Frequent promoter methylation of HOXD10 in endometrial carcinoma and its pathological significance

Abstract

Homeobox D 10 (HOXD10) is important in cell differentiation and morphogenesis and serves as a tumor suppressor gene (TSG) in a number of malignancies. The present study investigated its promoter methylation status and association with the clinicopathological features of endometrial cancer (EC), and measured HOXD10 protein expression levels. EC samples (n=62), including 50 endometroid adenocarcinoma (EA) and 12 mucinous endometrial carcinoma samples (EC) and 70 non-cancerous samples were collected. All samples were evaluated for the methylation status of several TSGs, including HOXD10, using methylation-specific PCR. HOXD10 expression level was evaluated using immunohistochemistry. 5-Aza-2-deoxycytidine treatment was performed in the EC cell line Ishikawa to observe the change in HOXD10 expression levels. HOXD10 promoter methylation was more frequent in cancer samples (P<0.001). Downregulation of HOXD10 in EC samples was confirmed at the protein level using immunohistochemistry (P<0.001) and immunohistochemical staining was negatively associated with methylation status (P<0.05). Less HOXD10 protein was expressed in MEC compared with EA samples (P<0.001). The HOXD10 promoter was hypermethylated in both EA and MEC, causing decreased HOXD10 protein expression levels in EC cells. HOXD10 expression levels were partially reversed by 5-Aza-2-deoxycytidine treatment. The results of the present study demonstrated that epigenetic silencing of HOXD10 putatively contributed to the tumorigenesis of EA. Although there was no significant difference in HOXD10 methylation between EA and MEC, HOXD10 protein expression levels differed between these two diseases, indicating that it may be a useful protein biomarker for distinguishing between these two lesions.

Keywords: DNA methylation; HOXD10; endometroid carcinoma; mucinous endometrial carcinoma.

Figure 1.

HOXD10 promoter hypermethylation in EC. Methylation statuses of eight out of 62 cancer samples and eight out of 70 non-cancerous samples. U, unmethylated HOXD10; M, methylated HOXD10; EC, endometrial carcinoma; N, non-cancerous tissue; T, tumor tissue. H₂O were used as negative control.

Figure 2.

Decreased expression levels of HOXD10 in EC. (A) Classic HOXD10 staining in EC (×100). (B) No HOXD10 staining was observed in MEC samples (×100). (C) For simple hyperplasia, EC tissue was strongly stained and HOXD10 is positively stained in the nucleus and cytoplasm (×400). (D) In complex hyperplasia, EC tissue was strongly stained, and HOXD10 was positively stained in the nucleus and cytoplasm (×100). HOX, homeobox; EC, endometrial carcinoma; MEC, mucinous endometrial carcinoma. Arrows indicate positively stained cells within the figure.

Figure 3.

Effects of 5-Aza-CdR on regulation of expression levels of HOXD10 in Ishikawa cell line. β-actin was used as endogenous control. The ratio of HOXD10 to β-actin is shown on the y-axis. *P<0.01. 5-aza-CdR, 5-aza-2′-deoxycytidine; HOX, homeobox.