Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model.

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17ß-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Role of SLC5A8 as a Tumor Suppressor in Cervical Cancer.

BACKGROUND: The SLC5A8 gene is silenced in various types of cancer, including cervical cancer; we recently demonstrated that the SLC5A8 gene is also silenced in cervical cancer by hypermethylation of the CpG island in the gene promoter. This study aims to analyze whether SLC5A8 could be a tumor suppressor in cervical cancer. METHODS: After ectopic expressing SLC5A8 in the HeLa cell line, we evaluated its effects on cell behavior both in vitro and in vivo by Confocal immunofluorescence, cell proliferation, migration assays, and xenograft transplants. RESULTS: Overexpression of SLC5A8 in the HeLa cell line decreased its proliferation by arresting cancer cells in the G1 phase and inhibiting cellular migration. Furthermore, we observed that pyruvate increased the SLC5A8 effect, inducing S-phase arrest and inhibiting the entry into mitosis. SLC5A8 decreased tumor growth in xenograft transplants, significantly reducing the volume and tumor weight at 35 days of analysis. CONCLUSIONS: In summary, our results indicate that SLC5A8 has a role as a tumor suppressor in cervical cancer.

Combined Inhibition of Indolamine-2,3-Dioxygenase 1 and C-X-C Chemokine Receptor Type 2 Exerts Antitumor Effects in a Preclinical Model of Cervical Cancer.

Cervical cancer is a public health problem diagnosed in advanced stages, and its main risk factor is persistent high-risk human papillomavirus infection. Today, it is necessary to study new treatment strategies, such as immunotherapy, that use different targets of the tumor microenvironment. In this study, the K14E7E2 mouse was used as a cervical cancer model to evaluate the inhibition of indolamine-2,3-dioxygenase 1 (IDO-1) and C-X-C chemokine receptor type 2 (CXCR-2) as potential anti-tumor targets. DL-1MT and SB225002 were administered for 30 days in two regimens (R1 and R2) based on combination and single therapy approaches to inhibit IDO-1 and CXCR-2, respectively. Subsequently, the reproductive tracts were resected and analyzed to determine the tumor areas, and IHCs were performed to assess proliferation, apoptosis, and CD8 cellular infiltration. Our results revealed that combined inhibition of IDO-1 and CXCR-2 significantly reduces the areas of cervical tumors (from 196.0 mm2 to 58.24 mm2 in R1 and 149.6 mm2 to 52.65 mm2 in R2), accompanied by regions of moderate dysplasia, decreased papillae, and reduced inflammation. Furthermore, the proliferation diminished, and apoptosis and intra-tumoral CD8 T cells increased. In conclusion, the combined inhibition of IDO-1 and CXCR-2 is helpful in the antitumor response against preclinical cervical cancer.

Ion Channels as Potential Tools for the Diagnosis, Prognosis, and Treatment of HPV-Associated Cancers.

The human papilloma virus (HPV) group comprises approximately 200 genetic types that have a special affinity for epithelial tissues and can vary from producing benign symptoms to developing into complicated pathologies, such as cancer. The HPV replicative cycle affects various cellular and molecular processes, including DNA insertions and methylation and relevant pathways related to pRb and p53, as well as ion channel expression or function. Ion channels are responsible for the flow of ions across cell membranes and play very important roles in human physiology, including the regulation of ion homeostasis, electrical excitability, and cell signaling. However, when ion channel function or expression is altered, the channels can trigger a wide range of channelopathies, including cancer. In consequence, the up- or down-regulation of ion channels in cancer makes them attractive molecular markers for the diagnosis, prognosis, and treatment of the disease. Interestingly, the activity or expression of several ion channels is dysregulated in HPV-associated cancers. Here, we review the status of ion channels and their regulation in HPV-associated cancers and discuss the potential molecular mechanisms involved. Understanding the dynamics of ion channels in these cancers should help to improve early diagnosis, prognosis, and treatment in the benefit of HPV-associated cancer patients.

Ion Channels and Personalized Medicine in Gynecological Cancers.

Targeted therapy against cancer plays a key role in delivering safer and more efficient treatments. In the last decades, ion channels have been studied for their participation in oncogenic processes because their aberrant expression and/or function have been associated with different types of malignancies, including ovarian, cervical, and endometrial cancer. The altered expression or function of several ion channels have been associated with tumor aggressiveness, increased proliferation, migration, invasion, and metastasis of cancer cells and with poor prognosis in gynecological cancer patients. Most ion channels are integral membrane proteins easily accessible by drugs. Interestingly, a plethora of ion channel blockers have demonstrated anticancer activity. Consequently, some ion channels have been proposed as oncogenes, cancer, and prognostic biomarkers, as well as therapeutic targets in gynecological cancers. Here, we review the association of ion channels with the properties of cancer cells in these tumors, which makes them very promising candidates to be exploited in personalized medicine. The detailed analysis of the expression pattern and function of ion channels could help to improve the clinical outcomes in gynecological cancer patients.

Global expression profiling of CD10 + /CD19 + pre-B lymphoblasts from Hispanic B-ALL patients correlates with comparative TARGET database analysis.

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.

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein.

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.

Vitamin A deficiency in K14E7HPV expressing transgenic mice facilitates the formation of malignant cervical lesions.

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 (RARß), 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.

The vaginal and fecal microbiota of a murine cervical carcinoma model under synergistic effect of 17ß-Estradiol and E7 oncogene expression.

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 17ß-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.

Circulating miR-15b, miR-34a and miR-218 as promising novel early low-invasive biomarkers of cervical carcinogenesis.

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.

E7 oncoprotein from human papillomavirus 16 alters claudins expression and the sealing of epithelial tight junctions.

Tight junctions (TJs) are cell­cell 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 Madin­Darby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins ­1 and ­10 in the cervix of 7­month­old transgenic K14E7 mice treated with 17ß­estradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin­1 expression in the cervix of 2­month­old K14E7 mice, and claudin­10 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 7­month­old 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 claudin­4. 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.

Genes Involved in the Transcriptional Regulation of Pluripotency Are Expressed in Malignant Tumors of the Uterine Cervix and Can Induce Tumorigenic Capacity in a Nontumorigenic Cell Line.

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.

Sodium-coupled monocarboxylate transporter is a target of epigenetic repression in cervical cancer.

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 bisulphite­modified sequencing. Additionally, SLC5A8 reactivation was examined in the CC cell lines following treatment with DNA methylation (5­aza­2'­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 bisulphite­sequencing 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 5­aza­2'­deoxycytidine alone, or in combination with trichostatin A or pyruvate, suggesting that DNA methylation and histone deacetylation contribute to its inhibition in a cell line­dependent 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.

Mutant p53R248Q downregulates oxidative phosphorylation and upregulates glycolysis under normoxia and hypoxia in human cervix cancer cells.

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 ß-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.

Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin.

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.

HPV16-E6 Oncoprotein Activates TGF-ß and Wnt/ß-Catenin Pathways in the Epithelium-Mesenchymal Transition of Cataracts in a Transgenic Mouse Model.

OBJECTIVE: This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-ß and Wnt/ß-catenin signaling activation. MATERIALS AND METHODS: Cataracts of K14E6 mice were analysed histologically; and components of TGF-ß and Wnt/ß-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-ß and Wnt/ß-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-ß and Wnt/ß-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

Early synergistic interactions between the HPV16­E7 oncoprotein and 17ß-oestradiol for repressing the expression of Granzyme B in a cervical cancer model.

Although high-risk human papillomavirus (HR­HPV) 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 HR­HPV in the early stages of cervical carcinogenesis is poorly understood. Since 17ß-oestradiol (E2) and the HPV type 16­E7 oncoprotein induce CC in transgenic mice, a microarray analysis was performed in the present study to generate global gene expression profiles from 2­month­old FVB (non­transgenic) 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 HR­HPV and oestrogen.

Calcium-activated potassium channels as potential early markers of human cervical cancer.

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.

Inhibition of RAD51 by siRNA and Resveratrol Sensitizes Cancer Stem Cells Derived from HeLa Cell Cultures to Apoptosis.

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.

The PDZ-Binding Motif of HPV16-E6 Oncoprotein Modulates the Keratinization and Stemness Transcriptional Profile In Vivo.

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.