Silencing of RASSF3 by DNA hypermethylation is associated with tumorigenesis in somatotroph adenomas

Abstract

The pathogenic mechanisms underlying pituitary somatotroph adenoma formation, progression are poorly understood. To identify candidate tumor suppressor genes involved in pituitary somatotroph adenoma tumorigenesis, we used HG18 CpG plus Promoter Microarray in 27 human somatotroph adenomas and 4 normal human adenohypophyses. RASSF3 was found with frequent methylation of CpG island in its promoter region in somatotroph adenomas but rarely in adenohypophyses. This result was confirmed by pyrosequencing analysis. We also found that RASSF3 mRNA level correlated negatively to its gene promoter methylation level. RASSF3 hypermethylation and downregulation was also observed in rat GH3 and mouse GT1.1 somatotroph adenoma cell lines. 5-Aza-2' deoxycytidine and trichostatin-A treatment induced RASSF3 promoter demethylation, and restored its expression in GH3 and GT1.1 cell lines. RASSF3 overexpression in GH3 and GT1.1 cells inhibited proliferation, induced apoptosis accompanied by increased Bax, p53, and caspase-3 protein and decreased Bcl-2 protein expression. We also found that the antitumor effect of RASSF3 was p53 dependent, and p53 knockdown blocked RASSF3-induced apoptosis and growth inhibition. Taken together, our results suggest that hypermethylation-induced RASSF3 silencing plays an important role in the tumorigenesis of pituitary somatotroph adenomas.

Figure 1. RASSF3 is hypermethylated in pituitary adenomas.

(A) Average signal value in RASSF3 promoter of 27 tumors and 4 adenohypophyses. Signal values of the probes were graded from 1 to 32. The average signal values in every grade of tumor were significantly higher than in 4 adenohypophyses. However, there was no significant difference between different tumor grades. (B) Pyrosequencing technology demomstrates that RASSF3 promoter methylation level is higher in human somatotroph adenomas (P<0.01). The average methylation level of the 10 CpG sites in RASSF3 promoter of the 4 normal samples and 27 tumor samples is shown.

Figure 2. RASSF3 downregulation correlates with its promoter hypermethylation in human somatotroph adenomas.

(A) The average relative RASSF3 mRNA level in 4 normal samples and 27 tumor samples is shown. RASSF3 expression level was significantly lower in human somatotroph adenomas than in normal pituitary samples (P<0.01). (B) There was no significant difference in RASSF3 methylation and expression levels between different tumor grades. (C) Spearman analysis of all normal and tumor samples with RASSF3 mRNA level and promoter region methylation level demonstrated a significant inverse correlation (r = –0.6655) between DNA hypermethylation and gene expression.

Figure 3. Demethylation treatment increased RASSF3 gene expression in somatotroph adenoma cell lines with RASSF3 hypomethylation.

(A) DNA methylation levels of corresponding samples are shown. 5-Aza treatment induced RASSF3 demethylation, and administration of TSA following 5-Aza led to a further decrease in the methylation level of RASSF3. (B) Relative GT1.1 expression ratios normalized to β-actin are shown. Decreased methylation and increased mRNA level of RASSF3 were observed after 5-Aza treatment. In GH3 cells, TSA treatment after 5-Aza had a significant additive effect in restoring RASSF3 expression. This additive effect of TSA was also observed in GT1.1 cells, athough it was not significant. (C) RASSF3 protein level was increased after 5-Aza and TSA treatment.

Figure 4. Effects of RASSF3 transfection on somatotroph cell proliferation and apoptosis.

(A) Compared with cells stably transfected with Lenti-GFP, qRT-PCR analysis demonstrated that RASSF3 mRNA was significantly improved in somatotroph cells stably transfected with Lenti-RASSF3, and decreased in somatotroph cells stably transfected with Lenti-RASSF3 shRNA. (B) Effect of RASSF3 overexpression and suppression on GH3 cell proliferation was measured by MTT assay. Absorbance was read at 570 nm and the average value was calculated from 3 wells. (P<0.05 at 48, 72, 96, and 120 h for Lenti-RASSF3 or Lenti-RASSF3 shRNA versus Lenti-GFP or nontransfected GH3 cells). The results in GT1.1 cells were similar to GH3 cells. (C) Apoptosis in GH3 cells was measured by Annexin V/PI staining following Lenti-RASSF3 transfection. Early apoptotic cell populations were significantly increased after Lenti-RASSF3 transfection, and decreased after Lenti-RASSF3 shRNA transfection. The results in GT1.1 cells were similar to GH3 cells. RF3: abbreviation of RASSF3.

Figure 5. Western blotting analysis for RASSF3, p53, Bax, Bcl-2, and caspase-3.

β-actin served as an endogenous control. Increased expression of p53, Bax, and caspase-3 and decreased expression of Bcl-2 were observed in GH3 and GT1.1 cells 72 h after Lenti-RASSF3 transfection as compared with the control groups (P<0.05).

Figure 6. RASSF3-induced apoptosis is p53 dependent.

(A) Compared with the Lenti-RASSF3-transfected GH3 cells, qRT-PCR analysis demonstrated that Lenti-P53 shRNA reduced p53 expression by 81%. Compared with Lenti- RASSF3 shRNA GH3 cells, Lenti-p53 increased p53 mRNA expression by 17.1-fold in Lenti-RASSF3 shRNA-transfected GH3 cells. (B) Western blotting analysis for RASSF3, p53. (C) Proliferation in GH3 cells was measured by MTT assay after Lenti-p53 shRNA transfection of Lenti-RASSF3-transfected GH3 cells, and Lenti-p53 transfection of Lenti-RASSF3 shRNA-transfected GH3 cells. Absorbance was read at 570 nm and the average was calculated for 3 wells. p53 suppression blocked the effect of RASSF3 on cell proliferation, and p53 overexpression showed a similar effect to RASSF3. (D) Apoptosis in GH3 cells was measured by Annexin V/PI staining following Lenti-p53 shRNA transfection of Lenti-RASSF3-transfected GH3 cells, and Lenti-p53 transfection of Lenti-RASSF3 shRNA-transfected GH3 cells. Early apoptotic cell populations were significantly decreased after Lenti-p53 shRNA transfection, and increased after Lenti-p53 transfection.

Figure 7. Effect of RASSF3 expression on cell invasive capacity in the Matrigel model.

Matrigel assays showing the effect of Lenti-RASSF3 shRNA and Lenti-RASSF3 on the invasive potential of GH3 and GT1.1 cells. The bar graph indicates the mean number of invaded cells at 24 h after top-side cell seeding. Lenti-RASSF3 or Lenti-shRNA did not affect invasion of GH3 and GT1.1 cells.