Tumor volume and cancer stem cell expression as prognostic markers for high-dose loco-regional failure in head and neck squamous cell carcinoma - A DAHANCA 19 study.

BACKGROUND AND PURPOSE: Reliable and accessible biomarkers for patients with Head and Neck Squamous Cell Carcinoma (HNSCC) are warranted for biologically driven radiotherapy (RT). This study aimed to investigate the prognostic value of putative cancer stem cell (CSC) markers, hypoxia, and tumor volume using loco-regional high-dose failure (HDF) as endpoint. MATERIALS AND METHODS: Tumor tissue was retrieved from patients treated with primary chemo-(C-)RT and nimorazole for HNSCC in the Danish Head and Neck Cancer Study Group (DAHANCA) 19 study. Tumor volume, hypoxic classification, and expression of CSC markers CD44, SLC3A2, and MET were analyzed. For patients with eligible data on all parameters (n = 340), the risk of HDF following primary chemo-(C-)RT were analyzed by these biomarkers as a whole and stratified for p16-positive oropharynx (p16 + OPSCC) vs p16-negative (p16-) tumors (oral cavity, p16- oropharynx, hypopharynx and larynx). RESULTS: Higher risk of HDF was seen for patients with larger primary and nodal volume (>25 cm3, Hazard Ratio (HR): 3.00 [95 % CI: 1.73-5.18]), high SLC3A2 (HR: 2.99 [1.28-6.99]), CD44 (>30 % positive, HR: 2.29 [1.05-5.00]), and p16- tumors (HR: 2.53 [1.05-6.11]). p16- tumors had a higher CSC marker expression than p16 + OPSCC. The factors associated with the highest risk of HDF were larger volume (HR: 3.29 [1.79-6.04]) for p16- tumors (n = 178) and high SLC3A2 (HR: 6.19 [1.58-24.23]) for p16 + OPSCC (n = 162). CONCLUSION: Tumor volume, p16, and CSC markers are potential biomarkers for HDF for patients with HNSCC treated with (C-)RT. Lower expression of CSC in p16 + OPSCC may contribute to better tumor control.

Trichorhinophalangeal Syndrome Type 1 Immunohistochemical Expression in Carcinomas of Gynecologic Origin.

Trichorhinophalangeal syndrome type 1 (TRPS1) is a new reportedly sensitive and specific immunohistochemical marker for carcinomas of breast origin, including triple-negative (estrogen receptor, progesterone receptor, and HER2) tumors. In our practice, we have observed a subset of cases of nonmammary carcinomas that are positive for TRPS1, with higher frequency in cytology effusion samples with metastatic gynecologic malignancies. This study aimed to evaluate the expression of TRPS1 in a large tissue cohort of Müllerian carcinomas. We retrospectively retrieved 105 cases of formalin-fixed paraffin-embedded gynecologic tumors from our surgical pathology archives. Cases corresponded to tumors of tubo-ovarian (17 high-grade serous carcinomas, 3 low-grade serous carcinomas, 2 clear cell carcinomas, and 8 endometrioid adenocarcinomas), endometrial (25 endometrioid adenocarcinomas, 8 serous carcinomas, 6 clear cell carcinomas, 12 carcinosarcomas, 1 dedifferentiated carcinoma, and 1 mesonephric-like adenocarcinoma), cervical (6 human papillomavirus [HPV]-associated squamous cell carcinomas [SCCs], 11 HPV-associated endocervical adenocarcinomas, and 2 HPV-independent gastric-type endocervical adenocarcinomas), and vulvar (2 HPV-independent SCCs and 1 HPV-associated SCC) origins. Immunohistochemistry for TRPS1 was performed in whole tissue sections and assessed for positivity (≥5% of nuclear labeling), distribution (focal: 5% to 49%, diffuse: 50% to 100%), and intensity (1+, 2+, 3+) in tumor cells. Positive TRPS1 staining was observed in 51.4% (54/105) of cases. Most tumors (64.8%) demonstrated diffuse labeling, while focal in 35.2%. Among positive cases, the intensity was predominantly 1+ (57.4%), followed by 2+ (33.3%) and 3+ (9.2%). Tumors with a high percentage of positivity overall consisted of tubo-ovarian (70%) and endometrial carcinomas (58.4%). TRPS1 immunostain is often expressed in gynecologic carcinomas. Awareness of this phenomenon is crucial when evaluating challenging cases in which the differential diagnosis includes a malignancy of breast origin, to avoid misclassification of the primary site.

ErbB2/HER2 receptor tyrosine kinase regulates human papillomavirus promoter activity.

Human papillomaviruses (HPVs) are a major cause of cancer. While surgical intervention remains effective for a majority of HPV-caused cancers, the urgent need for medical treatments targeting HPV-infected cells persists. The pivotal early genes E6 and E7, which are under the control of the viral genome's long control region (LCR), play a crucial role in infection and HPV-induced oncogenesis, as well as immune evasion. In this study, proteomic analysis of endosomes uncovered the co-internalization of ErbB2 receptor tyrosine kinase, also called HER2/neu, with HPV16 particles from the plasma membrane. Although ErbB2 overexpression has been associated with cervical cancer, its influence on HPV infection stages was previously unknown. Therefore, we investigated the role of ErbB2 in HPV infection, focusing on HPV16. Through siRNA-mediated knockdown and pharmacological inhibition studies, we found that HPV16 entry is independent of ErbB2. Instead, our signal transduction and promoter assays unveiled a concentration- and activation-dependent regulatory role of ErbB2 on the HPV16 LCR by supporting viral promoter activity. We also found that ErbB2's nuclear localization signal was not essential for LCR activity, but rather the cellular ErbB2 protein level and activation status that were inhibited by tucatinib and CP-724714. These ErbB2-specific tyrosine kinase inhibitors as well as ErbB2 depletion significantly influenced the downstream Akt and ERK signaling pathways and LCR activity. Experiments encompassing low-risk HPV11 and high-risk HPV18 LCRs uncovered, beyond HPV16, the importance of ErbB2 in the general regulation of the HPV early promoter. Expanding our investigation to directly assess the impact of ErbB2 on viral gene expression, quantitative analysis of E6 and E7 transcript levels in HPV16 and HPV18 transformed cell lines unveiled a noteworthy decrease in oncogene expression following ErbB2 depletion, concomitant with the downregulation of Akt and ERK signaling pathways. In light of these findings, we propose that ErbB2 holds promise as potential target for treating HPV infections and HPV-associated malignancies by silencing viral gene expression.

HPV-Related Oropharyngeal Cancer in Southern Thailand: Proportion Trend and Survival Outcome.

BACKGROUND: Persistent high-risk human papillomavirus (HPV) infection is one of the major etiologies of oropharyngeal squamous cell carcinoma (OPSCC). This study aimed to determine the proportion, temporal trend, and prognostic significance of HPV-related OPSCC in Thai patients. METHODS: The study included patients with OPSCC who were treated at Songklanagarind Hospital (Songkhla, Southern Thailand) from 2009 to 2020. HPV status was screened by p16 expression using immunohistochemistry and confirmed by real-time polymerase chain reaction. Cox regression was used to determine prognostic significance. RESULTS: The overall proportion of HPV+ OPSCC was 15.3% (95% confidence interval [CI]: 12.1-18.5) with a slightly increased proportion from 10.6% in 2009-2010 to 16.5% (2019-2020) (P for trend = 0.166). Among the HPV+ cases, HPV16 was detected in 65.3%, HPV18 in 34.7%, and other high-risk HPV types in 24%. Patients with P16+ or HPV+ OPSCC had significantly better overall survival (hazard ratio [HR]: 0.63, 95% CI: 0.45-0.90 and HR: 0.63, 95% CI: 0.45-0.88, respectively). CONCLUSION: Thai patients in the southern region have a low proportion of HPV-related OPSCC with an increasing trend. Both P16 expression and HPV DNA status are strong independent prognostic factors of OPSCC.

Human papillomavirus and p16INK4a in oropharyngeal squamous cell carcinomas: A systematic review and meta-analysis.

We intended to update human papillomavirus (HPV) prevalence and p16INK4a positivity in oropharyngeal squamous cell carcinomars (SCC), and calculate HPV attributable fraction (AF) for oropharyngeal SCC by geographic region. We searched Medline, Embase, and the Cochrane Library to identify published studies of HPV prevalence and p16INK4a positivity alone or together in oropharyngeal SCC before December 28, 2021. Studies that reported type-specific HPV DNA prevalence using broad-spectrum PCR-based testing methods were included. We estimated pooled HPV prevalence, type-specific HPV prevalence, and p16INK4a positivity. AF of HPV was calculated by geographic region. One hundred and thirty-four studies including 12 139 cases were included in our analysis. The pooled HPV prevalence estimate for oropharyngeal SCC was 48.1% (95% confidence interval [CI] 43.2-53.0). HPV prevalence varied significantly by geographic region, and the highest HPV prevalence in oropharyngeal SCC was noted in North America (72.6%, 95% CI 63.8-80.6). Among HPV positive cases, HPV 16 was the most common type with a prevalence of 40.2% (95% CI 35.7-44.7). The pooled p16INK4a positivity in HPV positive and HPV16 positive oropharyngeal SCC cases was 87.2% (95% CI 81.6-91.2) and 91.7% (84.3-97.2). The highest AFs of HPV and HPV16 were noted in North America at 69.6% (95% CI 53.0-91.5) and 63.0% (48.0-82.7). [Correction added on 31 October 2023, after first online publication: the percentage symbol (%) was missing and has been added to 63.0% (48.0-82.7) in the Abstract and Conclusion.] A significant proportion of oropharyngeal SCC was attributable to HPV. HPV16 accounts for the majority of HPV positive oropharyngeal SCC cases. These findings highlight the importance of HPV vaccination in the prevention of a substantial proportion of oropharyngeal SCC cases.

Evaluation of programmed cell death ligand 1 expression in a contemporary cohort of penile squamous cell carcinoma and its correlation with clinicopathologic and survival parameters: A study of 134 patients.

OBJECTIVES: Penile squamous cell carcinomas (PCs) are rare malignancies with a dismal prognosis in a metastatic setting; therefore, novel immunotherapeutic modalities are an unmet need. One such modality is the immune checkpoint molecule programmed cell death ligand 1 (PD-L1). We sought to analyze PD-L1 expression and its correlation with various clinicopathologic parameters in a contemporary cohort of 134 patients with PC. METHODS: A cohort of 134 patients with PC was studied for PD-L1 immunohistochemistry. The PD-L1 expression was evaluated using a combined proportion score with a cutoff of 1 or higher to define positivity. The results were correlated with various clinicopathologic parameters. RESULTS: Overall, 77 (57%) patients had positive PD-L1 expression. Significantly high PD-L1 expression was observed in high-grade tumors (P = .006). We found that 37% of human papillomavirus (HPV)-associated subtypes and 73% of other histotype tumors expressed PD-L1, while 63% of HPV-associated tumors and 27% of other histotype tumors did not (odds ratio, 1.35; P = .002 when compared for HPV-associated groups vs all others). Similarly, PD-L1-positive tumors had a 3.61-times higher chance of being node positive than PD-L1-negative tumors (P = .0009). In addition, PD-L1 high-positive tumors had a 5-times higher chance of being p16ink4a negative than PD-L1 low-positive tumors (P = .004). The PD-L1-positive tumors had a lower overall survival and cancer-specific survival than PD-L1-negative tumors. CONCLUSIONS: Overall, PD-L1 expression is associated with high-grade and metastatic tumors. Lower PD-L1 expression is observed more frequently in HPV-associated (warty or basaloid) subtypes than in other, predominantly HPV-independent types. As a result, PD-L1 positivity, including higher expression, portends lower overall and cancer-specific survival. These data provide a rational for further investigating PD-L1-based immunotherapeutics in PC.

Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes.

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.

Importance of tumor volume, overall treatment time and fractionation sensitivity for p16-positive and p16-negative oropharyngeal tumors.

BACKGROUND: Analyses of clinical outcomes following radiotherapy (RT) have advanced our understanding of fundamental radiobiological characteristics in head and neck squamous cell carcinoma (HNSCC). Low fractionation sensitivity appears to be a common feature, as well as susceptibility to changes in overall treatment time (OTT). Large tumors should be harder to cure if a successful RT requires the sterilization of all clonogenic cells. Congruently, primary tumor volume has proven to be an important parameter. However, most findings come from an era when p16-negative HNSCC was the dominant tumor type. HPV-associated, p16-positive, oropharyngeal tumors (OPSCC) are more radiosensitive and have better outcome. The current study aims to investigate the role of primary tumor volume, OTT and estimate α/ß-ratio for p16-positive OPSCC, and to quantify the differences in radiosensitivity depending on p16-status. METHODS: A cohort of 523 patients treated with RT was studied using a tumor control probability (TCP)-model that incorporates primary tumor volume (V) raised to an exponent c, OTT and α/ß-estimation. The significance of V was also investigated in Cox-regression models. RESULTS: In the p16-positive cohort (n = 433), the volume exponent c was 1.44 (95%CI 1.06-1.91), compared to 0.90 (0.54-1.32) for p16-negative tumors (n = 90). Hazard ratios per tumor volume doubling were 2.37 (1.72-3.28) and 1.83 (1.28-2.62) for p16-positive and p16-negative, respectively. The estimated α/ß-ratio was 9.7 Gy (-2.3-21.6), and a non-significant daily loss of 0.30 Gy (-0.17-0.92) was found. An additional dose of 6.8 Gy (interquartile range 4.8-9.1) may theoretically counteract the more radioresistant behavior of p16-negative tumors. CONCLUSION: Primary tumor volume plays a crucial role in predicting local tumor response, particularly in p16-positive OPSCC. The estimated α/ß-ratio for p16-positive oropharyngeal tumors aligns with previous HNSCC studies, whereas the impact of prolonged OTT was slightly less than previously reported. The differences in radiosensitivity depending on p16-status were quantified. The findings should be validated in independent cohorts.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.

Expression and Significance of MTA2 and CPNE1 in Cervical Squamous Cell Carcinoma.

The aim of this study was to investigate the expression and clinical significance of MTA2 and CPNE1 proteins in cervical squamous cell carcinoma. In this study, high-risk human papillomavirus (HPV) typing was performed on cervical cancer tissues. Reverse transcription polymerase chain reaction and immunochemical EliVision method were used to examine the expressions of MTA2 and CPNE1 in the cervix, and their relationship with clinicopathologic features. We found that it is mainly distributed in these types, namely HPV-16 (23.8%), HPV-18 (20.9%), HPV-53 (17.1%), HPV-52 (15.5%), HPV-82 (11.7%), HPV-56 (10.8%). The expressions of MTA2 and CPNE1 in cervical squamous cell carcinoma tissues were significantly higher than those in normal tissues ( P <0.01). The expressions of MTA2 and CPNE1 were correlated with FIGO stage, degree of differentiation, and lymph node metastasis of cervical cancer ( P <0.05), but not with the patient's age ( P >0.05). The rank correlation coefficient of MTA2 and CPNE1 protein expression in cervical squamous cell carcinoma was 0.668 ( P <0.01), and the 2 expressions were positively correlated. MTA2 and CPNE1 are closely related to the occurrence and development of cervical squamous cell carcinoma and may play a synergistic role in the evolution of cervical squamous cell carcinoma.

Phosphorylation of E2 Serine Residue 402 Is Required for the Transcription and Replication of the HPV5 Genome.

Cutaneous human papillomavirus type 5 (HPV5) belongs to the supposedly oncogenic ß-HPVs associated with specific types of skin and oral cavity cancers. Three viral proteins, namely, helicase E1 and transcription factors E2 and E8^E2, are master regulators of the viral life cycle. HPV5 E2 is a transcriptional activator that also participates in the E1-dependent replication and nuclear retention of the viral genome, whereas E8^E2 counterbalances the activity of E2 and inhibits HPV transcription and replication. In the present study, we demonstrate that the HPV5 E2 protein is extensively phosphorylated by cellular protein kinases, and serine residue 402 (S402) is the highest scoring phosphoacceptor site. This residue is located within a motif conserved among many ß-HPVs and in the oncogenic HPV31 α-type. Using the nonphosphorylatable and phosphomimetic mutants, we demonstrate that phosphorylation of the E2 S402 residue is required for the transcription and replication of the HPV5 genome in U2OS cells and human primary keratinocytes. Mechanistically, the E2-S402-phopshodeficient protein is unable to trigger viral gene transcription and has an impaired ability to support E1-dependent replication, but the respective E8^E2-S213 mutant displays no phenotype. However, phosphorylation of the E2 S402 residue has no impact on the E2 stability, subcellular localization, self-assembly, DNA-binding capacity, and affinity to the E1 and BRD4 proteins. Further studies are needed to identify the protein kinase(s) responsible for this phosphorylation. IMPORTANCE Human papillomavirus type 5 (HPV5) may play a role in the development of specific types of cutaneous and head and neck cancers. The persistence of the HPV genome in host cells depends on the activity of its proteins, namely, a helicase E1 and transcription/replication factor E2. The latter also facilitates the attachment of episomal viral genomes to host cell chromosomes. In the present study, we show that the HPV5 E2 protein is extensively phosphorylated by host cell protein kinases, and we identify serine residue 402 as the highest scoring phosphoacceptor site of E2. We demonstrate that the replication of the HPV5 genome may be blocked by a single point mutation that prevents phosphorylation of this serine residue and switches off the transcriptional activity of the E2 protein. The present study contributes to a better understanding of ß-HPV5 replication and its regulation by host cell protein kinases.

N-Acetyl-L-Cysteine Reduces Cervical Carcinogenesis by Promoting Apoptosis.

BACKGROUND AND OBJECTIVE: Cervical cancer is the fourth leading cause of cancer death in women, and is one of the most common malignant tumors of the reproductive system. However, more effective treatment for cervical cancer is needed. In this study, we aim to investigate whether N-acetyl-L-cysteine (NAC) could inhibit the proliferation of human papillomavirus (HPV)-positive cells, and reduce cervical carcinogenesis. METHODS: The cervical cancer cell lines SiHa, HeLa, HPV-negative cell line C33A, and the immortalized human cervical keratinocyte cells S12 were used. The protein expression was determined using Western blot assay. mRNA expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was determined by Cell Counting Kit-8 assay. Cell apoptosis was evaluated using Annexin V-FITC apoptosis kits. The numbers of colonies were measured using colony-forming assay. Xenograft tumor necrosis and HPV16 E7 expression were determined using hematoxylin and eosin (H&E) staining and immunohistochemistry. RESULTS: Our results showed that NAC treatment at the concentration of 1.5 mM significantly promoted cell apoptosis and reduced cell growth by inhibiting HPV16 E7 expression. NAC inhibited HPV16-oncoprotein-induced hypoxia-inducible factor (HIF)-1α protein expression and Akt activation in vitro. Additionally, NAC suppressed tumor growth, as evidenced by the smaller tumor size in the xenograft mouse model and decreased HPV16 E7 expression in tumor tissues. CONCLUSION: Our findings demonstrate that NAC exhibits the potential to promote HPV-positive cell apoptosis, and suppress the proliferation of HPV-positive cells by inhibiting cell inhibitor of apoptosis protein 2 and HIF-1α.

Human papillomavirus-driven repression of NRF2 signalling confers chemo-radio sensitivity and predicts prognosis in head and neck squamous cell carcinoma.

PURPOSE: To investigate the role of NRF2 signalling in conferring superior prognosis in patients with HPV positive (HPV+ve) head & neck squamous cell carcinomas (HNSCC) compared to HPV negative (HPV-ve) HNSCC and develop molecular markers for selection of HPV+ve HNSCC patients for treatment de-escalation trials. METHODS: NRF2 activity (NRF2, KEAP1, and NRF2-transcriptional targets), p16, and p53 levels between HPV+ve HNSCC and HPV-ve HNSCC in prospective and retrospective tumor samples as well as from TCGA database were compared. Cancer cells were transfected with HPV-E6/E7 plasmid to elucidate if HPV infection represses NRF2 activity and sensitizes to chemo-radiotherapy. RESULTS: Prospective analysis revealed a marked reduction in expression of NRF2, and its downstream genes in HPV+ve tumors compared to HPV-ve tumors. A retrospective analysis by IHC revealed significantly lower NQO1 in p16high tumors compared to p16low tumors and the NQO1 expression correlated negatively with p16 and positively with p53. Analysis of the TCGA database confirmed low constitutive NRF2 activity in HPV+ve HNSCC compared to HPV-ve HNSCC and revealed that HPV+ve HNSCC patients with 'low NQO1' expression showed better overall survival compared to HPV+ve HNSCC patients with 'high NQO1' expression. Ectopic expression of HPV-E6/E7 plasmid in various cancer cells repressed constitutive NRF2 activity, reduced total GSH, increased ROS levels, and sensitized the cancer cells to cisplatin and ionizing radiation. CONCLUSION: Low constitutive NRF2 activity contributes to better prognosis of HPV+ve HNSCC patients. Co-expression of p16high, NQO1low, and p53low could serve as a predictive biomarker for the selection of HPV + ve HNSCC patients for de-escalation trials.

HPV E6 promotes cell proliferation of cervical cancer cell by accelerating accumulation of RBM15 dependently of autophagy inhibition.

The mechanism of m6A modification in HPV-related cervical cancer remains unclear. This study explored the role of methyltransferase components in HPV-related cervical cancer and the mechanism. The levels of methyltransferase components and autophagy, ubiquitylation of RBM15 protein and the co-localization of lysosomal markers LAMP2A and RBM15 were measured. CCK-8 assay, flow cytometry, clone formation experiment and immunofluorescence assay were conducted to measure cell proliferation. The mouse tumor model was developed to study the cell growth in vivo. The binding of RBM15 to c-myc mRNA and m6A modifcation of c-myc mRNA were analyzed. The expressions of METTL3, RBM15 and WTAP were higher in HPV-positive cervical cancer cell lines than those in HPV-negative cells, especially RBM15. HPV-E6 knock-down inhibited the expression of RBM15 protein and promoted its degradation, but couldn't change its mRNA level. Autophagy inhibitor and proteasome inhibitor could reverse those effects. HPV-E6 siRNA could not enhance ubiquitylation modification of RBM15, but could enhance autophagy and the co-localization of RBM15 and LAMP2A. RBM15 overexpression could enhance cell proliferation, block the inhibitory effects of HPV-E6 siRNA on cell growth, and these effects could be reserved by cycloeucine. RBM15 could bind to c-myc mRNA, resulting in an increase to m6A level and protein expression of c-myc, which could be blocked by cycloeucine. HPV-E6 can downregulate autophagy, inhibit the degradation of RBM15 protein, induce the accumulation of intracellular RBM15, and increase the m6A modification on c-myc mRNA, resulting in an increase of c-myc protein and a growth promotion for cervical cancer cells.

Quantitative Proteomics for the Identification of Differentially Expressed Proteins in the Extracellular Vesicles of Cervical Cancer Cells.

The extracellular vesicles (EVs) in a tumoral microenvironment can exert different functions by transferring their content, which has been poorly described in cervical cancer. Here, we tried to clarify the proteomic content of these EVs, comparing those derived from cancerous HPV (+) keratinocytes (HeLa) versus those derived from normal HPV (-) keratinocytes (HaCaT). We performed a quantitative proteomic analysis, using LC-MS/MS, of the EVs from HeLa and HaCaT cell lines. The up- and downregulated proteins in the EVs from the HeLa cell line were established, along with the cellular component, molecular function, biological processes, and signaling pathways in which they participate. The biological processes with the highest number of upregulated proteins are cell adhesion, proteolysis, lipid metabolic process, and immune system processes. Interestingly, three of the top five signaling pathways with more up- and downregulated proteins are part of the immune response. Due to their content, we can infer that EVs can have a significant role in migration, invasion, metastasis, and the activation or suppression of immune system cells in cancer.

HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

Structural analysis of human papillomavirus E6 interactions with Scribble PDZ domains.

The cell polarity regulator Scribble has been shown to be a critical regulator of the establishment and development of tissue architecture, and its dysregulation promotes or suppresses tumour development in a context-dependent manner. Scribble activity is subverted by numerous viruses. This includes human papillomaviruses (HPVs), who target Scribble via the E6 protein. Binding of E6 from high-risk HPV strains to Scribble via a C-terminal PDZ-binding motif leads to Scribble degradation in vivo. However, the precise molecular basis for Scribble-E6 interactions remains to be defined. We now show that Scribble PDZ1 and PDZ3 are the major interactors of HPV E6 from multiple high-risk strains, with each E6 protein displaying a unique interaction profile. We then determined crystal structures of Scribble PDZ1 and PDZ3 domains in complex with the PDZ-binding motif (PBM) motifs of E6 from HPV strains 16, 18 and 66. Our findings reveal distinct interaction patterns for each E6 PBM motif from a given HPV strain, suggesting that a complex molecular interplay exists that underpins the overt Scribble-HPV E6 interaction and controls E6 carcinogenic potential.

Evaluation of the proteomic landscape of HPV E7­induced alterations in human keratinocytes reveal therapeutically relevant pathways for cervical cancer.

The lack of specific and accurate therapeutic targets poses a challenge in the treatment of cervical cancer (CC). Global proteomics has the potential to characterize the underlying and intricate molecular mechanisms that drive the identification of therapeutic candidates for CC in an unbiased manner. The present study assessed human papillomavirus (HPV)­induced proteomic alterations to identify key cancer hallmark pathways and protein­protein interaction (PPI) networks, which offered the opportunity to evaluate the possibility of using these for targeted therapy in CC. Comparative proteomic profiling of HPV­transfected (HPV16/18 E7), HPV­transformed (CaSki and HeLa) and normal human keratinocyte (HaCaT) cells was performed using the liquid chromatography­tandem mass spectrometry (LC­MS/MS) technique. Both label­free quantification and differential expression analysis were performed to assess differentially regulated proteins in HPV­transformed and ­transfected cells. The present study demonstrated that protein expression was upregulated in HPV­transfected cells compared with in HPV­transformed cells. This was probably due to the ectopic expression of E7 protein in the former cell type, in contrast to its constitutive expression in the latter cell type. Subsequent pathway visualization and network construction demonstrated that the upregulated proteins in HPV16/18 E7­transfected cells were predominantly associated with a diverse array of cancer hallmarks, including the mTORC1 signaling pathway, MYC targets V1, hypoxia and glycolysis. Among the various proteins present in the cancer hallmark enrichment pathways, phosphoglycerate kinase 1 (PGK1) was present across all pathways. Therefore, PGK1 may be considered as a potential biomarker. PPI analysis demonstrated a direct interaction between p130 and polyubiquitin B, which may lead to the degradation of p130 via the ubiquitin­proteasome proteolytic pathway. In summary, elucidation of the key signaling pathways in HPV16/18­transfected and ­transformed cells may aid in the design of novel therapeutic strategies for clinical application such as targeted therapy and immunotherapy against cervical cancer.

Chlamydia trachomatis infection co-operatively enhances HPV E6-E7 oncogenes mediated tumorigenesis and immunosuppression.

Chlamydia trachomatis and human papilloma virus (HPV) are the two most common sexually transmitted infections among women. HPV infection can increase the risk of cervical cancer and infertility while C. trachomatis induces pelvic inflammatory disease. Here, we elucidate the molecular conundrum of the co-infection of HPV and C. trachomatis infection and their outcome with respect to cervical cancer. HPV infection was mimicked by overexpression of HPV 16 E6-E7 or using human cervical cell lines SiHa and C33a (with and without HPV 16 respectively). HPV transfected co-infection increased cell proliferation and resistance to H202 and TNFα-induced cell death compared to individual infections. These changes are brought by alteration in the cell cycle proteins (CDK2, CDK6 and Bcl2) and thus increasing the stemness of the epithelial cells as observed by increased colony forming units and CD133 expression. The co-infection also induces change in the mRNA levels of cells which are involved in mesenchymal phenotype. C. trachomatis in presence of E6-E7 overexpression caused cervical epithelial neoplasm in mice with increased Ki67 expression and decreased P53 levels. Stem cell marker, CD133 expression also increased in the cervical tissues of both infected and co-infected group of mice. The cells obtained from the cervix were able to grow continuously in ex vivo cultures. All these results indicate the co-existence of the C. trachomatis and HPV 16 might increase the risk of cervical cancer.

Detection of HPV RNA in Extracellular Vesicles from Neuroendocrine Cervical Cancer Cells.

Background: Neuroendocrine carcinoma of the cervix (NECC) is an aggressive and rare type of cervical cancer. The five-year overall survival is low at 30% and there is no standardized therapy based on controlled trials for this type of tumour. Most are locally advanced or metastasized at the time of the diagnosis. Extracellular vesicles (EVs) could be a carrier of viral DNA/RNA, given their vital role in cellular communication. The content of EV derived from NECC cells has not been investigated due to the lack of cell line, and it is not known whether they contain human papillomaviruses (HPV) DNA/RNA or not. Methods: The presence of viral E7 DNA/RNA in EVs purified from a culture of a recently established NECC cell line, GUMC-395, was evaluated by using droplet digital polymerase chain reaction (ddPCR). These EVs were characterized using nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, Western blot for CD63, and bioanalyser for RNA quantity and quality. Results: HPV16 viral-RNA, but not DNA, was detected in EVs from GUMC-395 using ddPCR. NTA identified EVs with a mean diameter of 105.0 nm, TEM confirmed normal morphological shape and size, and Western blot analysis confirmed the presence of EV-associated proteins CD63. The EVs were found to be enriched with small RNAs using a bioanalyser. Conclusions: HPV16 RNA is found in EVs from a neuroendocrine cervical cancer and could be involved in the pathogenesis of the disease and used as a diagnostic biomarker.