| To gain insight into molecular alterations in oral squamous cell carcinoma (OSCC), we performed deep sequencing (RNA-Seq) 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 genes are dysregulated in OSCC patients. Polycomb repressive complexes (PRC) and retinoic acid (RA) 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 H3K27me3 mark at these genes in OKF6-TERTIR, but not in SCC-9 cells. In contrast, IRXI, 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. We investigated additional histone modifications at these selected homeobox genes in OKF6-TERT1R and SCC-9 cells. We detected the H3K9me3 mark at HOXB7,HOXC10, HOXC13 and HOXD8 genes at levels higher in OKF6-TERT1R than SCC-9 cells; and at IRX1 and SIX2 at levels higher in SCC-9 than in OKF6-TERT1R cells. The H3K79me3 mark was detectable only at IRX1 in OKF6-TERT1R cells and at IRX4 in SCC-9 cells. We detected the H3K4me3 mark at HOXB7, HOXC10, HOXC13 and HOXD8 at levels higher in SCC-9 than in OKF6-TERT1R cells. The levels of the H3K4me3 mark were higher in OKF6-TERT1R than in SCC-9 cells at IRX1, IRX4, S1X2, and TSHZ3. Similarly, the levels of H3K36me3 mark were higher in SCC-9 than in OKF6-TERT1R cells at HOXC13; and higher in OKF6-TERT1R than in SCC-9 cells at 1RXI, 1RX4, SIX2, and TSHZ3. Finally, we performed a sequencing-based DNA methylation analysis in OKF6-TERT1R and SCC-9 cells. This study also highlighted homeobox genes. We conclude that altered activity of PRC2 and changes in DNA methylation are associated with dysregulation of homeobox gene expression in human OSCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. |
