Estrogens and estrogen receptors (ERs), such as ERα and ERβ, prolactin (PRL) and prolactin receptor (PRLR) have been reported to be involved in the physiopathology of uterine cervical cancer (UCC). The 60 kDa PRL is an isoform of PRL, which is produced by UCC-derived cells. The present study aimed to evaluate the expression of hormonal receptors in different degrees of cervical lesions, and to determine whether 60 kDa PRL and 17β-estradiol (E2) modulated cell survival and metabolism in UCC cells, and in HaCaT cells transduced with human papillomavirus (HPV) 16 and 18 E6/E7 oncogenes. ERα, ERβ, PRLR, Ki67 and B-cell lymphoma 2 expression levels were analyzed in biopsies of precursor lesions and UCC using immunohistochemistry. In addition, HeLa, SiHa and C33A cells, and transduced HaCaT cells, were stimulated with 60 kDa PRL, E2 or a combination of both. Proliferation was evaluated using the xCELLigence platform, apoptosis was analyzed by flow cytometry and cell metabolism was determined using the MTT assay. The results revealed that ERα, ERβ, PRLR and Ki67 expression levels were increased during the progression of cancer. In vitro, 60 kDa PRL alone significantly increased proliferation of SiHa cells. Furthermore, E2 alone or in combination with 60 kDa PRL increased the sensitivity of SiHa cells to cisplatin and increased the percentage of apoptosis; in HaCaT cells, these treatment strategies had the opposite effect on cisplatin sensitivity. Treatment with E2 increased mitochondrial activity in HeLa and SiHa cells, and in HaCaT cells transduced with HPV 16 E6/E7 and HPV 18 E6 oncogenes. PRL had a similar effect on HeLa cells, and on HaCaT cells transduced with HPV 18 E6 and HPV 16 E7. The co-expression of these receptors demonstrated the hormonal dependence of UCC. In addition, E2 and the 60 kDa PRL significantly impacted the metabolism, but not the survival, of cells.

Keratinocyte infection with high-risk human papillomavirus genotypes has been linked to cancer development. In cervix, the alpha HPV16 and HPV18 have been reported as the mayor causative agents of cervical cancer. Oncogenic progression and chronic inflammation are closely related processes, with IL-6 as one of the main pro-inflammatory cytokines involved. However, there are limited studies about the regulation of IL-6 by low and high risk HPVs and the HPV proteins implicated in this modulation. In this work, we report the overexpression of IL-6 in HPV infected cervical cancer derived cell lines (HeLa and SiHa) compared to non-tumorigenic keratinocytes (HaCaT), and in Cervical Intraepithelial Neoplasia grade 1 HPV16 and HPV18 positive cervical samples compared to HPV negative samples without lesions. Moreover, we generated HaCaT keratinocytes that express E5, E6, and E7 from high risk (16 or 18) or low risk (62 and 84) HPVs. E5 proteins do not modify IL-6 expression, while E7 modestly increase it. Interestingly, E6 proteins in HaCaT cells upregulate IL-6 mRNA expression and protein secretion. Indeed, in HaCaT cells that express high risk HPV16E6 or HPV18E6 proteins, only the truncated E6 * isoforms were expressed, showing the stronger IL-6 overexpression, while in HaCaT cells that express low risk HPV62 and HPV84 full length E6 proteins, IL-6 was also upregulated but not so drastically. Since HaCaT cells have a mutated p53 form that is not degraded by the introduction of E6 or E6/E7, it seems that E6/E7 regulate IL-6 by an additional mechanism independent of p53. In addition, basal keratinocytes showed a strong expression of IL-6R using immunohistochemistry, suggesting an autocrine mechanism over proliferative cells. Altogether, IL-6 cytokine expression in keratinocytes is upregulated by E6 and E7 proteins from HPVs 16, 18, 62, and 84, especially by high risk HPV16 and HPV18 E6 * , which may contribute to promote a pro-inflammatory and highly proliferative microenvironment that can persist over time and lead to cervical tumorigenesis.

Background: Cervical cancer (CC) is associated to high-risk human papillomavirus (HPV) infections, for this reason it is crucial to have sensitive and accurate HPV diagnostic tests. To date, most research is focused on HPVs within the Alphapapillomavirus (α-PVs) genus and little attention has been paid to cervical infections with other HPV genotypes, like those of the Betapapillomavirus (β-PVs) and Gammapapillomavirus (γ-PVs) genera. The aim of this study was to determine the HPV genotypes from different genera in women with CC using Next-Generation Sequencing (NGS). Methods: The study comprised 48 HPV positive CC samples evaluated with the Linear Array HPV Genotyping test and individually sequenced by 454 NGS using PGMY09/11 and FAP primers. To determine the HPV genotypes present in each sample, the obtained sequences were compared with all HPV L1 gene reference sequences from the Papillomavirus Episteme database (PaVE). Moreover, 50 HPV positive low-grade cervical lesion samples individually genotyped with NGS were also included to determine the genotypes present preferentially in CC patients.

SynopsisHEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a member of the basic helix-loop-helix-orange (bHLH-O) family of transcription repressors that mediate Notch signalling. HEY1 acts as a positive regulator of the tumour suppressor p53 via still unknown mechanisms. A MALDI-TOF/TOF MS analysis has uncovered a novel HEY1 regulatory phosphorylation event at Ser-68. Strikingly, this single phosphorylation event controls HEY1 stability and function: simulation of HEY1 Ser-68 phosphorylation increases HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity. Unlike wild-type HEY1, expression of the phosphomimetic mutant HEY1-S68D failed to induce p53-dependent cell cycle arrest and it did not sensitize U2OS cells to p53-activating chemotherapeutic drugs. We have identified two related kinases, STK38 (serine/threonine kinase 38) and STK38L (serine/threonine kinase 38 like), which interact with and phosphorylate HEY1 at Ser-68. HEY1 is phosphorylated at Ser-68 during mitosis and it accumulates in the centrosomes of mitotic cells, suggesting a possible integration of HEY1-dependent signalling in centrosome function. Moreover, HEY1 interacts with a subset of p53-activating ribosomal proteins. Ribosomal stress causes HEY1 relocalization from the nucleoplasm to perinucleolar structures termed nucleolar caps. HEY1 interacts physically with at least one of the ribosomal proteins, RPL11, and both proteins cooperate in the inhibition of MDM2-mediated p53 degradation resulting in a synergistic positive effect on p53 transcriptional activity. HEY1 itself also interacts directly with MDM2 and it is subjected to MDM2-mediated degradation. Simulation of HEY1 Ser-68 phosphorylation prevents its interaction with p53, RPL11 and MDM2 and abolishes HEY1 migration to nucleolar caps upon ribosomal stress. Our findings uncover a novel mechanism for cross-talk between Notch signalling and nucleolar stress.

BackgroundHuman papillomavirus (HPV) is the main etiological agent of cervical cancer, the third most common cancer among women globally and the second most frequent in Mexico. Persistent infection with high-risk HPV genotypes is associated with premalignant lesions and cervical cancer development. HPVs considered as low risk or not yet classified, are often found in coinfection with different HPV genotypes. Indeed, HPV62 is one of the most prevalent HPV detected in some countries, but there is limited information about its prevalence in other regions and there are no HPV62 variants currently described. The aim of this study was to determine the prevalence of HPV62 in cervical samples from Mexican women and to identify mutations in the L1, E6 and E7 genes, which have never been reported in our population.MethodsHPV screening was performed by Cobas HPV Test in women who attended prevention health programs and dysplasia clinics. All HPV positive samples (n = 491) and 87 additional cervical cancer samples were then genotyped with Linear Array HPV Genotyping test. Some samples were selected to corroborate genotyping by Next-Generation sequencing. On the other hand, nucleotide changes in L1, E6 and E7 genes were determined using PCR, Sanger sequencing and analysis with the CLC-MainWorkbench 7.6.1 software. L1 protein structure was predicted with the I-TASSER server.ResultsUsing Linear Array, HPV62 prevalence was 7.6% in general population, 8% in Cervical Intraepithelial Neoplasia grade 1 (CIN1) samples and 4.6% in cervical samples. The presence of HPV62 was confirmed with Next-Generation sequencing. Regarding L1 gene, novel sequence variations were detected, but they did not alter the tertiary structure of the protein. Moreover, several nucleotide substitutions were found in E6 and E7 genes compared to reference HPV62 genomic sequence. Specifically, three non-synonymous sequence variations were detected, two in E6 and one in E7.ConclusionsHPV62 is a frequent HPV genotype found mainly in general population and in women with CIN1, and in 90.5% of the cases it was found in coinfection with other HPVs. Novel nucleotide changes in its L1, E6 and E7 genes were detected, some of them lead to changes in the protein sequence.