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. 2014 Jan 1;320(1):128-43.
doi: 10.1016/j.yexcr.2013.09.011. Epub 2013 Sep 25.

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

Katarzyna M Marcinkiewicz  1 , Lorraine J Gudas
Affiliations

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

Katarzyna M Marcinkiewicz et al. Exp Cell Res. .

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.

Keywords: ChIP; Chromatin; Epigenetic silencing; GAPDH; GO; H3K27me3; HNSCC; HOX; HPRT1; Head and neck squamous cell carcinoma; Homeobox; OSCC; Oral squamous cell carcinoma; PRC; Polycomb; RA; RAR; RARE; RNA sequencing; RNA-seq; RNAseq; RXR; Retinoic acid; SCC; SUZ12; TNM; TNM classification of malignant tumours; Tumorigenesis; WCMC; Weill Cornell Medical College; chromatin immunoprecipitation; gene ontology; glyceraldehyde 3-phosphate dehydrogenase; head and neck squamous cell carcinoma; histone 3 lysine 27 trimethyl; homeobox; hypoxanthine phosphoribosyltransferase 1; oral squamous cell carcinoma; polycomb repressive complexes; qRT-PCR; quantitative real time polymerase chain reaction; retinoic acid; retinoic acid receptor; retinoic acid response element; retinoid X receptor; shRNA; short hairpin RNA; squamous cell carcinoma.

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Conflict of interest statement

Conflict of interest

The authors disclose no potential conflicts of interest.

Figures

Fig. 1
Fig. 1
RNAseq analyses reveal differential expression of large numbers of genes in non- tumorigenic vs. tumorigenic oral keratinocytes. (A) Pie chart showing the distribution of genes with at least a 3 fold difference in transcript levels betweeen OKF6-TERT1R and SCC-9 cells. Lines indicate the part of gene list used in gene ontology (GO) analysis in (B) and (C). (B) and (C) Results of GO analysis for (B) the genes with transcript levels at least 3 fold higher in SCC-9 than OKF6-TERT1R cells and (C) the genes with transcript levels at least 3 fold higher in OKF6-TERT1R than SCC-9 cells. Ten GO terms with the lowest p values are shown. GO terms associated with homeobox genes are in highlighted in bold italics.
Fig. 2
Fig. 2
(a) mRNA levels of homeobox genes expressed differentially in non-tumorigenic and tumorigenic oral keratinocyte cell lines and (b) levels of SUZ12 and the H3K27me3 epigenetic mark proximal to homeodomain genes expressed differentially between OKF6-TERT1R and SCC-9 cells. n=3 independent biological repeats; * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001; note differences in scales of y-axes in different panels (a) mRNA levels are normalized to HPRT1 expression and represented relative to transcript levels in control treated OKF6-TERT1R cells (set as 1); differences in transcript levels between the cell lines were analyzed by one-way ANOVA followed by Dunnett’s posttest, setting the result obtained for the OKF6-TERT1cells as control value to which all other samples were comapred: A–D mRNA levels of HOX genes expressed at higher levels in SCC-9 vs. OKF6-TERT1R cells. E–H mRNA levels of genes expressed at lower levels in SCC-9 vs. OKF6-TERT1R cells. (b) Data are represented as percent of chromatin used as input in corresponding IPs; differences in immunoprecipitated chromatin levels between the cell lines were analyzed two-way ANOVA followed by Bonferroni post-test: A–D Levels of the H3K27me3 mark (clear bars) and SUZ12 (black bars) at HOX genes expressed at higher levels in SCC-9 vs. OKF6-TERT1R cells, as assessed by ChIP; E to H: Levels of H3K27me3 and SUZ12 at HOX genes expressed at lower levels in SCC-9 vs. OKF6-TERT1R cells, as assessed by ChIP; I: Negative control: association of H3K27me3 mark and SUZ12 with with an intergenic region, as assessed by ChIP J and K: Negative control: chromatin immunoprecipitated using normal rabbit IgG along with antibodies specific to SUZ12 and H3K27me3; sequences amplified are indicated by the graph legend.
Fig. 3
Fig. 3
(a) SUZ12 mRNA and protein levels in SUZ12 depleted OKF6-TERT1R and SCC-9 cell populations; (b) mRNA levels of homeobox genes expressed differentially in SUZ12 depleted OKF6-TERT1R and SCC-9 cell populations; and (c) proliferation of SUZ12 depleted OKF6-TERT1R and SCC-9 cell populations. (a) (A) mRNA levels are normalized to GAPDH mRNA levels and are represented relative to transcript levels in OKF6-TERT1R cells (set as 1); y-axis, arbitrary units. Differences in transcript levels between the cell populations were analyzed by unpaired t test, significant differences are indicated. (B) Western blot analysis of SUZ12 protein levels in cells expressing control “scrambled” or SUZ12 targeting shRNA sequence and parental cells. A representative result is shown. (b) PCR results presented and analyzed as in panel (a)(A); A–D mRNA levels of HOX genes expressed at higher levels in parental SCC-9 vs. OKF6-TERT1R cells. E–H mRNA levels of genes expressed at lower levels in parental SCC-9 vs. OKF6-TERT1R cells; (c) cell proliferation assays; data analyzed using two- way ANOVA followed by Bonferroni’s post test, equal numbers of parental and shRNA sequence expressing cell populations were seeded, allowed to attach for 24 h and counted 1, 4 and 7 days following plating (corresponding to 0, 3 and 6 days time points on the graph). n=3 independent biological repeats; note differences in scales of y-axes in different panels, * indicates p<0.05, * indicates p<0.01, *** indicates p<0.001 and **** indicates p<0.0001.
Fig. 4
Fig. 4
(a) mRNA levels of homeobox genes expressed differentially in vehicle and RA treated, non-tumorigenic and tumorigenic oral keratinocyte cell lines and (b) levels of SUZ12 and H3K27me3 epigenetic marks proximal to homeodomain genes expressed differentially between non-tumorigenic and tumorigenic oral keratinocyte cells in vehicle and RA treated OKF6-TERT1R and SCC-9 cells. n=3 independent biological repeats; * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001; note differences in scales of y-axes in different panels (a) mRNA levels are normalized to HPRT1 expression and represented relative to transcript levels in control, untreated OKF6-TERT1R cells (set as 1); differences in transcript levels between the vehicle and RA-treated samples for each cell line were analyzed by two-way ANOVA followed by Bonferroni post-test A–D mRNA levels of HOX genes expressed at higher levels in SCC-9 vs. OKF6-TERT1R cells after 48 h of treatment with 1 µM RA. The cells were treated for 48 h with 1 µM RA or 0.1% ethanol (vehicle control). E–H mRNA levels of HOX genes expressed at lower levels in SCC-9 vs. OKF6-TERT1R cell line after 48 h of treatment with 1 µM RA or 0.1% ethanol (vehicle control). (b) Data are represented as percent of chromatin used as input in corresponding IPs; differences in immunoprecipitated chromatin levels between the cell lines were analyzed by two-way ANOVA followed by Bonferroni post-test; A–D Levels of the H3K27me3 mark and SUZ12 at HOX genes expressed at higher levels in SCC-9 vs. OKF6-TERT1R cell line after 48 h of treatment with 1 µM RA, as assessed by ChIP; E–H Levels of the H3K27me3 mark and SUZ12 at HOX genes expressed at lower levels in SCC-9 vs. OKF6-TERT1R cell line after 48 h of treatment with 1 µM RA, as assessed by ChIP; I Association of H3K27me3 mark and SUZ12 with with an intergenic region after 48 h of treatment with 1 µM RA., as assessed by ChIP. J and K Chromatin immunoprecipitated using normal rabbit IgG along with antibodies specific to SUZ12 and H3K27me3; sequences amplified are indicated by the graph legend.
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References

    1. Curado MP, Hashibe M. Recent changes in the epidemiology of head and neck cancer. Curr. Opin. Oncol. 2009;21:194–200. - PubMed
    1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: Cancer J. Clin. 2013;63:11–30. - PubMed
    1. Means AL, Gudas LJ. The roles of retinoids in vertebrate development. Annu. Rev. Biochem. 1995;64:201–233. - PubMed
    1. Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J. 1996;10:940–954. - PubMed
    1. Langston AW, Gudas LJ. Identification of a retinoic acid responsive enhancer 3′ of the murine homeobox gene Hox-1.6. Mech. Dev. 1992;38:217–227. - PubMed

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