Transcriptional regulation of genes involved in keratinocyte differentiation by human papillomavirus 16 oncoproteins.

The life cycle of human papillomaviruses (HPVs) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes; however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to downregulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm the microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to downregulate the expression of several genes involved in keratinocyte differentiation (such as desmocollin 1, keratin 4, S100 calcium-binding protein A8 and small proline-rich protein 1A), at least partially by downregulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus-induced carcinogenesis.

Association of DSC3 mRNA down-regulation in prostate cancer with promoter hypermethylation and poor prognosis.

BACKGROUND: Desmocollin 3 (DSC3), a member of the cadherin gene superfamily, is associated with pathogenesis of some cancers, but its role in prostate cancer (PCa) remains largely unknown. METHODS: DSC3 gene expression level in available PCa microarray dataset was examined using the Oncomine database. DSC3 transcript expression in prostate cell line panel and an independent tissue cohort (n = 52) was estimated by quantitative PCR (Q-PCR). Epigenetic status of DSC3 gene promoter in PCa was investigated by uploading three dataset (ENCODE Infinium 450K array data and two methylation sequencing) in UCSC genome browser. While pyrosequencing analysis measured promoter DNA methylation, Q-PCR estimates were obtained for DSC3 transcript re-expression after 5-Aza-deoxycytidine (5-Aza) treatment. Clinical relevance of DSC3 expression was studied by Kaplan-Meier survival analysis. Finally, functional studies monitoring cell proliferation, migration and invasion were performed in prostate cell lines after siRNA mediated DSC3 knockdown or following 5-Aza induced re-expression. EMT markers Vimentin and E-cadherin expression was measured by Western Blot. RESULTS: Microarray data analyses revealed a significant decrease in DSC3 transcript expression in PCa, compared to benign samples. Q-PCR analysis of an independent cohort revealed DSC3 transcript down-regulation, both in PCa cell lines and tumor tissues but not in their benign counterpart. Examination of available NGS and Infinium data identified a role for epigenetic regulation DSC3 mRNA reduction in PCa. Pyrosequencing confirmed the increased DSC3 promoter methylation in cancer cell lines and restoration of transcript expression upon 5-Aza treatment further corroborated this epigenetic silencing mechanism. Importantly Kaplan-Meier analysis of an outcome cohort showed an association between loss of DSC3 expression and significantly increased risk of biochemical recurrence. Functional studies indicate a role for epithelial-mesenchymal transition in DSC3 regulated cell migration/invasion. CONCLUSION: Taken together, our data suggests that DNA methylation contributes to down-regulation of DSC3 in prostate cancer, and loss of DSC3 predicts poor clinical outcome.

Reduced membranous and ectopic cytoplasmic expression of DSC2 in esophageal squamous cell carcinoma: an independent prognostic factor.

Desmocollin 2, a desmosomal component, is a key membrane glycoprotein critically involved in cell-cell adhesion and the maintenance of normal tissue architectures in epithelia. Reports exploring the link of desmocollin expression to cancers are limited. The aim of this study was to investigate the expression of desmocollin 2 in esophageal squamous cell carcinoma and, in particular, to determine the extent to which the patterns of desmocollin 2 expression correlated with the clinical parameters. Desmocollin 2 expression was evaluated in 308 cases of esophageal squamous cell carcinoma using immunohistochemistry. Western blotting and reverse transcriptase polymerase chain reaction were performed to characterize the relative expression levels of desmocollin 2 isoforms. The results indicated that desmocollin 2 expression was reduced significantly in esophageal cancer in both protein and messenger RNA levels and that this reduction was associated with poor survival (P = .011). The expression of desmocollin 2 was prominent in normal esophageal epithelia and highly differentiated esophageal tumors, but was reduced or absent in poorly differentiated tumor specimens. Furthermore, in 74.7% of tumor tissues, desmocollin 2 immunoreactivity displayed an abnormal cytoplasmic localization that was correlated with poor tumor differentiation (P < .001), regional lymph node metastasis (P < .001), pathologic tumor-node-metastasis stages (P < .001), and poor prognosis (P = .048). Multivariate analysis showed that desmocollin 2 expression level was an independent prognostic factor for esophageal squamous cell carcinoma. These data suggest that desmocollin 2 is involved in the transformation and development of esophageal tumors and that desmocollin 2 expression level and intracellular localization may serve as a predictor for patient outcomes.

Desmocollin-3: a new marker of squamous differentiation in undifferentiated large-cell carcinoma of the lung.

Lung cancer classification in small-cell and non-small-cell types was recently challenged by data on the differential efficacy of new cytotoxic agents in specific histotypes. An accurate histotype definition has therefore gained interest in both preoperative and surgical materials, but is a hard task especially in undifferentiated large-cell tumors lacking morphological signs of squamous or glandular differentiation. The responsiveness of these latter subtypes to new drugs apparently more selective for adenocarcinomas or squamous carcinomas is not fully understood, also due to the heterogeneity of diagnostic criteria for this tumor entity. Current immunohistochemical markers are not fully specific and new molecules are to be explored. On the basis of gene expression profiling data, reporting a remarkable differential expression of desmocollin-3 (a protein localized in desmosomal junctions of stratified epithelial) between adeno- and squamous cancers, we immunostained 62 cases of resected undifferentiated large-cell lung carcinomas for desmocollin-3 (and for TTF-1, p63 and mucin stain), to test its ability to identify a (residual) squamous phenotype, if present. Desmocollin-3 was expressed in almost half of the undifferentiated large-cell cancers and was mutually exclusive with TTF-1 (positive in 39%; the remaining 18 % of cases was double negative). Special large-cell carcinoma variants expressed desmocollin-3 in 6 of 6 basaloid, 7 of 12 clear-cell types, again mutually exclusive with TTF-1 expression. None of seven sarcomatoid carcinomas reacted for either marker. In 31 cytological samples diagnosed as 'non-small-cell lung carcinoma', desmocollin-3 was again mutually exclusive with TTF-1 and stained all squamous carcinomas, 1 of 19 adenocarcinoma only, and 50% of large-cell carcinoma (all histologically confirmed). This combined morphophenotypic approach may represent a valid adjunct (for both surgical and cytological samples) in the selection of patients with lung cancer to medical treatments tailored according to different efficacy in different lung carcinomas of the squamous, adeno- and large-cell types.

Effects of insulin-like growth factor-1 on cellular and molecular characteristics of bovine blastocysts produced in vitro.

Addition of insulin-like growth factor-1 (IGF-1) to culture medium increases the proportion of bovine embryos that develop to the blastocyst stage and increases embryo survival following transfer to heat-stressed, lactating dairy cows. The objective of the present study was to determine molecular and cellular correlates of these actions of IGF-1. Embryos were produced in vitro and cultured for 7 days with or without 100 ng/ml IGF-1. On d 7 after insemination, grade 1 expanded blastocysts were harvested and used to determine total cell number, percent apoptosis, cell allocation to the inner cell mass and trophectoderm, and the relative abundance of several developmentally important gene transcripts. There was no significant effect of IGF-1 treatment on blastocyst cell number, the proportion of blastomeres that were apoptotic, or the number of cells in the inner cell mass and trophectoderm. However, differences in the relative abundance of several mRNA transcripts were observed between control and IGF-1 treated embryos. Addition of IGF-1 increased (P < 0.02) amounts of mRNA for IGF binding protein-3 and desmocollin II and tended (P < 0.08) to increase amounts of mRNA for Na/K ATPase and Bax. Moreover, IGF-1 treatment decreased (P < 0.05) steady-state amounts of transcripts for heat shock protein 70 and tended (P < 0.08) to reduce amounts of IGF-1 receptor mRNA. In conclusion, increased survival of embryos treated with IGF-1 does not appear due to effects on cell number, percent apoptosis, or cell allocation. Addition of IGF-1 to culture can, however, alter expression of several transcripts which may be important for embryo development and survival following transfer.

Altered expression of desmocollin 3, desmoglein 3, and beta-catenin in oral squamous cell carcinoma: correlation with lymph node metastasis and cell proliferation.

Desmocollin 3 (Dsc3) and desmoglein 3 (Dsg3) are both transmembrane glycoproteins that belong to the cadherin family of calcium-dependent cell adhesion molecules. beta-Catenin is a member of the cadherin-catenin complex that mediates homotypic cell-cell adhesion and is also an important molecule in the wnt signaling pathway. In this study, we examined the simultaneous expression level of Dsc3, Dsg3, and beta-catenin in oral squamous cell carcinomas (OSCCs) and normal oral epithelia using immunohistochemistry. There was a significant correlation (p < 0.05) among the following variables in OSCCs: reduced or loss of expression of Dsc3, Dsg3, and beta-catenin compared to normal oral epithelium, reduced or loss of expression of Dsc3 and histological grade (moderately or poorly differentiated), and reduced or loss of expression of beta-catenin and lymph node metastasis. Furthermore, a positive correlation was found between reduced or loss of beta-catenin staining and reduced or loss of Dsc3 staining in lymph node metastatic cancer tissue (r = 0.734, p < 0.05). These results suggest an abnormal expression of Dsc3, Dsg3, and beta-catenin induced in the progression of oral carcinomas and that the Dsc3 expression level might be related to the regulation of beta-catenin in lymph node metastasis and cell proliferation in OSCCs.