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BACKGROUND: Metformin, a widely prescribed antidiabetic drug, has shown several promising effects for cancer treatment. These effects have been shown to be mediated by dual modulation of the AMPK-mTORC1 axis, where AMPK acts upstream of mTORC1 to decrease its activity. Nevertheless, alternative pathways have been recently discovered suggesting that metformin can act through of different targets regulation. METHODS: We performed a transcriptome screening analysis using HeLa xenograft tumors generated in NOD-SCID mice treated with or without metformin to examine genes regulated by metformin. Western Blot analysis, Immunohistochemical staining, and RT-qPCR were used to confirm alterations in gene expression. The TNMplot and GEPIA2 platform were used for in silico analysis of genes found up-regulated by metformin, in cervical cancer patients. We performed an AMPK knock-down using AMPK-targeted siRNAs and mTOR inhibition with rapamycin to investigate the molecular mechanisms underlying the effect of metformin in cervical cancer cell lines. RESULTS: We shown that metformin decreases tumor growth and increased the expression of a group of antitumoral genes involved in DNA-binding transcription activator activity, hormonal response, and Dcp1-Dcp2 mRNA-decapping complex. We demonstrated that ZFP36 could act as a new molecular target increased by metformin. mTORC1 inhibition using rapamycin induces ZFP36 expression, which could suggest that metformin increases ZFP36 expression and requires mTORC1 inhibition for such effect. Surprisingly, in HeLa cells AMPK inhibition did not affect ZFP36 expression, suggesting that additional signal transducers related to suppressing mTORC1 activity, could be involved. CONCLUSIONS: These results highlight the importance of ZFP36 activation in response to metformin treatment involving mTORC1 inhibition.
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Alvo Mecanístico do Complexo 1 de Rapamicina , Metformina , Neoplasias do Colo do Útero , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Metformina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/genética , Feminino , Animais , Camundongos , Células HeLa , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos SCID , Camundongos Endogâmicos NOD , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
This study aimed to describe the prevalence of high-risk human papillomavirus (HR-HPV) types in the anal canal in a cohort of people living with HIV (PLWHIV) with a history of malignancy. SETTING: Referral tertiary care hospital for adult patients with cancer. METHODS: We reviewed data of patients from the AIDS Cancer Clinic on antiretroviral therapy in chronic control who were consecutively referred for high-resolution anoscopy (HRA), where they underwent anal evaluation, collection of specimens for anal cytology and anal human papillomavirus (HPV) followed by HRA with directed biopsy if needed. RESULTS: A total of 155 patients were included; 149 (96.1%) were men, all of them men who have sex with men (MSM); the median age was 39 (IQR 32-47) years; 105 (67.7%) with Kaposi sarcoma, 40 (25.8%) with non-Hodgkin lymphoma and 10 (6.4%) with other neoplasms; only 7 (4.5%) had active cancer. The prevalence of HR-HPV infection was 89% (n=138) (95% CI 83-93) with at least one HR-HPV infection, and 62% (96) had coinfection with at least two types; the median HR-HPV types of coinfection were 3 (IQR 2-4). The number of patients infected with HPV 16 was 64 (41.3%, 95% CI 33.8-49.3), HPV 18 was 74 (47.7%, 95% CI 39.9-55.7) and with both 35 (22.6%). Some 59 patients (38%) had high-grade squamous intraepithelial lesions (HSIL) and 49 (31.6%) had low-grade squamous intraepithelial lesions (LSIL). The prevalence of HR-HPV and HSIL among patients aged ≤35 and >35 years was the same. CONCLUSIONS: In this cohort of PLWHIV with a history of malignancy we found a high prevalence of HR-HPV 16 and 18 and anal HSIL, even in persons aged ≤35 years. These data highlight the importance of anal cancer screening in PLWHIV and history of malignancy.
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Aberrant canonical Wnt signaling is a hallmark of colon cancer. The TP53 tumor suppressor gene is altered in many solid tumors, including colorectal cancer, resulting in mutant versions of p53 (mut-p53) that lose their tumor suppressor capacities and acquire new-oncogenic functions (GOFs) critical for disease progression. Although the mechanisms related to mut-p53 GOF have been explored extensively, the relevance of mut-p53 in the canonical Wnt pathway is not well defined. This work investigated the influence of mut-p53 compared to wt-p53 in ß-catenin-dependent Wnt signaling. Using the TCGA public data from Pan-Cancer and the GEPIA2 platform, an in silico analysis of wt-p53 versus mut-p53 genotyped colorectal cancer patients showed that TP53 (p53) and CTNNB1 (ß-catenin) are significantly overexpressed in colorectal cancer, compared with normal tissue. Using p53 overexpression or p53 knockdown assays of wt-p53 or mut-p53, we found that while wt-p53 antagonizes canonical Wnt signaling, mut-p53 induces the opposite effect, improving the ß-catenin-dependent transcriptional activity and colony formation ability of colon cancer cells, which were both decreased by mut-p53 knockdown expression. The mechanism involved in mut-p53-induced activation of canonical Wnt appears to be via AKT-mediated phosphorylation of Ser 552 of ß-catenin, which is known to stabilize and enhance its transcriptional activity. We also found that while wt-p53 expression contributes to 5-FU sensitivity in colon cancer cells, the RITA p53 reactivating molecule counteracted the resistance against 5-FU in cells expressing mut-p53. Our results indicate that mut-p53 GOF acts as a positive regulator of canonical Wnt signaling and participates in the induction of resistance to 5-FU in colon cancer cells.
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BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Specific and thorough identification of cancer cell subsets with higher tumorigenicity and chemoresistance, such as cancer stem cells (CSCs), could lead to the development of new and promising therapeutic targets. For better CSC identification, a complete or extended surface marker phenotype is needed to provide increased specificity for new cell targeting approaches. Our goal is to identify and characterize a putative extended phenotype for CSCs derived from patients with GC before treatment, as well as to evaluate its clinical value. In addition, we aim to ensure that cells with this phenotype have stemness and self-renewal capabilities. METHODS: This is a cohort study including 127 treatment-naïve patients with GC who attended the Instituto Nacional de Cancerología. Multiparametric flow cytometry analysis was performed to determine the extended phenotype of cells derived from gastric biopsies. The tumorigenic capability of cells identified in patients was assessed in a zebrafish model. RESULTS: CD24+CD44+CD54+EpCAM+ cells were present in all treatment-naïve patients included, with a median abundance of 1.16% (0.57-1.89%). The percentage of CD24+CD44+CD54+EpCAM+ cells was categorized as high or low using 1.19% as the cutoff for the CD24+CD44+CD54+EpCAM+ cell subset. Additionally, a higher TNM stage correlated with a higher percentage of CD24+CD44+CD54+EpCAM+ cells (Rho coefficient 0.369; p < 0.0001). We also demonstrated that a higher percentage of CD24+CD44+CD54+EpCAM+ cells was positively associated with metastasis. The metastatic potential of these cells was confirmed in a zebrafish model. Ultimately, under our conditions, we conclude that CD24+CD44+CD54+EpCAM+ cells are true gastric cancer stem cells (GCSCs). CONCLUSION: The CD24+CD44+CD54+EpCAM+ cells present in tissue samples from patients are true GCSCs. This extended phenotype results in better and more specific characterization of these highly tumorigenic cells. The relative quantification of CD24+CD44+CD54+EpCAM+ cells has potential clinical value, as these cells are associated with metastatic disease, making their presence an additional prognostic marker and possibly a target for the design of new antineoplastic treatments in the era of precision oncology. Overall, the extended CD24+CD44+CD54+EpCAM+ phenotype of GCSCs could support their isolation for the study of their stemness mechanisms, leading to the identification of better molecular targets for the development of both new therapeutic approaches such as oncoimmunotherapy and new diagnostic and clinical prognostic strategies for GC.
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Neoplasias Gástricas , Peixe-Zebra , Animais , Biomarcadores Tumorais/metabolismo , Antígeno CD24/genética , Linhagem Celular Tumoral , Estudos de Coortes , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Medicina de Precisão , Neoplasias Gástricas/metabolismo , Peixe-Zebra/metabolismo , Molécula 1 de Adesão Intercelular , HumanosRESUMO
BACKGROUND: Worldwide prevalence of Oropharyngeal Squamous Cell Carcinoma (OPSCC) has increased, affecting mostly young males. OPSCC associated with Human Papillomavirus (HPV) infection exhibits particular characteristics in terms of response to treatment, hence HPV has been proposed as a prognostic factor. The impact of HPV positivity and associated biomarkers on OPSCC in the Mexican population has not been addressed. Therefore, the analysis of OPSCC prognostic markers in the Mexican population is necessary. METHODS: Retrolective study in Mexican OPSCC patients, where HPV prevalence, p16 and EGFR levels were assessed using INNO-LiPA and immunohistochemistry. RESULTS: We found an HPV prevalence of 57.6% in OPSCC cases treated at a reference center in Mexico. HPV and p16 positivity, as well as EGFR, associate with better outcomes in OPSCC patients, and they also promote reduced death risk. Notably, HPV presence and p16 positivity showed a significant association with disease-free survival (DFS), with a HR of 0.15 (p = 0.006) and a HR of 0.17 (p = 0.012), respectively, indicating a possible role as predictive biomarkers in Mexican OPSCC patients. CONCLUSIONS: Our results reflect the clinical utility of p16 analysis to improve overall survival (OS) and to predict recurrence in oropharyngeal cancer. These results position p16 and HPV as predictive biomarkers for OPSCC.
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Purpose: Cervical cancer (CC) is the second most frequent cancer in undeveloped countries. Serum biomarkers could be useful for evaluation of the treatment response and as a complementary means to improve diagnosis. The expression of galectin-9 is altered in cancer tissue, and higher concentrations are found in the serum of cancer patients. The objectives of this study were (a) to determine the serum galectin-9 concentration in patients with intraepithelial lesions and CC, (b) to determine if the concentration was related to the clinicopathological characteristics and (c) to determine if the galectin-9 concentration was related to its expression level in tumour tissue. Patients and Methods: In all, 222 serum samples from women with different diagnoses, including premalignant lesions and CC, as well as samples from women with normal cytology were included in the study. The serum galectin-9 concentration was determined by ELISA. To evaluate the expression level of galectin-9 in CC tissue, immunohistochemistry was performed in 34 CC biopsy specimens. Results: The galectin-9 concentration in the serum of CC patients (8.171 ng/mL) was increased compared with serum from women with normal epithelia (4.654 ng/mL) and those with low-grade (4.806 ng/mL) and high-grade (5.354 ng/mL) intraepithelial lesions (p value < 0.0001). The area under the ROC curve considering the CC group and the control group was 0.882. The optimal cut-off value was ≥6.88 ng/mL, the specificity obtained was 100%, and the sensitivity was 68.2%. In the CC group, the analysis of the clinical stage showed an increase of galectin-9 in the advanced stage IV group. Serum galectin-9 was not related to the level of galectin-9 expression in tissue, which suggests that galectin-9 is not secreted by tumour cells. Conclusion: The serum galectin-9 concentration is related to cancer progression, as the level of this protein is higher in patients with advanced-stage disease.
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Cell lines have been considered excellent research models in many areas of biomedicine and, specifically, in the study of carcinogenesis. However, they cease to be effective models if their behavior changes. Although studies on the cross-contamination of cell lines originating from different tissues have been performed, little is known about cell lines derived from cervical neoplasia. We know that high-risk HPV (HR-HPV) is associated with the development of this type of cancer. This link between HPV infection and cancer was first established over 35 years ago when HPV16 DNA was found to be present in a large proportion of cervical cancer biopsies. The present review paper aims to report the status of the establishment, authenticity, and characterization of cervical cancer (CC) cell lines. This is a systematic review of articles on the establishment, authenticity, and characterization of CC cell lines, published from 1960 to date in the databases and in cell repository databases. 52 cell lines were identified in the literature. Only 25 cell lines were derived from cervical neoplasia, of which only 45.8% have a reported identity test (genomic fingerprint). Despite the increase in the establishment of cell lines of cervical neoplasia and the standards for the regulation of these study models, the criteria for their characterization continue to be diverse.
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OBJECTIVE: To investigate the association of high-risk hu-man papilloma virus (HR-HPV) and other risk factors with ocular surface squamous cell neoplasia (OSSN). MATERIALS AND METHODS: We obtained DNA from 22 fresh frozen OSSN tissues and 22 pterygia as controls, we used a broad-spectrum HPV DNA amplification short PCR fragment to identify HPV infection in all specimens and then genotyped HPV by a reverse hybridization line probe assay. We also obtained demographic, sun exposure, and tobacco consump-tion information. RESULTS: HR-HPV frequency was 40.9% in the OSSN group and 4.5% in the pterygia group (p=0.009). After covariate adjustment, OSSN was associated with HR-HPV (OR=16.3, 95%CI=1.2,218.1, p=0.03) and sunburn (OR=10.8, 95%CI=1.8,86.0, p=0.02). CONCLUSIONS: Ocular surface squamous cell neoplasia is a multifactorial disease. The strong association between HR-HPV and OSSN, suggests that HR-HPV could play an etiological role in OSSN development.
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Alphapapillomavirus , Carcinoma de Células Escamosas , Neoplasias da Túnica Conjuntiva , Neoplasias Oculares , Infecções por Papillomavirus , Carcinoma de Células Escamosas/epidemiologia , Estudos de Casos e Controles , Túnica Conjuntiva/anormalidades , Neoplasias da Túnica Conjuntiva/complicações , Neoplasias da Túnica Conjuntiva/epidemiologia , Neoplasias Oculares/complicações , Neoplasias Oculares/epidemiologia , Humanos , México/epidemiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , PterígioRESUMO
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.
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Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Apoptose , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Feminino , Células HeLa , Papillomavirus Humano 16 , Humanos , Isoformas de Proteínas , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/metabolismoRESUMO
Abstract: Objective: To investigate the association of high-risk human papilloma virus (HR-HPV) and other risk factors with ocular surface squamous cell neoplasia (OSSN). Materials and methods: We obtained DNA from 22 fresh frozen OSSN tissues and 22 pterygia as controls, we used a broad-spectrum HPV DNA amplification short PCR fragment to identify HPV infection in all specimens and then genotyped HPV by a reverse hybridization line probe assay. We also obtained demographic, sun exposure, and tobacco consumption information. Results: HR-HPV frequency was 40.9% in the OSSN group and 4.5% in the pterygia group (p=0.009). After covariate adjustment, OSSN was associated with HR-HPV (OR=16.3, 95%CI=1.2,218.1, p=0.03) and sunburn (OR=10.8, 95%CI=1.8,86.0, p=0.02). Conclusions: Ocular surface squamous cell neoplasia is a multifactorial disease. The strong association between HR-HPV and OSSN, suggests that HR-HPV could play an etiological role in OSSN development.
Resumen: Objetivo: Investigar la asociación del virus del papiloma humano de alto riesgo (VPH-AR), así como de otros factores, con neoplasia escamosa de la superficie ocular (NESO). Material y métodos: Se obtuvieron 22 especímenes de tejido fresco de NESO y 22 de pterigión como controles; se utilizó una técnica molecular altamente sensible para identificar la infección por VPH en todos los especímenes, así como la genotipificación del VPH. También se obtuvo información demográfica sobre exposición a la luz solar y tabaquismo. Resultados: La frecuencia de infección por VPH-AR fue de 40.9% en el grupo de NESO y de 4.5% en el grupo control (p=0.009). Después de ajustar por covariables, NESO se asoció con el VPH-AR (OR=16.3, IC95%=1.2,218.1, p=0.03) y el eritema solar (OR=10.8, IC95%=1.8,86.0, p=0.02). Conclusiones: La neoplasia escamosa de superficie ocular en una neoplasia multifactorial. Los presentes resultados sugieren que el VPH-AR podría tener un papel etiológico en el desarrollo de NESO.
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BACKGROUND/AIM: To date, several proteomics studies in cervical cancer (CC) have focused mainly on squamous cervical cancer (SCC). Our study aimed to discover and clarify differences in SCC and CAD that may provide valuable information for the identification of proteins involved in tumor progression, in CC as a whole, or specific for SCC or CAD. MATERIALS AND METHODS: Total protein extracts from 15 individual samples corresponding to 5 different CC tissue types were compared with a non-cancerous control group using bidimensional liquid chromatography-mass spectrometry (2D LC-MS/MS), isobaric tags for relative and absolute quantitation (ITRAQ), principal component analysis (PCA) and gene set enrichment analysis (GSEA). RESULTS: A total of 622 statistically significant different proteins were detected. Exocytosis-related proteins were the most over-represented, accounting for 25% of the identified and quantified proteins. Based on the experimental results, reticulocalbin 3 (RCN3) and Ras-related protein Rab-14 (RAB14) were chosen for further downstream in vitro and vivo analyses. RCN3 was overexpressed in all CC tissues compared to the control and RAB14 was overexpressed in squamous cervical cancer (SCC) compared to invasive cervical adenocarcinoma (CAD). In the tumor xenograft experiment, RAB14 protein expression was positively correlated with increased tumor size. In addition, RCN3-expressing HeLa cells induced a discrete size increment compared to control, at day 47 after inoculation. CONCLUSION: RAB14 and RCN3 are suggested as potential biomarkers and therapeutic targets in the treatment of CC.
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Proteômica , Neoplasias do Colo do Útero , Cromatografia Líquida/métodos , Feminino , Células HeLa , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Neoplasias do Colo do Útero/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Polyphenols constitute an important group of natural products that are traditionally associated with a wide range of bioactivities. These are usually found in low concentrations in natural products and are now available in nutraceuticals or dietary supplements. A group of polyphenols that include apigenin, quercetin, curcumin, resveratrol, EGCG, and kaempferol have been shown to regulate signaling pathways that are central for cancer development, progression, and metastasis. Here, we describe novel mechanistic insights on the effect of this group of polyphenols on key elements of the signaling pathways impacting cancer. We describe the protein modifications induced by these polyphenols and their effect on the central elements of several signaling pathways including PI3K, Akt, mTOR, RAS, and MAPK and particularly those affecting the tumor suppressor p53 protein. Modifications of p53 induced by these polyphenols regulate p53 gene expression and protein levels and posttranslational modifications such as phosphorylation, acetylation, and ubiquitination that influence stability, subcellular location, activation of new transcriptional targets, and the role of p53 in response to DNA damage, apoptosis control, cell- cycle regulation, senescence, and cell fate. Thus, deep understanding of the effects that polyphenols have on these key players in cancer-driving signaling pathways will certainly lead to better designed targeted therapies, with less toxicity for cancer treatment. The scope of this review centers on the regulation of key elements of cancer signaling pathways by the most studied polyphenols and highlights the importance of a profound understanding of these regulations in order to improve cancer treatment and control with natural products.
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Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.
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Proteínas de Ligação a DNA/genética , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/genética , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Códon de Iniciação/genética , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação Viral da Expressão Gênica/genética , Células HEK293 , Células HaCaT , Células HeLa , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogênicas Virais/biossíntese , Iniciação Traducional da Cadeia Peptídica/genética , RNA Viral/genética , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
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.
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Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteína Homeobox Nanog/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Humanos , Proteína Homeobox Nanog/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
INTRODUCTION: Astrocytomas are the most common and aggressive primary brain tumors, and they are classified according to the degree of malignancy on a scale of I to IV, in which grade I is the least malignant and grade IV the highest. Many factors are related to astrocytomas progression as progesterone receptor (PR), whose transcriptional activity could be regulated by phosphorylation by protein kinase C alpha (PKCα) at the residue Ser400. Our aim was to investigate if PR phosphorylation together with PKCα expression could be used as a prognostic factor for astrocytomas malignancy. METHODS: By immunofluorescence, we detected the content of PKCα, PR and its phosphorylation at Ser400 in 46 biopsies from Mexican patients with different astrocytoma malignancy grades; by bioinformatic tools using TCGA data, we evaluated the expression of PR and PKCα mRNA according to astrocytoma malignancy grades. For all statistical analyses, significance was p<0.05. RESULTS: We detected a positive correlation between the tumor grade and the content of PKCα, PR and its phosphorylation at Ser400, as well as the intracellular colocalization of these proteins. Interestingly, using an in silico assay, we found that the PR and PKCα expression at mRNA level has an inverse ratio with astrocytomas tumor grade. DISCUSSION: These results indicate that PR and its phosphorylation at Ser400 site, as well as PKCα and their colocalization, could be considered as possible malignancy biomarkers for astrocytomas grades I-IV.
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Circulating biological markers, such as miRNAs, hold the greatest possibilities to complement tissue biopsy and clinical diagnostic tests. The objective of this study was to evaluate the relative abundance of three circulating miRNAs in serum from 17 HPV16-positive patients with early cervical lesions known as Low-Grade Squamous Intraepithelial Lesions (LSILs). The expression of circulating microRNAs miR-15b, miR-34a and miR-218 in patients with LSILs was compared to 23 HPV-negative individuals showing normal cervical epithelium (healthy women) and 23 Squamous Cell Carcinoma (SCC) samples. The expression levels of miR-15b remained unchanged while those of miRNAs 34a and 218 were relatively high in serum obtained from LSIL patients in comparison with healthy women (results were statistically significant with a p of < 0.01 or < 0.001). According to previous findings, miR-15b was overexpressed and miRNAs 34a and 218 were underexpressed in serum from SCC patients. Additionally, the mRNA expression levels of some selected gene targets were determined [Cyclin D1 (CCND1), Cyclin E1 (CCNE1), B-cell lymphoma 2 (Bcl-2) and MutS homolog 2 (MSH-2)]. All serum results correlated with tissue samples from the same patients. We propose that circulating microRNAs can be valuable as molecular markers for the early follow-up of cervical carcinogenesis risk.
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MicroRNA Circulante/sangue , MicroRNAs/sangue , Displasia do Colo do Útero/genética , Neoplasias do Colo do Útero/genética , Adulto , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Colo do Útero/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Infecções por Papillomavirus/genética , Neoplasias do Colo do Útero/sangue , Adulto Jovem , Displasia do Colo do Útero/sangueRESUMO
Our aim was to analyze the prevalence of high-risk human papillomavirus (HR-HPV) and its association with risk factors related to cervical lesions. We used 362 cervical samples from a transversal study to detect nineteen types from the high-risk HPV clade by highly sensitive PCR. Unexpectedly, we found a very high prevalence of HPV type 66 (32.8%), particularly in low-grade squamous intraepithelial lesions. A significant association of HPV66 with previously sexually transmitted disease was observed (p < 0.05). Our results strongly suggest that HPV66 might be indicative of cervical lesions that will not progress to cancer. HPV genotyping by methods that grouped type 66 with other HR-HPV clade types should be interpreted with caution.
Assuntos
Colo do Útero/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Displasia do Colo do Útero/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Detecção Precoce de Câncer , Feminino , Genótipo , Técnicas de Genotipagem , Humanos , Modelos Logísticos , México/epidemiologia , Pessoa de Meia-Idade , Papillomaviridae/classificação , Fatores de Risco , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal , Adulto Jovem , Displasia do Colo do Útero/patologiaRESUMO
ALDH is an enzyme involved in different cellular processes, including cancer. It has been shown that a cellular subpopulation with high ALDH activity (ALDHHIGH) within a tumor is related to functional capabilities such as stemness, chemoresistance, and tumorigenicity. However, few studies have focused on determining the mechanisms behind ALDH activity within the cells. Previously, our group reported that ALDHHIGH cells have higher tumorigenicity in Cervical Cancer (CC) cell lines. Based on this, we were interested to know the molecular mediators of the ALDHHIGH cells, specifically ß-catenin, inasmuch as ß-catenin is regulated through different pathways, such as Wnt signaling, and that it acts as a transcriptional co-activator involved in cancer progression. In this work, we show that the increase in ALDHHIGH cell percentage is reverted by ß-catenin knockdown. Consistently, upon GSK3-ß inactivation, a negative regulator of ß-catenin, we observed an increase in ALDHHIGH cells. Additionally, we observed a low percentage of cells positive for Fzd receptor, suggesting that in our model there is a low capacity to respond to Wnt ligands. The analysis of ALDHHIGH cells in a sphere formation model demonstrated the active state of AKT. In accordance with this, impairment of AKT activity not only reduced ß-catenin active state, but also the percentage of ALDHHIGH cells. This corroborates that AKT acts upstream of ß-catenin, thus affecting the percentage of ALDHHIGH cells. In conclusion, our results show that ALDHHIGH cells are dependent on ß-catenin, in spite of the Wnt pathway seems to be dispensable, while AKT emerges as central player supporting a mechanism in this important axis that is not yet well known but its analysis improves our understanding of ALDH activity on CC.
RESUMO
A common characteristic of cancer types associated with viruses is the dysregulated expression of the CDH1 gene, which encodes Ecadherin, in general by activation of DNA methyltransferases (Dnmts). In cervical cancer, E7 protein from high risk human papillomaviruses (HPVs) has been demonstrated to interact with Dnmt1 and histone deacetylase type 1 (HDAC1). The present study proposed that E7 may regulate the expression of CDH1 through two pathways: i) Epigenetic, including DNA methylation; and ii) Epigeneticindependent, including the induction of negative regulators of CDH1 expression, such as Snail family transcriptional repressor Snai1 and Snai2. To test this hypothesis, HPV16 and HPV18positive cell lines were used to determine the methylation pattern of the CDH1 promoter and its expression in association with its negative regulators. Different methylation frequencies were identified in the CDH1 promoter in HeLa (88.24%) compared with SiHa (17.65%) and Ca Ski (0%) cell lines. Significant differences in the expression of SNAI1 were observed between these cell lines, and an inverse association was identified between the expression levels of SNAI1 and CDH1. In addition, suppressing E7 not only increased the expression of CDH1, but notably decreased the expression of SNAI1 and modified the methylation pattern of the CDH1 promoter. These results suggested that the expression of CDH1 was dependent on the expression of SNAI1 and was inversely associated with the expression of E7. The present results indicated that E7 from HPV16/18 regulated the expression of CDH1 by the two following pathways in which Snai1 is involved: i) Hypermethylation of the CDH1 promoter region and increasing expression of SNAI1, as observed in HeLa; and ii) Hypomethylation of the CDH1 promoter region and expression of SNAI1, as observed in SiHa. Therefore, the suppression of CDH1 and expression of SNAI1 may be considered to be biomarkers of metastasis in uterine cervical cancer.