To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.

Histone deacetylases inhibitors (HDACi) have recently emerged as potent antitumor treatment modality. They are currently tested in many phase I, II and III clinical trials as single agents as wells as in combination schemes. They have demonstrated promising antitumor activity and favorable clinical outcome. Histone deacetylases (HDACs) are involved in the process of epigenetic regulation of gene expression. Epigenetic changes are believed to be crucial for the onset and progression of cancer and have recently gained remarkable attention. Since epigenetic regulation of gene expression is a reversible process, targeting histone deacetylases provides a good rationale for anticancer therapy. The acetylation status of histones regulates the organization of chromatin and the access of transcription factors. Moreover, functions of many non-histone proteins are controlled by acetylation. The broad and complicated influences of HDACi on various molecular processes may account for the observed pleiotropic effects. In this review we summarize recent advances in the understanding of biology of HDACs and mechanism of action of their inhibitors.

We recently reported a role of Polycomb repressive complex 2 (PRC2) and PRC2 trimethylation of histone 3 lysine 27 (H3K27me3) in the regulation of homeobox (HOX) (Marcinkiewicz and Gudas, 2013) gene transcript levels in human oral keratinocytes (OKF6-TERT1R) and tongue squamous cell carcinoma (SCC) cells. Here, we assessed both the levels of various histone modifications at a subset of homeobox genes and genome wide DNA methylation patterns in OKF6-TERT1R and SCC-9 cells by using ERRBS (enhanced reduced representation bisulfite sequencing). We detected the H3K9me3 mark at HOXB7, HOXC10, HOXC13 and HOXD8 at levels higher in OKF6-TERT1R than in SCC-9 cells; at IRX1 and SIX2 the H3K9me3 levels were conversely higher in SCC-9 than in OKF6-TERT1R. The H3K79me3 mark was detectable only at IRX1 in OKF6-TERT1R and at IRX4 in SCC-9 cells. The levels of H3K4me3 and H3K36me3 marks correlate with the transcript levels of the assessed homeobox genes in both OKF6-TERT1R and SCC-9. We detected generally lower CpG methylation levels on DNA in SCC-9 cells at annotated genomic regions which were differentially methylated between OKF6-TERT1R and SCC-9 cells; however, some genomic regions, including the HOX gene clusters, showed DNA methylation at higher levels in SCC-9 than OKF6-TERT1R. Thus, both altered histone modification patterns and changes in DNA methylation are associated with dysregulation of homeobox gene expression in human oral cavity SCC cells, and this dysregulation potentially plays a role in the neoplastic phenotype of oral keratinocytes.

A role for estrogen signaling in urothelial carcinoma of the bladder (UCB) is suggested to be associated with more advanced disease with worse outcomes in women. Estrogen receptor β (ERβ) is the predominant receptor in bladder tissues. We aimed to ascertain whether ERβ correlates with clinicopathological predictors of aggressive bladder cancer and worse survival outcomes. ERβ was measured by immunohistochemistry in malignant and adjacent benign bladder tissues in patients (N=72) with UCB who underwent radical cystectomy. ERβ expression was tested for statistical association with clinicopathological variables and patient survival. ERβ expression was determined in bladder cancer cell lines, and the effects of the selective estrogen modulator tamoxifen and the ERβ agonist diarylpropionitrile on cell growth were determined. The ERβ level was significantly higher in malignant vs. benign urothelium (P<0.001) and was strongly associated with aggressive tumor histology characterized by lymphovascular (P=0.008) and perineural (P=0.006) invasion, and clinical histories of pelvic irradiation (P=0.005), hydronephrosis (P=0.022) and no intravesical chemotherapy (P=0.038). All patients with a high (>70%) percentage of ERβ positivity in tissue with >3-month follow-up developed recurrent disease (P=0.009). Higher ERβ level was predictive of worse recurrence-free and overall survival following cystectomy, after adjustment for tumor stage, and remained significantly associated with recurrence-free survival in the multivariable analysis including tumor stage, nodal stage and lymphovascular invasion. Activation of ERβ in bladder cancer cell lines led to significant increases in proliferation, while pharmacological inhibition with tamoxifen blocked cell growth. Our study supports a role for ERβ in aggressive UCB. Pharmacological targeting of ERβ warrants further investigation as a therapeutic strategy in UCB.

Bladder cancer is three times more common in men than in women. However, the physiological basis of the male predominance of bladder cancer remains poorly understood. A higher than expected association of prostate and bladder cancers has also been reported which may indicate a common mechanism of carcinogenesis. Consistent with this, androgens and the androgen receptor (AR) play essential roles in prostate carcinogenesis and are believed to play a role in bladder carcinogenesis. There is also evidence implicating cancer stem cells in prostate and bladder cancers. Indeed putative prostate and bladder cancer stem cells share some common molecular features. We highlight key proteins (CD49f, CD133, PTEN, CD44) which are implicated in both prostate and bladder cancers and are enriched in putative prostate and bladder cancer stem cells. We examine published chromatin immuno-precipitation studies analyzing the genome-wide distribution of the AR to identify AR association with, and by inference potential AR-regulation of, these loci. We discuss recent evidence indicating a role for the AR in the splicing of the key urological stem cell protein CD44. We propose a model whereby aberrant AR regulation of these putative stem cell proteins contributes to malignant transformation of prostate and bladder cells. For these reasons we propose that the relationship between androgens and cancer stem cell associated proteins warrants further investigation.