HPV Oncogenes Research Articles

Abstract 3511: Sustained adrenergic signaling promotes cervical cancer progression

Abstract Objective: Chronic stress and sustained adrenergic signaling promote tumor progression. The underlying mechanisms behind this process are not well understood. We examined the effects of sustained adrenergic signaling on cervical cancer progression through increased expression of HPV oncogenes, E6 and E7. Methods: Beta-adrenergic receptor (ADRB) positive cervical cancer cell lines (CaSki and SiHa) were treated with norepinephrine (NE) or isoproterenol (ISO) to analyze intracellular responses. Migration, invasion and anoikis assays were performed to elucidate the resultant biological effect of NE and ISO. ADRB expression levels were examined from cervical cancer patient tumor samples. Results: ADRB expression was determined for cervical cell lines (CaSki, SiHa, and C33A) using qRT-PCR. CaSki and SiHa cells express ADRB1, ADRB2, and ABRB3. Cells exposed to NE for 30 min showed elevated cyclic AMP activity. After treatment with NE or ISO, mRNA levels from HPV oncogenes, E6 and E7, were significantly elevated in the SiHa and CaSki cells. NE exposure resulted in a significant increase in invasion and migration of cervical cancer cells, while E6 siRNA abrogated these effects. After 48 hours of NE or ISO exposure, CaSki cells showed a 30% reduction in anoikis. Among 166 tumor samples evaluated from cervical cancer patients, 85% had increased ADRB1 expression and 61% had increased ADRB2 expression. Tumor stage or grade was not related to ADRB expression. ADRB1 expression was not correlated with patient survival outcomes (p = 0.86); however, patients with high ADRB2 expressed had decreased overall survival (p=0.038). Conclusion: Increased adrenergic signaling promotes cervical cancer progression. Disruption of this pathway could provide a novel complement to current therapies. Citation Format: Nouara C. Sadaoui, Guillermo N. Armaiz-Pena, Archana S. Nagaraja, Rajesha Rupaimoole, Rebecca A. Previs, Heather J. Dalton, Mangala S. Lingegowda, Lois M. Ramondetta, Anil K. Sood, Susan K. Lutgendorf, Steve W. Cole. Sustained adrenergic signaling promotes cervical cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3511. doi:10.1158/1538-7445.AM2014-3511

Abstract 3176: The EGFR pathway as the Achilles’ heel for human papillomavirus-induced tumors: EGFR/MAPK pathway inhibitors exhibit antiviral activities and limit tumor growth in vivo

Abstract Human papillomaviruses (HPVs) are etiological agents of many anogenital and oropharyngeal cancers. HPV[+] oropharyngeal squamous cell carcinomas (OPSCCs) typically respond more favorably to current treatment regimens (including radiotherapy combined with cisplatin or cetuximab, an EGFR monoclonal antibody), than do HPV-negative OPSCCs. The discrepancy in patient outcomes has been attributed, in part, to substantially fewer genetic mutations present in HPV[+] cancers. Paradoxically, an increasing body of literature notes lower EGFR expression in HPV[+] vs. HPV[-] OPSCC. Yet, HPV oncoproteins E5, E6 and E7 each work to increase EGFR signaling, and MAPK-regulated AP-1 transcription factors direct HPV early gene expression. The goal of this work is to test the hypothesis that upon infection, HPV establishes a feed-forward loop with the EGFR pathway to drive viral gene expression independent of EGFR levels. We modeled early neoplasia with cell lines maintaining episomal HPV16 genomes, and cancers using SCC lines with integrated HPV16 genomes. Cells treated with EGF, EGFR inhibitors (cetuximab, erlotinib), or MEK antagonists (PD98059, trametinib) were evaluated for signaling and viral responses. Consistent with our hypothesis, we found increased EGF-dependent EGFR activation levels in HPV[+] cell lines, as well as heightened MEK1/2 activity independent of EGFR activation in HPV[+] cell lines compared to uninfected cells. RT-qPCR revealed that HPV oncogene transcription was enhanced by EGFR activation, whereas EGFR and MEK inhibitors led to significant declines in HPV transcription. In cells with episomal HPV genomes, lower viral genome levels accompanied inhibitor-reduced viral transcription. These data suggest EGFR/MAPK pathway interference leads to anti-viral affects, which are expected to restore p53 and pRb function and prevent tumorigenesis in vivo. As predicted, cell proliferation and tumor growth in NOD/SCID-gamma mouse HPV[+] SCC xenografts were significantly reduced with EGFR and MEK inhibitors. Experiments are underway to quantify the restored function of p53 and pRb in drug-treated cells and determine if these cells become sensitized to lower doses of cisplatin or radiation. In summary, our results reveal HPV infection creates a feed-forward loop with the EGFR/MAPK proliferation pathway, and EGFR and MEK antagonists have anti-viral effects, including reduction in the E6 and E7 oncoproteins needed to maintain the transformed phenotype in vitro and in vivo. Additionally, the ability of the drugs to reduce episomal viral genome levels suggests a potential curative effect if administered prior to viral genome integration. Lastly, this work reveals an anti-viral molecular mechanism which may account for the favorable outcomes of patients with HPV[+] OPSCC treated with cetuximab-radiotherapy. Citation Format: Anastacia M. Griego, Pamela Barraza, Chelin Hu, Agnieszka Dziduszko, Brianna K. Crowley, Helen J. Hathaway, Julie E. Bauman, Michelle A. Ozbun. The EGFR pathway as the Achilles’ heel for human papillomavirus-induced tumors: EGFR/MAPK pathway inhibitors exhibit antiviral activities and limit tumor growth in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2014-3176

Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage

Background and purposePatients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV−) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV− HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown. Material and methodsUsing immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot. ResultsHPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation. ConclusionsOur findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV− HNC.

High-risk human papillomavirus E6 protein promotes reprogramming of Fanconi anemia patient cells through repression of p53 but does not allow for sustained growth of induced pluripotent stem cells.

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway, which promotes error-free repair of DNA double-strand breaks, is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus, cells from Fanconi anemia patients, which lack this critical pathway, fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6, but not E7, recovers FA iPSC colony formation and, furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.

IGF expression in HPV-related and HPV-unrelated human cancer cells.

The human Igf-1 gene not only produces insulin-like growth factor-I (IGF-I), but also different carboxy-terminal extensions, known as E peptides, through alternative splicing. We and others have shown that human Eb peptide (hEb) derived from Igf-1 has intrinsic biological activity and is localized to nuclei of transfected cells. Since hEb actions can complement the activity of IGF-I itself, the aim of the present study was to compare IGF-I isoforms at the endogenous protein and transcript level in cancer cell lines, including HeLa, U2OS, HepG2 and K562 cells. Quantitative real-time PCR (qRT-PCR) using Igf-1 isoform specific primers was performed to determine expression patterns, using β-actin as a reference gene. The overall relative Igf-1 transcript level was different across the cell lines, with ~80-fold higher expression in K562 (130.2±31.2) than in U2OS cells (1.7±1.1). The relative copy number of Igf-1b was the highest in HepG2 (69.9±28.6) and K562 cells (28.3±6.7), whereas the relative copy numbers of Igf-1a and Igf-1c were significantly higher in K562 cells compared to all other cell lines. Immunoblotting using total cell lysates, cytoplasmic and nuclear fractions were carried out to determine the level and distribution of IGF-I proteins. K562 cells exhibited the highest level of hEb in total cell lysates and nuclear fractions and no cell lines displayed hEb in the cytoplasmic fractions. In contrast, IGF-IA was the highest in HeLa cells and was enriched only in the cytoplasmic fraction. Since relatively low IGF-1A transcript level but relatively high pro-IGF-1A protein level is plausible, we hypothesized that these transcripts could be processed with higher efficiency and/or the protein product may be stabilized by viral HPV oncogenes in HeLa cells. We assert that while it is important to analyze Igf-1 transcript level, it may be more relevant to determine the IGF isoforms at the protein level.

Multiple-integrations of HPV16 genome and altered transcription of viral oncogenes and cellular genes are associated with the development of cervical cancer.

The constitutive expression of the high-risk HPV E6 and E7 viral oncogenes is the major cause of cervical cancer. To comprehensively explore the composition of HPV16 early transcripts and their genomic annotation, cervical squamous epithelial tissues from 40 HPV16-infected patients were collected for analysis of papillomavirus oncogene transcripts (APOT). We observed different transcription patterns of HPV16 oncogenes in progression of cervical lesions to cervical cancer and identified one novel transcript. Multiple-integration events in the tissues of cervical carcinoma (CxCa) are significantly more often than those of low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). Moreover, most cellular genes within or near these integration sites are cancer-associated genes. Taken together, this study suggests that the multiple-integrations of HPV genome during persistent viral infection, which thereby alters the expression patterns of viral oncogenes and integration-related cellular genes, play a crucial role in progression of cervical lesions to cervix cancer.

Mate pair sequencing of oropharyngeal squamous cell carcinomas reveals that HPV integration occurs much less frequently than in cervical cancer

BackgroundHuman papillomavirus (HPV) is now recognized to be very important in the pathogenesis of oropharyngeal squamous cell carcinoma (OPSCC). It is not clear yet whether the physical status of HPV in OPSCC is similar to what is found in cervical cancer. Study designWe performed genome-wide mate pair next generation sequencing from 20 OPSCCs patients, thirteen of which were positive for HPV16 to determine the HPV physical status and its relationship to HPV oncogene E6 and E7 expression. ResultsThis high throughput approach detected HPV integration events and also determined the bridged HPV coverage in each sample. Two of the HPV16-positive OPSCCs had HPV integration and one of the HPV16-negative OPSCCs had an HPV26 integration. We mapped the site of integration in the HPV genome in all integration events and the integrations were located at E1, E5, E6 and L2 region respectively. One HPV positive OPSCC had two integration events but also had a very high bridged HPV coverage, while the other two just had HPV integrated into the human genome. ConclusionOur results are thus different from what is routinely observed in cervical cancer where HPV is almost always integrated into the human genome with loss of episomal HPV sequences. Thus more investigation should be carried out to study how episomal HPV alone can contribute to the development of most OPSCCs.

Detection of E6-AP as a potential therapeutic target in cervical specimen of HPV-infected women

Silencing of HPV oncogenes or their human host client proteins using topically applied small interfering RNA (siRNA) may be an attractive nonsurgical strategy for CIN treatment. An exploratory clinical investigation was designed to evaluate E6-AP mRNA expression levels in different stages of cervical intraepithelial neoplasia and during the menstrual cycle. In 38 premenopausal women aged 18-45 years referred to colposcopy clinic, analysis of serum hormones, cervical smears for cytology and HPV DNA, cervical biopsy, p16 immunohistochemistry and E6-AP mRNA expression levels in cervical smears and biopsies were performed. The intra-subject variability in E6-AP mRNA expression of vaginal smears was assessed and compared to cervical biopsy specimens. RNA of sufficient quantity and quality was available for E6-AP expression analysis from 97 % of the collected cervical smears and from 56 % of the collected biopsy samples. The normalized RNA levels from cervical smears were approximately tenfold higher compared to biopsies. There was little influence by the phase of the menstrual cycle or by CIN stage. Real-time PCR showed that the expression level of E6-AP is in a range (<28 C t) that would allow for detection of at least 100-fold modulation by a therapeutic agent (based on an assay LOD of C t = 36). Our findings suggest a potential therapeutic approach using E6-AP siRNA as a specific molecular target in cervical intraepithelial neoplasia.

Induction of heparanase by HPV E6 oncogene in head and neck squamous cell carcinoma

High-risk human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCCs) are highly invasive; however the identity of downstream effectors responsible for their aggressive phenotype remains underinvestigated. Here, we report that HPV-mediated up-regulation of heparanase enzyme can provide mechanistic explanation for augmented invasiveness of HPV-positive HNSCCs. Heparanase is the sole mammalian enzyme (endo-β-d-glucuronidase) degrading heparan sulphate glycosaminoglycan, key polysaccharide of the extracellular matrix. Cleavage of heparan sulphate by heparanase leads to disassembly of extracellular barriers, enabling local invasion and metastatic spread of the tumour, and releases heparan sulphate-bound growth factors from the extracellular depots. Heparanase is tightly implicated in head and neck cancer progression; yet, molecular mechanisms underlying transcriptional activation of the heparanase gene in HNSCC are largely unknown. We found that HPV16 oncogene E6 is capable of inducing overexpression of heparanase in HNSCC. Notably, radiation treatment dose-dependently suppresses E6-induced heparanase expression in vitro. Our results provide the first evidence for a functional involvement of HPV in heparanase induction in head and neck tumourigenesis and, given ongoing clinical testing of several heparanase-inhibiting compounds, offer important avenue for future therapeutic exploration in HNSCC, as well as other HPV-associated malignancies (i.e. cervical carcinoma).

Requirement of estrogen receptor alpha DNA-binding domain for HPV oncogene-induced cervical carcinogenesis in mice

Cervical cancer is caused by human papillomavirus (HPV) in collaboration with other non-viral factors. The uterine cervix is hormone responsive and female hormones have been implicated in the pathogenesis of the disease. HPV transgenic mice expressing HPV16 oncogenes E6 (K14E6) and/or E7 (K14E7) have been employed to study a mechanism of estrogen and estrogen receptor α (ERα) in cervical carcinogenesis. A chronic exposure to physiological levels of exogenous estrogen leads to cervical cancer in the HPV transgenic mice, which depends on ERα. The receptor is composed of multiple functional domains including a DNA-binding domain (DBD), which mediates its binding to estrogen-responsive elements (EREs) on target genes. A transcriptional control of genes by ERα is mediated by either DBD-dependent (classical) or DBD-independent (non-classical) pathway. Although molecular mechanisms of ERα in cancer have been characterized extensively, studies investigating importance of each pathway for carcinogenesis are scarce. In this study, we employ knock-in mice expressing an ERα DBD mutant (E207A/G208A) that is defective specifically for ERE binding. We demonstrate that the ERα DBD mutant fails to support estrogen-induced epithelial cell proliferation and carcinogenesis in the cervix of K14E7 transgenic mice. We also demonstrate that cervical diseases are absent in K14E7 mice when one ERα DBD mutant allele and one wild-type allele are present. We conclude that the ERα classical pathway is required for cervical carcinogenesis in a mouse model.

High Incidence of HPV-Associated Head and Neck Cancers in FA Deficient Mice Is Associated with E7’s Induction of DNA Damage through Its Inactivation of Pocket Proteins

Fanconi anemia (FA) patients are highly susceptible to solid tumors at multiple anatomical sites including head and neck region. A subset of head and neck cancers (HNCs) is associated with ‘high-risk’ HPVs, particularly HPV16. However, the correlation between HPV oncogenes and cancers in FA patients is still unclear. We previously learned that FA deficiency in mice predisposes HPV16 E7 transgenic mice to HNCs. To address HPV16 E6’s oncogenic potential under FA deficiency in HNCs, we utilized HPV16 E6-transgenic mice (K14E6) and HPV16 E6/E7-bi-transgenic mice (K14E6E7) on genetic backgrounds sufficient or deficient for one of the fanc genes, fancD2 and monitored their susceptibility to HNCs. K14E6 mice failed to develop tumor. However, E6 and fancD2-deficiency accelerated E7-driven tumor development in K14E6E7 mice. The increased tumor incidence was more correlated with E7-driven DNA damage than proliferation. We also found that deficiency of pocket proteins, pRb, p107, and p130 that are well-established targets of E7, could recapitulate E7’s induction of DNA damage. Our findings support the hypothesis that E7 induces HPV-associated HNCs by promoting DNA damage through the inactivation of pocket proteins, which explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer. Our results further demonstrate the unexpected finding that FA deficiency does not predispose E6 transgenic mice to HNCs, indicating a specificity in the synergy between FA deficiency and HPV oncogenes in causing HNCs.

Retrieval of HPV oncogenes E6 and E7 mRNA from cervical specimens using a manual open technology protocol

BackgroundHPV oncogenes mRNA detection gains momentum as an adjuvant for HPV-related cervical abnormalities diagnosis, but is based on costly detection assays not allowing viral type targeting.ObjectiveTo assess detection rate of HPV oncogenes E6/E7 mRNA from cervical specimens using a manual, open technology, fully customizable protocol and determine whether HPV-related epidemiological features influence mRNA retrieval. We reviewed literature and compared our retrieval rate with automated technologies.MethodsWe used 60 samples positive for HPV DNA types 16, 18, 31 and/or 45. We extracted mRNA with a TRizol-based protocol, and tested mRNA purity and concentration using light absorbance. We reverse-transcribed mRNA into cDNA for E6/7 detection.ResultsHPV oncogenes E6/E7 mRNA was retrieved from 36 (60%) out of 60 specimens. No HPV load-related clinical or epidemiological feature was significantly associated with mRNA retrieval. Presence of HPV-DNA 16/18 was associated with mRNA retrieval (OR = 9.08; 95% CI 1.26 to 65.32 for HPV 16; and 18.2; IC95% 1.86 to 391.44 for HPV 18).ConclusionsThe open-technology protocol yielded an mRNA detection rate similar to that of automated technologies. Advantages are lower costs and target HPV type customization.

High incidence of female reproductive tract cancers in FA-deficient HPV16-transgenic mice correlates with E7's induction of DNA damage response, an activity mediated by E7's inactivation of pocket proteins.

Fanconi anemia (FA) is a rare genetic disorder caused by defects in a DNA damage repair system, the FA pathway. FA patients frequently develop squamous cell carcinoma (SCC) at sites that are associated with HPV-driven cancer including the female reproductive tract. To assess experimentally whether FA deficiency increases susceptibility to HPV-associated cervical/vaginal cancer, we monitored cancer incidence in the female lower reproductive tract of FA-deficient mice expressing HPV16 oncogenes, E6 and/or E7. FA deficiency specifically increased the incidence of cancers in mice expressing E7; but, this effect was not observed in mice just expressing E6. We also observed that E7, but not E6, induced DNA damage as scored by induction of γ-H2AX and 53BP1 nuclear-foci, and this induction was heightened in FA-deficient tissue. Finally, we discovered that this induction of DNA damage responses was recapitulated in mice deficient in expression of ‘pocket’ proteins, pRb, p107, and p130, which are established targets of E7. Our findings support the hypothesis that E7 induces cancer by causing DNA damage at least in part through the inactivation of pocket proteins. This hypothesis explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer.

The HPV16 oncogenes cause aberrant stem cell mobilization

Human Papilloma Virus related epithelial cancers have been speculated to derive from virus-infected tissue stem cells. Stem cells also are thought to provide a reservoir of latently infected cells that can persist for long periods. In this study we have examined the effects of HPV16 E6 and E7 oncogenes on multipotent epithelial stem cells, using in vivo systems. Our results show that expression of HPV16 oncogenes reduces the number of bulge label-retaining cells within hair follicles at telogen suggesting aberrant mobilization, a result supported by increased mobilization upon acute anagen induction. Importantly the loss of relative quiescence, a hallmark feature of stem cells, occurs in the absence of a reduction in other stem cell markers. This points to an atypical stem cell compartment in the context of E6 and E7 expression. We hypothesize that this aberrant compartment may have important roles in the viral life cycle and/or ensuing carcinogenesis.

OP194: Novel mucosal orthotopic pre-clinical tumor model for the validation of therapeutic vaccines for HPV-associated head and neck cancers

Purpose Papillomavirus (HPV)-16 infection has been recently associated with oropharyngeal head and neck cancers (HNCs) that express the E6 and E7 oncoproteins. The licensed vaccines are efficient for the prevention of HPV infection, but not for established tumors. Therefore innovative therapeutic vaccines targeting HPV oncogenes are required. Previously, we have developed a DNA-based vaccine strategy using plasmo-VLP carrying a non-oncogenic mutated, but immunogenic, form of E7 from HPV16 (pVLP-E7). pVLPs allow to form in vivo VLPs (viral-like particles formed by structural proteins devoid of viral genome). Thus, we showed that pVLP-E7 can elicit specific anti-E7 immune responses and cure mice from E6/E7 HPV16-positive tumors injected subscutaneously in the flank of animals. The present study aims at developing an orthotopic model of HNCs in mice as a pre-clinical model. Material and methods TC-1 tumor cells, an epithelial murine cell line that express E6 and E7 of HPV 16, were injected into the tongue or cheek (orthotopic models) or in the flank (ectopic model) of C57BL/6 mice; different routes of vaccination (intranasal, intra-cheek or intradermal) with pVLP-E7 were tested in order to induce local and systemic immune responses; tumor growth, anti-E7 immune responses, and anti-tumor effects against established tumors were compared between the different tumor models and routes of vaccination. Results The cheek model was better suited for future test of immunization than intralingual or subcutaneous tumor models, due to better survival rates and slower growth kinetics. Intradermal vaccination with pVLP-E7 was the better route of administration to achieve a systemic anti-E7 T-cell response. Interestingly, intradermal and intra-cheek vaccinations appeared to be both equally efficient to stimulate local immune responses as well as anti-tumoral responses by using the orthotopic model. Conclusions DNA vaccinations with pVLP-E7 can induce therapeutic anti-tumoral effects using an intra-cheek orthotopic model of HPV-induced HNCs.

OP197: MI-RNA expression profiling of HPV-positive and -negative oropharyngeal carcinomas

Purpose HPV is detected in 40–80% of oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive OPSCCs have a different biology from that of HPV-negative cancers, with distinct phenotypic features, including poor differentiation, and are associated with improved prognosis. Recent studies have shown evidences of abnormal expression of miRNAs in human tumors. MiRNAs play an important role in fundamental cellular processes and are biological indicators for disease outcome. Little is known about miRNA expression in OPSCC. Methods PCR and immunohistochemistry was used to determine HPV-status and p16 immunopositivity of 65 OPSCCs collected at the University Clinic of Cologne. To characterize the expression profile of miRNAs, 20 HPV-positive and 20 HPV-negative OPSCC samples were analyzed by miRNA-Chip-Array. Significantly deregulated miRNAs were selected for further analysis including qRT-PCR and in situ hybridization. Expression of these selected miRNAs was also studied in HPV16-E6, -E7 and -E6/E7 positive primary human keratinocytes (PHK). Results By chip analysis, several miRNAs (e.g. miR-9, miR-9 ∗ , miR-16-2, miR-135a and miR-363) were found to be significantly overexpressed in HPV-positive OPSCCs. Their deregulation in tissue could be confirmed by qRT-PCR. In PHK qRT-PCR showed a viral influence especially on miR-9 and miR-363. Conclusion These data show distinctive miRNA expression profiles in HPV-positive and HPV-negative OPSCCs which may help to distinguish between these two types of cancers using miRNA profiles as indicators. Further studies on regulation of transcription of these miRNAs by HPV oncogenes E6, E7 and E6/E7 are necessary to understand their role in OPSCC development.

Live-attenuated Listeria-based immunotherapy

For decades Listeria monocytogenes has been used as a model of host–disease immunology, and a considerable body of knowledge has been amassed regarding the complex immune response to L. monocytogenes. Attenuated strains of L. monocytogenes are currently being assessed as therapeutic bacterial vectors to present tumor-associated antigens to the immune system for the clinical treatment of cancer. L. monocytogenes immunotherapy utilizes many synchronous and disparate action mechanisms that stimulate innate and cell-mediated adaptive immunity while reducing immunosuppressive influences in the tumor microenvironment. Other effects not typically associated with immunotherapy include the stimulation of myeloid hematopoiesis and vascular changes that enable chemotaxis. Preliminary clinical results using L. monocytogenes bearing the HPV oncogene E7 indicate good tolerability and a strong efficacy signal, warranting further development. This article reviews the current status of L. monocytogenes as a cancer immunotherapeutic and the complex immune responses that underlie L. monocytogenes immunotherapy.

Abstract 3611: Benzo(a)pyrene exposure increases expression of HPV oncoproteins: a potential co-factor for increased cervical cancer among Northern Plains American Indian women.

Abstract Introduction: Cervical cancer remains one of the most common and deadly cancers in women worldwide. There is a significant health disparity in the incidence and mortality of cervical cancer between American Indian and Caucasian women residing on the Northern Plains. The development of cervical cancer is closely associated with persistent infection with high risk HPV genotypes. Additionally, exposure to cigarette smoke is also a risk factor for cervical cancer. Our studies indicate that Northern Plains American Indian women have a high rate of both HPV infection and smoking. Our studies also show that American Indian women who tested positive for HPV were more likely to have an abnormal PAP test than HPV positive Caucasian women. While smoking is known to increase the risk of developing cervical cancer, the molecular interactions between HPV and smoke carcinogens are not well known. Herein, we determine the cellular and molecular effects of benzo-a-pyrene (BaP) (a carcinogen in cigarette smoke) on HPV oncoprotein expression in cervical cancer cells and elucidate the role of the aryl hydrocarbon receptor (AhR) in this process. Methods: The effects of BaP on HPV oncoproteins and AhR mediated molecular events was determined using cervical cancer cell line models and real time PCR, immunoblotting, immunostaining and flow cytometry analyses. The level of AhR activation in normal cervix and in cervical cancer tissues was also determined with immunohistochemical analysis. Results: Our results indicate that exposure to BaP, a tobacco carcinogen, activates the AhR pathway in a time and dose dependent manner, as determined by an increase in nuclear localization of AhR and increased expression of cytochrome p450 1A. Expsoure to BaP ultimately results in increased expression of HPV oncogenes. Importantly, curcumin, a natural compound, attenuated the BaP induced increase in the expression of HPV E7 oncoprotein. Interestingly, both cytoplasmic and nuclear localization of AhR is increased in cervical cancer tissue compared to normal cervical epithelium. Conclusion: Taken together this data imply that a high prevalence of HPV infection and a high exposure rate to BaP may synergize to increase the incidence and mortality of cervical cancer in American Indian women residing in the Northern Plains. We have identified curcumin as a potent compound that may be effective in attenuating oncogenic HPV infection. However, increased cervical cancer screening is warranted in addition to HPV vaccine and use of chemopreventive agents, such as curcumin, to effectively decrease the cervical cancer health disparity in Northern Plains American Indian women. Citation Format: Diane M. Maher, Maria Bell, Amanda Schaefer, Meena Jaggi, Subhash C. Chauhan. Benzo(a)pyrene exposure increases expression of HPV oncoproteins: a potential co-factor for increased cervical cancer among Northern Plains American Indian women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3611. doi:10.1158/1538-7445.AM2013-3611

Abstract 4383: Transglutaminase-2 mediates the pro-malignant effects of oncostatin-M receptor overexpression in cervical squamous cell carcinoma.

Abstract An important feature of cervical squamous carcinogenesis is genomic instability caused by deregulated expression of HPV oncogenes in proliferating epithelial cells. A genomic imbalance that is commonly selected in advanced cervical squamous cell carcinoma (SCC) is copy number gain of chromosome 5p. One gene that is likely to drive selection of this abnormality is the oncostatin-M receptor (OSMR; located at 5p13.1), which is commonly up-regulated in cervical SCC and produces a significantly worse clinical outcome when over-expressed. Cervical SCC cells that over-expressed OSMR showed enhanced responsiveness to the major ligand OSM, which induced multiple pro-malignant effects, including increased cell migration and invasiveness. We now show that transglutaminase-2 (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in cervical carcinoma cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of SCC of the cervix and head and neck. TGM2 depletion in cervical SCC cells abrogated the increased migration and invasiveness induced by OSM. TGM2 was shown to interact with integrin-α5β1 and fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin-α5β1 and fibronectin were over-expressed in cervical SCC samples, where levels correlated with those of OSMR. We conclude that an OSMR-TGM2-integrin-α5β1 pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting. Citation Format: Maria M. Caffarel, Anasuya Chattopadhyay, Cinzia G. Scarpini, Nicholas Coleman. Transglutaminase-2 mediates the pro-malignant effects of oncostatin-M receptor overexpression in cervical squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4383. doi:10.1158/1538-7445.AM2013-4383

Abstract 3482: Directly Detect High Risk HPV Oncogenes E6/E7 mRNAs from Pap Smear Samples without RNA Purification and RT-PCR.

Abstract Cervical cancer is one of the most common cancers in women, with about 450,000 new cases diagnosed each year. Nearly all the cervical cancers are from Papillomaviruses (HPV) infection. HPV are DNA-based viruses that infect the skin and mucous membranes of humans and many other animals. Most cancers of the vulva and vagina are induced by oncogenic HPV types. In precancerous lesions, most HPV genomes persist in an episomal state whereas, in many high-grade lesions and carcinomas, genomes are found integrated into the host chromosome. Two viral genes, E6 and E7, are invariably expressed in HPV-positive cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, E6 oncoprotein is also capable to up regulate the expression of inhibitors of apoptosis, and E6 and E7 cooperate to effectively immortalise primary epithelial cells. It has been demonstrated that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix via the deregulation of cellular genes controlling tumour cell proliferation. HPV E6/E7 oncogenes have been proved to be good biological markers for prognosis assessment and specific therapy of the disease. We have developed a sandwich nucleic acid hybridization assay using branched DNA to amplify the signals. The assay can simultaneously detect E6/E7 mRNAs of all 14 high risk HPV subtypes directly from Pap smear samples without RNA purification and RT-PCR. All HPV mRNA targets are captured through cooperative hybridization of multiple probes and probe set design determines the specificity of each HPV subtype of E6/E7 mRNA. Probe set oligonucleotides bind a contiguous region of the target E6/E7 mRNAs and selectively capture target RNAs to a solid surface during hybridization. Signal amplification is performed via sequential hybridization of Pre-Amplifier, Amplifier and label probe. The assay is highly specific and sensitive, which can detect as low as 50 transcript molecules. Citation Format: Aiguo Zhang, Lulu Zhang, Rachel Chuang, Xining Zhu, Rachel Diaz, Lily Chen. Directly Detect High Risk HPV Oncogenes E6/E7 mRNAs from Pap Smear Samples without RNA Purification and RT-PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3482. doi:10.1158/1538-7445.AM2013-3482