Expressing HPV16 E7 Research Articles

HPV-16 E6 and E7 expression drives transcription of p14(ARF) and p16(INK4a) prior to Intragenic methylation of CDKN2A.

Human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinomas (OPSCC), associated with improved patient outcome, exhibit increased expression of CDKN2A tumor suppressor p16(INK4a), while inactivation of the CDKN2A locus is frequently observed in HPV negative HNSCC. We recently identified a novel region of intragenic DNA hypermethylation within the CDKN2A locus that correlates with RNA expression of two CDKN2A transcripts and improved patient outcome in oropharyngeal and laryngeal SCC. Given the importance of CDKN2A expression in OPSCC, we chose an in vitro model using primary keratinocytes transduced with HPV-16 oncogenes E6 and E7 to study the relationship between CDKN2A methylation and expression. RNA levels for CDKN2A transcripts, p14(ARF) and p16(INK4a), and intragenic CDKN2A methylation were assessed in the primary cells and after transduction with E6 and E7 at two time points. CDKN2A expression increased early post-transduction, while there was a clear delay in intragenic methylation. Methylation eventually increased suggesting that intragenic methylation is not required for transcription at the CDKN2A locus, and that methylation may be a consequence of open and active chromatin.

DNA demethylation agent 5azadC downregulates HPV16 E6 expression in cervical cancer cell lines independently of TBX2 expression.

HPV16 is the most carcinogenic human papillomavirus and causes >50% of cervical cancers, the majority of anal cancers and 30% of oropharyngeal squamous cell carcinomas. HPV carcinogenesis relies on the continuous expression of the two main viral oncoproteins E6 and E7 that target >150 cellular proteins. Among them, epigenetic modifiers, including DNA Methyl Transferases (DNMT), are dysregulated, promoting an aberrant methylation pattern in HPV-positive cancer cells. It has been previously reported that the treatment of HPV-positive cervical cancer cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5azadC) caused the downregulation of E6 expression due to mRNA destabilization that was mediated by miR-375. Recently, the T-box transcription factor 2 (TBX2) has been demonstrated to repress HPV LCR activity. In the current study, the role of TBX2 in E6 repression was investigated in HPV16 cervical cancer cell lines following 5azadC treatment. A decrease of E6 expression was accompanied by p53 and p21 restoration. While TBX2 mRNA was upregulated in 5azadC-treated SiHa and Ca Ski cells, TBX2 protein was not detectable. Furthermore, the overexpression of TBX2 protein in cervical cancer cells did not allow the repression of E6 expression. The TBX2 transcription factor is therefore unlikely to be associated with the repression of E6 following 5azadC treatment of SiHa and Ca Ski cells.

58 Targeting Aurora Kinase to Enhance the Curative Potential of Radiotherapy in HPV Related Cancers

Purpose: Cervical cancer is caused by human papilloma virus (HPV), a sexually transmitted environmental agent. Approximately 40% of cervical cancer patients treated with radio(chemo)therapy (RTCT) develop recurrence that can be difficult to treat. New approaches for overcoming treatment failures are needed. Alisertib is a clinically approved, oral selective inhibitor of Aurora kinase A (AurkA,) which causes G2/M cell cycle arrest and apoptosis. High E7 oncogene expressing tumours and/or those carrying the ARID1A mutation are found in several disease sites, including cervix, and are more sensitive to AurkA inhibition. Our goal is to identify a clinically relevant treatment strategy, using AurkA as a therapeutic target in patients, to advance curative combination treatments with RTCT for HPVE7 related cancers. We hypothesize Alisertib influences the sensitivity to RT to improve primary tumour response and reduce lymph nodal disease compared to RT or drug alone. The aims are: 1) to determine the efficacy of fractionated RT combination with Alisertib in HPVE7/ARID1A expressing orthotopic cervical cancer PDXs on tumour growth delay response; and 2) to determine the effect on metastases development in response to AurkA inhibition with RT treatment. Materials and Methods: HPVE7 expression profiles of the cervix PDX models were determined by qRT-PCR. An orthotopic patient derived cervix cancer xenograft was treated with RT (30Gy;2Gy/day) with or without Alisertib (30mg/kg/day) given concurrently with RT daily (3wks). Expression of anti-apoptotic and DNA damage response proteins was evaluated by western blot at the end of treatment. Tumour growth delay and lymph node metastasis was/will be assessed. Results: The PDX HPV subtypes reflect the patient’s clinical HPV status at diagnosis. In an E7 expressing PDX model, RT combined with Alisertib treatment shows prolonged tumour growth delay compared with RT alone. Reduced lymph node metastasis was observed with Alisertib alone compared to control. These studies are still in progress. Tumours analysed at end of treatment suggest that AurkA inhibition resulted in the loss of RT-induced anti-apoptotic expression and γ-H2AX phosphorylation was enhanced by the combined treatment relative to RT alone. This suggests that Alisertib may reduce repair of DNA damage induced by RT treatment, which is consistent with its expected action on AurkA. Further investigation is ongoing to assess mechanisms underlying the RT induced effects with Alisertib on tumour response. Conclusions: Alisertib may enhance the curative potential of RT in patients with high E7 expressing cancers, and/or ARID1A mutations, which impacts on the sensitivity to treatment response. This study presents a promising approach to treating aggressive HPV cancers and may apply to other HPV-related cancers where RT plays a curative role and supports successful translation of new radiation-drug combinations to the clinic.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer

BackgroundThe US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients.MethodsWe used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy.ResultsMonotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC).ConclusionsThese results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Targeting Aurora Kinase to Enhance the Curative Potential of Radiotherapy in HPV Related Cancers

Purpose/Objective(s): Cervical cancer is caused by human papilloma virus (HPV), a sexually transmitted environmental agent. Approximately 40% of cervical cancer patients treated with radio(chemo)therapy (RTCT) develop recurrence that can be difficult to treat. New approaches for overcoming treatment failures are needed. Alisertib is a clinically approved, oral selective inhibitor of Aurora kinase A (AurkA), which causes G2/M cell cycle arrest and apoptosis. High E7 oncogene expressing tumors and/or those carrying the ARID1A mutation are found in several disease sites, including cervix, and are more sensitive to AurkA inhibition. The goal is to identify a clinically relevant treatment strategy, using AurkA as a therapeutic target in patients, to advance curative combination treatments with RTCT for HPVE7 related cancers. We hypothesize Alisertib influences the sensitivity to RT to improve primary tumor response and reduce lymph nodal disease compared to RT or drug alone. The aims are: Aim 1: To determine the efficacy of fractionated RT combination with Alisertib in HPVE7/ARID1A expressing orthotopic cervical cancer PDXs on tumor growth delay response. Aim 2: To determine the effect on metastases development in response to AurkA inhibition with RT treatment. Materials/Methods: HPVE7 expression profiles of the cervix PDX models were determined by qRT-PCR. An orthotopic patient derived cervix cancer xenograft was treated with RT (30Gy;2Gy/day) with or without Alisertib (30mg/kg/day) given concurrently with RT daily (3wks). Expression of anti-apoptotic and DNA damage response proteins was evaluated by western blot at the end of treatment. Tumor growth delay and lymph node metastasis was/will be assessed. Results: The PDX HPV subtypes reflect the patient’s clinical HPV status at diagnosis. In an E7 expressing PDX model, RT combined with Alisertib treatment shows prolonged tumor growth delay compared with RT alone. Reduced lymph node metastasis was observed with Alisertib alone compared to control. These studies are still in progress. Tumors analyzed at end of treatment suggest that AurkA inhibition resulted in the loss of RT-induced anti-apoptotic expression and g-H2AX phosphorylation was enhanced by the combined treatment relative to RT alone. This suggests that Alisertib may reduce repair of DNA damage induced by RT treatment, which is consistent with its expected action on AurkA. Further investigation is ongoing to assess mechanisms underlying the RT induced effects with Alisertib on tumor response. Conclusion: Alisertib may enhance the curative potential of RT in patients with high E7 expressing cancers, and/or ARID1A mutations, which impacts on the sensitivity to treatment response. This study presents a promising approach to treating aggressive HPV cancers and may apply to other HPVrelated cancers where RT plays a curative role and supports successful translation of new radiation-drug combinations to the clinic.

Transcription factor homeobox D9 is involved in the malignant phenotype of cervical cancer through direct binding to the human papillomavirus oncogene promoter

ObjectiveTo determine the involvement of homeobox D9 (HOXD9) in the survival, proliferation, and metastasis of cervical cancer cells through regulating the expression of human papillomavirus (HPV) 16 E6/E7 genes using the P97 promoter. MethodsOne hundred cases of cervical cancer (CC), CC cell lines SKG-I, SKG-II, SKG-IIIa, SKG-IIIb, HeLa, and SiHa, and a human tumor xenograft mouse model were used to examine the roles of HOXD9 in CC. Knockdown experiments employed RNA interference of HOXD9. qPCR, functional assays, western blotting, DNA microarray, and luciferase and ChIP assays were applied for assessments. ResultsAll CC cell lines expressed HOXD9 mRNA and protein. In uterine CC, HOXD9 gene expression was significantly higher than in normal cervical tissues. A positive correlation of lymphovascular space invasion and lymph node metastasis with high levels of HOXD9 expression was found in patient samples. HOXD9-knockdown cells in the mouse xenograft model only formed small or no tumors. Knockdown of HOXD9 markedly reduced CC cell proliferation, migration and invasion, induced apoptosis, increased P53 protein expression, and suppressed HPV E6/E7 expression by directly binding to the P97 promoter of HPV16 E6/E7 genes. A positive correlation between HOXD9 and HPV16 E6 expression was found in CC patients. ConclusionsHOXD9 promotes HPV16 E6 and E7 expression by direct binding to the P97 promoter, which enhances proliferation, migration, and metastasis of CCr cells. Our results suggest that HOXD9 could be a prognostic biomarker and potential therapeutic target in CC.

Tanshinone IIA inhibits glucose metabolism leading to apoptosis in cervical cancer.

Cancer requires aerobic glycolysis to supply the energy required for proliferation. Existing evidence has revealed that blocking glycolysis results in apoptosis of cancer cells. Tanshinone IIA (Tan IIA) is a diterpenoid naphthoquinone found in traditional Chinese medicine, Danshen (Salvia sp.). Tan IIA exhibits potential anticancer activity. However, its effect on cell viability of human cervical cancer cells and its mechanism are unknown. The aim of the present study was to determine the effect of Tan IIA on proliferation and glucose metabolism in cervical cancer cells. Cell viability was measured by MTT assay, apoptosis was determined using flow cytometry and glucose uptake, lactate production, and adenosine triphosphate content were measured to assess glucose metabolism. The expression of apoptosis-associated proteins was detected by western blotting and the antitumor activity of Tan IIA in vivo was evaluated in cervical carcinoma-bearing mice. The results revealed Tan IIA treatment resulted in a considerable reduction in the viability of SiHa cells. Tan IIA decreased the expression of HPV oncogenes E6 and E7, induced apoptosis and also decreased glycolysis by suppressing the activity of the intracellular AKT/mTOR and HIF-1α. In vivo, treatment with Tan IIA resulted in a 72.7% reduction in tumor volume. The present study highlights the potential therapeutic value of Tan IIA, which functions by inducing apoptosis and may be associated with inhibition of glycolysis.

A triphenylethylene nonsteroidal SERM attenuates cervical cancer growth

Selective estrogen receptor modulator drug molecules of triphenylethylene family have gained considerable attention as anti-cancer agents. Despite recent advances in screening and development of HPV vaccines, cervical cancer remains one of the deadliest malignancies as advanced stage metastatic disease is mostly untreatable, thus warrants newer therapeutic strategies. Ormeloxifene (ORM) is a well-known SERM of triphenylethylene family that has been approved for human use, thus represents an ideal molecule for repurposing. In this study, we for the first time have demonstrated the anti-cancerous properties of ormeloxifene in cervical cancer. Ormeloxifene efficiently attenuated tumorigenic and metastatic properties of cervical cancer cells via arresting cell cycle at G1-S transition, inducing apoptosis, decreasing PI3K and Akt phosphorylation, mitochondrial membrane potential, and modulating G1-S transition related proteins (p21, cyclin E and Cdk2). Moreover, ORM repressed the expression of HPV E6/ E7 oncoproteins and restored the expression of their downstream target tumor suppressor proteins (p53, Rb and PTPN 13). As a result, ormeloxifene induces radio-sensitization in cervical cancer cells and caused potent tumor growth inhibition in orthotopic mouse model. Taken together, ormeloxifene represents an alternative therapeutic modality for cervical cancer which may have rapid clinical translation as it is already proven safe for human use.

Abstract LB-329: Molecular mechanisms of loss of E7 expression in HPV16-transformed human keratinocytes

Abstract Human papillomavirus (HPV) causes about 5% of all human cancers. The HPV oncoproteins E6/E7 are responsible for the transforming potential of the virus. Although continuous expression of the HPV oncogenes was considered indispensable for HPV-induced carcinogenesis, we and others have demonstrated that in a subset of HPV positive head and neck and cervical cancers, the HPV oncogenes are not expressed (HPV-inactive cancers). Based upon the observation that primary HPV-positive tumors express E6/E7, while metastases tend to be HPV-inactive, we hypothesized that HPV-inactive cancers begin as HPV-active lesions and lose their dependence on continuous E6/E7 expression during progression. This may be due to genetic and/or epigenetic modifications caused by the genomic instability and the additional carcinogens to which the tumor is exposed. We observed that HPV-inactive cancers of the cervix often have mutated p53, while HPV-active cancers do not. Therefore, we proposed that HPV positive tumors may become inactive if p53 becomes mutated. The CRISPR-Cas9 system was used to knock out the p53 gene in differentiation resistant HPV16 immortalized human keratinocytes (HKc-DR). The DNA deletions within the p53 gene were confirmed by PCR and gel electrophoresis, and further validated by Sanger sequencing. Using qPCR, we found that HPV16 E7 expression was significantly lower (5-fold) in the p53 knock out (KO) lines than in the p53 wild type (WT) lines Reduced E7 expression in p53 KO lines was reversed by using the demethylating agent 5 Aza 2 deoxycytidine, suggesting that DNA methylation plays a role in this process. Also, we used in situ hybridization to detect HPV16 E7 mRNA in p53 WT and KO lines grown as spheroids on an agarose cushion. Interestingly, while all p53 WT lines have a uniform distribution of E7 signal, the p53 KO lines showed some spheroids that were completely lacking E7 mRNA, and some had a mixed population of E7-positive and E7-negative cells. These results indicate that the p53 KO lines are a heterogenous population with regard to HPV16 E7 expression. We concluded that p53 loss of function mutation may be an important factor in driving HPV16 transformed cells to lose dependence on continuous expression of the HPV oncogenes and become HPV-inactive. However, complete loss of p53 alone is not sufficient to suppress E7 expression entirely. We also determined that loss of E7 expression may be due, at least in part, to DNA methylation. We are currently examining HPV URR methylation in the p53 WT and KO lines, and isolating pure lines with complete loss of HPV16 E7 expression for further studies of the molecular mechanisms that may lead to the HPV-inactive phenotype. Citation Format: Fadi F. Abboodi, Kim E. Creek, Lucia A. Pirisi. Molecular mechanisms of loss of E7 expression in HPV16-transformed human keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-329.

REBACIN® as a noninvasive clinical intervention for high-risk human papillomavirus persistent infection.

The development of highly sensitive HPV-genotyping tests has opened the possibility of treating HPV-infected women before high-grade lesions appear. The lack of efficient intervention for persistent high-risk HPV infection necessitates the need for development of novel therapeutic strategy. Here we demonstrate that REBACIN®, a proprietary antiviral biologics, has shown potent efficacy in the clearance of persistent HPV infections. Two independent parallel clinical studies were investigated, which a total of 199 patients were enrolled and randomly divided into a REBACIN®-test group and a control group without treatment. The viral clearance rates for the REBACIN® groups were 61.5% (24/39) and 62.5% (35/56), respectively, for the two independent parallel studies. In contrast, the nontreatment groups showed self-clearance rates at 20.0% (8/40) and 12.5% (8/64). We further found that REBACIN® was able to significantly repress the expression of HPV E6 and E7 oncogenes in TC-1 and Hela cells. The two viral genes are well known for the development of high-grade premalignancy lesion and cervical cancer. In a mouse model, REBACIN® was indicated to notably suppress E6/E7-induced tumor growth, suggesting E6 and E7 oncogenes as a potential target of REBACIN®. Taken together, our studies shed light into the development of a novel noninvasive therapeutic intervention for clearance of persistent HPV infection with significant efficacy.

Effects of hnRNP E1 and both early genes E2 and E6 of HPV16 together with their interactions on cervical carcinogenesis

Objective: To explore the effects of hnRNP E1 and both early genes E2 and E6 of HPV16 as well as their interactions in the progression of cervical carcinogenesis. Methods: Subjects of this study included 56 women with normal cervix (NC), 58 patients with low-grade cervical intraepithelial neoplasm (CINⅠ) and 50 patients with high-grade cervical intraepithelial neoplasm (CINⅡ/Ⅲ) who were all recruited from the 'Cervical Lesions Study Cohort Project' in Jiexiu of Shanxi province from June to September, 2014. Another 40 patients with cervical squamous cell carcinoma (SCC) were from the Shanxi Tumor Hospital during the same period. Information related to cervical lesions were collected, using a structured questionnaire, with cervical tissues and cervical exfoliated cells gathered from all the participants. HPV infection was detected by flow-through hybridization, while the levels of expression on hnRNP E1, HPV16 E2 and E6 protein were measured by Western Blot. Kruskal-Wallis H test, χ(2) test, trend χ(2) test were analyzed by SPSS 22.0 software, while interaction was evaluated by generalized multifactor dimensionality reduction (GMDR). Results: The overall infection rates of HPV16 related to CINⅠ (15.52%, 9/58), CINⅡ/Ⅲ (40.00%, 20/50) and SCC (67.50%, 27/40) groups were all higher than that of the NC group (8.93%, 5/56) and with an increasing trend on the severity of cervical lesions (trend χ(2)=43.613, P<0.001). The levels of expression on hnRNP E1 protein were significantly different in the groups with different cervical lesions (H=9.98, P=0.019), showing a decreasing trend with the severity of cervical lesions (trend χ(2)=9.495, P=0.002). The levels of expression on HPV16 E2 (H=16.20, P=0.001) and HPV16 E6 (H=15.44, P=0.001) were significantly different in groups with different cervical lesions. Results of GMDR showed that the best interaction model in both groups of CINⅡ/Ⅲ and SCC appeared as hnRNP E1 low expression, HPV16 E2 low expression and HPV16 E6 high expression. However, no similar interaction was seen in CINⅠ (P>0.05). Conclusions: Both low expressions of hnRNP E1 and abnormal expression of HPV16 E2 and E6 could increase the risk of high-grade CIN and cervical cancer. It seemed that they might have an important synergistic effect on the progression of cervical cancer.

Anti-tumor immunity induced by ectopic expression of viral antigens is transient and limited by immune escape

ABSTRACTImmunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus- or host- “driver” oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed in vivo tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in Rag1-/- mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an “inflamed” or immune-activated tumor microenvironment and greater infiltration of CD8+ T cells compared to MOC2. By real-time PCR, we detected downregulation of E6 and E7 genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8+ T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and ‘driver’ status of target antigens to be considered in cancer vaccine design.

Three Prime Repair Exonuclease 1 (TREX1) expression correlates with cervical cancer cells growth in vitro and disease progression in vivo

Alterations in specific DNA damage repair mechanisms in the presence of human papillomavirus (HPV) infection have been described in different experimental models. However, the global effect of HPV on the expression of genes involved in these pathways has not been analyzed in detail. In the present study, we compared the expression profile of 135 genes involved in DNA damage repair among primary human keratinocytes (PHK), HPV-positive (SiHa and HeLa) and HPV-negative (C33A) cervical cancer derived cell lines. We identified 9 genes which expression pattern distinguishes HPV-positive tumor cell lines from C33A. Moreover, we observed that Three Prime Repair Exonuclease 1 (TREX1) expression is upregulated exclusively in HPV-transformed cell lines and PHK expressing HPV16 E6 and E7 oncogenes. We demonstrated that TREX1 silencing greatly affects tumor cells clonogenic and anchorage independent growth potential. We showed that this effect is associated with p53 upregulation, accumulation of subG1 cells, and requires the expression of E7 from high-risk HPV types. Finally, we observed an increase in TREX1 levels in precancerous lesions, squamous carcinomas and adenocarcinomas clinical samples. Altogether, our results indicate that TREX1 upregulation is important for cervical tumor cells growth and may contribute with tumor establishment and progression.

Brd4 inhibition suppresses HPV16 E6 expression and enhances chemoresponse: A potential new target in cervical cancer therapy.

Although a vast amount of research underlines the roles of the HR HPV E6 and E7 oncogenes in HPV-induced carcinogenesis of cervical cancer, it remains unclear whether these oncogenes are also involved in the resistance of the cancer against chemotherapy. We examined the role of the HPV16 E6 oncogene in cisplatin resistance by analyzing its expression in newly established cisplatin-sensitive versus -resistant cervical cancer cell lines (CC7, CC10). Resistant variants were obtained by interval exposure treatment with 1-2 μM cisplatin for 8-9 months. Our results demonstrate that the expression level of HPV16 E6 directly correlates with the extent of cisplatin resistance in novel as well as established (SiHa) drug resistant cervical cancer cell lines. Overexpression of HPV16 E6 in cisplatin-naïve cells rendered these cells more resistant to cisplatin. Reducing E6 expression by JQ1 treatment reversed the drug resistant phenotype and strongly enhanced chemoresponse only in HPV-positive cisplatin-resistant variants and not in HPV-negative C33A cervical cancer cells. The level of E6 directly correlated with the extent of cisplatin sensitivity and was shown to be increased in newly established drug-resistant cell line variants, while reducing E6 expression using Brd4-inhibitors enhanced chemoresponse when co-delivered with cisplatin. Inhibition of Brd4 could represent a new therapeutic option by increasing treatment response in cervical cancer cells and might allow lower cisplatin dosages, thus reducing negative side effects.

Recruitment of Antigen Presenting Cells to Skin Draining Lymph Node From HPV16E7-Expressing Skin Requires E7-Rb Interaction.

“High-risk” human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRbΔL/ΔL mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRbΔL/ΔL skin grafts. Here, we further show that K14.E7xRbΔL/ΔL skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b+ antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRbΔL/ΔL grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRbΔL/ΔL grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRbΔL/ΔL keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro. We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression.

Suppression of miR-22, a tumor suppressor in cervical cancer, by human papillomavirus 16 E6 via a p53/miR-22/HDAC6 pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that function to down-regulate gene expression involving in various cellular processes related to carcinogenesis. Recently, miR-22 was identified as a tumor-suppressing miRNA in many human cancers. However, the regulatory mechanism and the specific function of this miRNA in cervical cancer remain unclear. In the present study, we carried out gene transfection, western blot and quantitative RT-PCR to explore the regulatory mechanism and the functional role of miR-22 in cervical cancer. We verified that miR-22 was down-regulated in cervical cancer tissues and cervical cancer cell lines relative to matched non-tumor tissues and normal human cervical keratinocyte line (HCK1T). By contrast, histone deacetylase 6 (HDAC6) was inversely correlated with miR-22 in both cervical tissues and cancer cell lines. Mechanically, HDAC6 was down-regulated by miR-22 at the post-transcriptional level, via a specific target site within the 3’UTR, identified by a luciferase reporter assay. Moreover, we also showed that the correlation between miR-22 and HDAC6 expression was regulated by an E6/p53 pathway in HCK1Ts expressing HPV16 E6. For functional study, an ectopic expression of miR-22 could inhibit cell proliferation and migration, and could induce apoptosis of cervical cancer cell lines. These findings demonstrated that miR-22 was down-regulated in cervical cancer and inversely collated with its downstream target HDAC6. MiR-22 acts as tumor suppressor by inhibiting proliferation and migration, and by inducing apoptosis of cervical cancer cell lines by targeting the 3’UTR of HDAC6. This newly identified E6/p53/miR-22/HDAC6 regulatory network might be a candidate therapeutic target for cervical cancer.

Realgar, a traditional Chinese medicine, induces apoptosis of HPV16-positive cervical cells through a HPV16 E7-related pathway.

PurposeIn this study, we investigated the effect of Realgar on the apoptosis of HPV16-positive cervical cells in vitro.MethodsThe effect of Realgar on the apoptosis of HPV16-positive cervical cells was investigated by annexin V-fluorescein isothiocyanate/propidium iodide staining and growth inhibition assays using HPV16-positive cervical cancer cell line SiHa and HPV16-positive immortalized cervical epithelial cell line S12. The expression of genes was measured by real-time PCR, and the expression of corresponding proteins was detected by Western blotting. The adhesion and invasion of cells were detected by adhesion assay and Transwell invasion assay, respectively.ResultsThe Realgar inhibited the proliferation and induced the apoptosis of SiHa and S12 cells in a dose- and time-dependent manner. The Realgar suppressed the expression of HPV16 E7 and caspase-3. The Realgar suppressed the adhesion and invasion of both cells.ConclusionThe Realgar induced apoptosis, inhibited the proliferation of HPV16-positive cell lines through a HPV16 E7-dependent pathway, and inhibited cell adhesion and invasion.

Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status

AimsApproximately 20% of head and neck squamous cell carcinomas (HNSCCs) are caused by human papillomavirus (HPV) infection. The effect of arsenic trioxide (ATO) on HPV oncogene expression of HNSCC cells remains unknown. In this study, we investigated the anti-cancer activity and possible molecular pathways of ATO on the six HNSCC cell lines (three HPV-positive and three HPV-negative). MethodsThe effects of ATO on the cell proliferation, apoptosis, cell cycle of HNSCC cells were analyzed using CCK-8 assay, colony formation and flow cytometry. Transwell assay was used to examine the effect of ATO on cell migration. The transcriptional and protein expression of key genes were determined by real-time PCR and Western blot, respectively. Using a xenograft model, we assessed the effects of ATO on HNSCC cells in vivo. Key findingsHPV-positive and -negative HNSCC cells had different expression of key genes. ATO inhibited HNSCC cell proliferation and migration and induced apoptosis and these effects were more significant in HPV-positive HNSCC cells than in HPV-negative HNSCC cells. ATO treatment reduced the expression of HPV16-E6/E7 and cyclin D1 proteins and enhanced the expression of p16, pRb, and p53 in HPV-positive HNSCC cells. By contrast, ATO treatment reduced the expression of epidermal growth factor receptor, cyclin D1 and mutant p53 and enhanced the expression of pRb in HPV-negative HNSCC cells. Anti-cancer effect of ATO on HNSCCs was confirmed by inhibiting xenograft growth in vivo. SignificanceOur data suggest that ATO is a potential therapeutic drug for HNSCCs, especially HPV-positive HNSCCs.

Mucosal HPV E6/E7 Peptide Vaccination in Combination with Immune Checkpoint Modulation Induces Regression of HPV+ Oral Cancers.

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV- SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR.

The association between human papillomavirus 16, 18 DNA load and E6 protein expression in cervical intraepithelial neoplasia and cancer

BackgroundIt has been reported that high HPV DNA load and elevated E6 protein expression correlate with cervical cancer, but no epidemiological study has been performed. ObjectivesTo assess the association between type-specific HPV DNA load and presence of E6 protein in cervical intraepithelial neoplasia and cancer. Study designThis study was a cross-sectional multicenter study performed between 2013 and 2017. A total of 1717 women (normal histopathology: n = 916; CIN1: n = 94; CIN2: n = 63; CIN3: n = 130; SCC: n = 474; adenocarcinoma: n = 40) were included. HPV DNA load and presence of E6 protein were detected. DNA load was measured as log copies/10,000 cells. ResultsThe HPV16/18-E6 positivity rates increased from negative for DNA to the highest load grade. Compared to the HPV16 DNA negatives, women with low (RR = 31.5, 95%CI = 18.9–52.5), medium-low (RR = 133.5, 95%CI = 77.3–230.7), medium-high (RR = 247.9, 95%CI = 134.9–455.6) and high DNA loads (RR = 677.9, 95%CI = 301.6–1523.7) had increasingly higher relative ratios of HPV16-E6 expression. The association of HPV18-E6 with its DNA load was also significant for low (RR = 27.9, 95%CI = 10.4–74.9), medium-low (RR = 89.0, 95%CI = 32.8–241.3), medium-high (RR = 276.8, 95%CI = 76.7–998.9) and high grade (RR = 441.2, 95%CI = 97.7–1992.4). The positivity rates of both HPV16 DNA and E6 protein increased consistently with the severity of diseases from normal histopathology to SCC. Unlike HPV16, the trends of HPV18 DNA and E6 protein fluctuated consistently among women from normal histopathology to cancer. DNA load in E6-positive women was significantly higher than E6-negative for both types. ConclusionsThere is a type-dependent association between HPV16/18 DNA load and E6 protein; our study furthers the understanding of the natural history of cervical cancer.