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PDF file - 108K, HPV16-E6 enhances canonical Wnt/�-catenin signaling pathway in primary human keratinocytes. Wnt/�-catenin pathway activation was assessed using the TOPFLASH construct. Human foreskin keratinocytes were co-transfected with, E6, HPV16-E6�141-151 (E6�PDZ), Dvl2 and B-cat expression vectors. Both, E6 and the mutated form lacking the PDZ-binding domain (E6�PDZ), minimally activated the canonical Wnt pathway. However, both of the constructs induced very significant enhancement when they were co-transfected with Dvl2 or �-catenin (B-cat), which suggests that this effect was independent of the PDZ-binding domain.

PDF file - 103K, The E6 oncoprotein interacts with Dvl2 but not with Dvl1 or Dvl3. COS7 cells were transiently transfected with HPV-E6 and one of the Dvl cDNAs. HPV-E6 was tagged with AU1, and the Dvl genes included Flag tags. Cell lysates were prepared 48 h after transfection, and immunoprecipitation was performed with either an anti-flag antibody or anti-AU1 antibody. The immunoprecipitates were separated on a 10% acrylamide gel and transferred to membranes, and western blots were performed using anti-Flag and anti-AU1 antibodies. The Dvl proteins are indicated by arrows and the E6 proteins are indicated by the arrowheads. All three of the Dvl proteins (upper left panel) and HPV-E6 (lower right panel) were expressed at the expected size. The upper-right and lower-left panels show that Dvl2 and E6 may exist in the same protein complex. The first lane on each panel represents the resulting transfection with the empty vector as the negative control.

PDF file - 109K, HPV16-E6 but not HPV16-E7 oncoprotein enhances canonical Wnt/�-catenin pathway in cultured cells. (A) COS7 cells were transiently transfected with the TCF/B-catenin dependent luciferase reporter construct (TOPFLASH) and Renilla together with one or two of the following expression vectors encoding the following: HPV16-E6 (E6), HPV16-E7 (E7), Dvl2 or �-catenin (B-cat). Luciferase activity from culture cells was analyzed 48 h after transfection and is shown as normalized to total protein. HPV16-E6 but not E7 significantly induced TOPFLASH activity with or without B-cat cotransfection (A) or Dvl2 cotransfection (B) co-transfection, which suggests that E6 may positively modulate canonical Wnt signaling. The Renilla signal and empty vector were used to normalize luciferase activity.

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

Tight junctions (TJs) are cell‑cell adhesion structures frequently altered by oncogenic transformation. In the present study the role of human papillomavirus (HPV) 16 E7 oncoprotein on the sealing of TJs was investigated and also the expression level of claudins in mouse cervix and in epithelial Madin‑Darby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins ‑1 and ‑10 in the cervix of 7‑month‑old transgenic K14E7 mice treated with 17β‑estradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin‑1 expression in the cervix of 2‑month‑old K14E7 mice, and claudin‑10 accumulated at the border of cells in the upper layer of the cervix in FvB mice treated with E2, and in K14E7 mice treated with or without E2. These changes were accompanied by an augmented paracellular permeability of the cervix in 2‑ and 7‑month‑old FvB mice treated with E2, which became more pronounced in K14E7 mice treated with or without E2. In MDCK cells the stable expression of E7 increased the space between adjacent cells and altered the architecture of the monolayers, induced the development of an acute peak of transepithelial electrical resistance accompanied by a reduced expression of claudins ‑1, ‑2 and ‑10, and an increase in claudin‑4. Moreover, E7 enhances the ability of MDCK cells to migrate through a 3D matrix and induces cell stiffening and stress fiber formation. These observations revealed that cell transformation induced by HPV16 E7 oncoprotein was accompanied by changes in the pattern of expression of claudins and the degree of sealing of epithelial TJs.

Mexico City has one of the highest incidences of acute lymphoblastic leukemia (ALL) globally, with patients showing low survival, and high relapse rates. To gain more insight into the molecular features of B-ALL in Mexican children, we isolated CD10 + /CD19 + precursor B lymphoblasts from four bone marrow and nine peripheral blood samples of B-ALL patients using a fluorescence-activated cell sorting protocol. The global gene expression profile (BM vs PB) revealed 136 differentially expressed genes; 62 were upregulated (45.6%) and 74 were downregulated (54.4%). Pearson’s correlation coefficient was calculated to determine the similarity between pre-B lymphoblast populations. We selected 26 highly significant genes and validated 21 by RT-qPCR (CNN3, STON2, CALN1, RUNX2, GADD45A, CDC45, CDC20, PLK1, AIDA, HCK, LY86, GPR65, PIK3CG, LILRB2, IL7R, TCL1A, DOCK1, HIST1H3G, PTPN14, CD72, and NT5E). The gene set enrichment analysis of the total expression matrix and the ingenuity pathway analysis of the 136 differentially expressed genes showed that the cell cycle was altered in the bone marrow with four overexpressed genes (PLK1, CDC20, CDC45, and GADD45A) and a low expression of IL7R and PIK3CG, which are involved in B cell differentiation. A comparative bioinformatics analysis of 15 bone marrow and 10 peripheral blood samples from Hispanic B-ALL patients collected by the TARGET program, corroborated the genes observed, except for PIK3CG. We conclude the Mexican and the Hispanic B-ALL patients studied present common driver alterations and histotype-specific mutations that could facilitate risk stratification and diagnostic accuracy and serve as potential therapeutic targets.

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.

The aim of this study was to evaluate the association between prostate cancer (PCa) and Human papillomavirus (HPV) infection in the Mexican population. We studied 356 paraffin-embedded tissues from unrelated Mexican men with PCa or benign prostatic hyperplasia (BPH), with the latter serving as control. HPV detection was performed by polymerase chain reaction (PCR) using universal primers, and viral genotypes were detected using sequencing or multiplex PCR. Light microscopy analyses enabled the identification of koilocytes in samples subsequently analyzed for HPV detection by in situ PCR and for p16-INK4A expression by immunohistochemistry. The results showed that high risk- (HR) HPVs were detected in 37/189 (19.6%) PCa specimens compared to 16/167 (9.6%) of BHP specimens (odds ratio 2.3; 95% CI= 1.2 to 4.3; p=0.01). These data suggest HR-HPV may play a role in PCa. HPV 52 and 58 were the most frequent genotypes (33 and 17%, respectively) detected in the population studied. Koilocytes were detected in all in situ PCR-HPV-positive samples, representing a pathognomonic feature of infection, and we observed the overexpression of p16-INK4A in HPV-positive samples compared to HPV-negative samples, indirectly suggesting the presence of HR-HPV E7 oncoprotein. These results suggest that HPV infection plays an important role in prostate cancer development.

Introduction. Cervical cancer is an important health issue worldwide. Many factors are related to this condition as persistent human papillomavirus infection, the use of hormonal contraceptives, pH changes, and bacterial vaginosis. The association between the microbiota and cervical cancer is an interesting issue; given that environmental and hormonal factors changing the vaginal microbiota may contribute to cancer. Methods Using High-throughput DNA sequencing of V3-16S rDNA libraries, we determined the bacterial diversity in cervicovaginal lavages and fecal samples at different stages of cervical cancer development in the K14HPV16E7 mice model under +/- 17β-estradiol (E2) stimulus and compared it with an isogenic control (FVB). Results. We found that continuous E2 administration during 6 months in the model with type 16 E7 expression causing the development of cancer, is associated with significant changes in the microbiota diversity in the cervicovaginal lavages. Similar results were not observed in the model when no E2 was administered neither in the FVB mice. Comparable changes in the microbiota diversity of fecal samples were not observed. Conclusions. The persistent expression of E7 oncogene or the 17β-estradiol for longer periods of time, cause changes in the vaginal microbiota diversity and the cervical epithelium not necessarily leading to cervical cancer; however, the combined action of both, causes cervical carcinoma and defined changes in the abundance of bacterial taxa in the cervicovaginal lavages, suggesting a specific organ effect of E7 expression associated to E2 on the vaginal microbiota. Acknowledgments. Work supported by Cinvestav and CONACyT 163235 INFR-2011-01, FONSEC SS/IMSS/ISSSTE-CONACYT-233361, and CONACyT 0201904 grants.

The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical processes such as cell proliferation, apoptosis, and immune response. This oncoprotein plays an essential role in cervical carcinogenesis, but other cofactors such as long-term use of hormonal contraceptives are necessary to modulate the risk of cervical cancer (CC). The role of HR-HPVs in the alteration of microRNA (miRNA) levels in persistent viral infections currently remains unclear. The aim of this study was to evaluate the miR-34a and miR-15b expression levels in the murine HPV16K14E7 (K14E7) transgenic model after chronic estrogen (E2) treatment and their involvement in CC. Interestingly, results showed that, although miR-34a expression is elevated by the HPVE7 oncogene, this expression was downregulated in the presence of both the E7 oncoprotein and chronic E2 in cervical carcinoma. On the other hand, miR-15b expression was upregulated along cervical carcinogenesis mainly by the effect of E2. These different changes in the expression levels of miR-34a and miR-15b along cervical carcinogenesis conduced to low apoptosis levels, high cell proliferation and finally, to cancerous cervical tissue development. In this work, we also determined the relative mRNA expression of Cyclin E2 (Ccne2), Cyclin A2 (Ccna2), and B cell lymphoma 2 (Bcl-2) (target genes of miR-34a and miR-15b); Sirtuin 1 (Sirt1), Cmyc, and Bax (miR-34a target genes); and p21/WAF1 (mir15b target gene) and the H-ras oncogene. Given the modifications in the expression levels of miR-34a and miR-15b during the development of cervical cancer, it will be useful to carry out further investigation to confirm them as molecular biomarkers of cancer.

Infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer (CC), but viral infection alone does not guarantee the development of this malignancy. Indeed, deficiencies of dietary micronutrients could favor cervical cancer development in individuals that harbor HR-HPV infections. The status of retinoid levels, natural and synthetic derivatives of vitamin A, is important in maintaining cellular differentiation of the cervical epithelium. Moreover, many studies show a link between deficient intake of retinoids or alteration of the retinoid receptors and CC development. In spite of this, the effect of vitamin A deficiency (VAD) in presence of HR-HPV oncoproteins on cervical carcinogenesis in vivo has not been reported. Transgenic mice expressing E6 or E7 oncoproteins (K14E6 or K14E7 mice, respectively) were used to evaluate the possible role of VAD in the development of malignant cervical lesions. The survival of the mice in VAD condition was studied, and histopathological analysis and immunohistochemical detection of molecular cancer markers such as the tumor suppressor retinoic acid receptor beta (RARβ), proliferating cell nuclear antigen (PCNA), cleaved caspase 3, and the tumor suppressor protein p16INK4A (inhibitor of CDK4) were performed. Our results show that K14E6/VAD mice showed moderate cervical dysplasia; notably, K14E7/VAD mice developed severe cervical dysplasia and cervical in situ carcinoma at an early age. VAD synergizes with HPV16E7 oncoprotein expression favoring cervical carcinogenesis in vivo.

Mutations in p53 are strongly associated with several highly malignant cancer phenotypes but its role in regulating energy metabolism has not been completely elucidated. The effect on glycolysis and oxidative phosphorylation (OxPhos) of mutant p53R248Q overexpression in HeLa cells (HeLa-M) was analyzed and compared with cells overexpressing wild-type p53 (HeLa-H) and nontransfected cells containing negligible p53 levels (HeLa-L). p53 R248Q overexpression induced early cell detachment during in vitro growth; however, detached HeLa-M cells showed high viability, shorter generation time and significant diminution in the adhesion proteins E-cadherin and β-catenin versus HeLa-H and HeLa-L cells. Under normoxia, a lower growth rate of attached HeLa-M cells correlated with decreased levels of proliferating cell nuclear antigen (PCNA), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), adenosine monophosphate-activated protein kinase (AMPK), mitochondrial proteins (20-80%) and OxPhos flux (69 ± 12%). On the contrary, HeLa-M also showed increased contents of CDKN1A, nuclear factor κB (NF-κB), c-MYC, hypoxia-inducible factor 1-α (HIF-1α; 1-4 times), glycolytic HIF-1α targets (2-4 times), and glycolysis flux (2-fold) versus HeLa-H. In consequence, glycolysis provided ~70% of the cellular adenosine triphosphate (ATP) in HeLa-M cells under normoxia whereas, OxPhos predominated (65-82%) in HeLa-H and HeLa-L cells. Pifithrin-α, a specific p53 inhibitor, did not alter the p53 R248Q target protein contents and OxPhos and glycolytic fluxes, and a poor HIF-1α-p53 R248Q interaction was attained, in HeLa-M cells. These observations suggested that p53 R248Q deficiently interacted with pifithrin-α and HIF-1α. Therefore, lower mitochondrial biogenesis, deficient HIF-1α/mutant p53 interaction, and development of a pseudohypoxic state under normoxia were the apparent biochemical mechanisms underlying glycolysis activation and OxPhos downregulation in HeLa-M cells.

Transcription factors OCT4, SOX2, KLF4, C-MYC, and NANOG (OSKM-N) regulate pluripotency and stemness, and their ectopic expression reprograms human and murine fibroblasts that constitute the key of regenerative medicine. To determine their contribution to cell transformation, we analyzed the gene expression profiles of these transcription factors in cervical cancer samples and found that they are preferentially expressed in the tumor component. Also, cancer stem cell-enriched cultures grown as sphere cultures showed overexpression of OSKM-N genes. Importantly, we observed that lentiviral-mediated transduction of these factors confers, to a nontumorigenic immortalized human cell line, properties of cancer stem cells as the ability to form tumors in a mouse model. When we performed a meta-analysis using microarray data from cervical cancer biopsies and normal tissues, we found that the expression of OSKM-N and some target genes allowed separating tumor and normal tissues between samples, which enhanced the importance of OSKM-N in the tumorigenesis. Finally, we analyzed and compared both transcript and protein expression profiles of these factors within a cohort of patients with cervical cancer. To our knowledge, this is the first time that the expression of OSKM-N is described to induce one of the main characteristics of the cancer stem cell, the tumorigenicity. And, more importantly, its exogenous expression in a nontumorigenic cell line is sufficient to induce a tumorigenic phenotype; furthermore, the differential expression of this transcription factor distinguishes tumor tissue and normal tissue in cervical samples.

Background HER2 over-expression is related with a poor prognosis in patients with invasive breast cancer tumors. Clinical associations have reported that somatic mutations of p53 more frequently detected in cases of sporadic breast cancer of the HER2 subtypes, besides a high percentage of HER2-amplifying tumors carry germline mutations of p53. The mechanisms responsible for the acquisition of oncogenic functions of p53 mutant proteins (mtp53), known as Gain of Function (GOF), over HER2 expression have not been reported. The objective of this study was to evaluate a possible relationship between p53 mutants and HER2 regulation. Methods HER2 expression (transcription and protein), as well as HER2 protein stabilization have been evaluated after inducing or silencing of p53 mutants’ expression in cell lines. Finally, we evaluated the interaction of the p53 mutants over the HER2 receptor promoter. Results Higher HER2 expression in cell lines harboring endogenous mtp53 compared with wt or null expression of p53 cell lines. Transfection of p53 mutants (R248Q and R273C) in cell lines increased the expression of HER2. Silencing of p53 mutants, decrease HER2 expression. The p53 mutants R248Q and R273C significantly increase the luciferase activity on the HER2 promoter, and both mutants also promote acetylation of H3 and H4 histones binding in it. Conclusions These findings show for the first time that p53 mutants induce over-expression of HER2 at transcriptional level of the HER2 protein. Our results could have clinical implications in breast cancer and other types of cancer where HER2 is over-expressed and used as a therapy target. Electronic supplementary material The online version of this article (10.1186/s12885-018-4613-1) contains supplementary material, which is available to authorized users.

Objective. This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-βand Wnt/β-catenin signaling activation.Materials and Methods. Cataracts of K14E6 mice were analysed histologically; and components of TGF-βand Wnt/β-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor.Results. The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses’ cell morphology were due to actin filaments reorganisation, activation of TGF-βand Wnt/β-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels.Conclusion. The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-βand Wnt/β-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

Previous studies revealed that some bioactive food components have anti-cancer effects. However epigenetic effects of dietary compound resveratrol are largely unknown in breast cancer cells (M.A. Dawson, T. Kouzarides, 2012) [1]. Here we provide novel data and comparisons of DNA methylation status of promoter gene regions in response to resveratrol treatment at 24 h and 48 h versus untreated MDA-MB-231 breast cancer cells. DNA methylation changes were measured using Array-PRIMES method (aPRIMES) followed by whole-genome hybridization using human DNA methylation promoter microarray NimbleGen HG18 Refseq Promoter 3×720 K array. Our data were associated to corresponding changes in mRNA expression in a set of cancer-related genes. Using gene ontology analysis we also identify cancer-related cellular processes and pathways that can be epigenetically reprogramed by resveratrol. Data in this article are associated to the research articles "Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol". Medina Aguilar et al., PLoS ONE 11(6): e0157866. doi:10.1371/journal.pone.0157866 2016 (A.R. Medina, P.C. Pérez, L.A. Marchat, P. Gariglio, M.J. García, C.S. Rodríguez, G.E. Ruíz, et al., 2016) [2]; and "Resveratrol inhibits cell cycle progression by targeting Aurora kinase A and Polo-like kinase 1 in breast cancer cells" in Oncology Reports. Medina Aguilar et al., 2016 Jun; 35(6):3696-704. doi: 10.3892/or.2016.4728 (A.R. Medina, P. Gariglio, M.J. García, O.E. Arechaga, S.N. Villegas, C.M. Martínez et al., 2016) [3].

The SLC5A8 gene encodes Na monocarboxylate transporter 1, which is epigenetically inactivated in various tumour types. This has been attributed to the fact that it prevents the entry of histone deacetylase (HDAC) inhibitors and favours the metabolic reprogramming of neoplastic cells. Nevertheless, its expression and regulation in cervical cancer (CC) have not been elucidated to date. The aim of the present study was to investigate whether SLC5A8 expression is silenced in CC and if epigenetic mechanisms are involved in its regulation. Using RNA and DNA from human CC cell lines and tumour tissues from patients with CC, the expression of SLC5A8 was analysed by reverse transcription polymerase chain reaction and the methylation status of its CpG island (CGI) by bisulphite‑modified sequencing. Additionally, SLC5A8 reactivation was examined in the CC cell lines following treatment with DNA methylation (5‑aza‑2'‑deoxycytidine) and HDAC inhibitors (trichostatin A and pyruvate). All the CC cell lines and a range of tumour tissues (65.5%) exhibited complete or partial loss of SLC5A8 transcription. The bisulphite‑sequencing revealed that hypermethylation of the CGI within SLC5A8 first exon was associated with its downregulation in the majority of cases. The transporter expression was restored in the CC cell lines following exposure to 5‑aza‑2'‑deoxycytidine alone, or in combination with trichostatin A or pyruvate, suggesting that DNA methylation and histone deacetylation contribute to its inhibition in a cell line‑dependent manner. Together, the results of the present study demonstrate the key role of DNA hypermethylation in the repression of SLC5A8 in CC, as well as the involvement of histone deacetylation, at least partially. This allows for research focused on the potential function of SLC5A8 as a tumour suppressor in CC, and as a biomarker or therapeutic target in this malignancy.

Cervical cancer is an important health issue worldwide. Many factors are related to this condition as the persistence of human papillomavirus (HPV) infection (e.g. type 16 and 18), the use of hormonal contraceptives for long periods of time, pH changes and bacterial vaginosis. The association between the microbiota and cervical human cancer is an interesting issue to be explored; given that environmental and hormonal factors may change the vaginal microbiota contributing to this condition. Our hypothesis was that changes in the microbiota diversity is associated with the development of cervical cancer. We evaluated the microbiota diversity in vaginal lavages and fecal samples at different stages of cervical cancer development in a mice model (K14HPV16E7) with type 16 E7 oncogene expression (E7), under continuous or not continuous stimulus of 17β-estradiol (E2) and compared it with a non-transgenic isogenic control (FVB) under same conditions. Our results indicate that continuous E2 administration during 6 months in the model with type 16 E7 expression causing development of cancer, is associated with significant changes in the microbiota diversity of the cervicovaginal lavages. Similar results were not observed in the same model when no E2 was administered to the mice. The FVB mice with no E7 expression which do not develop cervical cancer, did not show comparable changes in the microbiota diversity when E2 was administered during the same period. Normal evolution of the cervical epithelium and microbiota diversity were observed for the FVB mice with no E2 administration. Large changes in the microbiota diversity in fecal samples were not observed suggesting a specific organ effect of E7 expression associated to E2 on the vaginal microbiota.

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

It is well known that vitamin A and its receptors protect against cancer development and that Retinoid Acid Receptor β (RARβ) is epigenetically silenced during tumoral progression. Cervical Cancer (CC) has been causally linked to high risk human papillomavirus (HR-HPV) infection. However, host factors are important in determining the outcome of persistent HR-HPV infection as most cervical precancerous lesions containing HR-HPVs do not progress to invasive carcinomas. Increasing evidence suggests that low diet in vitamin A and their receptors participate in the development of CC. The aim of this study has been to investigate the effects of abated RARβ expression in the development of cervical premalignant lesions in 4 month-old conditional mice (RARβ(L-/L-)). Results demonstrated the development of spontaneous squamous metaplasia, inflammatory infiltrate, enhanced mitotic activity, loss of cell differentiation, as well as decreased apoptosis and p16(INK4a) protein levels in RARβ(L-/L-) mice cervix. All these changes are hallmarks of moderate dysplasia. Importantly, our results suggest that the low expression of RARβ, may induce the down regulation of p16(INK4a), chronic inflammation and decreased apoptosis and may be involved in vulnerability to HR-HPV and early stage cervical carcinogenesis.

Maintenance of telomere length is one function of human telomerase that is crucial for the survival of cancer cells and cancer progression. Both telomeres and telomerase have been proposed as possible biomarkers of cancer risk and cancer invasiveness; however, their clinical relevance is still under discussion. In order to improve our understanding of the relationship between telomere length and telomerase activity with cancer invasiveness, we studied telomere length as well as telomerase levels, activity, and intracellular localization in breast cancer cell lines with diverse invasive phenotypes. We found an apparently paradoxical coincidence of short telomeres and enhanced telomerase activity in the most invasive breast cancer cell lines. We also observed that hTERT intracellular localization could be correlated with its level of activity. There was no association between human telomerase reverse transcriptase (hTERT) protein expression levels and invasiveness. We propose that simultaneous evaluation of these two biomarkers-telomere length and telomerase activity-could be useful for the assessment of the invasive capacity and aggressiveness of tumor cells from breast cancer patients.

The role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2–5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O2 level.

Introduction. Cervical cancer is a public health problem in developing countries, and the fourth leading cause of cancer death in women worldwide. Most human cervical cancer cases are associated with high-risk human papillomavirus (HR-HPV) infection, but different cofactors including long-term use of oral contraceptives and multiple pregnancies increase the risk for cervical cancer in HPV-positive women. Ion channel expression and/or function differ between healthy and tumor cells. This alteration confers proliferation, progression, and survival advantages to tumor cells, making ion channels promising targets for cancer therapy and diagnosis. Here, we studied the mRNA expression of two potassium channels in human keratinocytes stably transfected with either E6 or both E6/E7 oncogenes of HPV16, and in a K14E7 transgenic mouse model of cervical cancer. These mice express the HPV16-E7 oncogene and develop cervical cancer after 6 months of treatment with 17β-estradiol (E2). Methods. Human normal keratinocytes and keratinocytes transfected with either E6 or E6/E7 oncogenes of HPV16 were cultured. In addition, we used the K14E7-transgenic mice-cervical cancer model; some animals were treated with E2. Continuous-release pellets delivering daily 0.05 mg of E2 were implanted into 4–6-week old female mice for either 3 months to produce low-grade cervical dysplasia or 6 months to produce cervical cancer. Total RNA extraction and subsequent quantification by qPCR of Kv3.4 and KCa3.1 mRNA channel levels were performed both from cultured cells and the mice cervical tissue. Results. Significantly higher KV3.4 and KCa3.1 channel mRNA expression was observed in keratinocytes transfected with either the E6 or E6/E7 HPV oncogenes in comparison with normal keratinocytes. In the cervical cancer mouse model, E2 treatment for 6 months increased the mRNA levels of KV3.4 and KCa3.1 channels both in the transgenic and the non-transgenic mice in comparison with the rest of the groups. The highest mRNA relative expression was observed for the KV3.4 channels in the transgenic mice displaying cervical cancer (K14E7+E2 group). Conclusion. KV3.4 channel expression may serve as a cervical cancer biomarker. This work was partially supported by Conacyt grant A1-S-9783. Citation Format: Ana Ramírez, María de Guadalupe Chávez-López, Eunice Vera, Jaime de la Garza, Patricio Gariglio, Javier Camacho. Potassium channel expression in cervical cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2766.

Resistance to cisplatin (CDDP) is a major cause of cancer treatment failure, including human breast cancer. The tumor suppressor protein p53 is a key factor in the induction of cell cycle arrest, DNA repair, and apoptosis in response to cellular stimuli. This protein is phosphorylated in serine 15 and serine 20 during DNA damage repair or in serine 46 to induce apoptosis. Resveratrol (Resv) is a natural compound representing a promising chemosensitizer for cancer treatment that has been shown to sensitize tumor cells through upregulation and phosphorylation of p53 and inhibition of RAD51. We developed a CDDP-resistant MCF-7 cell line variant (MCF-7R) to investigate the effect of Resv in vitro in combination with CDDP over the role of p53 in overcoming CDDP resistance in MCF-7R cells. We have shown that Resv induces sensitivity to CDDP in MCF-7 and MCF-7R cells and that the downregulation of p53 protein expression and inhibition of p53 protein activity enhances resistance to CDDP in both cell lines. On the other hand, we found that Resv induces serine 20 (S20) phosphorylation in chemoresistant cells to activate p53 target genes such as PUMA and BAX, restoring apoptosis. It also changed the ratio between BCL-2 and BAX, where BCL-2 protein expression was decreased and at the same time BAX protein was increased. Interestingly, Resv attenuates CDDP-induced p53 phosphorylation in serine 15 (S15) and serine 46 (S46) probably through dephosphorylation and deactivation of ATM. It also activates different kinases, such as CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20, suggesting a novel mechanism of p53 activation and chemosensitization to CDDP.

Mutations in p53 are strongly associated with several highly malignant cancer phenotypes but its role in regulating energy metabolism has not been completely elucidated. The effect on glycolysis and oxidative phosphorylation (OxPhos) of mutant p53

The role of testosterone in males has been extensively studied. However, studies in several species suggest that estrogens are also important for the development and function of male brain, bone, gonads as well as for spermatogenesis and sexual behavior. Data related to testicular P450-aromatase expression and estrogen receptors α and β in several species had been published. However, to our knowledge there is no information on testicular mRNA aromatase expression, estrogen synthesis, or estradiol-17β concentrations in the circulation of the male goat. Thus, the objective of this work was to conduct in vivo and in vitro studies related to estrogen synthesis and serum concentrations during the postnatal testicular development in goat bucks. To do this, circulating concentrations of estradiol-17β and testosterone were evaluated in male goat during the first year of life, starting at 15 days of age. Besides, in order to assess the estrogen-producing ability of testicles from prepubertal male goats (four months), testicular cells were incubated with tritiated androgen precursors ( 3 H-androstenedione and 3 H-testosterone) in the presence or absence of the P450-aromatase inhibitor formestane, using TLC to identify sex steroids. Finally, the expression and tissue distribution of P450-aromatase mRNA in the testis of 3–5 month old males was evaluated by in situ RT-PCR. Results indicate that estradiol-17β is present in the serum of male goats at 0.5 months of age, and that there are significant increases at 3 and 10 months of age. The in vitro synthesis of tritiated estradiol-17β was inhibited by the P450-aromatase inhibitor formestane. Messenger RNA for P450-aromatase was abundantly expressed both in Sertoli cells in the testis of goats of ages three, four and five months. The expression of P450 mRNA was also localized in Leydig cells. It is concluded that the prepubertal goat has measurable quantities of serum estradiol-17β and that their testis has the capacity to synthesize estrogens during this period.

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration.

Cervical cancer is a major cause of cancer-associated mortality in women in developing countries. Thus, novel early markers are required. Ion channels have gained great interest as tumor markers, including cervical cancer. The calcium-activated potassium channel KCNMA1 (subunit α-1 from subfamily M) has been associated with different malignancies, including tumors such as breast and ovarian cancer that are influenced by hormones. The KCNMA1 channel blocker iberiotoxin decreases the proliferation of HeLa cervical cancer cells. Nevertheless, KCNMA1 channel expression during cervical carcinogenesis remains elusive. Therefore, KCNMA1 expression was studied in cervical cancer development. FVB transgenic mice expressing the E7-oncogene of high-risk human papilloma virus, and non-transgenic mice were treated with estradiol-releasing pellets during 3 or 6 months to induce cervical lesions. Twenty-four human cervical biopsies from non-cancerous, low- or high-grade intraepithelial lesions, or cervical cancer were also studied. mRNA and protein expression was assessed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. Cervical dysplasia and carcinoma were observed only in the transgenic mice treated with estradiol for 3 and 6 months, respectively. Estradiol treatment increased KCNMA1 mRNA and protein expression in all groups; however, the highest levels were observed in the transgenic mice with carcinoma. KCNMA1 protein expression in the squamous cells of the transformation zone was observed only in the transgenic mice with cervical dysplasia or cancer. Human biopsies from non-cancerous cervix did not display KCNMA1 protein expression; in contrast, the majority of the tissues with cervical lesions (16/18) displayed KCNMA1 protein expression. The lowest channel immunostaining intensity was observed in biopsies from low-grade dysplasia and the strongest in the carcinoma tissues. These results suggest KCNMA1 channels as potential early cervical cancer markers.

Although high-risk human papillomavirus (HR-HPV) infection has a prominent role in the aetiology of cervical cancer (CC), sex steroid hormones may also be involved in this process; however, the cooperation between oestrogen and HR-HPV in the early stages of cervical carcinogenesis is poorly understood. Since 17β-oestradiol (E2) and the HPV type 16-E7 oncoprotein induce CC in transgenic mice, a microarray analysis was performed in the present study to generate global gene expression profiles from 2-month-old FVB (non-transgenic) and K14E7 (transgenic) mice who were left untreated or were treated for 1 month with E2. Upregulation of cancer-related genes that have not been previously reported in the context of CC, including glycerophosphodiester phosphodiesterase domain containing 3, interleukin 1 receptor type II, natriuretic peptide type C, MGAT4 family member C, lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) and glucoside xylosyltransferase 2, was observed. Notably, upregulation of the serine (or cysteine) peptidase inhibitor clade B member 9 gene and downregulation of the Granzyme gene family were observed; the repression of the Granzyme B pathway may be a novel mechanism of immune evasion by cancer cells. The present results provide the basis for further studies on early biomarkers of CC risk and synergistic interactions between HR-HPV and oestrogen.

Resveratrol (RES), a polyphenol compound with anti‑proliferative properties, has been previously evaluated for its beneficial effects against a variety of tumour cells. The current study elucidated the means by which RES enhances the anti‑proliferative effects of cisplatin (CIS) on MCF‑7 cells, focusing on the inhibitory effects on DNA repair of double‑strand breaks (DSBs). Chemoresistant MCF‑7 cells (MCF‑7R) were generated by continuous exposure to low concentrations of CIS (10 µM CIS‑IC40) during 5 passages, with the IC50 value increasing ~3‑fold. Using an MTT assay, we estimated the changes in IC50 for CIS in MCF‑7, T47‑D, MDA‑MB‑231 and MCF‑7R cells in the presence of RES. The relative transcript level of Nbs‑1, Mre‑11 and Rad‑50 genes was assessed using RT‑qPCR analysis. Rad51 and H2AX [pSer139] protein expression was determined by western blot analysis. RES at 50 and 100 µM significantly enhanced the anti‑proliferative effects of CIS in both MCF‑7 and MCF‑7R cells, decreasing the IC50 values for CIS to one‑tenth and one‑sixth, respectively. A total of 100 µM RES decreased the relative transcript levels of homologous recombination (HR) initiation complex components and the Rad51 protein level in MCF‑7 and MCF‑7R cells. After 48 h of CIS DNA damage, the levels of Rad51 protein increased, but this effect was inhibited by 100 µM RES. RES also maintained serine 139 phosphorylation of histone H2AX, suggesting that RES prevents the repair of DSBs. It was observed that RES exerts an antagonistic effect over CIS on the activation of Rad51 and sustained phosphorylation of H2AX. The results suggest that RES in combination with DNA damage‑based therapy has potential as a strategy to overcome resistance and provide much safer and more effective treatment for breast cancer.

Cervical cancer is the second most frequent tumor type in women worldwide with cases developing clinical recurrence, metastasis, and chemoresistance. The cancer stem cells (CSC) may be implicated in tumor resistance to therapy. RESveratrol (RES), a natural compound, is an antioxidant with multiple beneficial activities. We previously determined that the expression of RAD51 is decreased by RES. The aim of our study was to examine molecular mechanism by which CSC from HeLa cultures exhibit chemoresistance. We hypothesized CSC repair more efficiently DNA breaks and that RAD51 plays an important role in this mechanism. We found that CSC, derived from cervical cancer cell lines, overexpress RAD51 and are less sensitive to Etoposide (VP16). We inhibited RAD51 in CSC-enriched cultures using RES or siRNA against RAD51 messenger RNA and observed a decrease in cell viability and induction of apoptosis when treated simultaneously with VP16. In addition, we found that inhibition of RAD51 expression using RES also sensitizes CSC to VP16 treatment. Our results suggest that resveratrol is effective to sensitize cervical CSC because of RAD51 inhibition, targeting high RAD51 expressing CD49f-positive cells, which supports the possible therapeutic application of RES as a novel agent to treat cancer.

Objective. The aim of this work was to compare the early gene expression profiles in the skin of HPV16-E6 transgenic mice regulated by the E6 PDZ-binding motif.Materials and Methods.The global transcriptional profiles in dorsal skin biopsies from K14E6 and K14E6Δ146-151 transgenic mice were compared using microarrays. Relevant genes obtained from the most differentially expressed processes were further examined by RT-qPCR, in situ RT-PCR, Western blot, or immunofluorescence.Results.The transcriptomic landscape of K14E6 versus K14E6Δ146-151 shows that the most affected expression profiles were those related to keratinocyte differentiation, stem cell maintenance, and keratinization. Additionally, downregulation of epidermal stemness markers such as K15 and CD34, as well as the upregulation of cytokeratin 6b, appeared to be dependent on the E6 PDZ-binding motif. Finally, wound healing, a physiological process linked to stemness, is impaired in the K14E6 mice compared to K14E6Δ146-151.Conclusion.The E6 PDZ-binding motif appears to affect stemness and keratinization during early stages of skin carcinogenesis. As E6 plays a significant role in HPV-induced skin carcinogenesis, the K14E6 versus K14E6Δ146-151 transcriptional profile provides a source of valuable data to uncover novel E6 functions in the skin.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.

Cervical neoplasia is one of the most frequent cancers in women and is associated with high-risk human papillomavirus (HPV) infection. Resveratrol, a natural polyphenolic phytochemical, has received considerable interest on the basis of its potential as a chemopreventive agent against human cancer. In this work, we analyzed the type of cell death induced by resveratrol in several cervical cancer cell lines. Resveratrol treatment (150-250 µmol/l) for 48 h increased cell cycle arrest at the G1 phase in C33A (with mutation in p53) and HeLa cells (HPV18 positive), as well as in CaSki and SiHa cell lines (HPV16 positive). Resveratrol treatment induced apoptosis in all cell lines, particularly in CaSki cells, as measured by Annexin-V flow cytometry analysis. There was a decrease in the mitochondrial membrane potential (apoptosis) in HeLa, CaSki, and SiHa cells and an increased lysosomal permeability (autophagy) in C33A, CaLo (HPV18 positive), and HeLa cell lines. Furthermore, when we used the IC50 of each line, we found that resveratrol produces a similar effect, suggesting that this effect is not dependent on the concentration of resveratrol. Interestingly, after resveratrol treatment, the expression of p53 was decreased in HPV18-positive cell lines (CaLo and HeLa) and increased in HPV16-positive cell lines (CaSki and SiHa) and C33A cells. The expression of p65 (an NF-κB subunit) was decreased after treatment in all cell lines except SiHa cells. These data indicate that resveratrol uses different mechanisms to induce cell death in cell lines derived from cervical cancer.

The wide range of invasive and noninvasive lesion phenotypes associated with high-risk human papillomavirus (HR-HPV) infection in cervical cancer (CC) indicates that not only the virus but also specific cervical epithelial cells in the transformation zone (TZ), such as stem cells (SCs), play an important part in the development of cervical neoplasia. In this review, we focused in an expression signature that is specific to embryonic SCs and to poorly differentiated cervical malignant tumors and we hypothesize that this expression signature may play an important role to promote cell growth, survival, colony formation, lack of adhesion, as well as cell invasion and migration in CC.