Epigenetic inactivation of the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene in human prostate tumors

Abstract

Gene silencing via CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. Previous studies have shown that the tissue inhibitors of metalloproteinases (TIMP)-2 gene, which is an endogenous inhibitor of matrix metalloproteinases involved in cell invasion and tumorigenesis, is downregulated or silenced in a variety of human cancer cell lines. Here, we investigated the mechanism underlying TIMP-2 expression in prostate cancer cell lines and primary prostate tumor samples. We observed a strong correlation between promoter hypermethylation and lost expression of TIMP-2 gene, which was supported by other results demonstrating that promoter demethylation by 5-aza-2'-deoxycytidine and trichostatin A reactivated TIMP-2 and restored its expression in TIMP-2-silenced metastatic prostate cell lines. These results were further substantiated by a chromatin immunoprecipitation assay, showing the preferential binding of MeCP2 to methylated CpG island in TIMP-2-silenced metastatic prostate cell lines. In vitro Matrigel invasion assays showed that re-expression of TIMP-2 after a combined treatment with 5-aza and trichostatin-A in metastatic prostate cells resulted in a significant reduction of tumor cell invasion. Furthermore, CpG methylation of TIMP-2 promoter was also shown in primary prostate tumors that expressed decreased TIMP-2 protein levels. These results suggest that the downregulation of the TIMP-2 gene is associated with promoter methylation and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

Figure 1. Activity and expression levels of TIMP and MMP proteins in prostate cell lines

(A) A model for the balance between TIMP and MMP activity and the subsequent effects on tumor cell invasion. (B) MMP (top) and TIMP (bottom) activity levels in prostate cell lines RWPE1, RWPE2, LNCaP, DU145, PC3 and Tramp-C1 were assessed by zymography and reverse zymography, respectively. (C) Immunoblot analysis of TIMP and MMP proteins in the concentrated culture medium of prostate cell lines. TIMP-4 was used as a loading control. (D) Cells invading through the matrigel were counted under a microscope in three random fields at 200X magnification. Each bar represents the mean ± SD of three fields counted where significant differences from normal prostate cancer cell lines RWPE1 are represented by asterisks (*) (p<0.01).

Figure 2. Analysis of TIMP-2 promoter CpG methylation in prostate cancer cell lines

(A) Schematic diagram indicating the CpG island on TIMP-2 promoter region. Nucleotide sequence located in the promoter region of the human TIMP-2 gene from −295 to −145 with respect to the translation initiation codon (bent arrow) is shown. CpG dinucleotides are indicated in bold. (B) Bisulfite-modified DNA derived from prostate cancer cell lines were amplified with TIMP-2 primers specific for unmethylated and methylated DNA. TIMP-2-U, unmethylated PCR product; TIMP-2-M, methylated PCR product; NC, no template control. Positive and negative controls were described in Materials & Methods. (C) mRNA expression of TIMP-2 was analyzed using RT-PCR in prostate cancer cells treated with 20 μM 5-aza-CdR followed by 50 nM TSA, as described in Materials & Methods. GAPDH mRNA was amplified as a loading control and expression standard. Bar diagram showing densitometry quantified data of TIMP-2 mRNA/GAPDH mRNA ratios from three independent experiments. Asterisks (*) indicate significant differences from untreated control cells (p<0.01). (D) Assessment of MeCP2 binding on the TIMP-2 promoter by ChIP analysis. The bound MeCPs fraction (ChIP) shows binding to the TIMP-2 promoter. Aliquots of chromatin taken before immunoprecipitation were used as “input” controls whereas chromatin eluted from immunoprecipitations lacking antibody were used as “no antibody” (-Ab) controls.

Figure 3. Effects of TIMP-2 promoter CpG methylation on cellular invasion in DU145 and PC3 cells

(A)The invasive capacity of the untreated (Control) and 5-aza-treated DU145 and PC3 cells were assessed in vitro by matrigel invasion assay. Results are the mean ± SD of three different experiments. Significant difference from control is represented by asterisks (p<0.01). (B) Representative invasive potentials of the untreated (Control) and 5-aza-treated DU145 and PC3 cells by matrigel invasion assay.

Figure 4. Expression of TIMP-2 in human prostate tissue samples

(A) Compared with normal prostate tissues, the overall expression level of TIMP-2 in prostate cancer tissues was significantly lower (p<0.01). TIMP-2 expression by normal human prostate tissue (32 cases) and prostate cancer (42 cases) was analyzed. (B) Representative immunostaining photographs were taken at different magnifications: a, normal human prostate showing TIMP-2 in epithelial cells; b, high-power view of (a) showing membrane staining of TIMP-2; c, lack of TIMP-2 staining in prostate cancer tissues; d, high-power view of (c) showing lack of TIMP-2 expression in prostate cancer tissues (p<0.01); T, tumor cells; N, normal prostate epithelial cells. (C) Total cell extracts were further prepared from four-paired normal prostate (N) and prostate tumor tissue (T) specimens. The levels of TIMP-2 protein expression were determined by immunoblot analysis. The level of TIMP-2 protein expression was significantly lower in tumor tissue than in normal tissue, which was indicated by the ratio of TIMP-2/GAPDH (bottom). (D) Quantitative mRNA expression of TIMP-2 in human prostate tumor (green) compared to normal adjacent tissue of the same individuals (red, connected by a line). Significant downregulation of TIMP-2 expression in tumor tissue than in normal adjacent tissue samples.

Figure 5. Methylation status of TIMP-2 promoter in normal and prostate cancer tissue samples

MSP analysis was performed on (A) prostate cancer (B) normal prostate and (C) BPH tissue samples. (D) Summary of TIMP-2 methylation results in formalin-fixed prostate tissue consisting of 42 prostate cancers, 15 BPH samples and 32 normal human prostate tissues.

Figure 6. Methylation patterns of the CpG island of TIMP-2 in human prostate tissues

CpG positions are indicated relative to the translation start codon; each circle in the figure represents a single CpG site. Representative sequencing results of the MSP products. A filled circle represents a methylated CG dinucleotide, and an empty circle represents a demethylated CG dinucleotide. (A)DNAs of ten normal prostate and prostate tumor samples that displayed methylated TIMP-2 in tumor (T) and unmethylated TIMP-2 in normal prostate tissue (N) was selected for genomic sequencing; the selected prostate tumor samples were negative in TIMP-2 protein expression. (B) DNAs of ten normal adjacent tissue (NAT) samples that displayed unmethylated TIMP-2 in assays of MSP and their tumor counterparts displayed decreased TIMP-2 expression in real-time PCR analysis were selected for genomic sequencing.