Klotho is silenced through promoter hypermethylation in gastric cancer

Abstract

As one of major epigenetic changes to inactivate tumor suppressor genes in human carcinogenesis, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes and predict the prognosis of cancer patients. In the present study, we found KL (klotho) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. KL expression was downregulated in primary gastric carcinoma tissues (n=22, p<0.05) and all of gastric cancer cells lines examined. Ectopic expression of KL inhibited the growth of gastric cancer cells partially through the induction of apoptosis, demonstrating a tumor suppressive role of KL in gastric cancer. Demethylation with 5-aza-2'-deoxycytidine (Aza) increased KL expression and KL promoter was hypermethylated in gastric cancer cell lines as well as some of primary gastric carcinoma tissues (47/99) but none of normal gastric tissues. Importantly, promoter methylation of KL was significantly associated with the poor outcome of gastric cancer patients (p=0.025, Log-rank test), highlighting the relevance of epigenetic inactivation of KL in gastric carcinogenesis. As a summary, we found that KL is a novel tumor suppressor gene epigenetically inactivated in gastric cancer and promoter methylation of KL could be used to predict the prognosis of gastric cancer patients.

Figure 1

KL is downregulated in gastric cancer. (A) KL expression in primary gastric carcinoma tissues and adjacent non-tumor tissues were determined by real-time RT-PCR (n=22, p<0.05, Wilcoxon matched pairs test). GAPDH was used for the normalization. (B) KL expression in gastric cancer cell lines were determined by RT-PCR. GAPDH was used the loading control.

Figure 2

KL functions as a tumor suppressor in gastric cancer. (A) The growth of gastric cancer cell lines transiently transfected with pcDNA3.1-KL or empty vector were determined by MTS assay. The asterisks indicate statistical difference (p<0.05, Student's t test). (B) Colony formation assay was applied to analyze the long-term effect of KL expression on the growth of gastric cancer cells. The asterisks indicate statistical difference (p<0.05, Student's t test). (C) Cells stained with PI and FITC-Annexin-V were subjected to Flowcytometry analysis. (D) The amount of phosphory-lated Erk-1/2 and p21 were determined by western blotting. Pan-Erk-1/2 and GAPDH were used as the loading control respectively.

Figure 3

KL downregulation in gastric cancer cells was mediated by promoter hypermethylation. The schematic structure of the KL CGI was shown in (A) CGI was plotted by GeneTool program. Black triangles indicate BstU I sites. Promoter methylation of KL were analyzed by Cobra (B) and MSP (C), respectively. M: methylation specific PCR; U: un-methylation specific PCR. N1 and N2 are normal stomach tissues. (D) The expression of KL before and after Aza treatment were analyzed by RT-PCR. GAPDH was used as the loading control.

Figure 4

Methylation of KL promoter was associated with poor outcome of gastric cancer patients. (A) Methylation of KL promoter were analyzed by Cobra as Figure 3B. T1 and T2 are tumor tissues; A1 and A2 are adjacent non-tumor tissues; N1 and N2 are normal stomach tissues. (B) Survival curves were plotted based on Kaplan-Meier survival analysis. Methylation status of KL promoter was used as the variate to separate two lines (n=99, p=0.025, Log-rank test).