New insights into the interactions of HPV-16 E6*I and E6*II with p53 isoforms and induction of apoptosis in cancer-derived cell lines.

An important characteristic of cancers associated with high-risk human papillomaviruses (HR-HPV) is the inability of p53 to activate apoptosis due to the effect of the oncoprotein E6. However, the effect of HPV-16 E6 splice variant isoforms (namely E6*I and E6*II), their interaction with the existing p53 isoforms, and their influence on apoptosis is unclear. Here, we report the outcome of ectopic expression of HPV-16 E6, E6*I, and E6*II on the relative levels of p53 and p53 isoforms Δ40p53 and Δ133p53 and their interactions with these proteins. Additionally, we evaluated the effect of ectopic expression of p53, Δ40p53, and Δ133p53 on apoptosis in a p53 null pulmonary cell line (H1299) co-transfected with E6 isoforms and p53+/+ cell lines with HR-HPV (SiHa and HeLa), transfected with p53 isoforms and treated with cisplatin, a conventional drug used to treat cervical cancer. Our results show that E6 and E6*II induced a significant decrease in p53, but only E6 triggered a Δ40p53 decrease and that E6*II interacts with p53 but not with Δ40p53 and Δ133p53. On the other hand, E6*I did not show any effect or interaction with the p53 isoforms. We found that apoptosis was elevated in H1299 cells transfected with p53 (p = 0.0001) and Δ40p53 (p = 0.0001). A weak apoptotic effect was observed when Δ133p53 was ectopically expressed (p = 0.0195). We observed that both p53 (p = 0.0006) and Δ40p53 (p = 0.0014) induced apoptosis in cisplatin-treated SiHa cells; however in cisplatin-treated HeLa cells, only p53 induced apoptosis (p = 0.0029). No significant differences in apoptosis were observed upon ectopic expression of p53, Δ40p53, and Δ133p53 in SiHa and HeLa cells. Our findings suggest a possible therapeutic application for the combining of p53 or Δ40p53 with cisplatin to induce an increased apoptosis of cancer cells expressing E6 isoforms from HPV-16.

Prognostic importance of DNA from human papillomavirus in patients with oral squamous cell carcinoma

Background: Survival of patients with oral squamous cell carcinoma (OSCC) is generally low, with the likelihood of locoregional recurrence or disease progression (LR/DP). Knowledge of prognostic factors for survival is key to achieving an understanding and increased survival. The present study aimed to identify prognostic factors for patients with OSCC, especially the presence of DNA from human papillomavirus (HPV). Material and Methods: Retrospective cohort study including 119 patients with OSCC treated at the National Cancer Institute in Mexico City (2009-2013). Clinical information was obtained from patient records including LR/DP. Formalin-fixed, paraffin-embedded tissues were obtained and used for detecting DNA from different types of HPV. Potential prognostic factors for Overall Survival (OS) were analyzed using the Cox proportional hazards model. Results: After model adjustment, factors associated with longer OS were a pre-treatment platelet count above 400,000/mm3 (HR=0.09, p=0.026) and response to primary treatment (HR=0.26, p=0.001). HPV DNA was present in 23 (19.3%) of the patients and importantly, type 16 found in 19 of them. Although survival of HPV-positive patients was longer, difference was not significant. However, among patients with LR/DP, HPV positivity was significantly associated with increased survival (HR=0.23, p=0.034). Importantly, survival was significantly different for HPV-positive patients with LR/DP > 6 months (HR=0.20, p=0.002), had higher absolute lymphocyte count at start of treatment (HR=0.50, p=0.028) or had local rescue treatment (HR=0.24, p=0.019). Conclusions: Although HPV positivity was not associated with a longer OS of OSCC patients, a better prognosis was significantly associated with HPV positivity and recurring or progressing disease, particularly with HPV type 16.(AU)

Nanog, in Cooperation with AP1, Increases the Expression of E6/E7 Oncogenes from HPV Types 16/18.

Persistent infections with some types of human papillomavirus (HPV) constitute the major etiological factor for cervical cancer development. Nanog, a stem cell transcription factor has been shown to increase during cancer progression. We wanted to determine whether Nanog could modulate transcription of E6 and E7 oncogenes. We used luciferase reporters under the regulation of the long control region (LCR) of HPV types 16 and 18 (HPV16/18) and performed RT-qPCR. We found that Nanog increases activity of both viral regulatory regions and elevates endogenous E6/E7 mRNA levels in cervical cancer-derived cells. We demonstrated by in vitro mutagenesis that changes at Nanog-binding sites found in the HPV18 LCR significantly inhibit transcriptional activation. Chromatin immunoprecipitation (ChIP) assays showed that Nanog binds in vivo to the HPV18 LCR, and its overexpression increases its binding as well as that of c-Jun. Surprisingly, we observed that mutation of AP1-binding sites also affect Nanog's ability to activate transcription, suggesting cooperation between the two factors. We searched for putative Nanog-binding sites in the LCR of several HPVs and surprisingly found them only in those types associated with cancer development. Our study shows, for the first time, a role for Nanog in the regulation of E6/E7 transcription of HPV16/18.

High prevalent human papillomavirus infections of the oral cavity of asymptomatic HIV-positive men.

BACKGROUND: Incidence of anal and oral infections with Human Papillomavirus (HPV) is increasing, particularly among Human Immunodeficiency Virus-positive (HIV+) men. HPV type 16 has exhibited the highest incidence and only limited data is available on other prevalent types, variants of HPV16, as well as associated factors. We were interested in identifying prevalent HPV types, variants of type 16, as well as factors associated with HPV16 infections in the oral cavity of HIV+ men who have sex with men (MSM). METHODS: A cross-sectional study of oral cavity samples from HIV+ MSM, that in a previous study were identified as positive for HPV16 in the anal canal. Cells from the oral cavity (102 samples, paired with 102 from the anal canal of same patient) were used to extract DNA and detect HPV infections using INNO-LiPA HPV Genotyping Extra II, and PCR. From these, 80 samples (paired, 40 anal and 40 oral) were used to identify variants of type 16 by sequencing. Statistical differences were estimated by the X2 test, and p values equal to or less than 0.05 were considered significant. SPSS ver. Twenty-four statistical software (IBM Corp) was used. RESULTS: We found a high prevalence of High-Risk HPV (HR-HPV) and Low-Risk HPV (LR-HPV). Patients were positive in the oral cavity for HR types; 16, 39 and 18 (80.4, 61.8 and 52.9% respectively) and LR types 11 and 6 (53.9 and 34.3% respectively). Surprisingly, only European variants of type 16 were found in the oral cavity, although American Asian (22.5%) and African (2.5%) variants were identified in the anal canal. The analysis showed that CD4 counts could be the most important risk factor associated with HR-HPV infections in the oral cavity, anal canal or both anatomical regions. The risk of infection of the oral cavity with type 18 increased in men diagnosed with HIV for more than 6 years. CONCLUSIONS: Prevalence of both HR and LR HPV's in the oral cavity of Mexican HIV+ MSM is very high. The fact that only European variants of HPV16 were found in the oral cavity suggest a possible tropism not previously described.

HPV oral lesions in HIV-infected patients: the impact of long-term HAART.

BACKGROUND: Since the introduction of highly active antiretroviral therapy (HAART), an increase in the frequency of human papillomavirus-associated oral lesions (HPV-OL) has been observed. Thus, the aim of this study was to determine the prevalence and factors associated with HPV-OL in Mexican HIV-infected patients, as well as its genotyping, in the HAART era. METHODS: In a cross-sectional study developed at an HIV/AIDS referral center in Mexico City, HIV-infected patients were consecutively included from 2004 to 2011. An oral exam was performed; lymphocyte CD4(+) count, HIV-viral load, CDC-stage, and HAART use were recorded. HPV-OL samples were taken for routine histopathological analysis (H-E) and HPV-DNA amplification/sequencing. Logistic regression models were performed and the interactions tested using the STATA software. RESULTS: Among 787 HIV patients, 55 (6.9%) showed HPV-OL. HPV-OLs were independently associated with age (≥40 years) and with a longer time of HAART use (≥12 months). The most frequent lesion was squamous cell papilloma in 22 (40%) cases, followed by multifocal epithelial hyperplasia in 15 (27.3%) cases. Labial mucosa was the most common site involved (56.4%). Of the sequences obtained, 65.4% corresponded to low risk and 11.5% to high risk. Mixed high- and low-risk infection were identified in 7.7% of the cases. CONCLUSIONS: Human papillomavirus-associated oral lesions were associated with older age and longer HAART use. All lesions were benign in nature and most of the HPV sequences corresponded to low-risk types. The rise of HPV-OLs in HIV patients on HAART may be related with the longer life expectancy of individuals with an impaired immune system rather than a direct effect of HAART.

High association of human papillomavirus infection with oral cancer: a case-control study.

BACKGROUND: The aim of the present study was to determine the association of high-risk human papillomavirus (HR-HPV) in Mexican individuals with oral squamous cell carcinoma (OSCC) and their association with various risk factors. METHODS: We designed a matched case-control study. Cases were individuals with newly diagnosed OSCC, age- and sex-matched with controls (1:4). Demographic and clinical data were obtained; also a self-administered questionnaire about sexual behavior was included. DNA from oral brushing was purified to amplify HPV-DNA through MY09/MY11 and GP5+/GP6+ primers and subsequently subjected to sequencing. Conditional regression models were built to calculate odds ratios (ORs) and 95% confidence intervals (CI). RESULTS: Sixty two cases and 248 controls (53.2% males), median age 62 years (Q1-Q3=54-72 years) were included. HPV prevalence was 43.5% in cases and 17.3% in controls (HR-HPV: 37.1% cases, 9.7% controls). The most frequent types in cases were HPV-16 and HPV-18 (55.6 and 18.5%). The presence of HR-HPV was associated with OSCC (OR=6.2; 95% CI: 2.98-12.97) controlling for the most common risk factors. An interaction between smoking and drinking was detected, and family history of cancer was also significant (OR: 3.61; 95% CI=1.44-8.99). Early age at first sexual intercourse and large number of lifetime sexual partners showed an association with HR-HPV (p=0.019 and p=0.033, respectively). CONCLUSIONS: Oral HR-HPV was strongly associated with OSCC, suggesting that HPV-16 and -18 are risk factors for oral cancer in Mexican patients. A significant association of tobacco and alcohol was confirmed. In addition, family history of cancer was associated with OSCC. The results underline the role of HPV in OSCC and its multifactorial etiology.

Human papillomavirus-16 DNA methylation patterns support a causal association of the virus with oral squamous cell carcinomas.

Infection with human papillomavirus-16 (HPV-16) is the cause of most anogenital carcinomas. This virus is also detected in about 20% of all head and neck squamous cell carcinomas. While there is strong evidence for a causal etiological role in the case of tonsillar carcinomas, causal association with malignant lesions of the oral cavity is not yet conclusive. Our previous investigations of HPV-16 DNA methylation in anogenital sites have identified hypermethylation of the L1 gene and part of the long control region in many malignant lesions, but rarely in asymptomatic infections and low-grade precancerous lesions. Here, we report hypermethylation of this diagnostically important segment of the viral DNA in 10 out of 12 HPV-16 positive oral carcinomas from Mexican patients. These data indicate epigenetic changes of HPV-16 in oral carcinomas similar to those in anogenital carcinomas, suggesting carcinogenic processes under the influence of HPV-16 in most if not all of these oral malignant lesions.

Association between high-risk human papillomavirus DNA load and precursor lesions of cervical cancer in Mexican women.

OBJECTIVE: Our objective was to determine the association between viral load of high risk human papilloma virus (HPV) using the Hybrid Capture II (HC II) system and cervical intraepithelial neoplasia (CIN) lesion stage. METHODS: A total of 182 consecutive women with confirmed diagnoses of CIN 1-3 and 182 healthy women with negative Pap were included. All subjects underwent structured interviews focused on socioeconomic and reproductive factors. HC II testing was used to detect human papilloma virus (HPV) DNA. Viral load was measured by light measurements expressed as relative lights unit (RLU) ratio (specimens/control). Log(10)RLU ratios were categorized for analysis into four groups: negative (

[High-risk human papilloma virus and cervical intraepithelial neoplasia in women at 2 hospitals in Mexico City]. / Virus de papiloma humano de alto riesgo (VPH-AR) y neoplasia intraepitelial cervical (NIC) en mujeres de dos hospitales de la Ciudad de México.

OBJECTIVE: To determine the high risk HPV (HR-HPV) association with Cervical Intraepithelial Neoplasia (CIN) in women of two Dysplasia Clinics in Mexico City. MATERIAL AND METHODS: Prolective case-control study was done. Women with and without security affiliation attended in Instituto Mexicano del Seguro Social (Hospital 1) and Hospital General de México (Hospital 2) were included in the study. Cases were women with histopathologic diagnosis of CIN and controls were women with negative dysplasia in cytologic study (Pap). Information was obtained by direct interview. HR-HPV was determined by Hybrid Capture II assay, in cervical samples. Bivariate and logistic regression analysis was done. RESULTS: One hundred and two cases and 192 controls from Hospital 1 and 89 cases and 66 controls from Hospital 2 were included. 83.3% and 77.3% of women from Hospital 1 and 2 respectively were positive to HR-HPV. The association HR-HPV and CIN in Hospital 1 was ORa = 40.6, C.I. 95% = 17-96.8; while in Hospital 2 there was not association. Age was an effect modifier in the HR-HVP and CIN association, in Hospital 1. It was observed a correlation between viral load and CIN degree. CONCLUSIONS: The HR-HPV infection frequency in controls and CIN I was higher than the reported in other studies. Age was a modifier in the HR-HPV association and CIN. In dysplasia clinics without medical referral system of patients is possible to observe similar risk factors to cervical cancer.

Virus de papiloma humano de alto riesgo (VPH-AR) y neoplasia intraepitelial cervical (NIC) en mujeres de dos hospitales de la Ciudad de México / High-risk human papilloma virus and cervical intraepithelial neoplasia in women at 2 hospitals in Mexico City

OBJECTIVE: To determine the high risk HPV (HR-HPV) association with Cervical Intraepithelial Neoplasia (CIN) in women of two Dysplasia Clinics in Mexico City. MATERIAL AND METHODS: Prolective case-control study was done. Women with and without security affiliation attended in Instituto Mexicano del Seguro Social (Hospital 1) and Hospital General de MÚxico (Hospital 2) were included in the study. Cases were women with histopathologic diagnosis of CIN and controls were women with negative dysplasia in cytologic study (Pap). Information was obtained by direct interview. HR-HPV was determined by Hybrid Capture II assay, in cervical samples. Bivariate and logistic regression analysis was done. RESULTS: One hundred and two cases and 192 controls from Hospital 1 and 89 cases and 66 controls from Hospital 2 were included. 83.3 and 77.3 of women from Hospital 1 and 2 respectively were positive to HR-HPV. The association HR-HPV and CIN in Hospital 1 was ORa = 40.6, C.I. 95 = 17-96.8; while in Hospital 2 there was not association. Age was an effect modifier in the HR-HVP and CIN association, in Hospital 1. It was observed a correlation between viral load and CIN degree. CONCLUSIONS: The HR-HPV infection frequency in controls and CIN I was higher than the reported in other studies. Age was a modifier in the HR-HPV association and CIN. In dysplasia clinics without medical referral system of patients is possible to observe similar risk factors to cervical cancer.