Identification and validation of the high expression of pseudogene TCAM1P in cervical cancer via integrated bioinformatics analysis

Background HPV as the main cause of cervical cancer has long been revealed, but the detailed mechanism has not yet been elucidated. The role of testis/cancer antigen in cervical cancer has been revealed. However, there are no reports about the statement of testis/cancer-specific non-coding RNA. In this study, we first proposed TCAM1P as a testis/cancer-specific pseudogene, and used a series of experimental data to verify its relationship with HPV, and analyzed its diagnosis value of high-grade cervical lesions and the mechanism of their high expression in cervical cancer. This provides a new direction for the prevention and treatment of cervical cancer. Methods The specific expression of pseudogenes in each tissue was calculated by “TAU” formula. ROC curve was used to judge the diagnosed value of TCAM1P for high-grade lesions. The proliferation ability of cells was measured by CCK8. The expression of TCAM1P, HPV E6/E7 were detected by qRT-PCR. The binding for RBPs on TCAM1P was predicted by starbase v2.0 database, then RIP assay was used to verify. Besides, Gene Ontology (GO) and KEGG enrichment analysis were performed with “clusterprofiler” R package. Results TCAM1P was specifically high-expressed in normal testicular tissue and cervical cancer. Interesting, with the severity of cervical lesions increased, the expression of TCAM1P increased, and TCAM1P could effectively diagnose high-grade cervical lesions. Besides, the expression of TCAM1P was HPV dependent, with highest expression in HPV-positive cervical cancer tissues. Furthermore, RIP assay showed that EIF4A3 regulated the expression of TCAM1P through binding with it. CCK8 assay showed that TCAM1P promoted the proliferation and the Gene ontology (GO) and KEGG Pathway enrichment analysis same suggested that TCAM1P is involved in multiple ways in cell proliferation including Cell cycle, DNA replication and etc. Conclusions In this study, we firstly proposed that TCAM1P is cancer/testis pseudogene and is regulated by HPV E6/E7 and EIF4A3. TCAM1P promotes the proliferation of cervical cancer cells and acts as promoter in cervical cancer. Otherwise, TCAM1P promote proliferation through regulating cell cycle and DNA replication, but more evidence needs to be provided to reveal the mechanism by which TCAM1P plays a role in cervical cancer.

Local radiotherapy and E7 RNA-LPX vaccination show enhanced therapeutic efficacy in preclinical models of HPV16+ cancer

Human papilloma virus (HPV) infection is a causative agent for several cancers types (genital, anal and head and neck region). The HPV E6 and E7 proteins are oncogenic drivers and thus are ideal candidates for therapeutic vaccination. We recently reported that a novel ribonucleic acid lipoplex (RNA-LPX)-based HPV16 vaccine, E7 RNA-LPX, mediates regression of mouse HPV16+ tumors and establishes protective T cell memory. An HPV16 E6/E7 RNA-LPX vaccine is currently being investigated in two phase I and II clinical trials in various HPV-driven cancer types; however, it remains a high unmet medical need for treatments for patients with radiosensitive HPV16+ tumors. Therefore, we set out to investigate the therapeutic efficacy of E7 RNA-LPX vaccine combined with standard-of-care local radiotherapy (LRT). We demonstrate that E7 RNA-LPX synergizes with LRT in HPV16+ mouse tumors, with potent therapeutic effects exceeding those of either monotherapy. Mode of action studies revealed that the E7 RNA-LPX vaccine induced high numbers of intratumoral-E7-specific CD8+T cells, rendering cold tumors immunologically hot, whereas LRT primarily acted as a cytotoxic therapy, reducing tumor mass and intratumor hypoxia by predisposing tumor cells to antigen-specific T cell-mediated killing. Overall, LRT enhanced the effector function of E7 RNA-LPX-primed T cell responses. The therapeutic synergy was dependent on total radiation dose, rather than radiation dose-fractionation. Together, these results show that LRT synergizes with E7 RNA-LPX and enhances its anti-tumor activity against HPV16+ cancer models. This work paves into a new translational therapy for HPV16+ cancer patients.

The Efficacy of Therapeutic DNA Vaccines Expressing the Human Papillomavirus E6 and E7 Oncoproteins for Treatment of Cervical Cancer: Systematic Review

Cervical cancer is recognized as a serious public health problem since it remains one of the most common cancers with a high mortality rate among women despite existing preventative, screening, and treatment approaches. Since Human Papillomavirus (HPV) was recognized as the causative agent, the preventative HPV vaccines have made great progress over the last few years. However, people already infected with the virus require an effective treatment that would ensure long-term survival and a cure. Currently, clinical trials investigating HPV therapeutic vaccines show a promising vaccine-induced T-cell mediated immune response, resulting in cervical lesion regression and viral eradication. Among existing vaccine types (live vector, protein-based, nucleic acid-based, etc.), deoxyribonucleic acid (DNA) therapeutic vaccines are the focus of the study, since they are safe, cost-efficient, thermostable, easily produced in high purity and distributed. The aim of this study is to assess and compare existing DNA therapeutic vaccines in phase I and II trials, expressing HPV E6 and E7 oncoproteins for the prospective treatment of cervical cancer based on clinical efficacy, immunogenicity, viral clearance, and side effects. Five different DNA therapeutic vaccines (GX-188E, VGX-3100, pNGVL4a-CRT/E7(detox), pNGVL4a-Sig/E7(detox)/HSP70, MEDI0457) were well-tolerated and clinically effective. Clinical implementation of DNA therapeutic vaccines into treatment regimen as a sole approach or in combination with conservative treatment holds great potential for effective cancer treatment.

Genome-Wide Profiling Reveals HPV Integration Pattern and Activated Carcinogenic Pathways in Penile Squamous Cell Carcinoma

Human papillomavirus (HPV) is a significant etiologic driver of penile squamous cell carcinoma (PSCC). The integration pattern of HPV and its carcinogenic mechanism in PSCC remain largely unclear. We retrospectively reviewed 108 PSCC cases who received surgery between 2008 and 2017. Using high-throughput viral integration detection, we identified 35 HPV-integrated PSCCs. Unlike cervical cancer, the HPV E2 oncogene was not prone to involvement in integration. Eleven of the 35 (31.4%) HPV-integrated PSCCs harbored intact HPV E2; these tumors had lower HPV E6 and E7 expression and higher expression of p53 and pRb proteins than those with disrupted E2 did (p < 0.001 and p = 0.024). Integration breakpoints are preferentially distributed in or near host genes, including previously reported hotspots (KLF5, etc.) and newly identified hotspots (CADM2, etc.), which are mainly involved in oncogenic signaling pathways (MAPK, JAK/STAT, etc.). Regarding the phosphorylation levels of JNK, p38 was higher in HPV-positive tumors with MAPK-associated integration than those in HPV-positive tumors with other integration and those in HPV-negative tumors. In vitro, KLF5 knockdown inhibited proliferation and invasion of PSCC cells, while silencing CADM2 promoted migration and invasion. In conclusion, this study enhances our understanding of HPV-induced carcinogenesis in PSCC, which may not only rely on the E6/E7 oncogenes, but mat also affect the expression of critical genes and thus activate oncogenic pathways.

HPV E6/E7 mRNA in situ hybridization in endocervical adenocarcinoma: implications for prognosis and diagnosis

Background Human papillomavirus (HPV) E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) appears to be a sensitive and specific technique in detecting transcriptionally active HPV. We aimed to examine the diagnostic utility of this technique in endocervical adenocarcinoma (ECA), to explore the prognostic factors for ECA patients and develop a clinically useful nomogram based on clinicopathological parameters to predict it. Methods We retrospectively analyzed 200 patients with ECA who had undergone surgery at Sun Yat-sen University Cancer Center from 2010 and 2014. The diagnostic performance of HPV E6/E7 RNAscope were evaluated by receiver operating characteristic (ROC) curve. A prognostic nomogram model including HPV E6/E7 RNAscope was generated based on multivariate Cox regression analysis, then we compared the predictive accuracy of the prognostic model with FIGO staging and treatment using concordance index (C-index), time-dependent ROC (tdROC), and decision curve analysis (DCA). Results The sensitivity and specificity of HPV E6/E7 RNAscope for distinguishing HPV-associated adenocarcinoma (HPVA) from non-HPV-associated adenocarcinoma (NHPVA) in the whole cohort were 75.8% and 80%, respectively. According the univariate analysis and multivariate logistic regression analysis, age, lymphovascular invasion (LVI), lymph node involvement (LNI), and HPV E6/E7 RNAscope were valuable predictive factors for OS. These parameters were incorporated into the nomogram model (nomogram A) compared with FIGO stage and treatment. The C-index of nomogram A for predicting OS was 0.825, which was significantly higher than FIGO stage (C-index = 0.653, p = 0.002) and treatment (C-index = 0.578, p < 0.001). Conclusions HPV E6/E7 RNAscope is highly specific for ECA, and the 4-variable nomogram showed more accurate prognostic outcomes in patients with ECA.

Accuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer

BACKGROUNDThe incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. METHODSThe accuracy of three possible test strategies, i.e. a) hrHPV DNA PCR (DNA), b) p16INK4a immunohistochemistry (IHC) (p16), and c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. RESULTSNinety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16+DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16+DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. CONCLUSIONSSingle hrHPV DNA PCR and p16INK4a IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Since HPV-driven OPC cancer can be treated less aggressively, combined testing can reduce treatment sequelae at the expense of a small number of patients that may receive less effective therapy.

The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

Background: High-risk human papilloma virus (HR-HPV) persistent infection is the major tumorigenesis factor for cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). However, the incidence of HPV-negative CC is approximately 5%-30% with different HPV detection methods. HR-HPV E6/E7 mRNA in situ hybridization (RISH) can detect HPV-driven tumours. Our study aimed to explore whether HPV typing-negative CC was caused by HPV infection.Methods: The records of CC patients with HPV typing results who had undergone cervical biopsies, cervical conization or hysterectomies were collected from April 2018 to September 2021 at the Affiliated Hospital of Weifang Medical University. RISH was detected using RNAscope chromogenicin in the tissues of patients with CC. Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a and Ki67. CC patients with positive HPV typing in the same period were collected as controls.Results: A total of 308 women with HPV typing results were enrolled, and 30 (9.74%) cases of HPV typing were negative, including 22/30 (73.3%) cases of squamous cell carcinoma (SCC) and 8/30 (26.7%) cases of adenocarcinoma. In the CC patients who were HPV typing-negative, 28/30 (93.3%) were positive for RISH and p16INK4a block+ staining, which contained 22/22 (100%) SCCs and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) SCCs and 46/46 (100%) adenocarcinomas. While 273/278 (98.2%) showed p16INK4a block+ staining, the five negative cases were SCC. Positive RISH in HPV typing-negative CC was significantly lower than in the HPV typing-positive group (P=0.002, 95% CI: 0.848-1.027). However, this significant difference only existed in adenocarcinoma, and positive RISH in HPV typing-negative SCC was the same as in the HPV typing-positive group. No significant differences were seen in the expression of p16INK4a and Ki67 (P=0.291, 95% CI: 0.863-1.047 and P=0.174, 95% CI: 0.905-1.033).Conclusions: HPV typing may cause misdiagnosis in 9.74% of CC patients, and HPV E6/E7 mRNA can detect the majority of CC with HPV-related status even in HPV typing-negative patients. This approach could provide a novel option to accurately detect HR-HPVs in cervical tumours and help to eliminate the percentage of misdiagnosed HPV-related cases.

Corrigendum

Minor changes are required in the Funding information and the acknowledgement for the article entitled “Organosulphur Compounds Induce Apoptosis and Cell Cycle Arrest in Cervical Cancer Cells via Downregulation of HPV E6 and E7 Oncogenes” in “Anti-Cancer Agents in Medicinal Chemistry, 2021, Vol. 21, No. 3, pp. 393-405.” The correct Funding information and Acknowledgement is given below: FUNDING: This project was funded by the Council of Science and Technology (CST), Lucknow, Uttar Pradesh, India (Sanction No. CST/374). The financial support during this research was also provided by the Deanship of Scientific Research, King Khalid University, Abha, Saudia Arabia through the General Research Project under grant number R.G.P. 01/48/42. ACKNOWLEDGEMENT: IAA is greatly thankful to the Council of Science and Technology (CST), Uttar Pradesh, India, for providing him project as a Principal Investigator. Authors would also like to acknowledge the support of the King Khalid University through the General Research Project under grant number R.G.P. 01/48/42. under the Deanship of Scientific Research, King Khalid University, Abha, Saudia Arabia

Targeting Aberrant Expression of STAT3 and AP-1 Oncogenic Transcription Factors and HPV Oncoproteins in Cervical Cancer by Berberis aquifolium

Background: Present study examines phytochemical preparation that uses berberine’s plant source B. aquifolium root for availability of similar anti-cervical cancer (CaCx) and anti-HPV activities to facilitate repurposing of the B. aquifolium based drug in the treatment of CaCx.Purpose: To evaluate therapeutic potential of different concentrations of ethanolic extract of B. aquifolium root mother tincture (BAMT) against HPV-positive (HPV16: SiHa, HPV18: HeLa) and HPV-negative (C33a) CaCx cell lines at molecular oncogenic level.Materials and Methods: BAMT was screened for anti-proliferative activity by MTT assay. Cell cycle progression was analyzed by flowcytometry. Then, the expression level of STAT3, AP-1, HPV E6 and E7 was detected by immunoblotting, whereas nuclear localization was observed by fluorescence microscopy. Phytochemicals reportedly available in BAMT were examined for their inhibitory action on HPV16 E6 by in silico molecular docking.Results: BAMT induced a dose-dependent decline in CaCx cell viability in all cell types tested. Flowcytometric evaluation of BAMT-treated cells showed a small but specific cell growth arrest in G1-phase. BAMT-treatment resulted in reduced protein expression of key transcription factors, STAT3 with a decline of its active form pSTAT3 (Y705); and components of AP-1 complex, JunB and c-Jun. Immunocytochemistry revealed that BAMT did not prevent the entry of remnant active transcription factor to the nucleus, but loss of overall transcription factor activity resulted in reduced availability of transcription factors in the cancer cells. These changes were accompanied by gradual loss of HPV E6 and E7 protein in BAMT-treated HPV-positive cells. Molecular docking of reported active phytochemicals in B. aquifolium root was performed, which indicated a potential interference of HPV16 E6’s interaction with pivotal cellular targets p53, E6AP or both by constituent phytochemicals. Among these, berberine, palmatine and magnoflorine showed highest E6 inhibitory potential.Conclusion: Overall, BAMT showed multi-pronged therapeutic potential against HPV infection and cervical cancer and the study described the underlying molecular mechanism of its action.