Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

Abstract

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.

Clinical implications of epigenetic regulation in oral cancer

“…Further, Chromatin immunoprecipitation (ChIP) studies for H3K9me3 in keratinocyte cell line (OKF6-TERTIR) and tongue carcinoma cell line (SCC-9) corroborated differential expression in the normal and cancer cell lines. Besides, the downstream processes were associated with HOX genes (HOXB7, HOXC10, HOXC13 and HOXD8) in the cancer cell line and IRX1 and IRX4 genes in the keratinocytes [101,102]. The presence of Histone 2 variant c-H2A.X was reported in 30% oral cancer tissue (n = 42), and 61% oral dysplastic tissue (n = 34) [103].…”

The role of HOX genes in head and neck squamous cell carcinoma

“…The mechanism of this widespread dysregulation of HOX genes in HNSCC has not been clearly established. The alterations in promoter methylation and histone modifications mentioned earlier play a role , as do changes in microRNA expression, such as miR‐196 , and other transcription factors, such as POU2F1 . There are numerous reports of more widespread effects of alterations in the expression of miRNAs and lincRNAs encoded within the HOX gene cluster.…”

Altered Histone Mark Deposition and DNA Methylation at Homeobox Genes in Human Oral Squamous Cell Carcinoma

“…SUZ12 depletion by shRNA technology increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels in OKF6‐TERT1R cells, suggesting that the silencing of these HOX genes in non‐tumorigenic, oral keratinocytes results from the deposition of the H3K27me3 mark by EZH2, a component of PRC2 complex. In contrast, SUZ 12 depletion did not increase the transcript levels of the homeobox genes IRX1, IRX4, SIX2, and TSHZ3 in tumorigenic SCC‐9 cells (Marcinkiewicz and Gudas, ).…”

“…We recently discovered a role for the epigenetic mark H3K27me3 in the regulation of homeobox gene transcript levels in human oral keratinocytes and tongue squamous cell carcinoma (SCC) cells (Marcinkiewicz and Gudas, ). We found that the levels of the H3K27me3 marks at chromatin near homeobox genes were inversely correlated with the transcript levels in non‐tumorigenic, immortalized human oral keratinocytes (OKF6‐TERT1R) and tumorigenic oral SCC‐9 cells.…”

“…We found that the levels of the H3K27me3 marks at chromatin near homeobox genes were inversely correlated with the transcript levels in non‐tumorigenic, immortalized human oral keratinocytes (OKF6‐TERT1R) and tumorigenic oral SCC‐9 cells. Similarly, the association of the protein SUZ12, a core component of the Polycomb repressive complex 2 (PRC2), with chromatin near the homeobox genes was inversely correlated with the transcript levels in OKF6‐TERT1R and SCC‐9 cells (Marcinkiewicz and Gudas, ). SUZ12 depletion by shRNA technology increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels in OKF6‐TERT1R cells, suggesting that the silencing of these HOX genes in non‐tumorigenic, oral keratinocytes results from the deposition of the H3K27me3 mark by EZH2, a component of PRC2 complex.…”