and limits the production of type I

IFNs by binding with STING through its LXCXE motif (154). Additionally, HPV16 E7 protein interacts with IRF1, probably at its N-terminal DNA-binding domain, resulting in decreased expression of IRF1 responsive genes, including TAP1, IFN-β, and CCL2 (155-157). IFN signaling is further repressed by HPV16 E7 as it also binds to IRF9, another IFN regulatory factor (158). IFN signaling lies at the core of antiviral innate immune responses and appears to be heavily targeted by the HPV oncoproteins E6 and E7. Our previous studies have shown that IFN-β treatment significantly inhibits HPV infection in primary and immortalized keratinocytes (113, 117). These findings indicate that evasion of IFN signaling mediated through HPV E6 and E7 interactions with host proteins might be critical for HPV persistence.

The mechanisms of host protein degradation by the HPV oncoproteins E6 and E7 are well established (159). However, posttranslational modification of immunoregulatory proteins is relatively understudied. Karim et al. have shown that high-risk HPVs upregulate the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes (160).

UCHL1 inhibits K63-linked ubiquitination of tumor necrosis factor receptor-associated factor

3 (TRAF3), resulting in decreased TRAF3-TBK1 complex formation and IRF3 phosphorylation. UCHL1 also mediates degradation of the essential modulator of NF-κB,

NEMO, and suppresses p65 phosphorylation and NF-κB signaling. HPV16 E6 also induces degradation of pro-IL-1β in a proteasome-dependent manner (161). E6-mediated pro-IL-1β degradation is independent from caspase-1 activation, autophagy, or lysosomal degradation, but requires E6AP expression and poly-ubiquitination of pro-IL-1β. Since the proinflammatory cytokine IL-1β plays important roles in antiviral defense (162, 163), these results suggest that high-risk HPV suppresses the proinflammatory response by inducing proteasome-mediated degradation of intracellular cytokines. Interestingly, a number of studies have shown that polymorphism in the IL-1β gene cluster is tightly linked to CxCa and

HNC risk (164, 165). Specifically, women with IL-1β T-allele containing genotypes show

14 increased risk of CxCa (166). Our unpublished global gene expression data shows that IL-

1β expression is significantly downregulated in HPV-positive normal keratinocytes but upregulated in CxCa tissues (Cicchini et al., unpublished results). These results imply that

IL-1β may play dual roles in inhibiting HPV infection by its antiviral function in early stages of disease, but facilitating cancer progression through chronic inflammation in late stages.

Intracellular Sequestration of MHC Molecules by HPV

Antigen presentation of viral epitopes by MHC-I molecules is critical to elicit cell- mediated immune responses that eliminate virus-infected cells. In order to escape host recognition, many viruses have various strategies to suppress surface expression of MHC-I molecules (167-169), and HPV is no exception. HPV E5s and bovine papillomavirus (BPV)

E5s are small transmembrane proteins that bind to several host transmembrane proteins, including MHC-I. E5 binding to MHC-I leads to its downregulation at the host cell surface

(38, 170-172). Upon binding of antigenic peptides delivered by TAP1 in the ER, MHC-I molecules, composed of a heavy α chain and a soluble subunit β2-microglobulin (β2m), traffic through the secretory pathway and imbed within the cell membrane (173). Several quality control mechanisms are involved in this process for accurate antigen presentation.

HPV16 E5 physically interacts with the heavy chain of the MHC-I molecule through the hydrophobic region of E5 protein and retains MHC-I in the Golgi and ER (170, 171). While

HPV16 E5 does not affect expression of the heavy chain and TAP1 proteins, enhanced expression of the MHC I heavy chain by IFN-β treatment restores surface expression of

MHC-I molecules. Interestingly, HPV16 E5 specifically downregulates HLA-A and -B expression, but not HLA-C and HLA-E (171). Our unpublished data showed that HLA-C and

HLA-E are the two most transcriptionally downregulated MHC-I molecules in HPV-positive keratinocytes, and this downregulation is dependent on HPV16 E7 expression (Cicchini et al., unpublished results). Further, HPV E5 downregulation of MHC-I molecules at the cell surface correlates with poor CD8+ T cell responses in E5 expressing cells (174). An

15 additional study has described a correlation between surface expression of MHC-I molecules and decreased TAP1 expression, suggesting that TAP1 downregulation by E7 may also interfere with MHC-I trafficking (175). HPV16 E5 also downregulates the surface expression of MHC-II molecules by preventing degradation of the invariant chain that blocks peptide loading (176). Both high-risk HPV16 and low-risk HPV6 E5s also downregulate surface expression of the non-classical MHC molecule CD1d (177). HPV E5 directly interacts with calnexin in the ER and redirects CD1d to the cytosolic proteolytic pathway

(177). CD1d expression on the cell surface activates NKT cell responses to various viral, bacterial, and fungal infections (178). Thus, HPV may evade host immune responses by downregulating the surface expression of CD1d. Further investigation is necessary to determine if CD1d presents HPV-related antigens to induce protective antiviral activity.

APOBEC3A and HPV

APOBEC3 is a family of interferon inducible cytidine deaminase that convert cytidine

(C) to uridine (U) in single stranded DNA and RNA (179, 180). The human A3 family consists of seven members: A3A, A3B, A3C, A3D, A3F, A3G, and A3H (181, 182). Of these seven A3 family members, A3A, A3B, and A3H are expressed in skin keratinocytes (114). A3s were first discovered as important host restriction factors that block the replication of several retroviruses, including human immunodeficiency virus (HIV) (183-185). Several studies have shown that A3A can eliminate transfected foreign DNA (186) and restrict several DNA viruses (187, 188). Editing of episomal HPV genomes by A3A, A3B, and A3H in cervical lesions has been demonstrated (114, 115). Our recent study has revealed that A3A expression restricts HPV entry in a cytidine deaminase dependent manner (117). Despite its ability to restrict HPV infection, A3A expression is upregulated in HPV-infected keratinocytes as well as HPV-positive CxCa.

16 Rationale for Research

Infection with HPV is a source of enormous disease burden worldwide resulting in approximately 5% of all cancers. While most infections are cleared by the immune response within less than two years, some infections persist and develop into cancer. Understanding the mechanisms employed by the virus to evade the immune response, as well as immune mechanisms utilized by the host to clear these infections would greatly assist in identifying targets for therapy. The overall goal of this dissertation is to determine mechanisms of how the HPV oncoprotein E7 manipulates the immune response to promote persistence, and potentially utilizing these pathways against the virus. In chapter III, we evaluate the post translational interaction between E7 and a known HPV restriction factor, APOBEC3A (A3A).

We show that E7 from high-risk HPV genotypes stabilize A3A protein and does not limit is function. These results suggest that A3A stabilization by E7 may contribute to HPV- associated cancer progression. In chapter IV, we define how HPV E7 suppresses the expression of the chemokine CXCL14 though promoter methylation. We show that the re- expression of CXCL14 in an HPV-positive mouse tumor model facilitates tumor suppressive immune response. In chapter V, we further characterize the CXCL14 antitumor immune response by identifying necessary immune components promoting immune suppression.

We show that CD8+ T cells are necessary to enact the CXCL14 mediated immune suppression. Furthermore, we reveal that CXCL14 impacts the expression of MHC-I which acts to further reverse the HPV mediated immune suppression.

17

E6

LCR

P E E7

L1 P L

HPV16 7.9 BP

E1

E2 L2

E5 E4

Figure 1-1. Cartoon schematic of the HPV16 genome. The long control region (blue). The early (E) proteins are transcribed after activation of the early promoter (P ). The early proteins associated with E replication E1, E2, and E4 (green), and the oncoproteins E5, E6 and E7 (orange). The late (L) proteins L1 and L2 (purple) are transcribed after activation of the late promoter (P ). Modeled after L Faridi et. al Virol J. 2011

18

Figure 1-2. HPV life cycle in epithelial layer. HPV virions (small red dot) enter squamous epithelial layer through micro-abrasion and infect basal epithelial cells. The infected cell (orange nucleated cells) progress through cell differentiation. Early (E) proteins (green triangle) are expressed in initial stages of cell progression facilitating genome maintenance, cell proliferation and genome amplification. Late (L) proteins (purple triangle) are expressed in granular stage of epithelial layer promoting virus encapsulation and viral release. Figure modeled after Doorbar 2006.

19

Functional checkpoint HPV disrupted checkpoint

pRB

E7 E2F

CUL2 pRB

E2F

Figure 1-3. HPV E7 degradation of pRB. In uninfected cells pRB (green) interacts with E2F (purple) to prevent E2F transcriptional activity that promotes progression to S phase. In high-risk HPV infected cells E7 (red) interacts directly with pRB and CUL2 (blue) to facilitate pRb proteasomal degradation. The destruction of pRb allows E2F to freely activate expression of genes critical for progression to S phase.

20 CHAPTER II

MATERIALS AND METHODS

Cell Lines and Reagents

293FT cells were purchased from Life Technologies and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Normal human immortalized keratinocyte (NIKS) cells (obtained from Lynn Allen-Hoffman) (189) and NIKS-

16, -18, -31 (obtained from Paul Lambert) (190), W12E (derived from a low-grade precancerous cervical lesion with episomal HPV16)(191), and W12G (low-grade with integrated HPV16) (192) cells were established in the Paul Lambert laboratory were maintained with mitomycin C-treated NIH 3T3 cells (obtained from Paul Lambert) in E- medium, as previously described (193). Transformed W12GPXY cells derived from W12G cells were obtained from Dr. Sheila Graham (University of Glasgow) in 2011. NIKS cell lines expressing various wildtype and mutant E7 (NIKS-6E7, NIKS-11E7, NIKS-16E7, NIKS-

18E7, NIKS-16E7 H2P, ΔD21-24C (ΔDLYC), SS31-32AA (CKII), CVQ68-70AAA (CVQ), and

ΔL79-L83 (ΔLEDLL)) were established using lentiviral transduction and puromycin (3 μg/mL) selection. NIKS and NIKS derivatives were maintained under passage 50 and passage 10, respectively, and frequently validated by morphology, HPV early gene expression, and feeder cell dependency. CaSki (194) and C33A (195) cell lines were obtained from Paul

Lambert. SCC-25, SCC-90 and SCC-152 cell lines were purchased from American Type

Culture Collection (ATCC). All CxCa and HNC cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. All cell lines were confirmed mycoplasma- free before use. Mouse oropharyngeal epithelial (MOE) cell lines, MOE/shPTPN13

(transformed with Ras and shRNA against Ptpn13) and MOE/E6E7 (transformed with Ras and HPV16 E6/E7) were generated by Dr. John Lee in 2009 (196), and validated by assessing cytokeratin expression, the presence of the E6 and E7 expression vectors which confer resistance to puromycin, and activation of the MAPK pathway, a hallmark of E6

21 expression. All cell lines were cultured according to the suppliers’ recommendations.

MOE/E6E7 cell re-expressing CXCL14 were established using lentiviral transduction and clonal selection.

Plasmid Constructs

Hemagglutinin (HA)-tagged A3A (pcDNA3.1-A3A-HA) expression plasmid and its parental vector (pcDNA3.1) were prepared as previously described (197). Wildtype HPV16

E7 (pCMV-16E7) and the HPV16 E7 mutants (H2P, ΔDLYC, CKII, CVQ, and ΔLEDLL; obtained from Karl Munger) were cloned into a lentiviral plasmid, pCDH-CMV-MCS-EF1-

Puro (System Bioscience, CD510B-1). Green fluorescent protein (GFP) expression plasmid

(pEGFP-N3) was obtained from Clontech. High-risk (HPV16 and HPV18) and low-risk

(HPV6 and HPV11) E7 expression plasmids were obtained from Joe Mymryk (Western

University), and cloned into pCDH-CMV-MCS-EF1-Puro (198). Chicken ovalbumin

(pcDNA3-OVA) was purchased from Addgene. The lentiviral expression plasmids of V5- tagged CUL2 (pLKO.1-puro-CUL2-V5, ccsbBroad304_01930), V5-tagged CUL3 (pLKO.1- puro-CUL3-V5, ccsbBroad304_07243), and V5-tagged TRIB3 (pLKO.1-puro-TRIB3-V5, ccsbBroad304_03850), and the shRNA clones against TRIB3 (TRCN0000196756 and

TRCN0000295919), CUL2 (TRCN0000006522, TRCN0000006523, TRCN0000006525,

TRCN0000006526), and B2M

(TRCN0000066425, TRCN0000288438) were obtained through the Functional Genomics

Facility at the University of Colorado School of Medicine.

Lentivirus Production and Transduction

Lentiviruses were produced using 293FT cells transfected with the packaging constructs pMDG.2 (Addgene, 12259), psPAX2 (Addgene, 12260), and the transfer vector as previously described (117). At 48 and 72 h post transfection, cell culture supernatants were collected and virus was concentrated using ultracentrifugation at 25,000 x g for 3 h.

Keratinocytes were spinfected with 100X concentrated lentiviruses by centrifugation at 1,000

22 x g for 30 min at 37oC, followed by selection using 3 μg/mL puromycin or 10 μg/mL blasticidin.

Drug Treatment and Cell Viability Assays

To inhibit de novo protein synthesis, cells were treated with 50 μg/mL of CHX

(Sigma, 01810). For proteasome inhibition, keratinocytes were treated with 20 μM of MG132

(Cayman Chemical, 133407-82-6). To inhibit cullin neddylation, cells were treated with 33 nM to 3 μM of MLN4924 (Cayman Chemical, 905579-51-3). Cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the supplier’s instructions.

Western Blotting

Cell lysates were prepared and western blotting was performed as previously described (117). HA-tagged protein was detected using mouse anti-HA antibody (Abcam, ab9110) at 1:5,000 dilution. A3A was detected using rabbit anti-A3A antibody (Abcam, ab150369) at 1:3,000 dilution. GFP was detected using rabbit anti-GFP (Abcam, ab290) at

1:1,000 dilution. CUL2 was detected using rabbit anti-CUL2 antibody (Thermofisher,

50H17L12) at 1:500 dilution. TRIB3 was detected using rabbit anti-TRIB3 antibody (Abcam, ab75846) at 1:2,000 dilution. V5-tagged proteins were detected with mouse anti-V5 antibody

(ThermoFisher, MA5-15253) at 1:2,000 dilution. Mouse anti-HPV16 E7 (Santa Cruz

Biotechnology, sc-6981) and mouse anti-HPV18 (Santa Cruz Biotechnology, sc-365035) antibodies were used at a 1:100 dilution. Horseradish peroxidase-conjugated goat anti- rabbit (Jackson Laboratories, 111-035) and donkey anti-mouse (Jackson Laboratories, 715-

035) secondary antibodies were used at a 1:10,000 dilution. Band densities were determined using Bio-Rad Image Lab Versions 4.1 and normalized to β-actin.

Co-immunoprecipitation

Cell lysates were prepared and incubated with Protein A/G agarose beads

(ThermoFisher) at 4oC for 4 h to eliminate non-specific binding. The lysates were incubated

23 with 1 μg of specific antibody (anti-V5 or anti-HA) or isotype-matched antibody and incubated overnight, followed by incubation with Protein A/G agarose beads at 4oC for 15 min. The beads were washed five times and the bound proteins were analyzed by western blotting.

Cytidine Deaminase Assay

293FT cells were harvested at 72 h post transfection and cell lysates were prepared.

In each reaction, 5 μL of protein lysate was added to the DNA deaminase reaction mixture containing 5 pmol of 5' fluorescein labeled oligonucleotide with a single TC target motif

(5'[ATA]7X -TCC-[ATA]6X -3') in deaminase buffer (100 mM Tris HCl, 500 mM NaCl, and 10 mM DTT). The reaction mixture was incubated at 37oC for 2 h, then 1 unit of uracil DNA glycosylase was added, and further incubated at 37oC for 1 h. NaOH (100 nM) was added to the reaction mixture and incubated at 95oC for 10 min. Reaction products were analyzed on a denaturing 15% polyacrylamide-urea gel using Bio-Rad Molecular Imager FX, as previously described (199-201).

Reverse Transcriptase-Quantitative PCR

Total RNA was extracted using a RNeasy Mini kit (Qiagen) with on-column DNase digestion using the RNase-free DNase (Qiagen) according to the suppliers’ instructions.

First-strand cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit

(Roche) from 1 μg of total RNA. Using the Bio-Rad CFT Connect real-time system, real-time

PCR was performed in a 20 μL of reaction mixture containing FastStart Universal SYBR green master (Rox, Roche Applied Science), 0.5 μM of each primer, 10 μL of SYBR green

PCR master mix, 5 μL of cDNA template, and nuclease-free water. Primer sequences appear in (Appendix A). Data were normalized by the level of ß-actin mRNA.

Bisulfite Modification, Methylation-Specific PCR (MSP), and Bisulfate Sequencing

Genomic DNA was extracted from keratinocytes (Qiagen) and bisulfite converted using EZ DNA Methylation Kit (Zymo Research). Bisulfite sequencing products were cloned

24 into pGEM-T easy vector (Promega) and sequenced. Quantitative MSP (qMSP) was performed with bisulfite-converted genomic DNA using SYBR Green (Roche). Relative DNA methylation was calculated using the ΔCt equation using methylated DNA as target.

Cell Migration Assays

Confluent CaSki and MOE/E6E7 monolayers were scratched and the width of the gap was measured every 4 hrs. The transwell migration assay was performed using 1 X 105 cells per well of an 8 μm 24-well transwell permeable support, and incubated overnight using FBS as a chemoattractant. Spleens from C57BL/6 mice injected with MOE/E6E7 cells were harvested at 21 days post injection and mechanically disrupted through a 100-μm filter. Red blood cells (RBC) were cleared by RBC lysis buffer (Sigma) and remaining splenocytes were rested at 37°C in RPMI 1640 medium containing 10% FBS and 10 ng/mL of mouse recombinant IL-2 (mrIL-2, eBioscience) for 3 hours. Conditioned media (CM) from the culture of MOE cells re-expressing Cxcl14 (clones 8 and 16) or containing vector were added into the bottom chamber of a transwell (3 μm pore size; Costar). Isolated splenocytes (2 × 106 cells/mL) were resuspended in RPMI 1640 medium supplemented with mrIL-2 and added to the upper chamber of the transwell. Splenocytes in RPMI 1640 medium without mrIL-2 was used as a negative control. After 12 hour incubation at 37°C, splenocytes were harvested from the top and bottom chambers, stained with trypan blue, and counted using a hemocytometer. Cell populations were analyzed using flow cytometry as described below in Antibodies and Flow Cytometry. Total cell populations were determined by applying the cell counts to the cell population percentages. Migration index for each cell type was calculated by: Percent cell migration = migrated cell number (bottom chamber) / total cell number (upper chamber + bottom chamber).

Mice and Tumor Growth

Breeding pairs of C57BL/6J, OT-I mice (C57BL/6-Tg(TcraTcrb)1100Mjb/J), and CD8a knockout mice (B6.129S2-Cd8atm1Mak/J), were obtained from Jackson Laboratory and

25 were bred in house. Mice were genotyped according to recommended protocols. Rag1-/- mice were purchased as needed (The Jackson Laboratory). Four to eight week old, 20-25 g mice were maintained in accordance with the USDA guidelines. Tumors were initiated by injection of engineered MOE/E6E7 cells (1-5 × 105) subcutaneously into the rear right flank of mice (n = 10 per group unless otherwise indicated). Tumor growth was measured weekly using previously established techniques (202). Tumor volume was calculated using the equation: volume = (width)2 × depth. Animals were euthanized when tumor size was greater than 1.5 cm in any dimension. Conversely, mice were considered tumor free when no measurable tumor was detected for a period of 11 weeks. Survival graphs were calculated by standardizing for a tumor volume of 2,500 mm3.

Antibodies and Flow Cytometry

For each experimental group, TDLNs and spleens were harvested from wild type mice at 21 days post injection. The following anti-mouse antibodies were purchased from

Thermofisher and used according to manufacturer’s specifications: MHCII (FITC conjugate, clone M5/114.15.2), CD4 (eF450 conjugate, clone RM4-5), F4/80 (APC conjugate, clone

BM8), Gr1 (AF700 conjugate, RB6-8C5), H-2Kb (PE conjugate, clone AF6-88.5.5.3), H2Dd

(FITC conjugated, clone 28-14-8). Anti-mouse CD45 (PerCP conjugate, clone 30-F11) and

NKp46 (PECy7 conjugate, clone PC61.5) were purchased from Biolegend. All tissue samples were passed through a 100 μm cell strainer (Corning Life Sciences) and spleens were incubated in Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) for 3 min at room temperature. For detection of MHC-I on MOE cells, MOE cells were lifted with 1X citric saline (203) to prevent cleavage of MHC-I by trypsin. The isolated cells were incubated with the corresponding panel of antibodies conjugated with unique fluorophores for 0.5-1 hour at room temperature and washed with PBS. Samples were passed through a 35 μm cell strainer (Corning Life Sciences) immediately before analysis on an LSRII flow cytometer

(Becton Dickinson) using FACSDiva collection software. All cells were assessed for viability

26 by staining with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies). Analysis was performed using FlowJo software and the gating strategy is described in Figure 4-9.

In Vivo Immune Cell Depletion

Mice received i.p. administration of 100μg/treatment of anti-NK1.1 (BioXcell, clone

PK136) mAb or anti-CD8 mAb (BioXcell, clone GK 2.43), or control isotype IgG (mIgG2a, clone BE0085 or rat IgG2b, clone BE0090) starting at day -1 and repeated twice a week for

30 days. Tumor challenge was performed on day 0. On day 5, flow cytometric analysis was performed on pooled peripheral blood to verify depletion of an appropriate subset of T cells.

CD8 Depletion was detected with CD8a clone 53-6.7. Depletion and detection of NK epitopes do not overlap. CD8 depletion clone 53-6.7 does not compete for the epitope with

2.43 as has previously been shown (204-206)

Immunofluorescence

MOE/E6EVector and MOE/E6ECXCL14 tumors were collected and fixed overnight in 4% paraformaldehyde. All tissues were cryoprotected using 20% sucrose in PBS and subsequently frozen in optimal cutting temperature compound (OCT). Tissue was then cryosectioned at 12-μm increments into sections. Sections were blocked using lamb serum and permeabilized using triton X. Immunofluorescence was performed on tissue sections using the following antibodies: mouse anti-pan cytokeratin (1:100, Novus) and rat anti-CD8a

(1:200, Novus) antibodies. After incubation with primary antibodies, sections were incubated with appropriate Alexa Fluor–conjugated secondary antibodies: anti-mouse IgG Alexa Fluor

488 (Invitrogen), anti-rat IgG Alexa Fluor 594 (Invitrogen), and DAPI (Invitrogen).

Immunofluorescence (IF) images were captured using a Zeiss 780 LSM confocal microscope using the 40X objective with a 2X optical zoom. The laser percentage and gain settings were set on the control tissue.

27 T-Cell Purification and Cultures

T cells were purified from the spleens of mice, mechanically disrupted over a 100-μm filter, and subjected to magnetic bead negative enrichment for CD8+ T cells, using a CD8+

T-cell isolation kit (Stemcell). Purified CD8 T cells were cultured in RPMI (Gibco) containing

10% FBS, L-glutamine, penicillin/streptomycin, and β-mercaptoethanol (50 μM). All cultures were maintained at a concentration of 1 × 106 cells/mL. Cultures subjected to T-cell receptor stimulation, were stimulated with 1 μg/mL soluble anti-CD28 (clone 145-2C11;

ThermoFisher) in co-culture or were stimulated with anti-CD3/anti-CD28 microbeads (per manufacturers recommendation Dynabeads Mouse T-Activator CD3/CD28; Invitrogen).

Stimulated cultures were supplemented with 10 ng/mL of recombinant mouse IL-2

(ThermoFisher).

ELISA and In Vitro CTL Lactate Dehydrogenase Assay

CD8+ T cells were isolated from tumor bearing or tumor controlling mice. CD8+ T cells were co-cultured with mitomycin treated MOE/E6E7Vector or MOE/E6E7CXCL14 cells with

IL-2 and anti-CD28. After 5 days, supernatants and CD8+T cells were harvested. Culture supernatants were tested for IL-2, TNFa, and IFNg protein production by cytokine specific

ELISAs. IL-2 (R&D, M2000), TNFa (R&D, MTA00B), and IFNg (Biolegend, 430801) ELISAs were performed per manufacturers recommendations. Harvested CD8+T cells were incubated for 8 h with MOE/E6E7Vector or MOE/E6E7CXCL14 cells (10,000 per reaction).

Reactions were performed in quadruplicate at the indicated effector/target ratios.

Spontaneous lysis was assessed in the absence of effectors, and maximum lysis was detected by treating target cells with 1% SDS, specific lysis was determined by the lactate dehydrogenase kit (Roche) per the manufactures instructions.

Statistical Analysis

Student’s t test and one-way analysis of variance (ANOVA) were used to calculate significance for comparison of two matched groups and three or more unmatched groups,

28 respectively. The correlation coefficient (R2) was determined by linear regression using

Prism 6 (GraphPad). Results were considered statistically significant at a p-value of less than 0.05. Distributions of time to event outcomes (e.g. survival time) was summarized with

Kaplan-Meier curves, compared across groups using the log-rank test with α = 0.01.

29 CHAPTER III

HUMAN PAPILLOMAVIRUS 16 E7 STABILIZES APOBEC3A PROTEIN BY INHIBITING

CULLIN 2-DEPENDENT PROTEIN DEGRADATION2

Importance

Human papillomavirus (HPV) is causally associated with over 5% of all human malignancies. Several recent studies have shown that a subset of cancers, including HPV- positive head and neck and cervical cancers, have distinct mutational signatures potentially caused by members of the APOBEC3 cytidine deaminase family. However, the mechanism that induces APOBEC3 activity in cancer cells is poorly understood. Here, we report that the

HPV oncoprotein E7 stabilizes APOBEC3A (A3A) protein in human keratinocytes by inhibiting ubiquitin-dependent protein degradation in a cullin-dependent manner.

Interestingly, the HPV E7-stabilized A3A protein maintains its deaminase activity. These findings provide a new insight into cancer mutagenesis enhanced by virus-induced A3A protein stabilization.

Introduction

Human papillomaviruses (HPVs) are small, non-enveloped, double stranded DNA viruses causally associated with over 5% of all human cancers (207, 208). Persistent infection of high-risk HPV, such as HPV16 and HPV18, is required for HPV-associated cancer progression (209). The HPV oncoprotein E7 plays important roles in cancer progression and maintenance (Reviewed in (210, 211)). HPV E7, lacking inherent enzymatic activity, relies on protein-protein interactions with a myriad of host factors to promote virus replication and persistence (212). Previous studies have shown that high-risk HPV E7 modulates proteasome-mediated protein degradation of several host proteins including pRB, p107, p130, and PTPN14 (78, 213-216). These proteins are rapidly degraded by

2 This chapter was published with permission from Westrich et al. Human Papillomavirus 16 E7 Stabilizes APOBEC3A Protein by Inhibiting Cullin-2-Dependent Protein Degradation. JVI

30 ubiquitination through the cullin ubiquitin ligase complex. In contrast, HPV E7 also inhibits degradation of other proteins such as p53 and p21 (85, 87, 217).

The apolipoprotein-B mRNA editing enzyme, catalytic polypeptide like 3 (APOBEC3 or A3) family of interferon inducible cytidine deaminases functions as antiviral restriction factors (Reviewed in (218)). Humans express seven A3 family members: A3A, A3B, A3C,

A3D, A3F, A3G and A3H (181, 182). A3A is notable in that it specifically targets and restricts foreign DNA elements. A3A binds to single-stranded DNA with high affinity (219), mediates the catabolism of foreign DNA (186), and restricts infection of several DNA viruses, including

HPV (116, 117, 187, 188, 220). Another study has shown a strong correlation between A3A expression and HPV DNA integration in oropharyngeal cancer, suggesting that HPV episomes in persistent infection may also be targeted by A3A (221).

A3 mutational signatures have been observed in multiple human cancers (222-228).

Interestingly, cervical (CxCa) and head and neck (HNC) cancer genomes are enriched with somatic mutations related to A3 cytidine deamination, particularly by A3A and A3B (228-

231). We have shown that A3A and A3B mRNA expression is upregulated in HPV-positive keratinocytes and CxCa patient tissues by a mechanism involving the HPV oncoprotein E7

(117). Other studies have also shown that the HPV oncoprotein E6 increases A3B mRNA expression (117, 232-235). These findings imply that cytidine deamination by HPV-induced

A3A and/or A3B expression generates somatic mutations in the host genome.

Given that A3A restricts HPV infection (117), HPV must subvert the antiviral activities of A3A to complete its lifecycle. To evade A3G restriction, the HIV-1 Vif protein promotes degradation of A3G protein through cullin 5 and the 26S proteasome (236). This process is strikingly similar to HPV E7-mediated pRB degradation mediated by cullin 2 (CUL2) (78).

Therefore, we initially hypothesized that HPV counters A3A by promoting its degradation via an E7-dependent process. Contrary to our hypothesis, we found that A3A protein levels were highly elevated in HPV-positive keratinocytes and cancer cell lines. We further

31 revealed that high-risk HPV E7s inhibit the natural turnover of A3A protein by interfering with cullin-mediated degradation of A3A. Our findings suggest that A3A protein stabilized by HPV

E7 may contribute to the accumulation of A3 mutational signatures in host cells persistently infected with HPV.

Results

HPV16 E7 Increases A3A Protein Levels

Although A3A mRNA expression is upregulated in HPV-positive patient tissue samples and cultured keratinocytes (117), A3A mRNA levels may not precisely reflect A3A protein levels. Thus, we examined endogenous A3A protein levels in normal immortalized keratinocytes (NIKS), NIKS cells stably harboring episomal HPV16 genomes (NIKS-16), and

NIKS-16 cells lacking E7 expression (NIKS-16ΔE7). Our results demonstrated that A3A protein levels were increased by ~3-fold in the NIKS-16 cells when compared to NIKS cells

(Figure 3-1A and 3-1B). In contrast, A3A protein level in NIKS-16ΔE7 cells was not significantly different to NIKS cells (Figure 3-1A and 3-1B). This result is consistent with the

A3A mRNA levels in NIKS, NIKS-16, and NIKS-16ΔE7 cells shown in our previous study

(117). Next, to determine if HPV16 E7 expression is sufficient to increase A3A protein levels, 293FT cells were cotransfected with a constant amount of hemagglutinin (HA)-tagged

A3A expression plasmid (A3A-HA) and increasing concentrations of an HPV16 E7 expression plasmid. Transfection efficiency was determined by cotransfection of a fixed amount of a green fluorescent protein (GFP) expression plasmid. After 72 h, A3A-HA protein was analyzed by western blotting. As previously shown by ourselves and others, transfected

A3A, unlike endogenous A3A, shows two distinct bands by alternative initiation from a second start site found 36 bases after the canonical start site (Figure 3-1C) (237, 238). Our results showed that A3A protein levels were increased by HPV16 E7 expression in a dose- dependent manner (Figure 3-1C and 3-1D). These results suggest that HPV16 E7 expression is sufficient to increase A3A protein levels.

32 HPV16 E7 prevents A3A protein degradation

To determine if HPV16 E7 modulates A3A protein stability, we assessed the natural turnover of A3A protein in 293FT cells cotransfected with A3A-HA and HPV16 E7.

Cycloheximide (CHX), which prevents de novo protein synthesis, was used to assess the post-translational stability of A3A protein. Cotransfected 293FT cells were harvested at 0, 2,

4, 6, and 8 h post CHX treatment and A3A-HA protein was detected by western blotting. In the absence of HPV16 E7 expression, the majority of A3A protein was degraded within the 8 h time course (Figure 3-2A and 3-2B). In contrast, HPV16 E7 expression dramatically stabilized both the large and small isoforms of A3A-HA. To determine if HPV16 E7 similarly protects endogenous A3A protein from degradation, NIKS, NIKS-16, and NIKS-16ΔE7 cells were treated with CHX and A3A protein levels were determined. Consistent with our results from exogenous expression of A3A, NIKS-16 cells showed minimal degradation of A3A protein up to 8 h post CHX treatment, while A3A protein in both NIKS and NIKS-16ΔE7 cells was degraded over the time course (Figure 3-2C and 3-2D). We tested cell viability and found no significant effect of CHX treatment on the viability of NIKS cells (Figure 3-2E and

3-2F).

Next, to test if A3A is degraded by a proteasome-dependent mechanism, we treated

NIKS cells with the proteasome inhibitor MG132 and examined A3A protein levels over a time course. We found that blocking proteasome function rapidly accumulates A3A protein in

NIKS cells (Figure 3-2G and 3-2H), indicating that proteasome-dependent protein degradation plays a key role in the natural turnover of A3A protein. Taken together, our results suggest that HPV16 E7 expression stabilizes A3A protein levels in human keratinocytes by preventing proteasome-dependent A3A protein degradation.

High-risk HPV E7, but not Low-risk HPV E7, Prevents A3A Protein Degradation

Previous studies have shown that high- and low-risk E7 proteins differentially regulate the degradation of host proteins, most notably pRB (83, 239). Thus, we

33 hypothesized that E7s from high-risk HPV genotypes, such as HPV16 and HPV18, stabilize

A3A protein more efficiently than E7s from low-risk HPV genotypes, such as HPV6 and

HPV11. To test this hypothesis, we utilized NIKS cell lines engineered to stably express the

E7s of HPV6, HPV11, HPV16, or HPV18 (198). These cells were treated with CHX for 0, 2,

4, 6, and 8 h and endogenous A3A protein levels were analyzed by western blotting.

Consistent with the results shown for NIKS-16 cells (Figure 3-2C), both NIKS-16E7 and

NIKS-18E7 cells showed no detectable degradation of A3A protein up to 8 h post CHX treatment (Figure 3-3A and 3-3B). This result indicates that expression of high-risk HPV E7 is sufficient for preventing endogenous A3A protein degradation in human keratinocytes. In contrast, NIKS-6E7 and NIKS-11E7 cells expressing low-risk E7 showed rapid degradation of A3A protein similar to NIKS and NIKS-vector cells (Figure 3-3A and 3-3B). These results suggest that high-risk HPV E7s, but not low-risk E7s, prevent A3A protein degradation in human keratinocytes.

A3A Protein is Stabilized in HPV-Positive CxCa and HNC Cell Lines

To determine if stabilization of A3A protein also occurs in HPV-positive cancer cells, we evaluated the turnover of A3A protein in HPV-positive CxCa (CaSki) and HNC (SCC-90 and SCC-152) cell lines comparing to HPV-negative CxCa (C33A) and HNC (SCC-25) cell lines. The results showed that A3A protein was minimally degraded for 8 h post CHX treatment in all HPV-positive cancer cells (CaSki, SCC-90, and SCC-152) (Figure 3-4A and

3-4C). In contrast, A3A protein was gradually degraded over the time course in both HPV- negative CxCa (C33A) and HNC (SCC-25) cells (Figure 3-4B and 3-4C). These results suggest that A3A protein is stabilized in cancer cells by HPV consistent with our observation in normal immortalized keratinocytes. Notably, we found that HPV16 E7 protein in the HPV- positive cells is rapidly degraded within 2 h (Figure 3-4A). These results suggest that, although A3A stabilization requires high-risk HPV E7, stability of A3A is maintained after an initial action of HPV E7.

34 The CUL2 Binding Site in E7 is Important for A3A Protein Stabilization

To determine the mechanism by which high-risk HPV E7 stabilizes A3A protein, we tested five well-characterized HPV16 E7 mutants: H2P, ΔD21-24C (ΔDLYC), SS31-32AA

(CKII), CVQ68-70AAA (CVQ), and ΔL79-L83 (ΔLEDLL) (70). These HPV16 E7 mutants result in: loss of pRB degradation (H2P), the inability to bind pRB (ΔDLYC), lack of phosphorylation by casein kinase II (CKII), and failure to interact with host proteins including histone deacetylases (HDAC), p21 and CUL2 (CVQ and ΔLEDLL) (Figure 3-5A) (Reviewed in (212)). Stable NIKS cell lines expressing each of these HPV16 E7 mutants were established by lentiviral transduction and puromycin selection. The expression of HPV16 E7 mutants was validated by RT-qPCR (Figure 3-5B). NIKS cells stably expressing E7 mutants or containing vector alone were treated with CHX and endogenous A3A protein levels were analyzed by western blotting. Interestingly, NIKS-16E7(CVQ) and NIKS-16E7(ΔLEDLL) cells showed gradual degradation of A3A protein over the time course, while NIKS cells expressing the other HPV16 E7 mutants consistently stabilize A3A protein similar to cells expressing wildtype HPV16 E7 (Figure 3-5C and 3-5D). Unlike the NIKS-vector cells, A3A protein was not completely degraded in both NIKS-16E7(CVQ) and NIKS-16E7(ΔLEDLL) cells at 8 h post CHX treatment. This suggests that the mutations in each of these two E7 domains is not sufficient to completely block the E7-mediated A3A protein stabilization. To determine if the reduction of A3A stabilization observed for CVQ and ΔLEDLL was due to expression differences, E7 protein levels were determined in CHX treated NIKS-16, NIKS-

16E7, NIKS-16E7(CVQ) and NIKS-16E7(ΔLEDLL) cells. We noted that the various E7 proteins were stable throughout the time course in each of the groups (Figure 3-5E) unlike

HPV-positive CxCa and HNC cell lines (Figure 3-4A). However, the initial levels of E7 protein were comparable between wildtype and mutant E7, the decreased A3A stability in

NIKS-16E7(CVQ) and NIKS-16E7(ΔLEDLL) cells was not caused by different E7 expression levels. The regions spanning the CVQ and LEDLL domains in HPV16 E7 are responsible for

35 E7 interaction with CUL2, which is a core component of cullin-RING-based E3 ubiquitin ligase complexes involved in proteasome-mediated pRB degradation (78, 240). Since low- risk HPV E7 does not bind to CUL2 (78), these results are in accordance with the differential functions in A3A protein stabilization between high-risk and low-risk HPV E7s shown in

Figure 3-3. Thus, our findings imply that A3A protein stabilization by high-risk HPV E7 is, at least in part, mediated by the E7 interaction with CUL2 in the ubiquitin ligase complex.

CUL2 is Necessary for A3A Degradation

Previous studies have shown that activation of cullin activity requires neddylation, which is a post-translational modification that covalently conjugates a ubiquitin like protein

NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) (241).

Treatment of NIKS cells with the neddylation inhibitor MLN4924 leads to a dose dependent reduction in NEDD8-conjugated CUL2 (Figure 3-6A). To determine if inhibition of cullin neddylation abrogates A3A protein degradation, CHX treated NIKS cells were incubated with

MLN4924 for 0 to 8 h and A3A protein levels were assessed. Interestingly, MLN4924 treatment prevented A3A protein degradation similar to high-risk HPV E7 expression (Figure

3-6B). To further determine if CUL2 is required for A3A degradation, we knocked down

CUL2 expression in NIKS cells using lentiviral transduction of three shRNA clones against

CUL2 (shRNA-CUL2 #1-3). We confirmed that CUL2 expression in NIKS cells was significantly decreased by shRNA-CUL2 compared to the scrambled shRNA (shRNA-Scr)

(Figure 3-6C). Next, to determine if CUL2 knockdown abrogates A3A degradation, we treated the NIKS cells expressing shRNA-CUL2 or shRNA-Scr with CHX for 8 h and measured A3A protein levels. Interestingly, A3A protein degradation was inhibited by CUL2 knockdown in NIKS cells (Figure 3-6D). Particularly, shRNA-CUL2 #1 and #3, which knocked down CUL2 expression more efficiently than shRNA-CUL2 #2, showed significant inhibition of A3A protein degradation in NIKS cells (Figure 3-6C and 3-6D). Since high-risk

HPV E7 degrades other host proteins through CUL2-dependent mechanisms, it is possible

36 that A3A stabilization is a consequence of the limited CUL2 protein otherwise occupied by

E7. To determine if A3A stabilization is due to a shortage of CUL2, we overexpressed CUL2 protein in NIKS-16 cells and measured A3A protein levels. The result showed that CUL2 overexpression in NIKS-16 cells did not affect A3A protein stabilization by HPV16 E7

(Figure 3-6E). Taken together, our results suggest that CUL2 plays an important role in A3A protein degradation, which is inhibited by high-risk HPV E7.

CUL2 Interacts with A3A and HPV16 E7

HPV16 E7 directly interacts with CUL2 for pRB degradation (78). To determine if

CUL2 interacts with A3A in the presence or absence of HPV16 E7, 293FT cells were cotransfected with V5-tagged CUL2, A3A-HA, and/or HPV16 E7. At 72 h post transfection, cells were harvested and CUL2 and A3A proteins were pulled down using anti-V5 and anti-

HA antibodies, respectively. The results showed that CUL2 was co-immunoprecipitated with both A3A and HPV16 E7 (Figure 3-7A). However, A3A interacts only with CUL2, but not directly with HPV16 E7 (Figure 3-7B). The input A3A-HA, CUL2-V5 and HPV16 E7 proteins were confirmed by western blotting (Figure 3-7C). These results suggest that both A3A and

HPV16 E7 interact directly with CUL2, and that A3A protein stabilization by HPV16 E7 is not mediated through a direct protein interaction between A3A and HPV16 E7. Given that the

HPV16 E7 mutants CVQ and ΔLEDLL are less effective for A3A protein stabilization (Figure

3-5C and 3-5D), we next determined whether CUL2 interacts with these mutants.

Interestingly, our results revealed that CUL2 interaction with HPV16 E7 was significantly reduced by CVQ and ΔLEDLL mutations when compared to the wildtype HPV16 E7 (Figure

3-7D). This suggests that reduced A3A stabilization by CVQ and ΔLEDLL mutations was due to the poor ability of these mutants to interact with CUL2.

HPV18 E7 is also capable of stabilizing A3A protein (Figure 3-3A). However, a previous study showed that HPV18 E7 does not interact with CUL2 (78) and our data is consistent with this observation (Figure 3-7E). We next tested whether HPV18 E7 interacts

37 with another candidate cullin family member, CUL3, using cotransfection of CUL3-V5 and

HPV18 E7. Interestingly, we found that HPV18 E7 directly interacts with CUL3, although the binding capacity is modest (Figure 3-7F). This result suggests that CUL3, instead of CUL2, may contribute to A3A stabilization by HPV18 E7. Taken together, CUL2 appears to be integral in the stabilization of A3A by HPV16 E7 and additional cullins may be involved in

A3A stabilization by other high-risk HPV genotypes.

HPV16 E7-Stabilized A3A Maintains Deaminase Activity

To determine if the A3A protein stabilized by HPV16 E7 retains its cytidine deaminase activity, an in vitro DNA deamination assay was performed using a fluorescently tagged oligonucleotide containing a single A3 target motif (TC) (199, 232). First, to validate the cytidine deaminase assay, cell lysates were prepared from 293FT cells transiently transfected with wildtype A3A, a catalytically inactive A3A mutant (A3A/E72Q), or vector only. While the vector only and A3A/E72Q expression showed minimal cleavage of TC- containing oligonucleotides, wildtype A3A expression resulted in a significant increase of cleavage (Figure 3-8A). Expression of wildtype and mutant A3A protein in transfected cell lysates were validated by western blotting (Figure 3-8B). To determine the deaminase activity of HPV16 E7-stabilized A3A protein, 293FT cells were transfected with A3A alone or both A3A and HPV16 E7, followed by CHX treatment. In cell lysates where A3A protein was stabilized by HPV16 E7, we observed dramatically higher deaminase activity compared to control cell lysates (Figure 3-8C and 3-8D). These results suggest that A3A protein stabilized by high-risk HPV E7 is functionally active and is capable of deaminating target cytosine residues.

A3A Protein Stabilization by HPV16 E7 is Independent of TRIB3

Tribbles homolog 3 (TRIB3) is a pseudokinase that inhibits the AKT/mTOR

(mammalian target of rapamycin) pathway (242). A previous study showed that TRIB3 facilitates A3A protein degradation (243). To test whether A3A protein stabilization is caused

38 by HPV16 E7 inhibition of TRIB3 functions, we first determined endogenous TRIB3 protein levels in NIKS, NIKS-16, and NISK-16ΔE7 cells. Our result showed no detectable difference in the expression levels of TRIB3 protein in NIKS-16 cells compared to NIKS and NISK-

16ΔE7 cells (Figure 3-9A).

To determine the effect of TRIB3 on A3A protein levels in human keratinocytes, we knocked down TRIB3 expression in NIKS cells by lentiviral transduction of shRNA against

TRIB3 (shRNA-TRIB3). Consistent with the previous finding (243), endogenous A3A protein levels were increased in NIKS cells by TRIB3 knockdown (Figure 3-9B). Next, to determine if TRIB3 knockdown prevents A3A protein turnover in NIKS cells, NIKS cells containing shRNA-TRIB3 #2 were treated with CHX and A3A protein levels were analyzed by western blotting. The results showed that TRIB3 knockdown did not affect A3A protein degradation in

NIKS cells (Figure 3-9C). These results suggest that A3A protein stabilization by high-risk

HPV E7 is independent of TRIB3.

Discussion

HPVs are causally associated with multiple human cancers, including CxCa and a subset of HNC (30, 244). While the tumor suppressors p53 and pRB are rapidly inactivated by expression of the HPV oncoproteins E6 and E7, respectively, HPV-associated carcinogenesis requires decades of disease progression to develop invasive cancer (209).

During this process, HPV persists in host cells and continuously contributes to cancer progression (245, 246). The underlying mechanisms of this slow process, often taking decades, are largely unknown.

The majority of cancers are driven by somatic mutations that accumulate over decades. Several mechanisms of somatic mutagenesis in cancer are well understood. For example, smoking and ultraviolet light exposure induce mutations that contribute to lung and skin cancer development, respectively (225). In contrast, drivers of somatic mutation for other cancers, including HPV-positive HNC and CxCa, were completely unknown until only

39 recently. Several recent studies have shown that A3-associated mutation signatures are highly enriched in multiple human cancers, including HNC and CxCa (224, 225, 228, 247).

Our previous study revealed that A3A and A3B mRNA expression is upregulated in HPV- positive keratinocytes in an HPV oncoprotein-dependent manner (117). Interestingly, A3A, but not A3B, potently restricts HPV infection. A recent study by Chan and colleagues showed that A3A is likely the predominant mutagenic A3 in cancers, causing over ten-fold higher A3-specific mutations than A3B (248). Nevertheless, the increase of A3A mRNA expression in HPV-positive HNC and CxCa is modest compared to the increase of A3B mRNA expression levels (117, 232). This implies that there may be other mechanisms to enhance off-target A3A activity in HPV-positive cells.

Here, we report that A3A protein accumulates in HPV-positive cells through HPV E7- mediated protein stabilization. HPV E7 modulates protein degradation and stabilization through mechanisms dependent on or independent of the ubiquitin ligase complex (85, 87,

249, 250). While it is well known that the ubiquitin ligase complex is involved in promoting protein degradation, other studies have shown that the ubiquitin ligase complex also plays important roles in protein stabilization (251-253).

High-risk HPV E7 binds to pRB with high affinity, compared to low-risk HPV E7, and degrades pRB through the CUL2-based E3 ubiquitin ligase complex (78, 254, 255). Cullins are members of the ubiquitin ligase complex that play critical roles in mediating the degradation of a myriad of cellular proteins (78, 255). Interestingly, our results showed that high-risk HPV E7s, but not low-risk HPV E7s, inhibit proteasome-dependent degradation of

A3A. Further, HPV16 E7 mutants (CVQ and ∆LEDLL) responsible for interaction with CUL2 did not completely stabilize A3A protein. This diminished capacity to stabilize A3A may be due to a restricted interaction between HPV16 E7 and CUL2 by CVQ and ∆LEDLL mutations. These results suggest that CUL2 plays an important role in A3A protein stabilization by HPV16 E7.

40 Viral proteins frequently target ubiquitin ligase components to modulate protein degradation. Merkel cell polyomavirus small T antigen stabilizes viral large T antigen by inhibiting the cellular SCF(Fbw7) E3 ligase (256). To induce lymphoproliferation, murine γ- herpesvirus stabilizes host Myc protein through the viral E3 ubiquitin ligase mLANA (257).

Hepatitis B virus X protein also inhibits Myc protein degradation by targeting the SCF(Skp2) ubiquitin E3 ligase (258). Previous studies have shown that HPV16 E7 stabilizes several host proteins including HIF-1a, p53 and p21 (85, 87, 259). Interestingly, all these proteins stabilized by HPV16 E7 are naturally degraded through the CUL2-based E3 ubiquitin ligase complex (260). Degradation of pRB and HIF-1a proteins requires neddylation of CUL2 (241,

255). Our results also show that inhibition of neddylation stabilizes A3A protein, suggesting that cullin neddylation is required for A3A protein degradation. Further, knockdown of CUL2 expression inhibits A3A protein degradation, suggesting a pivotal role of CUL2 in A3A degradation. While the interaction of HPV16 E7 with CUL2 has been previously shown (78), no studies have determined if A3A protein interacts with HPV16 E7 and CUL2. Here, we show that CUL2 binds to both HPV16 E7 and A3A individually, but A3A does not directly interact with HPV16 E7. These results suggest that HPV16 E7 likely inhibits A3A protein degradation through a mechanism involving CUL2. However, we found that HPV18 E7 does not interact with CUL2, in agreement with a previous finding (78), indicating that A3A stabilization by HPV18 E7 may be mediated through other cullins. Indeed, we found that

HPV18 E7 interacts with CUL3 instead of CUL2. As their interaction is modest compared to the HPV16 E7 and CUL2 interaction, CUL3 alone may not be sufficient to account for the entire mechanism of A3A stabilization. Further investigations are necessary to understand the detailed mechanisms by which different high-risk HPV E7s stabilize A3A protein levels.

HPV E7 has been shown to increase the levels of p53 and p21 proteins while limiting their activity (85, 87, 217). In contrast, HPV16 E7 stabilizes A3A while maintaining A3A

41 deaminase activity. The deaminase activity of E7-stabilized A3A may facilitate mutagenesis of the host genome in persistently infected keratinocytes (223, 225, 227). Consistent with our finding of A3A protein stabilization in HPV-positive CxCa and HNC cells (Figure 3-4A), a recent study by Kondo et al. has shown highly increased A3A protein levels in HPV-positive

HNC tissues compared to HPV-negative HNC tissues (221).

Interestingly, the stability of the E7 protein varies extensively depending on different cell types. HPV16 E7 in HPV-positive CxCa and HNC cells showed a short half-life of less than 2 h in agreement with previous observations (Figure 3-4) (261, 262). In contrast,

HPV16 E7 protein is stable for 8 h in the NIKS cells (Figure 3-5E). The varied stability of E7 has been observed before as several studies have shown that the half-life of E7 proteins varies depending on different cell lines and cellular mechanisms involved in protein degradation (263). First, interactions with some cellular proteins such as HSP90, GRP78, and USP11 significantly increase the steady-state level of E7 (262, 263). Second, another study has shown that E7 protein stability varies depending on its subcellular localization.

While nuclear E7 is short-lived, cytosolic E7 forming oligomers is stable over 6 h (264).

Third, the half-life of phosphorylated E7 (Ser33phospho-E7) is over 24 h (265). Additionally, dual-specificity tyrosine phosphorylation-regulated kinase 1A stabilizes HPV16 E7 protein through phosphorylation of the threonine 5 and threonine 7 residues (266). Therefore, it is possible that E7 overexpressed in NIKS cells may have a long half-life due to one or more of these mechanisms. However, regardless of the differential E7 protein stability, A3A protein was consistently stabilized in HPV-positive cells. As HPV16 E7 does not directly bind to

A3A, this suggests that high-risk HPV E7 may initiate early steps of A3A protein stabilization.

Given that A3A restricts HPV infection (117), A3A protein stabilized by HPV E7 could be detrimental to the virus lifecycle. Thus, HPV must evade restriction by A3A to successfully establish infection. Strikingly, TC dinucleotides, the preferred target site of A3A, are significantly underrepresented in genomes of Alphapapillomaviruses, which includes all

42 high-risk HPV genotypes (267). Since the basal mRNA expression level of A3A is significantly higher in mucosal skin compared to cutaneous skin (267),

Alphapapillomaviruses may have evolved to survive in an environment with high A3A levels.

This implies that the HPV genome may be highly resistant to A3A-mediated mutagenesis, while elevated levels of A3A protein by high-risk E7 may contribute to off-target effects on the host. HPV may derive additional benefit from the increased A3A activity. It has been shown that HPV replication relies, in part, on the DNA damage response (DDR) (268-270).

Aberrant A3A activity has been shown to activate the DDR response (271), and thus may assist the virus in its replication. Notably, the antiviral effect of A3A appears to be most prominent in the next target cell due to a significant decrease in virion infectivity (117). This phenomenon may be explained by A3A encapsidation into HPV virions, similar to how

APOBEC3 proteins inhibit many other viruses. Thus, E7-mediated stabilization of A3A in the producer cell may not necessarily impact HPV replication if it does not affect encapsidation.

In fact, E7-CUL2-mediated sequestration may even serve as a viral countermeasure against

A3A.

Taken together, our findings imply that high-risk HPV E7 stabilizes A3A protein in persistent HPV infection. This stabilized A3A may contribute to cancer mutagenesis during

HPV-associated cancer progression, which generates A3 mutation signatures observed in

HPV-positive HNC and CxCa.

43

B 5 * A 4 NIKS NIKS16 NIKS16ΔE7 3 A3A n.s. 2 Relative A3A Relative HPV16 E7 band intensity 1

β-actin 0 E7 NIKS Δ NIKS16 NIKS16 D 10 C HPV16 E7:A3A-HA * 0:1 1:1 2:1 3:1 4:1 8

A3A-HA 6 * * 4 * HPV16 E7 Relative A3A Relative band intensity 2 * GFP 0 β-actin 0:1 1:1 2:1 3:1 4:1 0:1 1:1 2:1 3:1 4:1 Lower Band Upper Band HPV16 E7: A3A-HA

Figure 3-1. HPV16 E7 increases A3A protein levels. Protein levels of endogenous A3A in NIKS, NIKS-16, and NIKS-16ΔE7 cells (A and B) and exogenous A3A in 293FT cells (C and D) were determined by western blotting and densitometry. (C) 293FT cells were cotransfected with a fixed amount of pcDNA3.1-A3A-HA and pEGFP-N3 and increasing amounts of pCMV-16E7. (A and C) Cell lysates were prepared and A3A (endogenous A3A or transfected A3A-HA), HPV16 E7, and β-actin were detected by western blotting. (C) Transfection efficiency was determined by western blotting of GFP protein. (B and D) A3A band density was normalized to β-actin band density. Data are shown as fold changes of normalized A3A band density ± standard deviations relative to NIKS cells (B) or 293FT cells without E7 expression (D). Shown are representative of four independent experiments. P-values were calculated by the Student’s t-test. *P < 0.05, n.s., not significant. Data were collected in collaboration Cody Warren, PhD University of Colorado, Boulder.

44 B 2.0 A A3A-HA A3A-HA + HPV16 E7 1.5 CHX CHX 0 2 4 6 8 0 2 4 6 8 h 0 h 2 h 1.0 A3A-HA # 4 h * 6 h # 8 h 0.5 ∇ β-actin ∇ ∇

Relative A3A band intensity band A3A Relative 0.0 C A3A-HA A3A-HA A3A-HA A3A-HA CHX 0 2 4 6 8 h + E7 + E7 Lower Band Upper Band A3A NIKS D β-actin 1.8 CHX 1.5 0 h A3A 2 h NIKS16 1.2 4 h β-actin * 6 h 0.9 * * 8 h * # 0.6 A3A ∇ NIKS16ΔE7 0.3 ∇ β-actin Relative A3A band intensity band A3A Relative 0.0 NIKS NIKS16 NIKS16ΔE7 E F 100 100

n.s. n.s. 50 50 NIKS A3A-HA NIKS16 A3A-HA + HPV16 E7 Percent cell viability Percent cell viability NIKS16ΔE7 0 0 0 2 4 8 0 2 4 8 CHX treatment (h) CHX treatment (h) H # G 5 * 4 MG132 0 2 4 6 8 h * 3 * A3A NIKS 2 Relative A3A A3A Relative β-actin band intensity 1

0 0 2 4 6 8 MG132 treatment (h)

Figure 3-2. HPV16 prevents A3A protein degradation. (A and B) 293FT cells were cotransfected with pcDNA3.1-A3A-HA and pCMV-16E7 or a corresponding vector. Cotransfected 293FT cells (A, B, and E), and NIKS, NIKS-16 or NIKS-16ΔE7 cells (C, D, and F) were treated with 50 μg/mL cycloheximide (CHX) for the indicated time. NIKS cells were treated with 20 μM MG132 for the indicated time (G and H). A3A protein expression was analyzed as described in Figure 3-1. Transfected A3A-HA (A) or endogenous A3A (C and G) were detected by western blotting using anti- HA or anti-A3A antibodies, respectively, and quantified by densitometry as described in Figure 3-1 (B, D and H). Cell viability of CHX treated 293FT cells (E) or NIKS, NIKS-16, and NIKS-16ΔE7 cells (F) were assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Data are shown as percent cell viability ± standard deviations and normalized to untreated (0 h) cells (E and F). All experiments were repeated three times. Data are shown as fold changes to cells with 0 h treatment ± standard deviations. P-values were determined by the Student’s t-test (B, D and H) or one-way ANOVA (E and F). *P < 0.05, #P < 0.005, ∇P < 0.0005, n.s., not significant.

45

A CHX 0 2 4 6 8 h

A3A NIKS-vector β-actin

A3A NIKS-16E7 β-actin High-risk A3A NIKS-18E7 β-actin

A3A NIKS-6E7 β-actin Low-risk A3A NIKS-11E7 β-actin

B CHX 1.8 0 h 2 h 1.5 4 h 6 h 1.2 8 h 0.9 # 0.6 * * 0.3 # ∇ ∇ ∇ Relative A3A band intensity A3A Relative 0.0 NIKS- NIKS- NIKS- NIKS- NIKS- Vector 16E7 18E7 6E7 11E7 High-risk Low-risk

Figure 3-3. High risk HPV E7s, but not low-risk HPV E7s, prevent A3A protein degradation. NIKS cells stably expressing HPV6 (NIKS-6E7), HPV11 (NIKS-11E7), HPV16 (NIKS-16E7), or HPV18 (NIKS-18E7), or containing vector alone (NIKS-vector) were treated with 50 μg/mL CHX and analyzed by western blotting using anti-A3A and β-actin antibodies as described above. P-values were determined by the Student’s t-test. *P < 0.05, #P < 0.005, ∇P < 0.0005.

46 A CHX 0 2 4 6 8 h B A3A CHX 0 2 4 6 8 h A3A HPV16E7 CaSki HPV16E7 C33A β-actin β-actin A3A A3A HPV16E7 SCC-90 HPV16E7 SCC-25 β-actin β-actin A3A

HPV16E7 SCC-152

β-actin

C CHX 1.5 0 h 2 h 1.2 4 h * 6 h 0.9 * 8 h # * 0.6 * # 0.3 *

Relative A3A band intensity A3A Relative 0.0 CaSki SCC-90 SCC-152 C33A SCC-25 HPV+ HPV-

Figure 3-4. A3A protein is stabilized in HPV-positive CxCa and HNC cell lines. (A) HPV-positive CxCa (CaSki) and HNC (SCC-90 and SCC-152) cell lines and (B) HPV-negative CxCa (C33A) and HNC (SCC-25) cell lines were treated with 50 μg/mL CHX for the indicated time and analyzed by western blotting using anti-A3A, anti-HPV16 E7, and β-actin antibodies as described above. A3A band density was normalized to β-actin band density. Data are shown as fold changes to cells with 0 h treatment ± standard deviations. Shown are representative of at least 2 independent experiments. P-values were calculated by the Student’s t-test. *P < 0.05, #P < 0.005.

47 A CUL2 binding

HPV16 E7 CR1 CR2 CR3 ^ ^ ^ ^ H2P ΔDLYC CKII CVQ ΔLEDLL^ B 50 C 40 CHX 0 2 4 6 8 h

30 A3A NIKS-vector 20 β-actin -actin (1,000X) β A3A

to to 10

E7 expression relative relative expression E7 N.D. NIKS-16E7 0 β-actin

Wt H2P CKII CVQ A3A DLYC LEDLL NIKS-16 Δ Δ NIKS-16E7(H2P) NIKS-vector CHX β-actin NIKS-16E7 0 h D 2 h A3A NIKS-16E7(ΔDLYC) 1.6 4 h 6 h β-actin 8 h 1.2 A3A NIKS-16E7(CKII) # * * 0.8 β-actin * * * * 0.4 A3A ∇ NIKS-16E7(CVQ) 0.0 β-actin Relative A3A band intensity band A3A Relative NIKS- Wt H2P ΔDLYC CKII CVQ ΔLEDLL vector A3A NIKS-16E7 NIKS-16E7(ΔLEDLL) β-actin E CHX 0 2 4 6 8 h

NIKS-16

NIKS-16E7 HPV16 E7 NIKS-16E7(CVQ)

NIKS-16E7(ΔLEDLL)

Figure 3-5. The CUL2 binding site in HPV16 E7 is required for A3A protein stabilization. NIKS cells stably expressing wildtype or mutant HPV16 E7 (H2P, ΔDLYC, CKII, CVQ, and ΔLEDLL), or containing vector alone were generated by lentiviral transduction and 3 μg/mL puromycin selection. (A) Total RNA was extracted from established NIKS cells and mRNA expression levels of wildtype and mutant HPV16 E7s were measured by RT-qPCR using specific primers and normalized by β- actin mRNA levels. Data are shown as HPV16 E7 mRNA copy number relative to β-actin mRNA copy number ± standard deviations. N.D., not detected. (B, C and D) Established NIKS cells were treated with 50 μg/mL CHX and A3A protein levels were analyzed as described above. (E) HPV16 E7 protein levels from CHX treated NIKS-16, NIKS-16E7, NIKS-16E7(CVQ) and NIKS-16E7(ΔLEDLL) cells were analyzed as described above. Data are shown as fold changes to cells with 0 h treatment ± standard deviations. Shown are representative of two independent experiments. P-values were determined by the Student’s t-test. *P < 0.05, #P < 0.005, ∇P < 0.0005.

48 A MLN4924 (nM): 0 33 110 330 1000 3000 NEDD8-CUL2 CUL2 β-actin

B - MLN4924 CHX 0 2 4 6 8 0 2 4 6 8 h

A3A

β-actin

-Scr C shRNA-CUL2 -Scr shRNA-CUL2 D shRNA #1 #2 #3 shRNA #1 #2 #3 CUL2 CHX - + - + - + - +

β-actin A3A

β-actin E - CUL2 ORF CHX - + - + A3A

CUL2-V5 NIKS-16E7

Actin

Figure 3-6. A3A degradation requires CUL2 expression and neddylation. (A) NIKS cells were treated with MLN4924 at indicated concentrations or vehicle (DMSO) for 8 h. Neddylated CUL2 (NEDD8-CUL2) and CUL2 proteins were analyzed by western blotting using anti-CUL2 antibody. (B) NIKS cells were treated with 50 μg/mL CHX and either vehicle (DMSO) or 330 nM MLN4924 for 0, 2, 4, 6, and 8 h. Endogenous A3A and β-actin proteins were analyzed by western blotting using anti- A3A and anti-β-actin antibodies, respectively. (C) NIKS cells stably expressing scrambled (shRNA- Scr) or three clones of CUL2 shRNA (shRNA-CUL2 #1-3) were generated by lentiviral transduction and 10 μg/mL blasticidin selection. CUL2 and β-actin protein expression was detected using anti- CUL2 and β-actin antibodies, respectively. (D) Established NIKS cells were treated with 50 μg/mL CHX, and A3A and β-actin proteins were analyzed by western blotting as described above. (E) NIKS- 16 cells stably expressing CUL2 were generated by lentiviral transduction of the CUL2-V5 open reading frame, followed by 3 μg/mL puromycin selection. Established NIKS-16 cells were treated with 50 μg/mL CHX for 8 h, and CUL2, A3A, and β-actin proteins were analyzed by western blotting using anti-V5, anti-A3A, and anti-β-actin antibodies, respectively. Shown are representative of two independent experiments.

49 D A CUL2-V5 - - + - + + + HPV16 E7 Wt CVQ ΔLEDLL CUL2-V5 A3A-HA + - - + + - + + - + - + - +

HPV16 E7 - + - + - + + V5 IP:V5 V5 HPV16-E7

HA IP: V5 V5 Input HPV16 E7 HPV16-E7 B V5 E CUL2-V5 + - + + - + HPV18 E7 - + + - + + HA IP: HA V5 HPV16 E7 HPV18 E7 C V5 Input IP: V5

HA Input F CUL3-V5 + - + + - + HPV18 E7 - + + - + + HPV16 E7 V5

HPV18 E7

Input IP: V5

Figure 3-7. CUL2 interacts with A3A and HPV16 E7 proteins. 293FT cells were cotransfected with expression plasmids of A3A-HA (A-C), CUL2-V5 (A-E), CUL3-V5 (F), HPV16 E7 (A-D), HPV16 E7(CVQ) (D), HPV16 E7(ΔLEDLL) (D), and/or HPV18 E7 (E and F). At 72 h post transfection, cells were lysed and CUL-V5 and A3A-HA proteins were pulled down using an anti-V5 (A, D-F) and anti- HA (B) antibodies, respectively. Each protein in the input and pulldown samples were detected by western blotting using anti-V5, anti-HA, anti-HPV16 E7, and anti-HPV18 E7, as indicated. Shown are representative of at least two independent experiments.

50 A B No lysate Vector A3A/E72QWildtype A3A

Vector A3A/E72QWildtype A3A

Substrate A3A-HA

Product β-actin

% Cleaved: N.D 10.67 9.53 72.93

CDA3A + A3A + A3A HPV16 E7 A3A HPV16 E7 CHX (8 h): - + - + CHX (8 h): - + - +

A3A-HA Substrate

Product β-actin % Cleaved: 68.29 10.50 80.60 71.53

Figure 3-8. HPV16 E7-stabilized A3A maintains deaminase activity. (A and B) 293FT cells were transfected with vector only, wildtype A3A-HA, or A3A/E72Q-HA for 72 h. Phosphate buffered saline was used as for the no lysate negative control. (B) Wildtype and mutant A3A expression was validated by western blotting. (C) 293FT cells were transfected with A3A-HA alone or both A3A-HA and HPV16 E7 for 72 h, and treated with vehicle or 50 μg/mL CHX as described above. Cell lysates were prepared and an in vitro cytidine deaminase assay was performed using TC-containing oligonucleotides as described in Materials and Methods (A and C). The reaction products were analyzed on 15% polyacrylamide-urea gel and percent cleavage was determined by densitometry using the formula: % cleaved = (product / (substrate + product)) ´ 100. (D) A3A expression was validated by western blotting. Shown are representative of two independent experiments. Data were collected in collaboration Michael Klausner, University of Colorado.

51 A NIKS NIKS16 NIKS16ΔE7 TRIB3

β-actin

B shRNA shRNA-TRIB3 -Scr #1 #2 TRIB3

A3A

β-actin C CHX 0 2 4 6 8 h

A3A shRNA-TRIB3 β-actin

Figure 3-9. A3A protein stabilization by high-risk HPV E7 is independent of TRIB3. (A) Endogenous TRIB3 protein in NIKS, NIKS-16, and NIKS-16ΔE7 cells was detected by western blotting using anti-TRIB3 antibody. NIKS cells stably expressing scrambled (shRNA-Scr) or two TRIB3 shRNA clones (shR-TRIB3 #1 and #2) were generated by lentiviral transduction and 10 μg/mL blasticidin selection. (B) TRIB3, A3A, and β-actin proteins were detected using anti-TRIB3, anti-A3A, and β-actin antibodies, respectively. (C) NIKS cells with TRIB3 knockdown were treated with 50 μg/mL CHX, and A3A and β-actin proteins were analyzed by western blotting.

52 CHAPTER IV

SUPPRESSION OF ANTITUMOR IMMUNE RESPONSES BY HUMAN PAPILLOMAVIRUS

THROUGH EPIGENETIC DOWNREGULATION OF CXCL143

Importance

Human papillomaviruses (HPVs) are causally associated with over 5% of all cancers.

During decades of cancer progression, HPV persists, evading host surveillance. However, little is known about the immune evasion mechanisms driven by HPV. Here, we report that the chemokine CXCL14 is significantly downregulated in HPV-positive head/neck and cervical cancers. Using patient tissues and cultured keratinocytes, we found that CXCL14 downregulation is linked to CXCL14 promoter hypermethylation induced by the HPV oncoprotein E7. Restoration of CXCL14 expression in HPV-positive cancer cells clears tumors in immunocompetent syngeneic mice, but not in immunodeficient mice. Mice with

Cxcl14 expression show dramatically increased natural killer and T cells in the tumor draining lymph nodes. These results suggest that epigenetic downregulation of CXCL14 by

HPV plays an important role to suppress antitumor immune responses. Our findings may offer novel insights to develop preventive and therapeutic tools for restoring antitumor immune responses in HPV-infected individuals.

Introduction

Human papillomaviruses (HPVs) are causally associated with multiple human cancers, including cervical cancer (CxCa) and head and neck cancer (HNC), and result in about half a million deaths worldwide each year (30). Persistent infection of HPV is required for HPV-associated cancer development and therefore HPV must evade host immune surveillance (272). To evade host immune surveillance, HPV creates a local immune suppressive environment by inducing chemokine expression and diminishing the cytotoxic T

3 This chapter was published with permission from Cicchini et al. 2016. Suppression of Antitumor Immune Responses by Human Papillomavirus through Epigenetic Downregulation of CXCL14. mBio, http://mbio.asm.org/content/7/3/e00270-16.

53 cell response (272, 273). However, little is known about the mechanisms of disease progression driven by HPV-induced immune suppression.

To better understand the roles of host immunity in HPV-associated cancer progression, we analyzed expression levels of all known chemokines and chemokine receptors using our global gene expression datasets of CxCa progression (274) and HPV- positive and -negative HNCs (193). Deregulated chemokine networks in the tumor microenvironment (TME) alter immune cell infiltration, angiogenesis, tumor cell growth, survival, and migration, leading to cancer progression (275). Recent laboratory studies and clinical trials have shown that restoring antitumor immune responses may be a promising therapeutic strategy to treat several cancers including HNCs (276-278). While initial studies have begun to explore relations between HPV infection and chemokine regulation, little is yet known about chemokine expression patterns altered by HPV during cancer progression.

Here we show that, while expression of many proinflammatory chemokines is increased,

CXCL14 expression is significantly decreased in HPV-associated cancer progression.

CXCL14 is a chemokine distantly related to other CXC chemokines, showing 30% identity with CXCL2 and CXCL3 (279). CXCL14 functions as a potent angiogenesis inhibitor and a chemotactic factor for dendritic cells (DCs) and natural killer (NK) cells (280, 281).

While normal human epithelial cells constitutively express CXCL14, its expression is frequently reduced in cervical, prostate, and oral cancers (142, 282, 283). Restoration of

Cxcl14 expression recruits DCs into tumors in vivo and in vitro (142, 284) and induces tumor necrosis (285). Importantly, Cxcl14 expression in HNC cells suppresses tumor growth from xenografts in athymic nude and SCID mice (286, 287). In addition, the rate of colorectal tumor formation and metastasis was significantly lower in Cxcl14 transgenic mice compared to isogenic wild type mice (288). Previous studies have shown that CXCL14 inhibits signaling of proinflammatory chemokines IL-8 (280) and CXCL12 (289), which are known to promote cancer development and metastasis. Thus, CXCL14 has been suggested as a

54 potential tumor suppressor having anti-inflammatory functions. CXCL14 expression is epigenetically regulated by promoter hypermethylation in colorectal cancer cells (284). In the current investigation we show that the CXCL14 promoter is highly methylated and its expression is downregulated in HPV-positive tissues and cells in an E7-dependent manner.

Importantly, restoration of murine Cxcl14 expression in HPV-positive mouse oropharyngeal epithelial (MOE) cells increases NK, CD4+ T, and CD8 + T cell infiltration into the tumor- draining lymph nodes (TDLN), and results in significant clearance of implanted HPV-positive

HNC cells in immunocompetent syngeneic mice.

Results

Proinflammatory Chemokines are Upregulated during CxCa Progression

To understand the mechanisms by which HPV deregulates host immune responses in the TME, we analyzed gene expression changes of known chemokines and their receptors in tissue epithelium during CxCa progression using our global gene expression data from human cervical tissue specimens of normal, cervical intraepithelial neoplasia

(CIN) 1/2, CIN3, or tumor tissues (GEO accession #GSE63514) (274). The results showed that fourteen chemokines and chemokine receptors increased at least 3-fold in during cancer progression. Expression of IL-8, CXCL9, CXCL11, CCL3, and CCL19 mRNAs was progressively increased throughout disease progression (Figure 4-1A). In contrast, expression of CXCL1, CXCL2, CXCL5, CXCL6, and CCL20 mRNA was significantly upregulated during the early transition from normal to CIN1/2 (Figure 4-1B), while CXCL13 and CCL8 mRNA expression significantly increased only in the later transition to invasive tumors (Figure 4-1C). Among chemokine receptors, CXCR2 mRNA expression was decreased by 12-fold and CXCR4 mRNA expression was upregulated nearly 7-fold throughout cancer progression (Figure 4-1D). To identify HPV-specific chemokine deregulation, we analyzed our previously published gene expression data of HPV-positive and -negative HNCs (GEO accession # GSE6791) (193). This analysis revealed that

55 expression of CXCL9, CXCL10, CXCL13, and CCL19 as well as CXCR4 mRNA was significantly upregulated in HPV-positive HNCs compared to HPV-negative HNCs (Figure 4-

2A-E), suggesting that HPV infection specifically modulates chemokine expression. Unlike increased expression during cervical cancer progression, the expression level of IL-8 mRNA was lower in HPV-positive HNCs by 2-fold compared to HPV-negative HNCs (Figure 4-2F).

Although HPV-positive cancers exhibit lower levels of IL-8 expression compared to HPV- negative cancers, our previous study showed that IL-8 expression was significantly increased in all HNCs compared to normal tissues (193). These results indicate that HPV- negative HNCs robustly upregulate IL-8 and CXCL1 expression more than HPV-positive

HNCs by other mechanisms. To explore these changes of chemokine expression in vitro, we analyzed chemokine expression in cervical keratinocyte lines using reverse transcriptase quantitative PCR (RT-qPCR). We used W12E (derived from a low-grade precancerous cervical lesion with episomal HPV16), W12G (low-grade with integrated HPV16), and

W12GPXY (transformed) cells, which sequentially mimic CxCa progression (290). As expected, all W12 cell lines express high levels of the HPV16 early gene transcript (Figure

4-2G). Expression levels of proinflammatory chemokines IL-8, CXCL1, CXCL2, CXCL10, and CXCL11 were significantly increased in W12G and W12GPXY cells compared to a normal immortalized keratinocyte line (NIKS) (Figure 4-2H-L). These results from tissue specimens and cultured keratinocytes suggest that several proinflammatory chemokines, which are recognized as major players in cancer development, are upregulated during HPV- associated cancer progression.

CXCL14 Expression is Downregulated in HPV-Associated Cancer Progression

While over a dozen chemokines were highly upregulated, CXCL14 was the only chemokine decreased over 3-fold in CxCa progression (Figure 4-3A). CXCL14 mRNA expression was progressively decreased by about 21-fold from normal to cancer tissue. The downregulation of CXCL14 was consistently observed in the W12 cell culture model (Figure

56 4-3B). CXCL14 expression levels showed a significant inverse correlation with the expression levels of IL-8 and other proinflammatory chemokines in cervical tissue specimens and cultured keratinocytes (Figure 4-1A, Figure 4-2H to 4-2L, and Figure 4-3A,

4-3B). To determine whether CXCL14 downregulation is unique to HPV-positive cancers, we compared CXCL14 mRNA expression between HPV-positive and HPV-negative HNCs using the datasets from our previous global gene expression study (193). The results showed that

CXCL14 mRNA expression was significantly lower in HPV-positive HNC compared to HPV- negative HNC (Figure 4-3C). We also confirmed downregulation of CXCL14 mRNA expression in HPV-positive HNC and CxCa compared to HPV-negative HNC using the

TCGA RNA-seq data (291) (Figure 4-4A). A previous study reported that CXCL14 expression was significantly decreased in HNCs compared to normal tissue (282). Taken together, these results suggest that CXCL14 is further downregulated in HPV-positive HNCs compared to HPV-negative HNCs and normal keratinocytes. To validate these observations using homogeneous keratinocyte culture models, we analyzed CXCL14 mRNA expression in NIKS cell lines with and without high-risk HPV genomes. We found that each high-risk

HPV (HPV16, HPV18, or HPV31) was sufficient to inhibit CXCL14 expression (Figure 4-

3D). Of note, CXCL14 expression was not downregulated in NIKS-16∆E7 cells, which contain an E7-deficient HPV16 genome (292) (Figure 4-3D). Furthermore, CXCL14 mRNA expression was modestly but significantly downregulated in NIKS cells expressing only the

E7 oncoprotein from HPV16 or HPV18 (Figure 4-3E). To detect secretion of the CXCL14 protein in cell culture supernatant, we performed an ELISA using culture supernatant from

NIKS and W12 cells. NIKS cells secreted a high level of CXCL14 protein, consistent with the previous study showing that normal keratinocytes constitutively express CXCL14 (282). In contrast, CXCL14 levels secreted by NIKS-16 and W12 cells were significantly decreased, indicating that the CXCL14 mRNA levels in NIKS and W12 cells correlate with CXCL14 secretion in cell culture supernatant (Figure 4-3F). Taken together, these results suggest the

57 HPV oncoprotein E7 is sufficient to suppress CXCL14 expression. However, long term exposure is required for dramatic repression as seen in the W12GPXY cells and HPV- positive cancers.

Next, using HPV-positive and -negative MOE cells, we assessed the effect of the

HPV16 oncoproteins E6 and E7 on murine Cxcl14 expression. A protein sequence alignment demonstrated 98% homology between human CXCL14 and murine Cxcl14 within the C-X-C chemokine motif (data not shown). Two neutral amino acid substitutions are observed within the C-X-C motif: human CXCL14 I70 and V75, corresponding with murine

Cxcl14 V58 and M63, respectively. We determined expression levels of Cxcl14 mRNA in

MOE cell lines, MOE/shPTPN13 (Ras transformed, HPV-negative) and MOE/E6E7 (Ras transformed, expressing the HPV16 oncogenes E6 and E7) that form tumors in immunocompetent syngeneic C57BL/6 mice (196). Consistently, Cxcl14 expression was also significantly downregulated in MOE/E6E7 cells compared to MOE/shPTPN13 cells

(Figure 4-3G and 4-3H). Taken together, our results suggest that CXCL14 expression is specifically inhibited in HPV-positive cells, likely in an E7-dependent manner.

CXCL14 Downregulation in HPV-Positive Keratinocytes is Associated with Promoter

Hypermethylation

Previous studies have shown that CXCL14 expression is suppressed by DNA hypermethylation in the CXCL14 promoter region (285). To determine whether HPV induces

CXCL14 promoter hypermethylation, we analyzed the methylation status of the CXCL14 promoter in NIKS, NIKS-16, and W12 cell lines using methylation-specific PCR (MSP), as previously described (284). We found the CXCL14 promoter region was hypermethylated differentiated and senesced when confluent, and therefore could not be used. Instead, we established CaSki and MOE/E6E7 cell lines re-expressing human CXCL14 or murine in

NIKS-16, W12E, W12G, and W12GPXY cells, but not in NIKS cells (Figure 4-5A).

Consistent with our results from cervical tissue specimens, the cervical keratinocyte lines

58 W12E, W12G, and W12GPXY showed gradually increasing levels of CXCL14 promoter hypermethylation during cancer progression (Figure 4-5A). To determine whether the HPV oncoprotein E7 affects CXCL14 promoter hypermethylation, we examined the methylation status of the CXCL14 promoter in NIKS-16∆E7 cells. Importantly, CXCL14 promoter hypermethylation was considerably less frequent in NIKS-16∆E7 cells (Figure 4-5A). These results indicate that the HPV16 E7 oncoprotein is necessary for HPV-induced CXCL14 promoter hypermethylation. Next, we analyzed the DNA methylation status of the CXCL14 promoter in HPV-positive vs. -negative MOE cell lines. Consistent with our results from the keratinocyte culture models, the CXCL14 promoter was hypermethylated in HPV-positive

MOE cells, but not in HPV-negative MOE cells (Figure 4-5B).

We determined the methylation status of the CpG island within the promoter region of CXCL14, using bisulfite sequencing on genomic DNA from NIKS, NIKS-16, W12E and

W12GPXY cells. Promoter amplicons were cloned from genomic DNA and 24 clones from each cell type were sequenced. Consistent with the MSP results above, there were no methylated cytidine residues detected in NIKS cells (Figure 4-5C). Conversely, DNA methylation in the CXCL14 promoter region appeared in NIKS-16 and W12E cells. A significantly higher frequency of CXCL14 promoter methylation was detected in the

W12GPXY cell line, showing that ~25% of the CXCL14 promoter clones contained multiple sites with DNA methylation (Figure 4-5C). These results indicate that CXCL14 promoter hypermethylation is induced by high-risk HPVs and accumulated over the course of cancer progression. This implies that other unknown factors in addition to E7 may be necessary for accumulation of CXCL14 promoter hypermethylation in HPV-positive cells. To examine

CXCL14 promoter hypermethylation in HPV-positive cancer tissues, we analyzed CXCL14

DNA methylation data from 279 HNC and 309 CxCa tissue samples obtained from the

TCGA database (291). Consistent with our results from keratinocytes, CXCL14 DNA methylation is significantly increased in HPV-positive HNC and CxCa compared to HPV-

59 negative HNC (Figure 4-4B). CXCL14 downregulation is highly correlated with CXCL14

DNA methylation in HPV-positive HNC and CxCa, but not in HPV-negative HNC (Figure 4-

4C to 4-4E). These results indicate that CXCL14 mRNA expression is controlled by CXCL14 promoter methylation in HPV-positive cancers. To verify CXCL14 downregulation by promoter hypermethylation, we determined whether the methylation inhibitor decitabine (5- aza-2’-deoxycytidine) restores CXCL14 mRNA transcription (293). Unfortunately, decitabine was toxic to NIKS cells and ineffective in W12 cells at all concentrations tested. Therefore, we examined consequences of DNA demethylation using an HPV16-positive CxCa cell line

(CaSki), which expresses low levels of CXCL14 transcripts (Figure 4-5D). Decitabine treatment for 6 days significantly increased CXCL14 expression in CaSki cells, together with a 50% decrease in CXCL14 promoter methylation as determined by quantitative MSP

(qMSP) (Figure 4-5E and 4-5F). These results suggest that reversing methylation at the

CXCL14 promoter, even partially, drastically increases CXCL14 expression in HPV-positive cancer cells. Taken together, our results suggest that HPV downregulates CXCL14 expression in HPV-positive cells by facilitating promoter hypermethylation.

CXCL14 Expression Hinders Cell Migration in vitro

Previous studies have shown that CXCL14 interferes with IL-8 and CXCL12 signaling, which are important for tumor cell migration and invasion (280, 289). Consistently,

CXCL14 downregulation suppresses migration of colorectal and tongue cancer cell lines

(294, 295). To determine the effects of restoration of CXCL14 expression on HPV-positive cell migration, an in vitro scratch assay was performed. Unfortunately, NIKS and W12 cells differentiated and senesced when confluent, and therefore could not be used. Instead, we established CaSki and MOE/E6E7 cell lines reexpressing human CXCL14 and murine

Cxcl14, respectively, using lentiviral transduction (Figure 4-6A and 4-6B). The expression level of CXCL14 in CaSki cells was comparable to the level seen in NIKS (Figure 4-6A).

The results showed that restoration of CXCL14 expression in both CaSki and MOE/E6E7

60 cells significantly delayed wound closure (Figure 4-6C to 4-6E). While the gaps were filled within 8 hours with control CaSki and MOE/E6E7 cells, both CaSki and MOE/E6E7 cells re- expressing CXCL14 showed wide gaps of 50 to 200 μm at 12 hours post wounding. To further corroborate these results, we performed a transwell migration assay using CaSki cells re-expressing CXCL14 with FBS as a generic chemoattractant. The data revealed that

CXCL14 expression significantly reduced CaSki cell migration compared to the vector only control (Figure 4-6F). CXCL14 expression did not affect proliferation of CaSki and

MOE/E6E7 cells (data not shown). Taken together, these results suggest that CXCL14 downregulation in HPV-positive HNC and CxCa cells increases epithelial cell motility.

Restoration of Cxcl14 Expression Clears HPV-Positive Tumors in Immunocompetent

Mice, but not in Rag1-Deficient Mice

To determine whether CXCL14 suppresses HPV-positive tumor growth in vivo, we established ~20 clones of MOE/E6E7 cells expressing various levels of murine Cxcl14 using lentiviral transduction. Untransduced and vector-transduced MOE/E6E7 cells consistently showed an over 30-fold decrease of Cxcl14 mRNA expression compared to HPV-negative normal parental MOE cells (Figure 4-7A). To define the in vivo effects of restored Cxcl14 expression, we tested two clones (8 and 16) of our Cxcl14 re-expressing MOE/E6E7 cells that had physiological levels of Cxcl14 mRNA expression comparable to parental MOE cells

(Figure 4-7A). Cxcl14 expression did not affect proliferation of MOE/E6E7 cells (data not shown). Wild type C57BL/6 mice were injected with 1 × 105 MOE/E6E7 cells, from our established clones, in the rear right flank. Tumor growth was monitored by measuring tumor volume for up to 11 weeks. Strikingly, restored Cxcl14 expression in MOE/E6E7 cells significantly suppressed tumor growth in wild type C57BL/6 mice, while vector-transduced

MOE/E6E7 cells rapidly formed tumors (Figure 4-7B). All ten mice transplanted with vector- transduced MOE/E6E7 cells succumbed to tumor burden within 5 weeks post injection

(Figure 4-7C). In contrast, five and seven out of ten mice transplanted with Cxcl14 re-

61 expressing MOE/E6E7 clones 8 and 16, respectively, were tumor-free up to 11 weeks post injection (Figure 4-7C and Figure 4-8A to 4-8C). To determine whether adaptive immune responses are involved in Cxcl14-mediated tumor suppression, we examined the tumor growth from these clones in Rag1-deficient C57BL/6 mice (Rag1-/-). Tumor growth was moderately slowed by restored Cxcl14 expression in Rag1-/- mice up to 14 days post injection (Figure 4-7D). However, all fourteen Rag1-/- mice injected with clones 8 and 16 exhibited tumor growth and succumbed to tumor burden within 5 weeks post injection

(Figure 4-7D, 4-7E, and Figure 4-8D to 4-8F). The results demonstrate no significant difference in tumor growth between wild type and Rag1-/- mice transplanted with vector control MOE/E6E7 cells at 21 days post injection (Figure 4-7F). These results indicate that

Cxcl14 expression is critical to trigger an adaptive immune response to clear implanted cancer cells in vivo.

Restored Expression of Cxcl14 Increases Natural Killer (NK), CD4+ T, and CD8+ T Cells in Tumor-Draining Lymph Nodes in vivo

To characterize immune cell infiltration regulated by Cxcl14 expression, we analyzed various immune cells in TDLNs and spleens harvested from the wild type C57BL/6 mice at

21 days post injection with vector or Cxcl14 re-expressing MOE/E6E7 cells. Using flow cytometry, we assessed populations of hematopoietic cells (CD45+) including NK cells

(NKp46+), CD4+ T cells (CD4+), CD8+ T cells (CD8+), antigen presenting cells (MHCII+), neutrophils (Gr1high), monocytes (Gr1mid), and macrophages (MHCII+, F4/80+). Our gating strategy for all interrogated cell types was based on cell populations detected in spleens and lymph nodes from C57BL/6 mice (Figure 4-9). Our data showed that percentages of NK,

CD4+ T, and CD8 + T cells were highly increased in TDLNs of the mice transplanted with

MOE/E6E7 cells re-expressing Cxcl14 (Figure 4-10). These results suggest that Cxcl14 increases infiltration of NK, CD4+ T, and CD8 + T cells into TDLNs, which may be critical for tumor clearance. This is consistent with our tumor growth results showing a moderate delay

62 in tumor growth by restored Cxcl14 expression in Rag1-/- mice, in which NK cell infiltration is increased in the absence of T cells (data not shown). These results suggest that NK cells alone may not be sufficient to clear HPV-associated tumors (Figure 4-7D and 4-7E). In addition to increased NK, CD4+ T, and CD8 + T cell infiltration, monocytes were also modestly increased in TDLNs of the mice injected with MOE/E6E7 cells re-expressing Cxcl14.

Conversely, Cxcl14 expression did not change antigen presenting cells, neutrophils, and macrophages in TDLNs (Figure 4-11), and marginal or no changes of these immune cell populations were observed in spleens by Cxcl14 expression (Figure 4-12). These results indicate that Cxcl14 locally affects NK, CD4+ T, and CD8 + T cell infiltration near the TME. To determine any difference in local and systemic immune responses altered by Cxcl14, populations of NK, CD4+ T, and CD8 + T cells were compared between TDLNs and distal lymph nodes (LNs) in the same mice injected with MOE/E6E7 cells with or without restored

Cxcl14 expression. We found that NK, CD4+ T, and CD8 + T cell populations were significantly decreased in TDLNs compared to distal LNs in mice injected with control

MOE/E6E7 cells (Figure 4-13). In contrast, mice injected with MOE/E6E7 cells re- expressing Cxcl14 showed significantly restored NK, CD4+ T, and CD8 + T cell populations in

TDLNs comparable to distal LNs (Figure 4-13). These results indicate that restoration of

Cxcl14 expression reverses suppression of antitumor immune responses by locally recruiting NK, CD4+ T, and CD8 + T cells.

Expression of Cxcl14 Induces Chemotaxis of NK, CD4+ T, and CD8+ T Cells in vitro

To determine whether expression of Cxcl14 in MOE/E6E7 cells induces chemotaxis of NK, CD4+ T, and CD8 + T cells, we performed an immune cell migration assay using the transwell system and splenocytes isolated from C57BL/6 mice. The results showed that conditioned medium from cultured MOE/E6E7 cells re-expressing Cxcl14 (clones 8 and 16) significantly increased NK, CD4+ T, and CD8 + T cell chemotaxis, while conditioned medium from MOE/E6E7 cells containing vector only has little effect compared to the negative

63 control (Figure 4-14A to 4-14C). Consistent with the in vivo immune cell infiltration, neutrophil migration was not affected by Cxcl14 expression (Figure 4-14D). These results suggest that Cxcl14 plays an important role in recruitment of NK, CD4+ T, and CD8 + T cells, which may enhance antitumor immune responses.

Discussion

Like most cancers, HPV-associated cancer development requires decades to progress from HPV-infected cells to invasive disease. Recent cancer genomics studies of

HNCs have reported that HPV-positive HNCs have far fewer oncogenic mutations (~5 per tumor) compared to HPV-negative HNCs (>20 per tumor) (296). These findings indicate that viral factors replace oncogenic processes usually triggered by multiple somatic mutations in

HPV-unrelated cancer progression. Other studies showed that continuous expression of the

HPV oncogene E7 is required for cancer growth and maintenance in vitro and in vivo (211,

246), suggesting that HPV E7 has multiple functions in HPV-associated cancer progression.

However, the mechanism by which HPV infection contributes to multiple steps of decades- long cancer progression is poorly understood.

Several proinflammatory chemokines such as IL-8, CXCL1, and CXCL12 drive cancer progression by facilitating tumor cell growth, survival, and migration, as well as by inducing angiogenesis (297). In our study, expression of proinflammatory chemokines IL-8,

CXCL1, CXCL2, and CCL3 was upregulated in the early stages of cancer progression

(Figure 4-1A and 4-1B). These chemokines are also increased in HPV-negative HNCs but to higher levels than HPV-positive HNCs, suggesting that most HNCs might have increased levels of proinflammatory chemokine expression that is pivotal for tumor cell migration and angiogenesis (298).

We found that CXCL14 was significantly downregulated during CxCa progression and in HPV-positive HNCs compared to HPV-negative HNCs (Figure 4-3A and 4-3C).

Constitutively expressed CXCL14 is an important homeostatic chemokine in normal

64 epithelial and neural tissue of mammals (282, 299, 300). Additionally, by directly binding to

IL-8, CXCL14 inhibits the ability of IL-8 to recruit endothelial cells and promote angiogenesis

(280), which is known to be essential for cancer progression. While specific receptors of

CXCL14 have not been identified, a recent study showed that CXCL14 binds to CXCR4 as a decoy ligand, inhibiting CXCL12 signal transduction through CXCR4 (289). This is an important signaling pathway for cell growth, angiogenesis, and metastasis in many cancers.

CXCL14 expression is frequently downregulated in cervical, prostate, colorectal, lung, and oral cancers (142, 282-284, 286, 294, 301). Overexpression of CXCL14 has shown antitumor effects by suppressing tumor growth and cancer cell migration in breast, oral, lung, and liver cancers (139, 285-287, 302, 303) . Consistently, our results here show that restored CXCL14 expression in HPV-positive cells significantly suppresses tumor growth in vivo (Figure 4-7).

Additionally, the HPV oncoprotein E7 induces CXCL14 promoter hypermethylation and significantly downregulates CXCL14 expression (Figure 4-3 and 4-5). A previous study determined that HPV16 E7 activates the methyltransferase activity of DNMT1 (139). A preliminary experiment detected upregulation of DNMT1 expression in HPV-positive cancers and keratinocytes (data not shown). These observations suggest that CXCL14 promoter methylation may be mediated through interactions between E7 and DNMT1. CXCL14 expression in HPV-positive CaSki cells was significantly increased following treatment with decitabine, an FDA-approved DNMT inhibitor (304) (Figure 4-5E). Previous studies have shown that DNA hypermethylation is associated with suppression of various immune factors including downregulation of cancer testis antigen, and MHC class I and chemokine expression (305). Consistently, inhibition of DNA methylation by decitabine increases expression of cancer testis antigens and MHC molecules and enhances cytotoxic NK and T cell antitumor activity (306-308). Decitabine treatment also activates expression of several different chemokines in a murine ovarian cancer model (309). Similarly, a recent study

65 showed that decitabine treatment enhanced antitumor immune responses by increasing

CXCL9 and CXCL10 expression and effector T cell infiltration (310). Thus, reversing the promoter hypermethylation of CXCL14 could be a feasible approach for restoring antitumor immune responses to treat HPV-positive cancers.

In our current study, we assessed the potential for CXCL14 to alter immune cell infiltration in TDLNs. We showed that restoration of Cxcl14 expression increases percentages of NK, CD4+ T, and CD8 + T cell populations in TDLN (Figure 4-10). Because tumor growth is only partially suppressed by Cxcl14 expression in Rag1-/- mice, our results indicate that both innate and adaptive immune responses play important roles in the antitumor functions of CXCL14. Consistently, a marked reduction in NK cell activity in uterine walls was observed in Cxcl14-/- mice, compared to Cxcl14+/- mice (311). In addition, NK cell depletion increases the risk of colorectal cancer in Cxcl14 transgenic mice (288). On the other hand, the effects of CXCL14 on T cells are completely unknown. Both NK and CD8+ T cells are well known as effector killer cells capable of eliminating virus-infected cells as well as cancer cells (312-314). NK cell activation induces CD8+ T cell responses through priming

DCs, suggesting that NK cells may be the link between innate and adaptive immunity to induce antiviral and antitumor CD8+ T cell responses (314, 315). Thus, our findings suggest that CXCL14, secreted by epithelial cells, might be one of the key regulators for NK, CD4+ T and CD8+ T cells to drive tumor clearance during HPV-associated cancer progression.

In conclusion, our study suggests that CXCL14 plays an important role in antitumor immune responses to clear HPV-positive HNC. CXCL14 is a small, secreted protein that can be used as a therapeutic agent. Additionally, identification of the native CXCL14 receptor(s) would provide druggable targets to enhance CXCL14 functions. Thus, further studies of the effects of CXCL14 on NK and T cells may provide a novel means of anti-cancer immunotherapy to treat HNCs

66 A IL-8 CXCL9 CXCL11 CCL3 CCL19 15 15 10 16 * * 15 * ** * * * 8 * * 12 * 10 10 10 6 8 4

5 5 4 5 2

0 0 mRNA expression level (log2) level expression mRNA

CIN3 CIN3 CIN3 CIN3 CIN1/2 CIN3 CIN1/2 CIN1/2 Normal Cancer CIN1/2 Normal Cancer Normal CIN1/2 Cancer Normal Cancer Normal Cancer B CXCL1 CXCL2 CXCL5 CXCL6 CCL20 16 15 16 15 15 * * * * * 12 12 10 10 10 8 8 5 4 5 4 5 0 0 mRNA expression level (log2) level expression mRNA

CIN3 CIN3 CIN3 CIN3 CIN3 CIN1/2 Normal CIN1/2 Cancer Normal CIN1/2 Cancer Normal CIN1/2 Cancer Normal CIN1/2 Cancer Normal Cancer

CDCXCL13 CCL8 CXCR2 CXCR4 15 12 * * 15 * 8 * * * * 10

10 * 6 8 10 6 4 5 4 2 5 mRNA expression level (log2) level expression mRNA mRNA expression level (log2) level expression mRNA

CIN3 CIN3 CIN3 CIN3 Normal CIN1/2 Cancer Normal CIN1/2 Cancer Normal CIN1/2 Cancer Normal CIN1/2 Cancer

Figure 4-1. Chemokine Expression is Deregulated in HPV-Associated Cancer Progression. Chemokines and chemokine receptors with significant changes of expression in CxCa progression are shown: (A) IL-8, CXCL9, CXCL11, CCL3, CCL19; (B) CXCL1, CXCL2, CXCL5, CXCL6, CCL20; (C) CXCL13, CCL8; and (D) CXCR2, CXCR4. The gene expression data were analyzed from a global gene expression study of 128 cervical tissue samples in different disease stages: normal (n = 24); low-grade lesion (n = 36); high-grade lesion (n = 40); and cancer (n = 28) (111). Normalized fluorescence intensities (log2) of gene expression from each group are shown in box-and-whisker plots with Tukey's method for outliers (black triangles) noted as distinct data points. P-values were calculated between each transition (normal to CIN1/2, CIN1/2 to CIN3, and CIN3 to cancer) by the Student’s t-test. *p<0.05 (A-D). These data were generated by Dohun Pyeon PhD, and Tao Xu PhD, University of Colorado.

67 ABCCXCL9 CXCL10 CXCL13 14 16 15 p=0.02 p=0.036 14 p=0.0003 12 12 10 10 10

8 8 6 5 mRNA expression level (log2) level expression mRNA HPV- HNC HPV+ HNC (log2) level expression mRNA HPV- HNC HPV+ HNC (log2) level expression mRNA HPV- HNC HPV+ HNC DEFCCL19 CXCR4 IL-8 12 14 p=0.01 14 p=0.03 p<0.008

10 12 12

10 10 8

8 8 6 6 6 mRNA expression level (log2) level expression mRNA HPV- HNC HPV+ HNC (log2) level expression mRNA HPV- HNC HPV+ HNC (log2) level expression mRNA HPV- HNC HPV+ HNC

GHIHPV16 early gene IL-8 CXCL1 2.0 20 25 ***

-actin 1.5 *** 20 β 15 -actin(fold) -actin(fold)

*** β β 15 1.0 10 10 ** 0.5 5 5 * mRNArelative to 0.0 0 0 mRNArelative to mRNArelative to

NIKS W12E W12G NIKS NIKS W12E W12G W12E W12G W12GPXY W12GPXY W12GPXY JKLCXCL2 CXCL10 CXCL11 20 *** 8 2.5 *** *** 2.0 15 6 -actin(fold) -actin(fold) -actin(fold) β β β 1.5 * 10 *** 4 * ** 1.0 5 2 ** 0.5

0 mRNArelative to 0.0 0 mRNArelative to mRNArelative to

NIKS NIKS W12E W12G W12E W12G NIKS W12E W12G W12GPXY W12GPXY W12GPXY

Figure 4-2. Proinflammatory Chemokines are Upregulated in HPV-Positive HNCs and Keratinocytes. (A-F) Gene expression levels of chemokines and chemokine receptors in head and neck were analyzed with HPV-positive (n = 16) and HPV-negative (n = 26) HNCs from our previous global gene expression study (233), as described in Fig. 1 and Supplementary Figure S1. Normalized fluorescence intensities (log2) of gene expression from each group are shown in box-and-whisker plots with Tukey's method for outliers (black circle) noted as distinct data points. P-values shown on each panel were calculated between HPV-negative and HPV-positive HNCs by the Student’s t-test. (G-L) Total RNA was extracted from NIKS, W12E, W12G, and W12GPXY keratinocyte lines. (G) HPV16 early gene transcript E1^E4 was measured by RT-qPCR, . (H-L) mRNA expression of IL-8, CXCL1, CXCL2, CXCL10, and CXCL11 were measured by RT-qPCR using specific primers (Appendix A), and normalized by β-actin mRNA. Data are shown as fold changes (± SD) to the mRNA level in NIKS cells. P-values were determined by the Student’s t-test. *p<0.05, **p<0.001, ***p<0.0001. These data were generated by Dohun Pyeon, PhD, University of Colorado and Tao Xu, PhD, St Jude Hospital, TN.

68 HNC A Cervical tissues B 1.2 Keratinocytes C 15 16 p < 0.005 1.0 p < 0.0001 -actin β 0.8 12 12 0.6

8 0.4 9 p < 0.0001 0.2 4 to relative mRNA 0.0 6 mRNA expression level (log2) level expression mRNA

mRNA expression level (log2) level expression mRNA HPV- HPV+

NIKS HNC HNC CIN3 W12E W12G Normal CIN1/2 Cancer W12GPXY DECXCL14 2.5 1.4 CXCL14 * 1.2 2.0 -actin -actin β β 1.0 1.5 0.8 * * 1.0 0.6 * 0.4 0.5 * 0.2

mRNA relative to to relative mRNA * 0.0 to relative mRNA 0.0 NIKS NIKS-16 NIKS-18 NIKS-31 NIKS-16 NIKS NIKS- NIKS- ΔE7 16E7 18E7 F CXCL14 ELISA GH 600 0.5 Cxcl14 250 HPV16 E7

** 0.4 200 400 0.3 150 * 200 * 0.2 100

CXCL14 (pg/ml) CXCL14 * 0.1 50 * 0 NIKS NIKS-16 W12E W12G W12GPXY 0.0 0 HPV- HPV+ HPV- HPV+ mRNA relative to 1000 mGAPDH 1000 to relative mRNA mRNA relative to 1000 mGAPDH 1000 to relative mRNA MOE MOE MOE MOE

Figure 4-3. CXCL14 expression is downregulated during HPV-associated cancer progression. CXCL14 mRNA expression levels were analyzed from global gene expression data sets of (A) 128 cervical tissue samples in different disease stages (normal, n = 24; low-grade lesion, n = 36; high- grade lesion, n = 40; and cancer, n = 28) (274) and (C) 42 HNC (HPV-HNC, n = 26; HPV+HNC, n = 16) (193) tissue samples. Normalized fluorescence intensities (log2) of gene expression from each group are shown in box-and-whisker plots with Tukey's method for outliers (black triangles) noted as distinct data points. P-values were determined by one-way ANOVA analysis (A) or the Student’s t-test (C). Total RNA was extracted from (B) W12 cell lines and (D and E) NIKS keratinocyte lines. The expression levels of CXCL14 were measured by RT-qPCR. (F) Secreted CXCL14 was measured by ELISA using culture supernatant from NIKS, NIKS-16, W12E, W12G and W12GPXY cells. (G & H) Total RNA was extracted from NIKS, NIKS-HPV16E7, NIKS-HPV18E7 and mouse oropharyngeal epithelial (MOE) cell lines, MOE/shPTPN13 (HPV-negative) and MOE/E6E7 (HPV-positive). The expression levels of HPV16 E1^E4 mRNA transcript (G) and murine Cxcl14 mRNA (H) were measured by RT-qPCR. HPV16 E1^E4 and CXCL14 mRNA copy numbers were calculated using serially diluted standard plasmids and normalized by human ß-actin and murine Gapdh mRNA copy numbers. P-values were calculated by the Student’s t-test. *p < 0.0001, **p = 0.0002. Panels A and C were contributed by Dohun Pyeon, PhD, University of Colorado. Panels B and D were contributed by Tao Xu, PhD, St. Jude Hospital, TN. Panels E-H contributed by Louis Cicchini, PhD, University of Colorado

69 mRNA expression DNA methylation p < 0.0001 p < 0.0001 1.0×106 p < 0.0001 n.s. 1.0 p = 0.0001 p = 0.03 100000 0.8 10000 1000 0.6

100 0.4

10 values Beta 0.2 1

RSEM normalized counts normalized RSEM 0.1 0.0 HPV- HPV+ CxCa HPV- HPV+ CxCa HNC HNC HNC HNC

HPV+ HNC HPV- HNC 100000 1000000 R2 = 0.3853 R2 = 0.0672 100000 10000 10000 1000 1000 100 100 mRNA expression (counts) expression mRNA mRNA expression (counts) expression mRNA 10 10 0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8 DNA methylation (β-values) DNA methylation (β-values)

CxCa 100000 R2 = 0.1389 10000

1000

100

10

mRNA expression (counts) expression mRNA 1 0.0 0.2 0.4 0.6 0.8 1.0 DNA methylation (β-values)

Figure 4-4. CXCL14 Downregulation Correlates with Increased CXCL14 Promoter Methylation in HPV-Positive HNC and CxCa. The TCGA data sets of CXCL14 RNA-seq RSEM counts (mRNA expression) and beta-values (DNA methylation) were retrieved from cBioPortal (cbioportal.org): HPV- HNC, n = 243; HPV+ HNC, n = 36; CxCa, n = 309 (NCI, TCGA, Provisional). (A & B) Shown are box- and-whisker plots with Tukey's method for outliers (black triangles) noted as distinct data points. P- values were determined by the Student’s t-test. n.s., not significant. Correlations between CXCL14 mRNA expression and DNA methylation were analyzed within HPV+ HNC (C), HPV- HNC (D), and CxCa (E). The correlation coefficient (R2) was determined by linear regression using Prism software. These data were generated by Dohun Pyeon, PhD, University of Colorado.

70

Figure 4-5. CXCL14 downregulation in HPV-positive epithelial cells is associated with CXCL14 promoter hypermethylation. Genomic DNA was extracted from (A) NIKS, NIKS-16, NIKS-16∆E7, W12E, W12G, and W12GPXY keratinocyte lines and (B) MOE/shPTPN13 (HPV-negative) and MOE/E6E7 (HPV-positive) cells. MSP was performed using specific primers and analyzed in 1.2% agarose gel as described in Experimental Procedures. MSP products of the control CXCL14 promoter and the hypermethylated CXCL14 promoter are indicated as “C” and “M”, respectively (B). (C) Bisulfite PCR products were cloned into the pGEM-T easy vector and sequenced. (D) CXCL14 expression was measured as described in Figure 4-3. (E & F) CaSki cells were treated with 10 μM decitabine for 6 days or a vehicle (H2O) control. RT-qPCR (E) and qMSP (F) were performed using total RNA and genomic DNA, respectively. CXCL14 mRNA copy numbers were normalized by ß-actin -∆∆C mRNA (E). Changes of CXCL14 promoter methylation were calculated using the 2 T method, and shown as a fold ratio of methylated signal over total signal (F). P-values were determined by Student’s t-test. Panels A-C were contributed by Tao Xu, PhD, St. Jude Hospital, TN. Panels D-F contributed by Louis Cicchini, PhD, University of Colorado

71

Figure 4-6. CXCL14 expression hinders mobility of HPV-positive cancer cells. (A & B) CXCL14 re-expressing CaSki and MOE/E6E7 cell lines were established using lentiviral transduction of the human CXCL14 and murine Cxcl14 genes, respectively, and validated by RT-qPCR. CXCL14 and Cxcl14 mRNA copy numbers were normalized by human ß-actin or murine Gapdh mRNA, respectively. In vitro scratch assay was performed with the established CaSki (C & D) and MOE/E6E7 (E) cells. Images were captured at 0, 4, 8, and 12 hours post wounding, and the width of the wound gaps were measured using NIH Image J software. Representative data from three replicates of each group are shown. The initial wound gaps (white dashed bar) and representative gaps at indicated time points (solid white bar) are shown. The scale bars (black bar) indicate 500 μm. (F) Transwell migration assays were performed on CaSki cells re-expressing CXCL14 generated as in (A). The percentage of cells that migrated through the permeable supports is shown, using 0%, 2.5%, and 5% FBS as a generic chemoattractant. P-values were calculated using the Student’s t-test. *p < 0.0001, **p < 0.03. Panels A-C were contributed by Tao Xu, PhD, St. Jude Hospital, TN. Panels D-F contributed by Louis Cicchini, PhD, University of Colorado

72 AB 104 6000 Vector ) 3 Cxcl14-clone 8

X) Cxcl14-clone 16 6 103 4000

102 p < 0.0001

GAPDH (10 2000 Expressionrelative to 1

10 Tumor volume (mm Parental MOE Vector Cxcl14 Cxcl14 MOE /E6E7 Clone 8 Clone 16 0 0 7 14 21 28 35 MOE/E6E7 clones Days CD 4000 Vector )

100 3 Cxcl14-clone 8 Cxcl14-clone 16 3000 Cxcl14-clone 16

2000 50 *p < 0.0001 Cxcl14-clone 8 1000 Percentsurvival Vector p < 0.0001 Tumor volume (mm 0 0 0 20 40 60 80 0 7 14 21 28 35 Days Days

EF n.s. 3200 100 ) Cxcl14-clone 8 3 *p < 0.0001 2400

1600 50 Cxcl14-clone 16 **

800 Percentsurvival

Vector p = 0.0015 Tumor volume (mm 0 0 0 20 40 60 80 Days Rag1-/- Rag1-/- Rag1-/- Wildtype Wildtype Wildtype Vector Cxcl14 Cxcl14 clone 8 clone 16

Figure 4-7. Restoration of Cxcl14 expression clears HPV-positive tumor in immunocompetent mice, but not in Rag1-deficient mice. MOE/E6E7 cell clones containing the Cxcl14 gene or vector were established and Cxcl14 expression levels were determined by RT-qPCR (A). Two MOE/E6E7 cell clones re-expressing Cxcl14 (clones 8 and 16) and one vector containing MOE/E6E7 cell clone were injected into the rear right flank of C57BL/6 (B, D, & E) and Rag1-/- (C, D, & F) mice (n = 10, each group of wild type; n = 7, each group of Rag1-/-). Tumor growth was determined every week by the formula: volume = (width)2 × depth. P-value was determined by one-way ANOVA analysis (B & C) and the Student’s t-test (D). Survival rates of wild type and Rag1-/- mice were analyzed using a Kaplan-Meier estimator (E & F). Time-to-event was determined for each group (vector only, Cxcl14- clone 8, Cxcl14-clone 16) with the event defined as a tumor burden larger than 2,500 mm3. Deaths not associated with tumor (samples collected for flow cytometry) were censored. P-values were determined by the Log-rank test (E & F). Panel A was contributed by Louis Cicchini, PhD, University of Colorado. Experiments for panels B-F were completed in collaboration with Dan Vermeer, Sanford Health.

73 aVector bCxcl14-clone 8 c Cxcl14-clone16 3000 3000 3000 ) ) 3 3 ) 3

2000 2000 2000

1000 1000 1000 Tumor volume (mm Tumor volume (mm Tumor volume (mm 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Days Days Days

dRag-vector eRag-clone 8 f Rag-clone 16 3000 3000 3000 ) ) ) 3 3 3

2000 2000 2000

1000 1000 1000 Tumor volume (mm Tumor volume (mm Tumor volume (mm 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Days Days Days

Figure 4-8. Restoration of Cxcl14 Expression Suppresses Tumor Growth in vivo. MOE/E6E7 cell clones re-expressing Cxcl14 (clones 8 and 16) and a vector containing MOE/E6E7 cell clone were injected into the rear right flank of wild type C57BL/6 (A-C) and Rag1-/- (D-F) mice (n = 10 for wild type, n = 7 for Rag1-/- each group). Tumor growth was determined every week by the formula: volume = (width)2 × depth. Tumor growth curves of each mouse are shown. These data were collected in collaboration with Dan Vermeer, Sanford Health.

74

Figure 4-9. Gating Strategy for Flow Cytometry. Whole spleen from a C57BL/6 mouse was homogenized and stained with a panel of antibodies conjugated to unique fluorophores. Single stain and no stain controls were used for fluorescence compensation. A generous large cell gate (forward scatter vs. side scatter area), single cell gate (side scatter area vs. side scatter width), and CD45+ gate (side scatter area vs. CD45) were applied as parental gates before determining antigen presenting cell (side scatter area vs. MHCII), neutrophil (side scatter high, Gr1high), monocyte (side scatter low, Gr1mid), and macrophage (MHCII+, F4/80+) populations. A small cell lymphocyte gate (side scatter area vs. forward scatter), and single cell (side scatter area vs. side scatter width) were applied as parental gates to determine NK cell (CD45+, NKp46+), CD4+ T cell (CD45+, CD4+), and CD8+ T cell (CD45+, CD8+) populations. A representative example of the overall gating strategy is shown and was applied to TDLN and spleen harvested from C57BL/6 mice injected with MOE/E6E7 cells with Cxcl14 or vector.

75

Figure 4-10. Cxcl14 expression increases NK, CD4+ T and CD8+ T cells in tumor draining lymph nodes. MOE/E6E7 cells with Cxcl14 (clones 8 and 16) or vector were injected into the rear right flank of C57BL/6 mice (n = 10, each group). Tumor-draining lymph nodes (TDLNs) were harvested from the mice at 21 days post injection. Percentage of immune cell populations defines the frequency of lymphocytes that were single cells and either NK (CD45+, NKp46+), CD4+ T (CD45+, CD4+), or CD8+ T (CD45+, CD8+) cells. Gating for flow cytometry was based on splenocyte populations and applied to TDLN samples as described in Figure 4-9. Representative flow cytometry diagrams are shown (A-C) and quantification of the indicated immune cells in each mouse tested appears (D-F). P-values were determined between vector and either clone 8 or clone 16 by the Student’s t-test. Data were collected in collaboration with Louis Cicchini, University of Colorado.

76 A MHC II B Neutrophil 80 0.3

60 0.2

40 0.1

20 0.0 Cell percentage Cell Cell percentage Cell

0 -0.1 Vector Clone 8 Clone 16 Vector Clone 8 Clone 16

Cxcl14 Cxcl14 C D Monocyte Macrophage 0.5 2.5 p = 0.001 p = 0.004 0.4 2.0

0.3 1.5

0.2 1.0 Cell percentage Cell 0.1 percentage Cell 0.5

0.0 0.0 Vector Clone 8 Clone 16 Vector Clone 8 Clone 16

Cxcl14 Cxcl14

Figure 4-11. Immune Cell Populations in TDLN Altered by Cxcl14 Expression. MOE/E6E7 cells with Cxcl14 (clones 8 and 16) or the vector were injected into the right flank of C57BL/6 mice (n = 10, each group). TDLNs were harvested at 21 days post injection. Percentages of antigen presenting cell (A), neutrophil (B), monocyte (C), and macrophage (D) populations were determined by flow cytometry using specific antibodies as described in Materials and Methods. P-values were determined between vector alone and clone 8 or clone 16 by the Student’s t-test. These data were collected in collaboration with Louis Cicchini, PhD, University of Colorado.

77 NK CD4 25 30

20 20 15 p = 0.005 10 p = 0.08 10

Cell percentage Cell 5 percentage Cell

0 0 Vector Clone 8 Clone 16 Vector Clone 8 Clone 16

Cxcl14 Cxcl14

CD8 MHC II 30 80

60 20

40

10 p = 0.004 20 Cell percentage Cell percentage Cell

0 0 Vector Clone 8 Clone 16 Vector Clone 8 Clone 16

Cxcl14 Cxcl14

Neutrophil Monocyte 8 2.0

6 1.5 4 1.0 2 0.5 Cell percentage Cell Cell percentage Cell 0

-2 0.0 Vector Clone 8 Clone 16 Vector Clone 8 Clone 16

Cxcl14 Cxcl14

Macrophage 2.5

2.0

1.5

1.0

Cell percentage Cell 0.5

0.0 Vector Clone 8 Clone 16 Cxcl14

Figure 4-12. Immune Cell Populations in Spleen Altered by Cxcl14 Expression. MOE/E6E7 cells with Cxcl14 (clones 8 and 16) or the vector were injected into the right flank of C57BL/6 mice (n = 10, each group). Spleens were harvested at 21 days post injection. Percentages of NK cell (A), CD4+ T cell (B), CD8+ T cell (C), antigen presenting cell (D), neutrophil (E), monocyte (F), and macrophage (G) populations were determined by flow cytometry using specific antibodies as described in Materials and Methods. P-values were determined between vector alone and clone 8 or clone 16 by the Student’s t-test. These data were collected in collaboration with Louis Cicchini, PhD, University of Colorado.

78

NK 60

p < 0.0001 40

20 Cell percentage Cell ns ns 0 Distal TDLN Distal TDLN Distal TDLN LN LN LN Vector Clone 8 Clone 16

CD4 50

40

30

20

Cell percentage Cell 10 p = 0.0003 p = 0.007 p = 0.005

0 Distal TDLN Distal TDLN Distal TDLN LN LN LN Vector Clone 8 Clone 16

CD8 40

30

20

10 Cell percentage Cell p < 0.0001 p = 0.01 p = 0.003

0 Distal TDLN Distal TDLN Distal TDLN LN LN LN Vector Clone 8 Clone 16

Figure 4-13. Cxcl14 expression restores decreased populations of NK, CD4+ T and CD8+ T cells in TDLNs. TDLNs (closed circle) and distal lymph nodes (distal LNs, open circle) were harvested from the mice injected with MOE/E6E7 cells re-expressing Cxcl14 (clones 8 and 16) or containing vector only. Percentage of NK, CD4+ T and CD8+ T cell populations were analyzed as described in Figure 4-10. P-values were determined between TDLN and distal LNs by the Student’s t-test. Data were collected in collaboration Jennifer Berger, University of Colorado.

79 p = 0.01 p = 0.02 25 NK 25 CD4 p = 0.01 p = 0.01 20 20

15 15

10 10

5 5 Percentmigration cell Percentmigration cell 0 0 Negative Vector Clone 8 Clone 16 Negative Vector Clone 8 Clone 16 control CM CM CM control CM CM CM

p = 0.02 25 CD8 40 Neutrophil p = 0.01 20 30

15 20 10 10 5 Percentmigration cell Percentmigration cell 0 0 Negative Vector Clone 8 Clone 16 Negative Vector Clone 8 Clone 16 control CM CM CM control CM CM CM

Figure 4-14. Cxcl14 expression induces chemotaxis of NK, CD4+ T and CD8+ cells. Conditioned media (CM) from the culture of MOE cells with Cxcl14 (clones 8 and 16) or vector were added into the bottom chamber of a transwell and supplemented with IL-2. Splenocytes isolated from C57BL/6 mice were added to the upper chamber. After 12 hour incubation, migrated splenocytes to the bottom chamber were collected and analyzed by flow cytometry. Percentage of immune cell populations defines the frequency of immune cells that were single cells and either (A) NK (CD45+, NKp46+), (B) CD4+ T (CD45+, CD4+), (C) CD8+ T (CD45+, CD8+) cells, or (D) neutrophils (CD45+, Gr1high). P-values were determined between vector and CXCL14 expressing cells (clones 8 and 16) by the Student’s t- test.

80 CHAPTER V

ANTIGEN-SPECIFIC CD8+ T CELL RESPONSE IS REQUIRED FOR CXCL14-MEDIATED

TUMOR SUPPRESSION IN HPV-POSITIVE HEAD AND NECK CANCER

Importance

Expression of the homeostatic chemokine CXCL14 is suppressed by human papillomavirus (HPV) in HPV-positive cancers. We have previously shown that restoration of

CXCL14 expression in HPV-positive tumor cells results in suppression of tumor growth and increased survival through an immune dependent mechanism. CXCL14 expression resulted in higher NK and T cell populations of the tumor draining lymph node and directly promoted their cell migration. The contribution of NK and T cells to enact the CXCL14 mediated tumor suppression remained undefined. To understand the contribution of NK and T cells in

CXCL14 mediated tumor suppression, we utilized in vivo mouse models deficient or specifically depleted of NK or CD8+T cells. Here we report that depletion or elimination of

CD8+ T cells results in loss of CXCL14 mediated tumor suppression. Using a CD8+ T cell receptor transgenic model, we show that the CD8+ T cells require antigen specificity to promote tumor suppression. Furthermore, we show that CD8+ T cells are capable of responding to and eliminating tumor cell in vitro. Finally, we show that CXCL14 increases tumor cell expression of MHC-I, and knockdown of MHC-I in CXCL14 expressing cells results in loss of tumor control. These results suggest that CXCL14 promotes a CD8+ T cell anti-tumor response and antigen specific CD8+ T cells are the principal effector driving tumor suppression.

Introduction

Persistent infection with high-risk human papillomavirus (HPV) is necessary for development of HPV-associated malignancies (209, 316, 317). HPV is associated with several human cancers including nearly all cervical cancers (CxCa) and an increasing number of head and neck cancers (HNC) (36, 318). In total, HPV is estimated to be

81 associated with over five percent of all human cancer incidences (208). To establish persistence in host, HPV must evade antiviral host defense including innate and adaptive immune responses. It is well known that there is a great similarity between antiviral and immune mechanisms (319-322)

CD8+ T cells have been shown to play a critical role in effecting successful antitumor immune responses (323-327). Several anti-cancer immunotherapies focus on the enhancing the activity of cytotoxic CD8+ T cells to promote targeting and clearance of cancer cells

(277, 328-331). These immunotherapies have shown great efficacy in a spectrum of cancers

(332, 333). An effective CD8+ T cell response is also of critical importance in HPV persistence and related diseases (334, 335). Indeed, Trimble et al were able to predict if a

HPV infected lesion would regress based on quantity of CD8+ T cells infiltration of infected tissue (336). Furthermore, several studies have shown that the high level of CD8+ T cell infiltrates in tumor is a strong prognostic indicator for positive disease-free survival in HPV- associated cancers (331, 337). However, the mechanism of how CD8+ T cells are recruited to the tumor microenvironment to eliminate cancer cells remains elusive.

Chemokine (C-X-C motif) ligand 14 (Cxcl14) is a homeostatic chemokine that promotes immune surveillance of skin epithelia through recruitment of several immune cell types, including dendritic cells (DCs), natural killer (NK) cells, and T cells (140, 338, 339).

Downregulation of CXCL14 expression is frequently observed in several cancers including

HNC, CxCa, breast cancer, and renal cancer (282, 340). Previously, we have shown that the

HPV oncoprotein E7 is responsible for the epigenetic repression of CXCL14 transcription in

HPV-positive keratinocytes and cancer cells (140). We further revealed that restoration of

CXCL14 expression in HPV-positive HNC cells dramatically suppresses tumor growth in vivo.

Here, we report that the CXCL14-mediated tumor suppression is reliant on an antigen-specific CD8+ T cell response. Our results suggest that CXCL14 enacts effective

82 tumor suppression through recruitment of CD8+ T cells and restoration of MHC-I antigen presentation.

Results

CXCL14-Mediated Tumor Suppression Requires CD8+ T cells

We previously have shown that CXCL14 expression significantly enhances NK and T cell migration and infiltration into the tumor draining lymph nodes (140). To examine the contribution of NK and CD8+ T cells in the CXCL14-mediated antitumor immune response,

NK and CD8+ T cells were depleted in wildtype B6 mice using anti-NK1.1 and anti-CD8a neutralizing antibodies, respectively (Figure 5-1A). To validate the specific cell depletion, we evaluated NK and CD8+ T cell populations in peripheral blood by flow cytometry. The results showed that NK and CD8+ T cell populations were decreased to 0.8% and 0.4% of the

CD45+ cell population, respectively (Figure 5-1B). Next, tumor growth was monitored in the

NK and CD8+ T cell-depleted mice injected with MOE/E6E7Vector or MOE/E6E7CXCL14 cells.

As expected, mice injected with MOE/E6E7Vector cells showed robust tumor growth regardless of isotype control and either NK or CD8+ T cell depletion (Figure 5-1C and 5-

1F). In contrast, while the isotype control mice injected with MOE/E6E7CXCL14 cells showed significant tumor suppression, NK cell depletion facilitated tumor growth in mice injected with MOE/E6E7CXCL14 cells as 8 out of 10 mice eventually grow tumor despite the lack of a statistical significance (Figure 5-1D and 5-1G). This suggests that NK cells may contribute to the antitumor effect but are not required for CXCL14-mediated tumor suppression.

Interestingly, however, all CD8+ T cell-depleted mice injected with MOE/E6E7CXCL14 cells exhibited robust tumor growth (Figure 5-1D and 5-1G). All MOE/E6E7CXCL14 cell injected

CD8 + T cell depleted mice succumbed with in 60 days (Figure 5-1E). These results suggest that CD8+ T cells are required for CXCL14-mediated tumor suppression.

Although all CD8+ T cell-depleted mice grew tumors, the tumor growth was delayed as compared with the control mice injected with MOE/E6E7Vector cells. This observation

83 indicates two possibilities: 1) that, in addition to CD8+ T cells, another cell type such as NK cells are necessary for the full response of CXCL14-mediated tumor suppression; and/or 2) that CD8+ T cell depletion by the anti-CD8a antibody is incomplete and a subset CD8+ T cells still remain, particularly in the TME. To further determine whether CD8+ T cells are required for CXCL14-mediated tumor suppression, we tested tumor growth in wildtype and

CD8a knockout mice injected with MOE/E6E7Vector or MOE/E6E7CXCL14 cells. Absence of

CD8+ T cells was confirmed by flow cytometry (Figure 5-2). We found that all wildtype and

CD8a knockout mice injected with MOE/E6E7Vector cells robustly grew tumors and succumbed to tumor burden within 35 days post injection (Figure 5-3A and 5-3B).

Interestingly, while the majority of the wildtype mice injected with MOE/E6E7CXCL14 cells did not grow tumor, all CD8a knockout mice showed robust tumor growth (Figure 5-3A and 5-

3C). As a result, all CD8a knockout mice succumbed to tumor burden within 35 days post injection, showing similar tumor growth kinetics as mice injected with MOE/E6E7Vector cells

(Figure 5-3D). When interpreted in context of the delayed tumor growth observed with antibody-based CD8+ T cell depletion (Figure 5-3D and 3F), these results indicate that even a small population of CD8+ T cells responding to CXCL14 can slow tumor growth.

Taken together, our results suggest that CD8+ T cells are the predominant driver of

CXCL14-mediated tumor suppression in HPV-positive HNC.

As CD8+ T cells are necessary to enact CXCL14 mediated tumor suppression and

CXCL14 expression significantly enhances T cell migration and infiltration into the tumor draining lymph nodes (140), we next determined the presence and abundance of CD8+ T cells in tumors with and without CXCL14 expression. To determine if CXCL14 induces T cell infiltration into the TME, we analyzed CD8+ T cells in frozen tumor tissue from wildtype

C57BL/6 (B6) mice injected with MOE/E6E7Vector or MOE/E6E7CXCL14 cells. Using immune fluorescence, CD8+ T cells were detected by anti-CD8a antibody, along with cytokeratin

84 counterstaining for epithelial cells and the percentage of CD8+ T cells per total cells in the

HPV-positive tumors expressing CXCL14 was determined. The results showed that infiltration of CD8+ T cells significantly increased in tumors from mice injected with

MOE/E6E7CXCL14 cells as compared to tumors from mice injected with MOE/E6E7Vector cells

(Figure 5-4). These data suggest that CXCL14 expression increases the CD8+ T cell population in the TME, similar to what was observed in the TDLN. Furthermore, this increase in CD8+ T cells may be critical for CXCL14-mediated tumor suppression.

CXCL14-Mediated Tumor Suppression Requires Antigen-Specific CD8+ T Cells

The activation of CD8+ T cells require interaction of the T cell receptor (TCR) with its cognate peptide presented by major histocompatibility complex class I (MHC-I) proteins. To evaluate if antigen specificity of CD8+ T cells is required for CXCL14-mediated tumor suppression, we utilized the MHC-I restricted, chicken ovalbumin TCR transgenic (OT-1) mouse model (341). The typical T cell repertoire in wildtype mice is estimated to be responsive to 2 million different peptides (342, 343). In contrast, OT-1 mice are genetically modified to have their CD8+ T cell responsive repertoire highly restricted to the chicken ovalbumin peptide sequence, SIINFEKL. Although the CD8+ to CD4+ T cell ratio is skewed in favor of CD8+ T cells as compared to wildtype, all immune cell populations are present

(Figure 5-2) (344). We tested tumor growth and survival in a cohort of wildtype and OT-1 mice injected with MOE/E6E7Vector or MOE/E6E7CXCL14 cells. As with the CD8a knockout, both wildtype and OT-1 mice injected with MOE/E6E7Vector robustly grew tumors and all mice succumbed to tumor burden within 35 days post injection (Figure 5-5A and 5-5B).

Interestingly, while the majority of the wildtype mice injected with MOE/E6E7CXCL14 cells showed no or delayed tumor growth, all OT-1 mice robustly grew tumors regardless of

CXCL14 expression (Figure 5-5A and 5-5C). As was observed in the CD8a knockout mice, all OT-1 mice injected with MOE/E6E7CXCL14 cells succumbed to tumor burden within 35

85 days (Figure 5-5D). These results suggest that even in the presence of the CD8+ T cell population, antigen specificity of the CD8+ T cells is critical for CXCL14-mediated tumor suppression

CXCL14 Expression Restores MHC-I Expression on Tumor Cell Surface

We have shown that the CXCL14 mediated CD8+ T cell tumor suppression is reliant on CD8+ T cell recognition of antigen. Counter intuitively, it has been well established that the HPV oncoproteins suppress MHC-I expression and trafficking (170, 198, 345, 346).

These findings were recapitulated in our model system as MHC-I (H-2Db) expression was greatly decreased in MOE/E6E7 cells compared to HPV-negative normal immortalized MOE cells, the parental cell of MOE/E6E7 cells (Figure 5-6A). Given that CD8+ T cell activation depends on interaction with MHC-I, we evaluated if CXCL14 expression increases MHC-I expression in MOE/E6E7 cells by detection of the two MHC-I alloantigens in B6 mice, H-2Dd and H-2Kb, using flow cytometry (347). Strikingly, H-2Db expression was restored in both 8 and 16 of MOE/E6E7CXCL14 clones (CL8 and CL16) comparable to the level of HPV-negative parental MOE cells (Figure 5-6B and 5-6C). However, little change was observed in the H-

2Kb levels regardless of CXCL14 expression (Figure 5-6D). To ensure proper detection of

H-2Kb, we demonstrated a significant increase of H-2Kb expression by interferon gamma

(IFNγ) treatment (Figure 5-6E). This indicates that H-2Kb expression is not affected by

CXCL14 expression. Our results suggest that CXCL14, in addition to promoting the migration of CD8+ T cells, may also augment antitumor immune recognition of CD8+ T cells by restoring MHC-I expression in HPV-positive tumor cells.

CXCL14-Mediated Tumor Suppression is Abrogated by Knockdown of MHC-I

Expression in Tumor Cells

As CXCL14 expression restores MHC-I expression in HPV-positive tumor cells, we next determined if knockdown of MHC-I in CXCL14 expressing cells had impact on tumor growth. To determine the role of MHC-I in CXCL14-mediated tumor suppression, we

86 knocked down β2-microglobulin (B2M) in MOE/E6E7CXCL14 cells using short hairpin RNAs

(shRNAs). B2M is a core component of the MHC-I complex, which is necessary for the proper formation of the MHC-I complex on the cell surface (Reviewed in (348)). Disruption of

B2M blocks MHC-I surface expression and antigen presentation to CD8+ T cells (349). We transduced the MOE/E6E7CXCL14 cells with a pool of shRNA clones against B2M

(MOE/E6E7CXCL14/shB2M). To enrich cells with B2M knockdown, we sorted out

MOE/E6E7CXCL14/shB2M cells by selecting cells with the lowest 10% of MHC-I expression levels using fluorescence associated cell sorting. The sorted MOE/E6E7CXCL14/shB2M cells showed significantly decreased H-2Db expression similar to MOE/E6E7Vector cells despite

CXCL14 expression (Figure 5-7A). Interestingly, H-2Kb, which had previously shown nominal changes due to CXCL14 expression, was also slightly decreased in

MOE/E6E7CXCL14/shB2M cells (Figure 5-7B). MOE/E6E7CXCL14/shB2M cells exhibited no significant difference in doubling time, HPV oncoprotein expression, or CXCL14 expression, as compared to the parental MOE/E6E7CXCL14 cells (Figure 5-8A to 5-8C). To determine if

B2M knockdown abrogates CXCL14-mediated tumor suppression in vivo, we monitored tumor growth in wildtype B6 mice injected with MOE/E6E7CXCL14/shB2M cells. Interestingly, the majority of the mice injected with MOE/E6E7CXCL14/shB2M cells exhibited robust tumor growth

(Figure 5-7B and 5-7D). Further, nine out of ten mice injected with MOE/E6E7CXCL14/shB2M cells succumbed to tumor burden within 50 days post injection, while eight out of ten mice injected with MOE/E6E7CXCL14 cells survived without tumor over 80 days (Figure 5-7C).

These results suggest that upregulation of MHC-I expression by CXCL14 expression is critical for CXCL14-mediated tumor suppression through CD8+ T cell response.

CXCL14 expression in tumor cells induces CD8+ T cell activation.

Although CD8+ T cells are infiltrated into the TME, immune suppression induced by various immune checkpoint proteins such as PD-1 and CTLA-4 interferes with antitumor functions of CD8+ T cells (Reviewed in (325), (350, 351)). To test if CXCL14 expression

87 induces CD8+ T cell activation, we determined production of IFNγ, TNFα, and IL-2, and cytolytic activity of CD8+ T cells in co-culture of CD8+ T cells and MOE/E6E7 cells in vitro

(352-355). CD8+ T cells were isolated from mice injected with MOE/E6E7Vector cells (denoted

“null-CD8” as no tumor suppression was maintained) or MOE/E6E7CXCL14 cell (denoted

“primed-CD8” as tumor suppression was maintained). The isolated null- or primed-CD8+ T cells were co-cultured with the mitomycin-treated target cells (MOE/E6E7Vector or

MOE/E6E7CXCL14 cells). First, IFNγ, TNFα, and IL-2 protein levels in supernatants of co- cultured cells were determined by ELISA. CD8+ T cells isolated from OT-1 mice co-cultured with MOE/E6E7CXCL14 cells expressing the chicken ovalbumin (MOE/E6E7CXCL14/Ova) were used as a positive control for CD8+ T cell activation. We also included CD8+ T cells stimulated with phorbol-12-myristate-13-acetate and ionomycin (PMA/iono) as another positive control. As expected, IFNγ and TNFα production was significantly increased in both positive control (Figure 5-9A and 5-9B). Interestingly, IFNγ and TNFα production was highly induced when primed-CD8+ T cells were co-cultured with MOE/E6E7CXCL14 cells, but not with MOE/E6E7Vector cells. IFNγ, but not TNFα, was produced when null-CD8+ T cells were co-cultured with MOE/E6E7CXCL14 cells, but not with MOE/E6E7Vector cells (Figure 5-9A and

5-9B). No IL-2 production was detected in either null- nor primed-CD8+ T cells co-cultured with the target cells nor CD8+ T cells from OT-1 mice, while PMA/iono-treated CD8+ T cells produced the high level of IL-2 (Figure 5-9C). These results suggest that CXCL14 expression in tumor cells induces IFNγ and TNFα production in CD8+ T cells.

To assess the cytolytic capacity of CD8+ T cells affected by CXCL14 expression in target tumor cells, we performed a lactate dehydrogenase (LDH) release assay that measures cytoplasmic LDH in cell culture supernatant released by cell lysis (356). Briefly, null-CD8+ T, or primed-CD8+ T cells were added to prepared target cells, MOE/E6E7Vector,

MOE/E6E7CXCL14, or MOE/E6E7CXCL14/shB2M cells. After 24 hours of incubation, the cell culture supernatant was collected and LDH levels were measured. Interestingly, primed-CD8+ T

88 cells efficiently killed both MOE/E6E7Vector and MOE/E6E7CXCL14 cells, but not

MOE/E6E7CXCL14/shB2M cells, at 10:1 effector-target ratio (Figure 5-9D). These results suggest that the primed-CD8+ T cells are sufficient to kill tumor cells but require MHC-I expression. Surprisingly, null-CD8+ T cells still showed a significant cell killing activity against MOE/E6E7CXCL14 cells at 10:1 effector-target ratio (Figure 5-9E). In contrast, the null-CD8+ T cells could not kill MOE/E6E7Vector and MOE/E6E7CXCL14/shB2M cells. These data further reinforce our findings that CXCL14 expression substantially enhances CD8+ T cell activity through priming of CD8+ T cells and by increasing MHC-I expression in tumor cells.

Taken together, our results strongly suggest that CXCL14 expression in tumor cells induces tumor suppression by driving CD8+ T cell activation and facilitating tumor cell killing.

Discussion

The CD8+ T cell infiltration into tumors correlates positively with patient survival is appreciated in several contexts of cancer, including HPV-associated cancers. HPV- associated cancers often take decades to develop and HPV must persist within the infected cell by evading immune clearance (272). Although the majority of infections are ultimately cleared by the immune response, a small percentage of patients with a persistent infection of a high-risk HPV genotype progress to cancer (30).

It has been well established that T cell responses are required to eliminate HPV- infected cells (357-359). An indicator of immune mediated regression of HPV infections is the high infiltration of both CD4+ and CD8+ T cells in infected tissues (360). Furthermore, activated intraepithelial CD8+ T cells is highly correlative to the regression rate of cervical precancerous lesions (123). It has been shown in the mouse papillomavirus (MmuPV1) model, that CD4+ and CD8+ T cells were necessary for clearance of MmuPV1 infections

(125). Interestingly, only when both CD4+ and CD8+ T cell compartments were eliminated, did infection persist, demonstrating the potent capacity to eliminate the infection. In persistent infections, immune tolerance driven by HPV limits the function of these immune

89 cells to enact a productive immune response (361). Recently it has been shown that

Langerhans cells, DCs residents of the epithelium, isolated from women with persistent HPV infection cannot activate CD8+ T cells by presenting HPV antigens alone (362). Additional immune-stimulatory factors are necessary for CD8+ T cell activation. In HPV-associated

OPSCC, antigen specific CD8+ T cells TILs are highly correlative with positive patient outcomes (363). Like HPV infected tissues CD8+ T cells are limited by insufficient activation by either suppressed levels of antigen presentation or through E7 mediated expression of suppressor signal like PDL1 (364, 365). To combat this tolerance, several immunotherapies have been tested with mixed results (366). Thus, it is of great importance to find factors that can recruit and facilitate the activation of CD8+ T cells in these cancers.

Although it is well accepted that CD8+ T cells play an important role in immune responses against HPV-associated cancers, the mechanisms of recruiting and activating antitumor CD8+ T cells in the TME remain elusive. We have previously shown that restoration of CXCL14 expression in HPV-positive tumor cells is sufficient for NK and T cell recruitment and tumor suppression (140). Here, we report that antigen-specific CD8+ T cells are required for CXCL14-mediated tumor suppression. Additionally, we show that CXCL14 expression upregulates MHC-I expression on HPV-positive HNC cells, recruits CD8+ T cells to the TME and activates CD8+ T cells to kill syngeneic tumor cells.

CXCL14 is a is highly conserved chemokine normally expressed in the normal skin keratinocytes (367). Constitutive expression of CXCL14 in these tissues promote immune surveillance by the recruitment of immune cells (338). Previous studies have shown that

CXCL14 recruits various immune cells including NK cells, dendritic cells (DC), monocytes, and neutrophils (141, 142, 311, 367). Shurin et al. revealed that CXCL14 mediated the recruitment and activation of DCs to the TME in vivo (142). However, since this study used

SCID mice that lacks adaptive immunity, the roles of T cells in CXCL14-mediated tumor suppression had not been evaluated.

90 Our finding of CXCL14-mediated anti-tumor immune response suggests that

CXCL14 would be a useful tool to develop a novel immunotherapy for HPV-positive CxCa and HNC patients. Although efficacy needs to be determined, CXCL14 may induce tumor suppression in other cancers beyond HPV-positive cancers. CXCL14 expression has also been shown to be downregulated in multiple cancer types including gastric, lung, and prostate cancers (284, 285, 368). Indeed, restoration of CXCL14 expression triggers tumor suppressive effects in several models of HNC, lung, and colorectal cancers (142, 283, 285,

288, 369). In addition, CXCL14 could be used to treat HPV-positive premalignant lesions for cancer prevention. Although current HPV vaccines are effective to prevent new HPV infection, there is no means to treat individuals persistently infected with HPV. Because

CXCL14 expression restores MHC-I expression in HPV-infected keratinocytes and mediates

CD8+ T cell response, delivery of CXCL14 may be useful to remove HPV-infected cells and prevent cancer progression.

On the other hand, several studies have shown that CXCL14 expression levels are correlated to poor clinical outcome in pancreatic cancer, osteosarcomas, and a subtype of prostate cancers (370-372). As a homeostatic chemokine, CXCL14 is expressed constitutively in epithelial layers by dermal fibroblasts (367). Interestingly, CXCL14 expression from fibroblasts result in more severe disease pathologies in mouse tumor models (372, 373). In HPV cancer progression, peripheral expression of CXCL14 may promote a pro-tumor environment due to the conflicting stimuli. CXCL14 has been shown to contribute to a pro-inflammatory immune response (374). When the CXCL14 recruited immune cells are recruited to the microenvironment, the potent HPV mediated immune suppression would stunt the immune response and potentially, result in immune tolerance.

Thus, further studies are necessary to better understand the mechanisms of CXCL14- mediated immune regulations in the TME of different cancers.

The native receptor(s) of CXCL14 still remains unidentified, likely due to low binding

91 affinity of CXCL14 protein associated with a truncated N-terminus receptor binding sequence (338, 375). This is a major limitation of studying the mechanisms of CXCL14 and developing therapeutic methods based on CXCL14. Several studies have suggested that

CXCL14 may play a role in the CXCL12/CXCR4 signaling axis, although with highly different outcomes. Tanegashima et al. has shown that CXCL14 binds to the CXCR4 receptor with high affinity, preventing CXCL12 from interacting with CXCR4 (289). Another group has shown a conflicting result where CXCL14 did not affect the signaling properties of CXCL12

(376). A third observation is that CXCL14 synergizes with CXCL12 signaling through CXCR4

(377). This is accomplished through redistribution of the CXCR4 receptors on the cell surface. Without confirmation of the CXCL14 receptor, the field is limited in its capacity to clearly identify in which contexts therapy would be beneficial and restricts the development of highly specific treatments.

Many cancers have been shown to suppress MHC-I expression as a mechanism to evade an anti-tumor immunity (378-380). Particularly, a significant decrease of MHC-I repression is commonly observed in HPV-positive cells and cancer (381). The HPV oncoproteins utilize various mechanisms to downregulate MHC-I expression in infected cells. HPV E5s bind to MHC-I, trapping it the Golgi apparatus, preventing trafficking at the cell surface (38, 170, 171). Previous work from our lab has shown that all but one (HLA-F)

MHC-Ia subunits are repressed in HPV positive keratinocytes, likely through an E7 mechanism (198). Furthermore, the MHC-Ia subunit HLA-E expression is suppressed by E7 mediated promoter methylation. Other studies have implicated E7 mediates suppression of the TAP1 protein as a major factor in MHC-I suppression. (175). In addition to MHC-I, HPV

E5 downregulates MHCII expression of the non-classical MHC molecule CD1d (176, 177).

Signaling to CD4+ T cells and NKT, respectively (178).

Here we shown that CXCL14 expression in MOE/E6E7 cells increases the level of

92 MHC-I. While the mechanism of increased expression remains to be determined, these data suggest that CXCL14 may be impacting the tumor microenvironment by more than just promoting the recruitment of immune cells. A breakdown of the HPV mediated immune suppression is necessary for a productive CD8+ T cell response, and expression of CXCL14 appears to facilitate this. Thus, it would be of great interest to determine other changes that further promote tumor suppression. These findings would provide useful insight into the mechanism that increases CD8+ T cell recognition of tumor cells and boost the efficacy of current immunotherapies (378).

Taken together, our study shows that re-expression of CXCL14 in HPV-positive HNC, enacts a potent anti-tumor immune response reliant on recruitment and activation of antigen-specific CD8+ T cells. Furthermore, in addition to promoting cell migration, CXCL14 expression impacts the microenvironment by increasing MHC-I to further immune recognition of the tumor cells.

93 A Tumor implantation

Day 0 7 14 21 28 35 42

Antibody injections B NK cell CD8+ T cell 100 100 Isotype Anti-NK1.1 Isotype Anti-CD8α 75 75

50 50 % of Max %of % of Max %of 25 3.2% 0.8% 25 8.3% 0.4%

0 0 101 102 103 104 105 106 107 101 102 103 104 105 106 107 101 102 103 104 105 106 107 101 102 103 104 105 106 107 NKp46 NKp46 CD8a CD8a C D E IsotypeMOE/E6E7 control IsotypeMOE/E6E7 control Isotype control 4000 NK-depletedMOE/E6E7 4000 NK-depletedMOE/E6E7 NK-depleted 100 CD8-depletedCD8-depletedMOE/E6E7 CD8-depletedMOE/E6E7 Isotype control )

) 3000 3000 3 3 * 2000 n.s 2000 n.s 50

(mm n.s (mm 1000 1000 NK-depleted Tumor Volume Tumor Volume *

Percent survival CD8-depleted 0 0 0 0 10 20 30 40 50 0 10 20 30 40 50 0 20 40 60 80 100 Days Days Days F MOE/E6E7Vector cells: MOE/E6E7Vector cells: MOE/E6E7Vector cells: 4000 IsotypeMOE/E6E7 controlVector : 4000 NK NKdepleted depletion 4000 CD8CD8 depletiondepleted 3000 Isotype control 3000 3000 ) 3 2000 2000 2000 (mm 1000 1000 1000

Tumor Volume 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Days Days Days G MOE/E6E7CXCL14 cells: MOE/E6E7CXCL14 cells: MOE/E6E7CXCL14 cells: Isotype control NK depleted CD8 depleted 4000 4000 4000

) 3000 3000 3000 3 2000 2000 2000 (mm 1000 1000 1000 Tumor Volume 0 0 0 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Days Days Days

Figure 5-1. CD8+ T cell depletion abrogates CXCL14-mediated tumor suppression.

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Figure 5-1. CD8+ T cell depletion abrogates CXCL14-mediated tumor suppression. Wildtype B6 mice (n = 10 per group) were depleted of NK and CD8+ T cells using anti-NK1.1 and anti-CD8 antibodies, respectively, with biweekly intraperitoneal (i.p.) injections. Corresponding isotype antibodies were used as controls. Two days after the first antibody injection, MOE/E6E7Vector or MOE/E6E7CXCL14 cells (5 × 105 cells/mouse) were subcutaneously (s.c.) injected into the left flank of each mouse (A). NK and CD8+T cell depletion was validated using peripheral blood from the mice by flow cytometry (B). Tumor volume was measured twice a week in mice injected with either MOE/E6E7Vector (C) or MOE/E6E7CXCL14 (D) cells. Survival rates of MOE/E6E7CXCL14 cell injected mice were analyzed using a Kaplan-Meier estimator. The time to event was determined for each group (isotype, NK, and CD8+T cell depletion) with the event defined as a tumor burden larger than 2,500 mm3. Deaths not associated with tumor were censored. P values were determined by the log rank test (E). Values that were not significantly different (n.s.) are also shown. Shown are individual tumor growth curves injected with MOE/E6E7Vector (F) or MOE/E6E7CXCL14 (G) cells. P values of NK or CD8+T cell depleted mice compared to isotype injected mice were determined for tumor growth (C and D) and survival (E) by one-way ANOVA analysis. * = p < 0.0001.; n.s., not significant. Shown are representative of 2 independent experiments. Experiments was completed in collaboration with and by Dan Vermeer, Sanford Health.

95

Figure 5-2. Immune populations of Wildtype and OT-I transgenic mice. CD4+ and CD8+ T cell populations were determined in splenocytes from wildtype, OT-I, or CD8a-/- mice by flow cytometry

96 A MOE/E6E7Vector cells B MOE/E6E7CXCL14 cells Vector ) ) 4000 WT mice 4000 WTMOE/E6E7 mice 3

3 MOE/E6E7 : Wildtype CD8MOE/E6E7α-/- miceVector : CD8α -/- CD8MOE/E6E7α-/- mice 3000 3000

2000 2000

* 1000 n.s 1000 Tumor Volume (mm 0 Tumor Volume (mm 0 0 5 10 15 20 25 0 5 10 15 20 25 C Days Days

MOE/E6E7Vector cells : WT mice Vector -/- ) 4000 4000 MOE/E6E7 cells : CD8α mice 3

3000 3000

2000 2000

1000 1000 Tumor Volume (mm 0 0 0 20 40 60 80 0 20 40 60 80 D Days Days

CXCL14 CXCL14 -/- ) 4000 MOE/E6E7 cells : WT mice 4000 MOE/E6E7 cells: CD8α mice 3

3000 3000

2000 2000

1000 1000 Tumor Volume (mm 0 0 0 20 40 60 80 0 20 40 60 80 E Days Days MOE/E6E7Vector cells MOE/E6E7CXCL14 cells 100 100 WT mice

50 WT mice 50

-/- CD8α -/-

Percent survival CD8α Percent survival mice n.s mice * 0 0 0 20 40 60 80 0 20 40 60 80 Days Days

Figure 5-3. CXCL14-mediated tumor suppression disappears in CD8a knockout mice. Wildtype (WT) or CD8a-/- mice (n = 10 per group) were s.c. injected with MOE/E6E7Vector or MOE/E6E7CXCL14 cells (5 × 105 cells/mouse). Tumor volume was measured twice a week (A-D). Overall (A and B) and individual (C and D) tumor growth curves are shown for mice injected with MOE/E6E7Vector (A and C) or MOE/E6E7CXCL14 (B and D) cells. Survival rates were analyzed as was performed in Figure 1 (E). P value of WT compared to CD8a-/- was determined for tumor growth (A and B) and survival (E) by one-way ANOVA analysis. * = p < 0.0001.; n.s., not significant. Shown are representative of 2 independent experiments.

97

A C

4 * Vector 3 T cells + 2

MOE/E6E7 1 % CD8

B 0 (#CD8+ cells/ total DAPI cells) Vector CXCL14 CXCL14

MOE/E6E7 MOE/E6E7 MOE/E6E7

Figure 5-4. CXCL14 mediates CD8+ T cell infiltration into the tumor microenvironment Representative image of MOE/E6EVector (A) and MOE/E6ECXCL14 (B) tumor sections immunostained with anti-CD8a (red), anti-cytokeratin (green) and DAPI (blue) to identify CD8+ T cells and tumor tissues. CD8+ T cells were quantified as a number of CD8a positive cells per total number of DAPI positive cells across 5 representative images (C). Unpaired two-tailed t-tests were performed. * p < 0.05. Scale bars are 50 µm. Panels A and B were generated in collaboration with Stephanie Bonney, University of Colorado

98 A MOE/E6E7Vector cells MOE/E6E7CXCL14 cells

) 2500 WT mice 2500 WTWildtypeWi mice 3 Wildtype OT-IOT-I mice OT-IOT-IOT mice 2000 2000

1500 n.s 1500 ** 1000 1000

500 500

Tumor Volume (mm 0 0 0 5 10 15 20 0 5 10 15 20 25 B Days Days MOE/E6E7Vector cells: WT mice MOE/E6E7Vector cells: OT- I mice

) 2500 2500 3 2000 2000

1500 1500

1000 1000

500 500 Tumor Volume (mm 0 0 0 20 40 60 80 0 20 40 60 80 C Days Days CXCL14 CXCL14 ) 2500 MOE/E6E7 cells : WT mice 2500 MOE/E6E7 cells : OT- I mice 3 2000 2000

1500 1500 ★ 1000 1000 ★ 500 500 Tumor Volume (mm 0 0 0 20 40 60 80 0 20 40 60 80 Days Days D MOE/E6E7Vector cells MOE/E6E7CXCL14 cells 100 100

WT mice WT mice 50 50

OT- I n.s OT- I Percent survival Percent survival mice mice ** 0 0 0 20 40 60 80 0 20 40 60 80 Days Days

Figure 5-5. CXCL14-mediated tumor suppression requires antigen-specific CD8+ T cells. Wildtype (WT) or OT-I mice (n = 5 per group) were s.c. injected with MOE/E6E7Vector or MOE/E6E7CXCL14 cells (5 × 105 cells/mouse). Tumor volume was measured twice a week (A). Individual growth curves are shown for MOE/E6E7Vector cells injected (B) or MOE/E6E7CXCL14 cells injected WT or OT-I mice (C) Survival rates of WT or OT-I mice injected with MOE/E6E7Vector or MOE/E6E7CXCL14 (D) were analyzed as was performed in Figure 1. P value of WT compared to OT-I was determined for tumor growth (A) and survival (D) by one-way ANOVA analysis. ★ represent mice that exhibited tumor growth but had to be euthanized due to self-inflicted wounds. ** = p < 0.001. Shown are representative of two independent experiments.

99

A B C MOE/E6E7 MOE/E6E7Vector MOE/E6E7Vector 4 NI-MOE MOE/E6E7CXCL14-CL8 MOE/E6E7CXCL14-CL16 p = 0.007 100 100 100 3 80 80 80 60 60 60 2 40 40 40

% of Max %of Max %of 1 20 20 20 to unstained H-2Db MFI relative 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 Vector CXCL14-CL16 H-2Db H-2Db MOE/E6E7 D E Unstained MOE/E6E7Vector MOE/E6E7Vector Untreated MOE/E6E7CXCL14-CL8 MOE/E6E7CXCL14-CL16 IFNγ treated 100 100 100 80 80 80 60 60 60 40 40 40 % of Max %of % of Max %of 20 20 20 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 H-2Kb H-2Kb

Figure 5-6. CXCL14 expression restores surface expression of MHC-I proteins on HPV-positive HNC cells. Surface expression of MHC-I haplotype proteins, H-2Db (A-C) and H-2Kb (D and E), was determined in normal immortalized MOE (NI-MOE) cells (purple) (A), parental MOE/E6E7 cells (gray) (A), MOE/E6E7Vector cells (gray) (B), and two clones of MOE/E6E7CXCL14 cells (Clone 8-Red and Clone16- Blue) (B) by flow cytometry. Relative mean fluorescent intensity (MFI) values of MOE/E6E7Vector and MOE/E6E7CXCL14-CL16 were calculated (C). P value was determined by the Student’s t-test. INFg treatment was used as a positive control to validate H-2Kb expression in MOE/E6E7 CXCL14 cells (E). Shown are representative of 3 independent experiments.

100 A 100 100 100 100 MOE/E6E7Vector 80 80 80 80 MOE/E6E7CXCL14 CXCL14/shB2M 60 60 60 60 MOE/E6E7 % of Max % of Max 40 40 40 40

% of Max %of 20 20 20 20

0 0 0 0 0 102 2 1033 1044 105 5 0 102 2 1033 1044 105 5 0 10 :10 H-2Kd10 10 0 10 :10 H-2Kb10 10 H-2Db H-2Kb B C Vector MOE/E6E7Vector

) 3000 MOE/E6E7 3 CXCL14 100 MOE/E6E7CXCL14 MOE/E6E7CXCL14/shB2MCXCL14/shB2M ** *** 2000 MOE/E6E7MOE/E6E7Vector 50 MOE/E6E7MOE/E6E7CXCL14 1000 MOE/E6E7MOE/E6E7CXCL14/shB2M Percent survival * Tumor Volume (mm ** 0 0 0 10 20 30 0 20 40 60 80 Days Days D CXCL14 ) 4000 MOE/E6E7 CXCL14 4000 CXCL14/shB2MCXCL14/shB2M 3 MOE/E6E7 cells MOE/E6E7MOE/E6E7 cells

3000 3000

2000 2000

1000 1000 ★

Tumor Volume (mm 0 0 0 20 40 60 0 20 40 60 Days Days

Figure 5-7. CXCL14-mediated tumor suppression is abrogated by MHC-I knockdown in tumor cells. MOE/E6E7CXCL14 cells were transduced with shRNAs directed to B2M and enriched for knockdown by FACS sorting for the bottom 10% of MHC-I expressing cells (MOE/E6E7CXCL14/shB2M). Flow cytometric analysis of H-2Db (A) and H-2Kb MHC-I molecules in MOE/E6E7Vector, MOE/E6E7CXCL14, and MOE/E6E7CXCL14/shB2M cells by flow cytometry. Wildtype B6 mice (n = 10 per group) were injected s.c. with MOE/E6E7Vector, MOE/E6E7CXCL14, or MOE/E6E7CXCL14/shB2M cells (5 × 105 cells/mouse). Tumor volume was measured twice a week (B). Individual growth curves are shown for MOE/E6E7CXCL14 and MOE/E6E7CXCL14/shB2M injected mice (D) Survival rates of mice injected with MOE/E6E7Vector, MOE/E6E7CXCL14, and MOE/E6E7CXCL14/shB2M (C) were analyzed as was performed in Figure 1. P value of MOE/E6E7CXCL14/shB2M compared to MOE/E6E7Vector or MOE/E6E7CXCL14 was determined for represent mice that exhibited tumor growth but had to be euthanized due to self-inflicted wounds. * = p < 0.01, ** = p < 0.001, *** = p < 0.0001. Shown are representative of 2 independent experiments. Experiments were performed in collaboration with Alexa Silva

101 A B HPV16-E7 20 3) 100

15

10 10

5 Doubling time (hours)

0 1 mRNA relative to Gapdh (X10 mRNA

Vector CXCL14 CXCL14

CXCL14/shB2M CXCL14/shB2M

MOE/E6E7 MOE/E6E7 C MOE/E6E7 MOE/E6E7 MOE/E6E7 C Cxcl14 6) 100

10

1

0.1 mRNA relative to Gapdh (X10 mRNA

Vector CXCL14

CXCL14/shB2M

MOE/E6E7 MOE/E6E7

MOE/E6E7

Figure 5-8. B2M knockdown does not affect cell proliferation, HPV16 E7 expression, and CXCL14 expression in HPV-positive HNC cells. In vitro growth rate of MOE/E6E7CXCL14 cells compared to MOE/E6E7CXCL14/shB2M cells. Growth is shown as doubling time (calculated as DT = Tln(2)/ln(xE/xb) where DT is doubling time; T is the time period; and xE or xB is the number of cells at the ending or beginning of the time period), each bar represents the mean ± SEM, N = 4/group. Differences in growth between the clones did not show statistically significant differences based on Student’s t-test. Total RNA was extracted from MOE/E6E7 Vector, MOE/E6E7CXCL14, and MOE/E6E7CXCL14/shB2M cells. The mRNA expression of HPV16E7 (B) and CXCL14 (C) was determined by RT-qPCR using specific primers. CXCL14 and HPV16 E7 mRNA copy numbers were normalized by Gapdh mRNA used as a housekeeping gene. Differences in growth or transcripts between the MOE/E6E7CXCL14 cells and MOE/E6E7CXCL14/shB2M cells did not show statistically significant differences based on Student’s t-test. Shown are representative of 2 independent experiments.

102

A 1500

1000 * pg/mL pg/mL γ

IFN 500 *

L.O.D. 0 Target - - Vector CXCL14 Vector CXCL14 CXCL14- MOE/E6E7cells: OVA + CD8 T cells from: Naive Naive + MOE/E6E7Vector MOE/E6E7CXCL14 OT-1 PMA/Iono injected (Null) injected (Primed) B 600

400 pg/mL pg/mL α * 200 TNF

0 L.O.D. Target - - Vector CXCL14 Vector CXCL14 CXCL14- MOE/E6E7cells: OVA + CD8 T cells from: Naive Naive + MOE/E6E7Vector MOE/E6E7CXCL14 OT-1 PMA/Iono injected (Null) injected (Primed) C 150

100

IL-2 pg/mL IL-2 pg/mL 50

E L.O.D. 0 Target - - Vector CXCL14 Vector CXCL14 CXCL14- MOE/E6E7cells: OVA + CD8 T cells from: Naive Naive + MOE/E6E7Vector MOE/E6E7CXCL14 OT-1 PMA/Iono injected (Null) injected (Primed) D E Primed-CD8+ T cells effectors 50 + 50 Target Cells Null-CD8 T cells effectors Vector Target CellsVector MOE/E6E7 MOE/E6E7 40 CXCL14 40 VectorCXCL14 MOE/E6E7 MOE/E6E7MOE/E6E7MOE/E6E7 TargetCXCL14 Cells /shB2M CXCL14CXCL14 /shB2M MOE/E6E7 MOE/E6E7MOE/E6E7MOE/E6E7 MOE/E6E7Vector 30 CXCL14/shB2M 30 MOE/E6E7 MOE/E6E7MOE/E6E7 MOE/E6E7MOE/E6E7CXCL14 CXCL14/shB2M 20 20 MOE/E6E7MOE/E6E7

10 10 Percent Cell killing Percent Cell killing L.O.D. 0 L.O.D. 0 100:1 10:1 1:1 0 100:1 10:1 1:1 0 Effector : Target ratio Effector : Target ratio Figure 5-9. CXCL14 expression in tumor cells activates CD8+ T cells for IFNγ and TNFα production and tumor cell killing.

103 Figure 5-9. CXCL14 expression in tumor cells activates CD8+ T cells for IFNγ and TNFα production and tumor cell killing.CD8+T cells isolated from mice injected with MOE/E6E7Vector (Null-CD8+ T cells) or MOE/E6E7CXCL14 (Primed-CD8+ T cells) were co-cultured with mitomycin- treated MOE/E6E7Vector or MOE/E6E7CXCL14 cells in the presence of anti-CD28 antibody and IL-2 for 5 days (Naïve stim). Naïve CD8+ T cells alone acted as a negative control. Naïve CD8+ T cells cultured with anti-CD3/CD28 beads and IL-2 were stimulated with PMA and Ionomycin served as a positive control. CD8+ T cells from isolated from a OT-I mouse co-cultured with MOE/E6E7CXCL14 expressing OVA (MOE/E6E7CXCL14-OVA) served as an additional positive control. Supernatants were collected and measured for IFNγ (A), TNFα (B) and IL-2 (C) protein production using cytokine specific ELISAs. Shown are means ± standard deviations of quadruplicates. The cytotoxic activity of null- and primed- CD8+ T cells was determined by LDH release assay in co-culture with MOE/E6E7Vector and MOE/E6E7CXCL14 cells as target cells (E and F) at effector : target ratios, 100:1, 10:1, 1:1, and 0:1. Mean percent specific lysis values ± standard deviations of triplicates were normalized to the signal from total cell lysis by 1% SDS disruption. P value was determined by the Student’s t-test comparing target MOE/E6E7CXCL14 to target MOE/E6E7Vector (A-C). * = p < 0.0001 Shown are representative of 3 (A-C) and 2 (D and E) independent experiments.

104 CHAPTER VI

DISCUSSION AND CONCLUDING REMARKS4

Discussion

While expression of the HPV oncoproteins E6 and E7 inactivates several tumor suppressors, the development of invasive cancer requires many years of viral persistence and disease progression. Our previous global gene expression analysis of human cervical tissues in different disease stages (normal, early and late precancerous lesions, and cancer) has revealed dynamic gene expression changes in a series of cellular pathways, including the cell cycle, translation, mitochondrial energy metabolism, and estrogen signaling (274).

Aside from cell proliferation-related genes, which are significantly upregulated with high-risk

HPV E6 and E7 expression, a majority of host gene expression changes influenced by HPV persistence are slow progressing and accumulating throughout cancer progression. Many of the genes are altered slowly and continuously and are involved in immune responses and inflammation including several cytokines and chemokines (274). Our study has further revealed that restoration of the chemokine CXCL14 in mouse HNC cells, which is downregulated by HPV E7, significantly suppresses HPV-positive HNC growth in vivo and enhances NK and T cell infiltration into tumor-draining lymph nodes (140). These results suggest that HPV-mediated immune dysregulation during virus persistence is important to prevent the elimination of HPV-infected cells during cancer progression. Thus, furthering our understanding of virus-directed immune dysregulation would be critical to develop preventive and therapeutic tools for treating virus-associated cancer as well as eliminating virus-infected cells.

4 Portions of this chapter were published from Permission from Westrich J.A. and Pyeon D. 2017 Evasion of Host Immune Defenses by Human Papillomavirus. Virus research 231 21- 23 doi.org/10.1016/j.virusres.2016.11.023

105 HPV-E7 stabilization of A3A

Viral Stabilization of Host Proteins

Although the mechanism of viral mediated protein degradation has been well studied, the prevention of host protein degradation by viral proteins is a relatively nascent field that has now begun to emerge. Viral stabilization of host proteins typically occurs through disruption of the E3 ubiquitin ligase complex. Several viruses including gammaherpesvirus

(257), Merkel cell polyomavirus (256), and hepatitis B virus (258) enact mechanism to protect cellular factors. Interestingly, all of the protected cellular factors contribute to the viral life cycle in some way (e.g. replication, cellular survival, and oncogenesis). In HIV-1 infections, the host protein tetherin restricts HIV-1 infection by binding and preventing release of the HIV virions form the infected cell (382). This restriction is countered by the

HIV-1 Vpu-mediated tetherin degradation. Interestingly, it has recently been shown that monkey kidney cells, Vpu, can increase the expression and stabilize the tetherin protein

(383). Although stabilized, the function of tetherin is neutralized. In HPV infected cells, HPV

E7 has been shown to stabilize host proteins that are typically associated with preventing productive viral replication, p53 and p21cip1 (85, 87, 217). Although the mechanism of the stabilization remains undetermined, it has been shown that p53 and p21cip1 are functionally inactive. We have recently shown that HPV E7 stabilizes the restriction factor APOBEC3A

(A3A) (Figure 3-1). However, the stabilized A3A remains enzymatically activity (Figure 3-8).

A3A Stabilization by High-risk HPV E7

Members of the APOBEC3 (A3) family has been well established as restriction factors for a multitude of viruses including: human papillomavirus (117), transfusion transmitted virus (384), Herpes simplex virus 1 and Epstein-Barr virus (385), Human Immunodeficiency virus (386), Hepatitis B virus (220), adeno-associated virus, and retrotransposons (187). In

HPV infected tissues, A3 editing of HPV genomes was first observed in HPV16 infected cervical tissue and HPV1 infected plantar wart samples (114). We have previously shown

106 specific restriction of HPV infectivity by A3A and induction of A3A and A3B by HPV16 oncoproteins during persistence (117). Another study showed similar restriction of HPV16 infectivity by A3A and A3C (116). Given the potent restrictive capability of A3A on HPV infectivity, we initially hypothesized that HPV degrades A3A protein through ubiquitin ligation and proteasomal degradation to combat the increased expression of A3A, similar to HIV Vif- mediated A3G protein degradation (387). Interestingly, the degradation of HIV-1 Vif to degrade A3G is strikingly similar to the mechanism employed by HPV E7 degradation of pRB (236). Surprisingly, high-risk HPV E7 prevents A3A proteasomal degradation through direct interaction with CUL2, a core component of the ubiquitin ligase complex (UBL).

Although we have shown that A3A protein is stabilized through a posttranslational mechanism by HPV E7, the contribution of the stabilized A3A to somatic mutations in HPV- associated cancers remains to be identified (Discussed in Chapter III). Additional evidence continues to emerge indicating A3A as a major contributor to HPV associated cancers including further evidence for HNC (388), penile cancer (389) and anal cancer (390). Thus, targeting A3A protein stabilization by HPV E7 may prevent or slow down disease progression.

Mechanism of HPV E7-mediated A3A Protein Stabilization

The stabilization of A3A by E7 in HPV infected cells is paradoxical as A3A has been shown to be a potent restriction factor for HPV infection (Figure 6-2) (117). Nevertheless,

HPV E7 stabilizes A3A protein, which is enzymatically active (Figure 3-2 and Figure 3-8).

One explanation is that the stabilization of A3A is an intentional mechanism by HPV that uses the A3A enzymatic activity for its own benefit. One such benefit is that HPV could utilize the deamination activity of A3A to promote host DNA damage. In line with this reasoning, A3A has been shown to contribute to DNA damage during DNA replication (391).

Furthermore, HPV genome replication relies on the proteins involved with the DNA damage response (DDR) (Reviewed in (392)). Although A3A is capable of restricting the viral

107 infectivity, HPV may be capable of coping with a certain level of A3A activity through an underrepresentation of the target motifs of A3A (267). Although the necessity of the DDR in viral replication has been well established, the contributing role of A3A remained unidentified.

Whether the stabilization of A3A is intentional or an accidental event, it could be speculated that A3A stabilization may be a conserved function of related small DNA tumor viruses. In a model evaluating the role of A3A in the context of human adenovirus (HAdV) infection, the Schreiner group showed that A3A possesses potent restrictive capacity against

HAdV infectivity, but A3A is not targeted for degradation by viral proteins (393). Additionally, the large T antigen of SV40 modestly increases A3A expression in expressing cells, although it remains undetermined if A3A protein is stabilized (201). These results may suggest that the mechanism is conserved across several DNA tumor viruses. Interestingly, the CUL2 binding domain, which we have shown to be necessary for A3A stabilization, shows high homology between HPV16 E7, the E1A protein of HAdV-5, and the large T antigen of SV40 (Figure 3-5 and Figure 6-1). Interestingly, both proteins were shown to interact with members of the cullin family and inhibit the degradation of host proteins through the ubiquitin ligase complex (394, 395). It would be of great interest to see if the stabilization of A3A is maintained across these proteins, as A3A stabilization may suggest a necessary role for DNA viral lifecycle.

A key element necessary to understanding why A3A protein is stabilized in HPV infections is further definition of the mechanism involved. We have shown that the stabilization of A3A is reliant on E7 interaction with CUL2 (Figure 3-7). One potential mechanism involving CUL2 for E7 mediated A3A stabilization may involve the COP9 signalosome complex (CSN) (396). The CSN is involved in deneddylating cullin proteins, which we have shown that the degradation of A3A is reliant on the neddylation of CUL2

(Figure 3-6). Furthermore, the activity of CSN has been shown to differentially regulate

108 cullin activity in the presence of DNA damage, of which HPV facilitates during infection (397,

398). A compelling observation that may lend support to this hypothesis is CSN has been shown to be overexpressed in cervical cancers (399). Interestingly, a study identifying host proteins interactions with HPV E7 from several genotypes revealed several subunits of the

CSN complex interact with HPV16 and HPV18 E7s, but not HPV6 E7 ( (255) - Supplemental data 5). This finding is intriguing as we have shown A3A protein is stabilized by HPV16 and

HPV18 E7, but not HPV6 E7 (Figure 3-3). Identification of the mechanism of A3A protein stabilization may help us understand if the HPV mediated stabilization is an intentional path utilized by HPV, and if so what other host proteins are being stabilized.

The Contribution of A3A Stabilization in HPV Associated Cancer

In HPV infections, the progression to cancer takes decades of persistent infection.

Given the A3A mutational burden observed in HPV-associated cancers, it is possible that high-risk HPV E7 stabilization of A3A represents a mechanism that contributes to cancer progression. We have shown that A3A protein is stabilized in HPV-positive normal immortalized cells and cancer cells (Figure 3-2 and Figure 3-4). Furthermore, we have shown that the stabilized A3A protein remains enzymatically active (Figure 3-8). Thus, it is possible that A3A stabilization, over decades of HPV infection, may contribute to somatic mutagenesis and disease progression. In addition, A3A may contribute to disease progression through other mechanisms. In oropharyngeal cancers, high A3A expression has been correlated with integration of the HPV genome into the host genome (221). Integration of the HPV genome typically results in the dysregulation of E6 and E7 expression (12).

Higher E6 and E7 expression potentially results in increased A3A stabilization. Determining if A3A is a contributing factor in HPV-associated cancers would greatly enhance our knowledge of how HPV-positive cancers develop, and offer insights into potential therapies for persistently infected individuals. It would be of interest to utilize HPV-positive cells with or without A3A to evaluate the contributing effect of A3A stabilization on the host mutational

109 burden over time. This could be accomplished by evaluating monitoring A3A signature mutations in genes associated with HPV-cancer progression, such as phosphatidylinositol-

4,5 bisphosphate 3-kinase catalytic subunit alpha (PI3KCA). PI3KCA has been shown to be predominately targeted and mutated by A3A in HPV-associated cancers (400-402). This work would be important as it represents a bridge between A3A protein stabilization and A3A specific mutation signatures in HPV-associated cancers. By defining the contribution of A3A to these cancers, it opens up therapeutic options to target this pathway to prevent further disease progression.

These therapies must be approached with caution. The effects of A3A appears to be a doubled edged sword; it has been shown to have significant restrictive capabilities against viruses, but prolonged expression may have an impact on cancer progression. Recently,

A3A has been investigated as a potential therapeutic for cancers (403). Although shown to be efficacious at promoting tumor cell apoptosis at early stages of tumor development, A3A expression was ineffective in larger tumors. Without knowing the rate and extent of A3A mutagenic potential, and the potential consequences of increasing the mutagenic load in cancer cells, using A3A as therapy may not be wise. Thus, it is necessary to further understand the mechanisms of this protein both in infection as well as cancer to ensure the enzymatic activity is not perpetuating a more serious disease.

HPV-E7 suppression of CXCL14

Chemokines in HPV-Associated Cancer Progression

Chemokines initiate and orchestrate the immune responses crucial for antiviral defense (Reviewed in (404)). The principal function of chemokines is to control the migration of immune cells to sites of infection and/or inflammation. Chemokines act as early responders to recognize infections and play a key role in kicking off the immune response to combat the infection. Thus, chemokines represent prime targets for viral immune evasion mechanisms and many viruses have evolved elegant mechanisms to manipulate their

110 expression. In HPV-associated disease progression, the cytokine and chemokine environment become progressively more driven in favor of type 2 immune response (405-

409). Type 1 immunity is protective against most bacterial and viral infections, whereas type

2 responses are protective against large parasites, assist with the resolution of cell- mediated inflammation, and promote wound healing (410, 411). Thus, type 2 immunity represents a favorable condition for viral infections (410, 412). HPV-positive individuals ultimately clearing HPV-infected cells show a shift in immune response to a predominantly type 1 response (359, 413). Production of type 1 cytokines promotes cell-mediated immunity, which is effective at eliminating viral infections (110). CXCL14 has been shown to augment type 1 immune responses (374). Thus, it is not surprising that HPV has developed a mechanism to silence CXCL14 expression in HPV infected cells (Figure 4-5)

CXCL14 and Cancer

Several models of HNC, lung, and colorectal cancers have shown that re-expression of CXCL14 promotes tumor suppression (142, 283, 285, 369). Another study utilized

CXCL14-transgenic (CXCL14-Tg) mice that overexpress CXCL14 and showed marked tumor suppression as compared to its wildtype counterparts (288). Tumor suppression observed in the CXCL14-Tg mice was shown to be dependent on NK cells. Although full characterization of the T cell population was not performed, a separate study has shown that

T cells from CXCL14-Tg mice have enhanced T cell activation and proliferation in response to antigen stimulation (374). These studies would suggest that high CXCL14 expression is generally protective against tumor formation. In contrast, several other studies have shown that CXCL14 expression from neighboring fibroblasts may contribute to tumor growth in prostate and breast cancer (372, 373). A potential mechanism for this is the continual expression of low level type-1 immune chemokine and cytokines (413) combined with the lack of clearance due to the low immunogenicity of the tumor (414). By failing to clear the tumor the pro-inflammatory environment can persist and cause additional deleterious

111 damage (415).

Identification of CXCL14 signaling would be the key to understanding its antitumor functions vs. pro-tumor functions. A major limitation in studying CXCL14 functions is the lack of information about its native receptor. One study has shown that CXCL14 binds with high affinity to CXCR4 (289, 416). They have shown that by binding to CXCR4, CXCL14 antagonizes CXCL12 and prevents signaling though CXCR4. In contrast, another group found that CXCL14 binds to CXCR4 synergized with CXCL12 to increase CXCR4 signaling

(377). Finally, another group suggests that CXCL14 does not influence signaling through the

CXCL12-CXCR4 axis (376). These discrepancies may be caused by different experimental setting such as different types of cells and various sources of CXCL14 used in the studies

(338).

CD8+ T cells in HPV-Positive Cancer

CD8+ T cells appear to play a critical role in HPV-associated cancers as well. In patients with HPV-positive cancers, E6 and E7-specific CTLs can be stimulated by HPV16 peptides in vivo, suggesting that antigen-specific CTLs exist in the patients (417). Using

HPV16 E7 tetramer stain, it has been shown that HPV16 E7-specific CTLs exist in the peripheral blood of women with high-grade HPV-positive lesions (418). Interestingly, an in- depth analysis of tumor-infiltrating immune cells in HPV-positive and -negative OPSCCs found HPV-specific T cell infiltration in ~60% of HPV positive tumors (363). Although HPV- specific CD8+ T cells are present in patients with HPV-positive tumors, they are not sufficient to initiate an anti-tumor response.

The lack of immune responses in HPV-positive cancer patients is likely due to failure of CD8+ T cell activation. Previous studies have shown that low viral protein expression and disrupted MHC-I expression of infected cells led to insufficient antigen presentation to activate CD8+ T cells (364, 419, 420). Additionally, E7 induces immune suppression by up- regulating PD-L1 expression in cancer cells (365, 421). Consistently, a subset of HPV-

112 positive HNCs was effectively eliminated by immunotherapies using PD-1 inhibitors (422,

423), However, while current immunotherapies using immune checkpoint inhibitors show some efficacy, a majority of HPV-positive HNC patients did not respond to the therapies and the response rates are not significantly different between HPV-positive and -negative HNCs

(276, 366). The non-responders exhibit significantly reduced T cell infiltration into the

TME (424, 425). Thus, there is an urgent need to develop novel means to recruit antigen- specific T cells in the TME.

In our HPV-positive HNC mouse tumor model, we have shown that the CXCL14- mediated tumor suppression is reliant on the presence of antigen-specific CD8+ T cells

(Figure 5-5). Furthermore, we have shown that CD8+ T cells isolated from mice injected with HPV-positive HNC cells kills syngeneic tumor cells expressing CXCL14 in vitro (Figure

5-9E). This suggests that antigen-specific T cells exist in mice bearing tumor but cannot respond to tumor, as is seen in human patients with HPV-positive cancers. Thus, CXCL14 may play a key role to induce the migration of antigen-specific CD8+ T cells and facilitate antitumor immune responses in the TME.

Tumor Associated Antigens Recognized by CD8+ T cell Response

We have shown that the CXCL14 mediated anti-tumor immune response is reliant on antigen-specific CD8+ T cells (Figure 5-4, Figure 5-5). However, the specific antigen/s for

CD8+ T cell response remain undefined. The HPV oncoproteins E6 and E7 are likely candidates as presentation of E6 and E7 peptides have been shown to promote potent activation from CD8+ T cells (426-430). Identification of the tumor-associated antigen would provide us with the ability to ask questions we otherwise could not. What percentage of population of CD8+ T cells are specific for the tumor antigen in mice bearing tumors that do not express CXCL14 and cannot control tumor growth? Do mice that ultimately fail to control tumor growth, even with CXCL14 expression, have a deficit in this CD8+ T cell population?

These findings would help us further understand the role the CD8+T cell play in controlling

113 or failing to control HPV-positive tumor growth in our model.

A caveat to the identification of the predominate tumor associated antigen in our model is that it may not mirror exactly what is observed in HPV-positive human cancers.

Specifically, neoantigens arise in cancer due to mutations resulting in proteins that are no longer recognized as self and are able to be targeted by the immune response (431). The formation of neoantigens is based on the prevalence of somatic mutations in the cancer, and as such each cancer can be classified on rate of neoantigen formation: frequently, regularly, or occasionally (432). In HPV associated cancers, neoantigens are suspected to be regularly abundant, likely due to the decades of persistent infection (432). As such, given our forced transformation of the MOE/E6E7 cells, there may be an underrepresentation of the neoantigens. We can use this facet to our advantage as we can determine the sufficiency of potentially limited antigens in enacting a productive anti-tumor immune response.

Mechanism of MHC-I Expression Restored by CXCL14

We found that CXCL14 expression significantly increases expression of MHC-I alloantigen H-2Db in HPV-positive HNC cells (Figure 5-6). While we have shown that MHC-I expression is necessary for CXCL14-mediated tumor suppression (Figure 5-7), the mechanism of the CXCL14-mediated upregulation of H-2Db expression remains to be determined. A potential mechanism of MHC-I upregulation is by CXCL14 promoting NF-κB signaling. Several studies have shown that CXCL14 induces NF-κB (142, 338, 371) including in lung cancer cells (433). NF-κB has been shown to rescue MHC-I expression in neuroblastoma and breast cancer lines (434, 435). A caveat to this is that E7 has been shown to be effective at impairing the acetylation and nuclear translocation of the p65 subunit of NF-κB (436). Although it remains possible the CXCL14 promotion of NF-κB signaling can overcome E7 suppression, thus it would be worth evaluating downstream targets of NF-κB such as IL-12, IL-23 and RORγt in CXCL14 expressing or non-expressing

114 cells.

An interesting observation is that, unlike H-2Db, H-2Kb expression is not changed by

CXCL14 expression (Figure 5-6). Although it is unknown that expression of the two MHC-I alloantigens can be differentially regulated, there are notable differences in regulating protein stability that may explain our observation. H-2Db shows higher protein stability on cell surface, compared to H-2Kb (68% as compared to 50%, respectively) (437). In addition, the peptide binding affinity to the MHC-I regulates MHC-I protein stability on cell surface. A previous study using an independent HPV-associated cancer mouse model showed that the

E7 peptide aa49-57 (RAHYNIVTF) presented by H-2Db is critical to enact an antitumor

CD8+ T cell response (430). Other studies have revealed that the E7 peptide RAHYNIVTF binds to the H-2Db complex with high affinity (438). Thus, it is possible that our results showing H-2Db expression alone may be caused by the stable H-2Db complex remaining on the cell surface in sufficient time to be recognized by CD8+ T cells. In contrast, the H-2Kb complex with weak binding to a peptide may result in fast turnover and remain undetected.

Interestingly, the changes in MHC-I proteins seen in CXCL14 expressing cells in vitro may represent only a fraction of the MHC-I protein changes that occur in vivo. The recruitment of

NK and T cells to the TME was shown to increase the levels of both MHC-I alloantigens in vivo through expression of IFNγ (439). Given that CXCL14 expression also promotes the recruitment of NK and T cells, it is possible that additional enhancement of MHC-I could occur during in vivo interactions with these immune cells.

The Immunosuppressive Tumor Microenvironment in HPV Associated Cancers

The tumor microenvironment (TME) is created by complex interactions and communications of various cell types including epithelial, endothelial, stromal, and immune cells that support tumor growth and immune evasion. In HPV associated cancers, the HPV oncoproteins E6 and E7 play active roles for these processes of creating the TME (440).

Our recent studies have revealed that the chemokines CXCL1 and CXCL2 are progressively

115 upregulated during HPV-associated cancer progression (140, 274). Consistent with our findings, another study showed increased expression of CXCR2 ligands, including CXCL1 and CXCL2, in epithelial cells expressing the HPV oncoproteins E6 and E7 (441). CXCR2 signaling is involved in the support of a protumor microenvironment through promoting angiogenesis and recruiting immunosuppressive cells such as neutrophils and myeloid derived suppressor cells (MDSCs) (442, 443). Several recent publications have shown that

CXCR2 plays an important role in the development and maintenance of several cancers, including rhabdomyosarcoma, lung cancer, gastric cancer, and HNCs (444-

447). Interestingly, inhibition of CXCR2 reduces metastasis, prolongs tumor-free survival, and enhances T cell infiltration into tumor in a mouse model of pancreatic ductal adenocarcinoma (448). We recently tested if inhibition of CXCR2 synergistically suppresses tumor growth with CXCL14 expression in vivo, using a CXCR2 inhibitor, AZD5069 (obtained from AstraZeneca). Our results showed that AZD5069 treatment alone slightly delayed tumor growth, but all mice eventually died by tumor burden (Figure 6-3A). In contrast,

AZD5069 treatment in combination with CXCL14 completely suppressed tumor growth and all mice survived without any sign of tumor growth (Figure 6-3B). Given that 20% to 40% of mice with CXCL14 expression grow tumor and died in a delayed fashion, treatment of the

CXCR2 inhibitor synergistically suppresses tumor with CXCL14 expression, eliminating the non-responders of CXCL14 expression. These findings are in agreement with several other studies that show inhibition of CXCR2 boosts the efficacy of other cancer therapies such as

PD-1 inhibitor treatment (444, 448, 449). Our findings suggest that the microenvironment of

HPV-infected tissues may be curated by the HPV oncoproteins for immune evasion and virus persistence. Determining key immune regulators in HPV infection will be critical for the development of novel therapies to treat HPV-associated cancers.

116 Concluding remarks

HPV is the most prevalent sexually transmitted pathogen worldwide and is the etiological agent responsible for approximately 5% of all cancers. The HPV oncoproteins E6 and E7 play a critical role in promoting HPV-associated cancer progression by promoting cell survival and immune evasion. The HPV oncoprotein E7 is a key regulator in targeting numerous cellular pathways to support virus persistence. The goal of this dissertation is to determine how E7 dysregulates host immune responses to support cancer progression. In

Chapter 3, I discuss that the HPV oncoprotein E7 stabilizes A3A protein by an interaction with CUL2 of the ubiquitin ligase complex (Figure 6-2). These findings provide a novel insight about cancer mutagenesis driven by E7-stabilized A3A during HPV-associated cancer progression. In Chapter 4, I discuss that the HPV oncoprotein E7 suppresses antitumor immune responses through downregulation of CXCL14 expression by promoter methylation. We show that restoration of CXCL14 expression in HPV-positive tumor cells suppresses tumor growth in immunocompetent syngeneic mice. My findings have revealed a new epigenetic mechanism of HPV-induced immune suppression that gradually changes the microenvironment of virus-infected tissues for cancer progression. In Chapter 5, I further discuss that antigen-specific CD8+ T cells are necessary for CXCL14-mediated tumor suppression, in part through upregulating MHC-I expression on the HPV-positive tumor cells. These results suggest that CXCL14 may be one of the key regulators that coordinate potent antitumor immune responses (Figure 6-4). These findings may lead to developing novel immunotherapies to treat HPV-positive cancer patients, particularly for non- responders in the current immunotherapies using checkpoint inhibitors.

Overall, my studies presented in this dissertation shed light on the mechanisms by which the HPV oncoprotein E7 dysregulates host immune responses to promote virus persistence that eventually contribute to cancer progression.

117

CUL-2 binding box

MDKV-LNREESLQLMDLL SV40 Large T (65%) : : I : : : I : I I RLCVQSTHVDIRTLEDLL HPV16-E7

: : I : : : : : : I I I I AVRVGGRRQAVECIEDLL AdV-E1A (72%)

Figure 6-1. Sequence homology of the CUL2 binding domain of DNA viruses. HPV16 E7, SV40 Large T antigen and AdV E1A viral proteins sequences were acquired through uniprot.org and aligned using SerialCloner 2.6.1. “I” denotes conserved amino acid,“:” denotes semi-conserved amino acid. Percent homology to HPV16E7 was calculated by SerialCloner.

118

Figure 6-2. Model of E7 stabilized A3A role in HPV infection. A3A potently restricts HPV infectivity (1). E7 prevents the proteasomal degradation of A3A by the E3 ubiquitin ligase complex (2) resulting in an accumulation of enzymatically active A3A protein (3). The consequence of this stabilize remains unidentified but may result in host genome mutations, initiation of the DNA Damage response to promote viral replication, and integration of the HPV genome into the host genome (4).

119

Vector CXCL14 Figure 6-3. MOE/E6E7 and MOE/E6E7 tumor growth in mice with or without CXCR2 inhibition. Mice were administered either vehicle or AZD5069 (100mg/kg) twice daily by oral gavage. Vector CXCL14 Two days after initiation of treatment, MOE/E6E7 or MOE/E6E7 cells were injected into the rear right flank of wild-type C57BL/6 mice (n = 10 for each group) subcutaneous in the hip flank. Tumor growth was determined twice a week for 80 days. Mice were euthanized when tumor size exceeded 1.5 cm in any direction. Each tumor growth curve represents an individual mouse tumor growth

120

Figure 6-4. Model of CXCL14 promoting tumor suppression. HPV-positive tumor growth (left) is protected from immune recognition by E7 suppressing expression of CXCL14 and MHC-I. Re- expression of CXCL14 (right) initiates tumor suppression through recruitment of immune cells (CD4, CD8, and NK cell) as well as increases MHC-I on tumor cells.

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159 APPENDIX A

Primers Used for RT-qPCR

Appendix A Primers Used for RT-qPCR. Primer list provided by Louis Cicchini, PhD

160 APPENDIX B

Gene Expression Microarray Details and Methodology

MOE/E6E7Vector vs:

MOE/E6E7CXCL14 MOE/E6E7CXCL14 (CL8) (CL16)

1384 585 731

Appendix B-1. CXCL14 driven gene changes in HPV-positive tumor cells. MOE/E6E7Vector (Clone 2) and MOE/E6E7CXCL14 (Clone 8 and Clone 16) gene expression profiles were assessed by Affymetrix Mouse Gene 2.0 ST 2.0 arrays. Each experimental group was evaluated in quadruplicate obtained from total RNA isolated at cell pellets taken between passage 8 and 12 for all groups. Data were normalized using RMA log transformation and evaluated by a principal component analysis and array intensity distributions. A 2-group comparison was performed using linear model in R between MOE/E6E7Vector (Clone 2) vs. MOE/E6E7CXCL14 (Clone 8 and Clone 16) cells using genes with FDR < 0.05. Represented is a Venn diagram of genes that are differentially regulated between Clone 2 and Clone 8 (red circle) and Clone 2 and Clone 16 (blue circle). Data were analyzed and generated by Lauren Vanderlinden.

161 Annotation Mean Expression Value Annotation Mean Expression Value MOE/E6E7 MOE/E6E7 MOE/E6E7 MOE/E6E7 Transcript MOE/E6E7 Transcript MOE/E6E7 Gene Symbol CXCL14 CXCL14 Gene Symbol CXCL14 CXCL14 Cluster ID Vector CL2 Cluster ID Vector CL2 CL8 CL16 CL8 CL16 17211774 Plekhb2 9.52 8.49 8.56 17391899 Adra1d 4.24 5.45 5.05 17216753 Mgat5 7.87 6.83 6.52 17391997 Gpcpd1 7.22 7.62 7.47 17218186 Fam129a 5.99 4.77 4.65 17392056 Fermt1 9.51 7.74 7.67 17218321 Rgs16 10.56 8.83 9.17 17392076 Hao1 4.69 3.61 3.79 17219902 1700016C15Rik 4.11 5.07 5.28 17393173 Pxmp4 6.38 7.07 7.89 17220787 Batf3 5.53 4.56 4.91 17394153 Slpi 5.36 6.55 6.45 17221756 Ogfrl1 6.43 4.87 5.38 17395041 Bmp7 5.58 6.69 8.67 17222825 Nabp1 9.38 8.73 9.03 17395382 Gm14418 6.41 7.78 7.40 17222837 Myo1b 7.36 4.84 5.23 17397462 Rab33b 7.68 7.21 7.16 17224551 Ptprn 6.88 5.75 5.71 17397962 Rap2b 8.45 7.70 7.68 17225261 Ngef 7.00 5.57 5.97 17397990 Mme 4.69 8.06 6.36 17225683 2310007B03Rik 4.72 6.12 6.16 17398082 Tiparp 9.18 8.23 8.66 17226473 Tmem163 7.80 6.13 5.71 17399374 Muc1 7.06 6.10 5.74 17226771 Ikbke 6.57 5.87 5.46 17399802 S100a4 7.40 8.10 8.70 17227505 9230116N13Rik /// 923013.79 4.33 4.21 17399854 Sprr2a1 /// Sprr2a2 6.13 7.16 7.74 17227753 Trove2 9.10 8.73 8.54 17399910 Lce3c 5.50 6.12 6.85 17227828 Pla2g4a 9.53 8.47 8.17 17400449 Anp32e 8.25 7.96 7.75 17227861 Ptgs2os2 /// Ptgs2os2 5.86 4.26 4.30 17400486 Mtmr11 6.16 5.37 5.13 17228057 1700025G04Rik 8.92 8.62 8.60 17400545 Hist2h2aa1// Gm2063 12.77 12.56 12.39 17229096 Prrx1 6.19 6.94 7.17 17401673 Sort1 6.99 8.23 8.53 17230111 Ifi205 6.24 7.23 7.92 17402025 Dbt 7.72 6.76 7.16 17230225 Hmga2-ps1 7.46 6.13 6.15 17402361 Sec24d 8.81 8.24 7.99 17230484 Ephx1 /// Gm36961 8.04 9.24 9.42 17403282 Ccbl2 6.39 5.11 5.01 17230595 Degs1 8.54 9.26 9.02 17403439 Ddah1 10.69 10.08 9.69 17230823 Lyplal1 5.21 6.44 6.53 17403451 Bcl10 8.71 8.18 8.39 17231717 Fuca2 7.32 7.88 8.32 17403571 Ttll7 7.29 6.06 5.88 17232055 Sgk1 8.78 9.54 9.52 17403879 Ankrd13c 8.88 8.37 8.54 17232534 Frk 7.42 6.67 6.57 17404011 Hey1 /// Gm41994 5.21 5.56 5.67 17234192 Zwint 9.70 9.29 9.17 17404269 Pde7a 8.18 7.65 7.65 17234423 Derl3 6.25 5.40 5.22 17405082 Slc7a11 8.14 8.96 9.38 17234436 Chchd10 6.10 5.16 5.57 17405174 Cog6 8.62 8.21 8.43 17234955 Fstl3 6.28 7.32 8.05 17405435 Siah2 6.68 5.92 6.07 17235647 Apba3 /// Mir3057 /// Mi 6.53 6.05 6.04 17405506 B430305J03Rik 6.23 5.34 5.37 17235954 A230046K03Rik 10.93 8.87 8.57 17405541 Gpr149 5.35 4.44 4.32 17236102 Btbd11 7.14 6.05 5.87 17405737 Lxn 9.84 8.15 8.92 17236182 Timp3 4.69 9.19 8.38 17406615 Rrnad1 6.12 5.84 5.56 17236604 Ntn4 6.94 5.95 5.60 17406908 Fdps 11.09 10.72 10.59 17236688 Tmcc3 6.50 6.04 5.88 17407001 Efna3 5.87 9.01 8.81 17236900 Kitl 6.02 4.87 5.16 17407017 Adam15 7.93 8.98 8.53 17237280 Phlda1 9.09 8.35 8.49 17407333 --- 8.52 7.80 8.30 17238834 Syne1 5.32 4.45 4.46 17407738 Mllt11 9.03 8.31 8.53 17238846 Syne1 5.64 4.10 4.09 17407775 Anxa9 /// 6330562C20Rik6.55 5.57 5.97 17238866 Syne1 4.48 3.59 3.62 17407905 Prpf3 8.02 7.59 7.28 17238868 Syne1 4.52 3.46 3.60 17407969 Plekho1 7.69 7.08 6.76 17238880 Syne1 4.85 3.61 3.77 17408017 Hist2h2aa1 /// Hist2h2 12.77 12.56 12.39 17239040 Ulbp1 5.54 3.90 4.08 17408211 Pde4dip /// Mir7225 // 6.13 5.29 5.44 17240077 Hey2 6.42 7.37 7.32 17409463 Fam102b 8.13 7.74 7.57 17240089 Tpd52l1 6.22 7.11 7.48 17409633 Slc30a7 8.67 8.32 8.24 17240190 Dse 8.25 7.15 6.81 17409792 Palmd 6.17 7.87 8.09 17241409 Srgn 4.37 3.31 3.41 17409826 Snx7 8.09 7.04 7.19 17241420 Kif1bp 6.93 6.44 6.54 17410332 Ccdc109b 7.81 7.29 7.05 17242025 Prmt2 /// Mir678 /// Mi 7.57 6.82 6.89 17410617 Dapp1 4.62 7.15 7.41 17242207 Pcbp3 5.76 6.31 6.45 17410974 Cyr61 12.19 11.57 11.61 17242747 R3hdm4 8.96 8.13 8.49 17411751 Trp53inp1 7.45 7.81 8.08 17244514 Nudt4 10.67 11.04 11.17 17412705 Gm12371 /// Gm12371 3.66 3.09 3.09 17246091 Il23a 10.28 8.77 8.97 17414188 Slc44a1 8.83 9.09 9.37 17246310 Dgka 7.47 8.97 9.80 17414984 Lurap1l 6.35 6.97 6.88 17246810 Lif 8.94 8.43 8.36 17415979 Lepr 3.94 3.25 3.19 17247225 Upp1 8.88 7.02 7.58 17416234 Plpp3 5.46 7.37 7.27 17250567 Aldh3a1 5.00 6.10 6.67 17417290 Atpaf1 6.46 5.79 5.78 17253478 Rab34 7.52 8.21 8.48 17419553 Map3k6 /// LOC10264 7.07 6.16 6.14 17253648 Slc46a1 6.78 7.70 7.28 17420154 Wnt4 5.64 7.04 7.36 17255064 Mmd 6.35 5.41 5.39 17420347 Ece1 7.21 8.45 8.27 17255458 Zfp652 /// Phospho1 5.44 6.09 6.46 17421006 Arhgef19 5.12 6.60 6.49 17256129 Csf3 7.43 6.65 6.84 17421033 Epha2 11.14 10.73 10.43 17262357 Rufy1 7.77 8.14 8.19 17421384 Gm13166 /// Znf41-ps 4.36 4.95 5.37 17262600 Tcf7 5.73 6.11 6.37 17421444 Gm13248 /// Gt(pU21)1 5.66 6.61 7.30 17262816 Csf2 6.77 4.46 5.20 17421452 Gm30910 /// LOC1026410623.21 4.10 3.64

Appendix B-2. Genes significantly upregulated (FDR < 0.05) in CXCL14 expressing tumor cells compared to non-CXCL14 expressing tumor cells.

162 Annotation Mean Expression Value Annotation Mean Expression Value MOE/E6E7 MOE/E6E7 MOE/E6E7 MOE/E6E7 Transcript MOE/E6E7 Transcript MOE/E6E7 Gene Symbol CXCL14 CXCL14 Gene Symbol CXCL14 CXCL14 Cluster ID Vector CL2 Cluster ID Vector CL2 CL8 CL16 CL8 CL16 17264124 Arhgap44 5.67 6.35 6.02 17421821 Ctnnbip1 7.58 8.08 8.05 17264647 Tmem88 7.21 5.76 5.53 17421972 Errfi1 10.53 10.09 9.92 17265156 Slc16a13 5.52 6.76 7.21 17423463 Decr1 5.75 5.09 5.36 17267775 Utp18 8.85 9.34 9.23 17423939 Lingo2 4.80 3.68 3.86 17268088 Gm11543 /// Gm11543 3.98 3.08 3.20 17424279 Cntfr 5.01 6.27 6.41 17269085 Krt20 6.93 4.73 4.62 17424573 Fam214b 6.35 5.75 5.68 17269139 Krtap1-5 3.66 4.60 4.24 17424822 Gne 8.29 7.74 7.76 17269391 Krt14 7.57 9.36 9.55 17425533 Epb41l4b 4.70 5.65 6.21 17269486 P3h4 5.56 7.01 7.89 17425566 Ptpn3 7.84 7.01 7.18 17272926 Nptx1 6.13 4.16 4.23 17425816 Susd1 6.72 7.75 7.86 17273485 Ogfod3 6.74 7.42 7.51 17426181 Wdr31 4.92 5.50 5.65 17273591 Kif3c 7.50 6.96 7.10 17426791 Zdhhc21 7.26 6.74 6.79 17273948 Sdc1 8.36 6.83 6.92 17426929 Bnc2 5.54 6.43 6.54 17274249 Pdia6 /// LOC1026419 11.69 11.48 11.26 17427769 Pcsk9 8.25 6.85 6.60 17275436 Akap6 4.36 5.29 5.56 17429008 St3gal3 5.58 6.94 7.16 17276591 Plekhg3 9.63 9.08 8.74 17429057 Ptprf 7.76 9.53 9.36 17277387 Fos 9.12 9.63 10.14 17429933 Zc3h12a 6.97 6.09 6.21 17279499 Crip2 9.16 10.01 9.82 17430894 Smpdl3b 8.53 9.49 8.94 17280125 Pqlc3 7.64 6.97 7.19 17431181 Sh3bgrl3 9.36 8.87 8.91 17280310 Id2 7.13 9.40 9.36 17431234 Cnksr1 8.21 7.86 7.78 17280350 Sox11 4.63 5.22 5.26 17432387 Tmem51 7.49 6.53 6.79 17281084 Egln3 4.38 4.78 5.16 17432440 Pdpn 9.99 11.00 10.93 17281880 Six1 7.25 7.99 8.06 17433436 Espn 5.40 5.50 5.90 17283270 Rps6ka5 7.28 7.51 7.81 17434657 Gm8871 /// Gm6455 /// LOC1010559123.65 4.24 5.06 17283364 Tc2n 6.41 7.06 8.13 17434669 Gm8871 /// Gm8857 3.86 4.37 5.25 17283463 Itpk1 8.05 6.69 7.35 17434711 Gm6460 4.16 4.53 5.54 17284114 Ckb 7.66 8.43 9.96 17434742 Sema3d 4.74 3.92 3.89 17284194 Tmem179 6.15 5.36 5.43 17434813 Sema3e 9.35 9.78 10.00 17284795 Sp4 5.82 6.51 6.65 17436043 Trim54 6.23 7.12 6.70 17284876 2810429I04Rik /// 1700024F13Rik4.25 3.37 3.33 17436927 Psapl1 6.06 7.19 8.01 17286320 Foxq1 6.45 7.24 7.65 17436944 Ppp2r2c 5.04 9.12 8.90 17286973 Gmpr /// LOC102642477 6.08 5.07 5.20 17437156 Cc2d2a 5.76 6.47 6.42 17287081 Id4 6.87 8.34 8.98 17437558 Pcdh7 8.94 7.91 7.61 17287984 Dapk1 4.04 5.53 5.30 17437876 Uchl1 5.02 6.52 7.12 17288992 Edil3 /// Gm38504 4.84 5.35 6.36 17438062 Nipal1 9.14 7.35 6.75 17289037 Ssbp2 6.06 7.50 7.70 17438196 Fip1l1 10.40 10.13 10.17 17289196 Lhfpl2 4.92 4.10 4.35 17438963 Ppbp 9.28 11.60 11.29 17289329 Fam169a 4.35 5.33 5.71 17438995 Cxcl2 7.99 6.89 7.24 17289382 Lincenc1 /// Lncenc1 5.09 7.23 5.61 17439388 Fras1 4.14 4.73 4.58 17290155 Gm7120 6.41 7.39 7.60 17439464 Anxa3 11.76 12.27 12.10 17290674 Prl2c2 8.72 9.65 9.30 17439517 Fgf5 7.37 7.88 7.82 17290738 Hecw1 5.75 7.59 6.96 17439909 Lrrc8c 10.55 10.13 9.59 17291146 Btn1a1 8.03 6.95 7.01 17440312 Fgfrl1 7.19 7.62 7.94 17293573 Fancc 4.79 5.06 5.22 17443689 Zcwpw1 7.61 8.10 8.48 17293706 Ctsl 11.56 11.02 11.13 17444924 8430423G03Rik /// 8430423G03Rik5.72 5.26 5.27 17294135 Srd5a1 /// Gm17108 8.95 8.04 8.00 17444998 Tex26 4.78 5.05 5.41 17294899 Fam151b 4.51 5.41 5.65 17445044 B3glct 8.21 9.25 9.15 17295136 Iqgap2 6.60 8.56 8.25 17446269 Smarcd3 5.71 6.37 6.98 17295233 Hmgcr 10.50 10.23 10.15 17446767 Cgref1 4.75 7.16 6.97 17295278 Hexb 8.15 8.79 9.36 17447269 Mfsd10 9.30 8.88 8.74 17296355 Itga2 11.42 9.96 10.58 17447689 Zfp518b 5.04 7.02 6.90 17296436 Parp8 8.28 7.78 7.73 17448840 Sgcb 6.84 5.92 5.71 17298041 Arhgef3 8.05 6.15 6.70 17449084 Igfbp7 6.52 8.90 9.82 17298090 Erc2 4.16 3.47 3.42 17449301 Tmprss11e 11.43 9.74 10.54 17298775 Anxa8 7.83 8.87 8.85 17449562 Rassf6 6.57 5.46 5.64 17299217 Styx 6.20 5.67 5.75 17452378 Rad9b 6.02 6.63 6.78 17299575 Ang /// Rnase4 6.42 5.25 4.85 17452861 Rilpl1 7.11 7.56 7.80 17300247 Trac /// Trac 6.41 7.25 7.06 17453160 Gusb 8.85 8.55 8.43 17301823 Gfra2 8.44 6.31 5.38 17453406 Lat2 7.85 6.09 6.11 17302571 9330188P03Rik 6.70 5.39 5.50 17454428 Micall2 7.81 6.34 6.90 17302606 Tpm3-rs7 /// Tpm3 9.43 8.91 8.81 17455962 C1galt1 9.48 9.05 9.07 17303442 Oit1 5.08 5.72 6.00 17456247 Cftr 4.32 6.28 5.82 17303453 4930452B06Rik 7.08 5.71 6.05 17456569 Fam71f1 7.19 7.94 8.63 17304740 Sh3bp5 7.36 6.92 6.95 17456886 Cpa4 8.72 6.41 4.75 17305662 Fermt2 8.02 7.64 7.76 17456934 Mest 5.14 7.09 7.36 17305709 Bmp4 8.30 9.61 9.51 17457876 Gstk1 4.30 5.62 5.96 17306929 Sdr39u1 4.15 8.17 8.48 17458472 Fam221a 4.33 4.73 5.28 17307738 Fzd3 5.85 6.81 6.41 17459108 Ccser1 4.89 6.23 5.76

Appendix B-2 (continued). Genes significantly upregulated (FDR < 0.05) in CXCL14 expressing tumor cells compared to non-CXCL14 expressing tumor cells.

163 Annotation Mean Expression Value Annotation Mean Expression Value MOE/E6E7 MOE/E6E7 MOE/E6E7 MOE/E6E7 Transcript MOE/E6E7 Transcript MOE/E6E7 Gene Symbol CXCL14 CXCL14 Gene Symbol CXCL14 CXCL14 Cluster ID Vector CL2 Cluster ID Vector CL2 CL8 CL16 CL8 CL16 17308242 Pdlim2 7.61 8.42 8.71 17459276 Serbp1 12.11 11.89 11.78 17309675 Zic5 5.01 5.99 5.96 17459591 St3gal5 5.27 7.99 8.35 17309935 Dab2 6.74 7.78 7.73 17461433 Edem1 9.68 9.16 8.99 17310187 Nadk2 7.23 7.62 7.99 17461942 Pparg 6.93 8.22 8.34 17310673 Ank 5.52 6.94 7.32 17462373 Cecr2 4.20 5.18 5.41 17310893 Mtdh 9.84 10.67 10.50 17462694 Nanog 4.63 5.25 5.53 17310912 Laptm4b 9.83 10.34 10.62 17463761 Fam234b 4.53 5.34 5.10 17310982 Osr2 5.56 7.03 7.08 17464248 Gm15706 /// Gm15706 6.47 7.41 7.31 17311070 Spag1 4.75 5.88 5.67 17464588 Sgce 4.42 5.49 6.51 17311157 Atp6v1c1 9.11 9.67 9.87 17464626 Pon3 4.10 5.51 5.20 17311199 Dcaf13 9.07 9.70 9.67 17466204 E330009J07Rik 6.90 7.30 7.81 17311286 Oxr1 7.91 9.11 8.65 17466452 Tcaf1 6.34 4.75 4.97 17311315 Emc2 9.49 10.27 10.46 17466532 Pdia4 /// Mir704 /// Mir 9.55 9.03 8.69 17311343 Eny2 8.44 9.50 9.28 17466641 Igf2bp3 9.21 8.89 8.64 17311428 Ebag9 7.83 8.51 8.65 17467742 Vamp5 7.38 8.08 8.16 17311512 Mal2 10.03 10.64 10.45 17467806 Tcf7l1 5.25 7.41 7.32 17311633 Has2os /// Gm38516 /// Gm413274.70 5.60 6.24 17469016 Wnt7a 9.13 8.57 8.47 17311789 Rnf139 8.71 9.30 9.34 17469217 Lrig1 8.72 7.00 7.03 17311909 Efr3a /// Gm41338 8.75 9.27 9.39 17470187 Zfp9 8.41 7.38 7.33 17312186 Psca 4.95 7.33 7.45 17472085 Pbp2 8.51 7.56 7.93 17312480 Scx 6.29 7.72 8.27 17472903 Pthlh 8.50 9.71 9.41 17313433 Xrcc6 6.90 7.28 7.46 17474454 Dmpk 5.65 6.59 6.59 17314260 Lrrk2 7.14 8.17 8.33 17474558 Ercc1 8.56 7.91 7.71 17314840 Aqp5 5.71 8.73 8.72 17474941 Kcnn4 10.46 9.83 9.99 17315245 Krt18 12.33 11.78 11.72 17474974 Plaur 10.23 9.75 9.66 17315255 Eif4b 11.41 10.74 10.83 17475135 Arhgef1 9.34 8.21 8.18 17315438 Prr13 6.67 5.91 5.75 17477237 Klk8 6.74 7.70 7.75 17315447 Pcbp2 12.24 11.98 11.95 17477254 Klk6 5.01 5.87 6.40 17316043 Npr3 7.79 8.58 8.96 17478961 Pcsk6 5.05 6.35 6.58 17316120 Cdh6 6.38 8.52 7.65 17479497 Sema4b 8.87 7.15 7.29 17316365 Tspyl5 3.96 4.76 4.96 17479974 Nox4 7.35 8.60 8.62 17316392 Hrsp12 7.17 7.81 8.15 17480534 Gdpd5 7.47 6.38 6.50 17316402 Nipal2 7.45 8.23 8.17 17482897 Aqp8 8.72 5.71 6.03 17316480 Rnf19a 8.12 8.83 9.17 17483912 Htra1 9.97 9.68 9.47 17316564 Zfp706 9.66 10.41 10.25 17484233 Ptpre 7.42 6.71 6.54 17316690 Klf10 7.99 8.36 8.57 17484382 Inpp5a 9.04 8.54 8.61 17316831 Nudcd1 8.85 9.27 9.35 17485297 Cd81 8.03 11.24 11.85 17316996 Rad21 10.74 11.21 11.15 17485389 Shank2 5.49 6.52 6.16 17318013 Lynx1 6.48 7.30 7.61 17486110 Peg3 5.18 8.51 7.19 17319554 Pmm1 9.35 10.04 10.35 17487489 Pvr 10.84 9.63 9.71 17320583 Cpne8 8.14 8.48 8.59 17487952 Ceacam1 5.38 7.37 7.38 17320974 Rpap3 7.52 7.97 7.84 17488735 Spred3 8.39 7.15 6.93 17321761 Smagp 5.29 6.84 7.49 17490078 Gm5595 2.94 4.66 4.65 17322113 Krt8 11.75 11.32 11.23 17492870 Bnc1 8.03 6.97 7.22 17322125 --- 11.00 10.00 10.29 17493212 Prss23 6.37 7.10 6.85 17322200 Aaas 9.53 8.97 8.88 17493658 Serpinh1 7.82 8.56 8.82 17322327 Cbx5 9.60 9.24 8.86 17494510 Apbb1 5.20 6.11 6.13 17324623 Hes1 8.69 9.43 9.50 17495541 Syt17 6.09 4.95 5.26 17325109 Itgb5 7.85 8.75 8.85 17497091 Oat 9.49 9.15 9.06 17325373 Ildr1 4.84 6.03 5.70 17497718 Ifitm3 8.76 9.54 10.26 17325919 Atg3 8.51 9.13 9.01 17499279 Lamp1 11.39 11.79 11.86 17326188 G730013B05Rik /// G730013B05Rik4.86 3.90 3.97 17500108 Fgfr1 7.18 8.41 8.58 17327038 Ifnar2 /// Gm21970 7.49 6.91 6.81 17500172 Plpp5 9.28 8.74 8.91 17328610 2610318N02Rik 5.50 6.83 6.21 17501148 Aga 6.37 7.42 7.40 17328625 Sdf2l1 9.98 9.26 9.01 17501329 Aadat 3.28 4.50 4.47 17329479 Cldn1 6.86 8.53 8.14 17501367 Nek1 /// LOC102642008 7.04 7.65 7.98 17330443 Gap43 7.17 8.88 7.92 17501609 Ints10 7.84 8.20 8.28 17331162 Col8a1 9.11 9.87 10.12 17501810 Nr2c2ap 7.25 7.67 7.83 17331173 St3gal6 7.39 7.86 8.42 17501994 Lsm4 10.22 10.58 10.78 17331669 App 10.68 11.10 11.14 17502049 2010320M18Rik /// 20 6.05 6.71 6.87 17334097 Cldn6 9.94 7.01 6.80 17502440 Klf2 6.94 7.89 8.15 17334192 Prss27 6.17 8.01 9.67 17503841 Lpcat2 7.60 8.03 8.12 17334253 Eci1 8.06 8.76 8.83 17504190 Adgrg1 /// LOC1026414124.10 4.98 5.76 17334299 Rab26os /// Rab26os 7.35 8.54 8.46 17506991 Nrp1 7.20 9.40 8.77 17335770 Abcg1 5.39 6.80 6.82 17508380 Letm2 5.44 5.91 6.34 17335818 Slc37a1 4.85 6.21 6.75 17508804 Dlc1 5.88 7.26 6.82 17336906 Ddah2 5.73 6.77 7.04 17509537 Palld 5.89 7.06 7.01 17336939 Abhd16a 7.11 7.53 7.56 17510080 Uba52 /// Kxd1 5.91 6.42 6.59

Appendix B-2 (continued). Genes significantly upregulated (FDR < 0.05) in CXCL14 expressing tumor cells compared to non-CXCL14 expressing tumor cells.

164 Annotation Mean Expression Value Annotation Mean Expression Value MOE/E6E7 MOE/E6E7 MOE/E6E7 MOE/E6E7 Transcript MOE/E6E7 Transcript MOE/E6E7 Gene Symbol CXCL14 CXCL14 Gene Symbol CXCL14 CXCL14 Cluster ID Vector CL2 Cluster ID Vector CL2 CL8 CL16 CL8 CL16 17337276 Ppp1r18 7.78 7.41 7.40 17510823 Gab1 6.14 7.69 7.45 17337441 Trim26 7.61 7.17 7.29 17510948 Adgre5 7.86 9.13 9.62 17337796 Pla2g7 4.51 5.29 5.79 17514460 Pdgfd 3.60 4.55 4.59 17337844 Enpp5 4.44 5.50 5.46 17516030 Tmem218 6.03 6.80 7.33 17338965 Trip10 9.58 8.96 8.98 17516518 Mcam 7.72 9.16 8.49 17339889 Rmdn2 3.39 5.10 4.86 17516837 Bace1 6.75 8.10 7.47 17340050 Plekhh2 4.31 7.33 6.31 17516960 Cadm1 8.46 8.97 9.08 17341179 Dll1 6.96 5.89 5.83 17517132 Cryab 7.87 10.14 9.52 17343263 Sik1 8.22 9.81 9.36 17517349 Slc35f2 8.06 8.74 8.67 17344140 D17H6S56E-5 10.14 9.27 9.34 17517448 Hykk 4.83 3.95 3.98 17345202 Slc29a1 7.53 8.55 8.46 17517469 Chrna5 5.26 7.44 6.57 17347082 Alk 4.62 4.15 4.34 17517481 Ube2q2 9.67 10.18 10.19 17347435 Cdc42ep3 6.78 7.57 7.43 17517554 Pstpip1 5.71 6.48 7.59 17347448 Cyp1b1 5.48 8.29 8.49 17517812 Sema7a 8.00 6.90 7.13 17348791 Dsg3 5.55 8.10 8.77 17517947 Hexa 8.00 8.52 9.03 17348812 Dsg2 7.73 8.80 8.92 17519316 Wdr72 4.31 5.31 5.54 17348902 Dtna 7.01 6.64 6.42 17519438 Myo5c 5.88 5.35 5.29 17348965 Galnt1 9.30 8.77 8.65 17519550 Hmgcll1 8.20 7.34 6.12 17349304 Tslp 4.66 5.92 6.98 17519579 Gclc 7.86 9.12 8.54 17351178 Spink10 3.54 3.89 4.08 17519649 Gsta4 6.96 9.82 10.36 17351414 Pmaip1 7.72 7.39 7.13 17519673 Gsta1 /// Gm3776 5.51 8.49 7.65 17351634 Ccdc68 6.97 7.88 7.91 17519738 Cd109 5.39 7.35 7.36 17352549 Mpp7 6.77 7.32 7.65 17519898 Sh3bgrl2 9.67 9.36 9.28 17352933 Dsc2 4.25 6.33 7.19 17519940 Bckdhb 6.47 7.32 7.49 17353370 Nrep 5.69 6.09 6.51 17520851 Anapc13 10.92 11.38 11.35 17354434 Aldh7a1 8.83 7.42 7.81 17522488 Prss46 6.68 8.30 8.57 17354857 Adrb2 5.28 5.83 6.06 17523231 Entpd3 6.85 6.06 6.36 17355026 Atp8b1 7.09 8.45 8.14 17523531 Tmem42 6.01 6.82 7.04 17355304 4930503L19Rik 6.75 6.12 5.98 17523555 Exosc7 7.46 8.23 7.88 17356216 Pcx 6.31 7.58 7.75 17523921 Arhgap42 7.85 8.32 8.70 17356434 Fibp 8.27 7.60 7.93 17524858 Rgl3 6.06 6.75 6.65 17357207 Nxf1 /// Nxf1 /// Tmem 6.98 5.64 4.91 17525329 St3gal4 7.34 8.20 8.09 17357418 Fth1 12.76 12.91 13.01 17525342 Dcps 7.21 7.68 7.73 17357971 Gna14 5.35 7.67 6.98 17525492 A630095E13Rik 4.71 7.19 6.55 17360109 Ina 4.75 6.78 5.96 17525894 Sorl1 5.88 8.52 8.18 17360348 Dusp5 9.71 9.04 9.00 17526776 Ncam1 7.33 8.31 8.46 17360570 Plekhs1 6.48 8.57 8.38 17526813 Pts 5.90 6.80 7.22 17361996 Ppp2r5b 7.99 7.35 7.32 17526923 Hspb2 5.21 6.40 6.40 17362544 Asrgl1 6.17 6.96 7.02 17527421 Tspan3 9.02 9.48 9.57 17362784 Tmem132a 8.03 8.78 9.01 17527520 Scamp5 7.27 8.56 8.05 17363927 Asah2 8.23 7.25 7.50 17527963 Paqr5 4.40 3.77 4.05 17364565 Blnk 8.41 9.80 9.08 17527996 Coro2b 4.55 5.57 5.21 17364945 Gm20467 /// Gm20467 7.02 7.47 7.54 17528014 Fem1b 8.96 9.40 9.36 17367102 Mrc1 6.49 4.79 5.27 17528430 Tpm1 9.74 10.07 10.07 17367510 Gad2 5.51 4.81 4.89 17528775 Ccpg1os 8.01 8.43 9.16 17367726 Gm27193 /// Gm13415 /// 6.89Gm13415 7.61 7.64 17528850 Tmod3 9.89 10.31 10.62 17367868 Rnf208 6.07 6.78 6.71 17529099 Col12a1 4.38 7.73 6.85 17369286 Prrx2 5.88 6.92 7.42 17529276 Hmgn3 6.26 7.53 7.28 17369911 Ak1 6.53 7.51 7.14 17531001 Sema3f 7.77 9.13 9.13 17369952 Sh2d3c 7.02 6.06 6.33 17533336 Atp6ap2 8.82 9.05 9.23 17370659 Kif5c 4.88 5.97 5.75 17533474 Maoa 8.10 8.88 8.68 17371201 Gca 8.60 8.25 8.06 17533665 Usp11 6.63 5.25 5.36 17371427 Dhrs9 8.68 6.12 6.72 17533690 Araf 8.37 8.75 8.71 17372552 Fam171b 6.87 4.80 4.96 17534293 Rhox5 6.77 4.85 5.06 17374488 Thbs1 10.46 11.84 11.33 17535600 Dusp9 9.53 8.45 8.53 17375161 Tmem62 5.92 6.70 6.61 17536264 Pcyt1b 9.10 8.11 8.29 17375685 Fgf7 7.76 8.54 8.25 17536961 Chic1 4.23 5.17 4.94 17375841 Stard7 8.43 8.76 8.76 17539210 Sh3kbp1 8.99 7.98 8.40 17375859 Dusp2 6.16 7.66 8.38 17539271 Adgrg2 6.99 8.52 8.90 17376549 Prnd /// Prn 6.89 4.54 4.40 17539548 Piga 7.50 7.00 6.90 17376908 Ism1 7.15 5.82 5.42 17540739 Gm2005 /// Gm2005 4.02 5.75 5.82 17377144 Slc24a3 4.44 5.61 5.60 17541190 Cul4b 9.89 10.40 10.43 17377443 Gm14139 /// Gm14139 3.72 5.31 5.28 17542392 Idh3g 11.01 11.39 11.50 17377498 Pygb 9.22 8.73 8.57 17542419 L1cam 6.30 7.27 7.19 17377778 Id1 6.68 8.54 8.76 17543264 Maged1 10.62 10.21 10.18 17378072 Bpifb4 5.42 6.58 7.68 17543625 Cxcr3 8.06 6.32 6.61 17380766 Slco4a1 7.25 8.81 9.19 17543974 Magt1 9.70 9.25 9.07 17381404 Sec61a2 7.22 7.64 7.59 17544061 Cysltr1 4.11 4.91 5.62

Appendix B-2 (continued). Genes significantly upregulated (FDR < 0.05) in CXCL14 expressing tumor cells compared to non-CXCL14 expressing tumor cells.

165 Annotation Mean Expression Value Annotation Mean Expression Value MOE/E6E7 MOE/E6E7 MOE/E6E7 MOE/E6E7 Transcript MOE/E6E7 Transcript MOE/E6E7 Gene Symbol CXCL14 CXCL14 Gene Symbol CXCL14 CXCL14 Cluster ID Vector CL2 Cluster ID Vector CL2 CL8 CL16 CL8 CL16 17381752 Fam188a 10.61 11.46 11.74 17544164 Rps6ka6 9.11 8.42 8.17 17382038 Pip4k2a 8.35 7.91 7.73 17547680 --- 7.67 6.42 6.32 17383965 Cdk9 7.88 7.56 7.44 17547827 Gm5802 /// Gm35595 8.86 7.71 7.86 17385967 Galnt3 10.84 10.36 10.33 17547887 --- 4.92 5.10 5.17 17386540 Wipf1 4.84 5.47 5.41 17547994 --- 5.67 6.35 6.62 17386700 Nfe2l2 /// LOC1052442 9.94 10.29 10.27 17548228 Cisd3 /// Cisd3 /// Cisd3b7.12 7.59 7.59 17388454 Cd82 6.56 5.25 6.00 17548750 --- 5.42 7.46 7.04 17388974 Depdc7 7.36 7.93 8.22 17549476 --- 6.61 7.56 8.07 17390810 Gatm 3.88 7.19 6.91 17549514 --- 12.65 12.51 12.49 17390823 Slc30a4 10.11 10.42 10.74 17549620 --- 6.61 7.56 8.07 17391332 Mal 11.28 11.68 11.84 17550512 --- 4.54 3.36 3.54 17391554 Il1a 10.09 6.16 7.39 17550520 --- 4.46 3.51 3.61

Appendix B-2 (continued). Genes significantly upregulated (FDR < 0.05) in CXCL14 expressing tumor cells compared to non-CXCL14 expressing tumor cells.

166

MOE/E6E7Vector MOE/E6E7CXCL14 MOE/E6E7CXCL14 CL2 CL8 CL16

-2.5 0 2.7 Log2 fold

Appendix B-3. CXCL14 alters host gene expression in HPV-positive cancer cells. Gene expression profiles were assessed by Affymetrix Mouse Gene 2.0 ST 2.0 arrays in quadruplicate for MOE/E6E7 cell lines: Clone 2 (Vector), Clone 8 and Clone 16 (CXCL14), at different passages. Log2 fold changes of differentially expressed immune related genes in Clone 2 vs. Clone 8 and Clone 16 are shown by heat map (FDR adjusted p < 0.05). Transcript cluster IDs are listed in Appendix B-2. Figure generated by Dr. Dohun Pyeon, PhD.

167