Aberrant methylation inactivates transforming growth factor Beta receptor I in head and neck squamous cell carcinoma

Abstract

Background. Alterations in TGF-beta signaling are common in head and neck cancer (HNSCC). Mutations in TGF-beta type II receptor (TbetaR-II) occur frequently in HNSCC while TGF-beta type I receptor (TbetaR-I) mutations are rare, suggesting that other molecular alterations in the TGF-beta pathway are likely. To identify abnormalities in TbetaR-I expression we analyzed 50 HNSCCs and correlated the results with clinical-pathologic features. Methods. Hypermethylation of TbetaR-I was evaluated via methylation-specific PCR (MSP) and restriction enzyme-mediated PCR (MSRE). Mutations in exons 1 and 7, mRNA and protein expression were analyzed by direct sequencing, semiquantitative RT-PCR and immunohistochemistry, respectively. Results. TbetaR-I expression was lost in 83% HNSCCs and was linked to DNA hypermethylation of the CpG-rich promoter region in 62% of the tumors. The variants 9A/6A and Int7G24A were found in two patients. Conclusions. This study shows that suppression of TbetaR-I expression in HNSCC is associated with DNA hypermethylation.

Figure 1

Analysis of T β R-I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of BstUI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R-I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R-I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R-I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β-actin (153 bp). The length of the T β R-I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R-I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

Figure 2

Immunohistochemistry of T β R-I protein in HNSCCs (200X). (a) Immunohistochemical detection of T β R-I protein (brown signal in nonneoplastic epithelium adjacent to HNSCC). (b) Lack of T β R-I protein staining in the HNSCC.

Figure 3

Schematic depiction of T β R-I expression by semiquantitative RT-PCR, and hypermethylation by MSP. Tumors with methylated (red bars) and nonmethylated (blue bars) genes are depicted in association with T β R-I levels of expression. Columns (white, grey, and dark grey) correspond to relative expression levels (arbitrary units) of T β R-I determined by semiquantitative RT-PCR. Methylated genes are associated with lower expression levels of the T β R-I gene.