Higher expression of human telomerase reverse transcriptase (hTERT) and subsequent activation of telomerase occur during cellular immortalization and are maintained in cancer cells. To understand the mode of hTERT expression in cancer cells, we identified cancer-specific trans-regulatory proteins that interact with the hTERT promoter, using the promoter magnetic precipitation assay coupled with mass spectrometry. The identified proteins include MutS homolog 2 (MSH2), heterogeneous nuclear ribonucleoprotein (hnRNP) D, hnRNP K and grainyhead-like 2 (GRHL2). We noticed a higher expression of these proteins in human oral squamous cell carcinoma (OSCC) cells than in normal cells, which do not exhibit telomerase activity. Knockdown of MSH2, hnRNP D and GRHL2 resulted in a notable reduction of the hTERT promoter activity in tested cancer cells. Silencing of the above genes resulted in a significant reduction of the telomerase activity in OSCC cells. Interestingly, among the four identified genes, silencing of GRHL2 was essential in reducing telomerase activity and viability of tested cancer cells. These results suggest a possible role of GRHL2 in telomerase activation during cellular immortalization.

Summary: p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene‐induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age‐dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16^INK4A, induced G₂ cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells.

Introducing highly active antiretroviral therapy (HAART) has significantly decreased the morbidity and mortality in human immunodeficiency virus-positive (HIV+) individuals by decreasing the viral loads and increasing the CD4+ T-cell counts. Subsequently, the occurrence of many HIV-associated diseases has been dramatically declined except human papillomavirus (HPV)-associated lesions. Such notion suggests that immune response is not a major determinant, and that the direct interaction between HIV and HPV may be involved in the HPV-associated pathogenesis. In the current study, we investigated whether HIV plays a direct role in HPV-associated oral carcinogenesis by using HIV-1 transactivator protein (Tat), which is known to have oncogenic properties. We found that HIV-1 Tat not only increased the expression of HPV-16 E6 and E7 oncogenes in human oral keratinocytes harboring the HPV type-16 genome (HOK-16B), but also notably enhanced the proliferative capacity of the cells in vitro. Moreover, HOK-16B cells expressing HIV-1 Tat was capable of inducing cystic nodules in nude mice, while the control HOK-16B cells failed to produce nodules in the mice. Our results indicate that HIV could play a role in the HPV-associated pathogenesis by exerting oncogenic stimulus via Tat protein.

Alcohol consumption is associated with an increased risk of several cancers, including oral/oropharyngeal squamous cell carcinoma (OSCC). Alcohol also enhances the progression and aggressiveness of existing cancers; however, its underlying molecular mechanism remains elusive. Especially, the local carcinogenic effects of alcohol on OSCC in closest contact with ingestion of alcohol are poorly understood. We demonstrated that chronic ethanol exposure to OSCC increased cancer stem cell (CSC) populations and their stemness features, including self-renewal capacity, expression of stem cell markers, ALDH activity, and migration ability. The ethanol exposure also led to a significant increase in aerobic glycolysis. Moreover, increased aerobic glycolytic activity was required to support the stemness phenotype of ethanol-exposed OSCC, suggesting a molecular coupling between cancer stemness and metabolic reprogramming. We further demonstrated that chronic ethanol exposure activated NFAT (nuclear factor of activated T cells) signaling in OSCC. Functional studies revealed that pharmacological and genetic inhibition of NFAT suppressed CSC phenotype and aerobic glycolysis in ethanol-exposed OSCC. Collectively, chronic ethanol exposure promotes cancer stemness and aerobic glycolysis via activation of NFAT signaling. Our study provides a novel insight into the roles of cancer stemness and metabolic reprogramming in the molecular mechanism of alcohol-mediated carcinogenesis.