Frequent transcriptional inactivation of Kallikrein 10 gene by CpG island hypermethylation in non-small cell lung cancer

Abstract

The role of Kallikrein 10 gene (KLK10) in non-small cell lung cancer (NSCLC) remains largely unknown. We determined the frequency and functional significance of KLK10 hypermethylation in NSCLC. The mRNA expression and methylation status of KLK10 in 78 pairs NSCLC specimens was explored. The biological effects of KLK10 were analyzed by transfection. The results showed that, KLK10 was significantly downregulated in NSCLC (57.7%, 45/78) as compared to non-cancer samples (P = 0.010). CpG island hypermethylation of KLK10 was detected in 46.2% (36/78) NSCLC tissues and was closely correlated with loss of transcript (P < 0.001). KLK10 methylation was associated with advanced stage (P = 0.013) and lymph metastasis (P = 0.015). Furthermore, demethylation treatment restored the expression of KLK10 in two lung adencarcinoma cell lines (A549, SPC-A1). Forced expression of KLK10 in A549 and SPC-A1 remarkably suppressed cells proliferation, migration in vitro and oncogenicity in vivo. Additionally, methylated KLK10 was detected in 38.7% (30/78) of plasma samples from cancer patients but rare in cancer-free controls (P < 0.001). In conclusion, KLK10 acts as a functional tumor suppressor gene in NSCLC, epigenetic inactivation of KLK10 is a common event contributing to NSCLC pathogenesis and may be used as a potential biomarker.

Figure 1

Transcript expression of KLK10 in NSCLC tissues. (A) Typical RT‐PCR results of mRNA expression levels of KLK10 in five paired (patients 32, 34, 38–40) NSCLC tumor (T) and their adjacent normal lung tissues (N). GAPDH was used as an endogenous control. KLK10 gene was detected a low or absent of transcripts in most (57.7%, 45/78) tumor tissues, while widely expressed in adjacent normal tissues and benign control lung lesions. (B) mRNA expression levels of KLK10 in NSCLC samples (T, n = 78) and their adjacent normal tissues (N, n = 78), benign control lung lesions (B, n = 25) as determined by quantitative real‐time PCR. The results were expressed as the ratio of copies of target gene relevant to GAPDH form three independent experiments. Data are expressed as mean ± SD, *P < 0.05.

Figure 2

Methylation status of KLK10 gene in matched tumor tissues and plasma samples of NSCLC patients. (A) Typical agarose gel electrophoresis of MSP results in tissue samples. T, NSCLC tumor; N, adjacent normal lung tissues (patients 32, 34, 38–40). KLK10 showed an aberrant methylation in tumor tissues of #32, #34, #38, #39 and normal tissue of #32. (B) Typical agarose gel electrophoresis of MSP results in plasma samples. KLK10 methylation status in plasma samples was in accordance with corresponding tumor tissues (patients 32, 34, 38–40). The benign control, B1 was a 68‐year‐old man diagnosed to have tuberculosis and showed KLK10 unmethylation in plasma. Lymphocyte DNA, original or methylated in vitro by excessive CpG (SssI) methylase, was used as unmethylation and methylation positive control. Water blank was used as a negative control.

Figure 3

Restoration of KLK10 expression by 5‐Aza‐dC treatment in NSCLC cell lines. KLK10 was hypermethylated and silenced in two human lung adencarcinoma cell lines (A549 and SPC‐A1). After treatment with 10 μm of 5‐Aza‐dC, a methyltransferase inhibitor for 72 h, KLK10 expression was markedly induced in the two cell lines.

Figure 4

Exogenous expression of KLK10 in A549 and SPC‐A1 cells by transfection of recombinant vector. (A) Fluorescence photos of A549 and SPC‐A1 cells transfected with KLK10 gene (magnification ×200). The 1.0 kb KLK10 open‐reading frame was cloned into the pEGFP‐C1 vector to generate the pEGFP‐KLK10 recombinant vector. KLK10 was stable expression in transfected pEGFP‐KLK10‐A549 and pEGFP‐KLK10‐SPC‐A1 cells. (B) Protein expression of KLK10 gene in A549 and SPC‐A1 cells was examined by western blotting. Mouse anti‐human‐KLK10 monoclonal antibody diluted at 1:500. Lane 1–6 was as follows: A549, pEGFP‐A549, pEGFP‐KLK10‐A549, SPC‐A1, pEGFP‐SPC‐A1, pEGFP‐KLK10‐SPC‐A1.

Figure 5

Inhibition of colony formation in A549 (A) and SPC‐A1 (C) cells transfected with KLK10 gene. 1 × 10³ cells seeded into six‐well plates cultured in RPMI 1640 for 2 weeks, colonies were stained with Giemsa, counted and photographed. A significant decrease in colony number and volume was observed in transfected pEGFP‐KLK10‐A549 (B, P = 0.008) and pEGFP‐KLK10‐SPC‐A1 (D, P = 0.003) cells, compared with wild cells and empty‐vector transfected cells.

Figure 6

KLK10 inhibits growth of tumors derived from A549 and SPC‐A1 in vivo. 2 × 10⁶ cells transfected with pEGFP‐KLK10 recombinant vector or the empty vector were injected into nude mice (ten per group). (A) Representative picture of nude mice at week 4 injected with pEGFP‐KLK10‐A549 cells and pEGFP‐A549 cells. (C) Representative picture of nude mice at week 4 injected with pEGFP‐KLK10‐SPC‐A1 cells and pEGFP‐SPC‐A1 cells. The tumor volume of nude mice in each group was indicated as mean tumor volume ± SD(mm³). Tumor mean volume of pEGFP‐KLK10‐A549 mice (B, P < 0.001) and pEGFP‐KLK10‐SPC‐A1 (D, P < 0.001) were significantly smaller than empty‐vector mice group, respectively.

Figure 7

KLK10 inhibits cell migration in wound‐healing assay. Monolayer of wild, mock and transfected A549 (A) and SPC‐A1 (C) cells were scraped with micropipette tips and cultured in FBS‐free media. The representative picture showed repair of lesion by cell migration was photographed 48 h later (magnification ×100). The uncovered area of transfected pEGFP‐KLK10‐A549 cells (B, P < 0.001) and pEGFP‐KLK10‐SPC‐A1 (D, P < 0.001) cells were significantly greater than the wild cells and empty‐vector transfected cells, respectively.