The Influence Of Lysine-specific Demethylase1(LSD1)on Invasion And Metastasis Of Colon Cancer And The Related Mechanisms

Backgrounds:The incidence of colon cancer is increasing rapidly, and it has become the third most common malignancy and the second leading cause of cancer deaths worldwide. Recurrence and metastasis are the leading cause of death among colon cancer patients. Epithelial mesenchymal transition (EMT) is now considered to be the initial and necessary step in the metastatic cascade. During EMT, epithelial cells acquire fibroblast-like characteristics, including loss of cell polarity, reduced intercellular adhesion, increased motility and invasive capacity. Transcription factors, such as snail, slug, zeb1, zeb2and twist can promote EMT by suppressing E-cadherin expression and consequently contribute to cancer metastasis.Lysine-specific demethylase1(LSD1), the first discovered histone demethylase, is required in Snail/Slug-mediated transcriptional repression during EMT, in the absence of LSD1, Snail and Slug fail to repress E-cadherin transcription. As a lysine-specific demethylase belonging to the flavin-dependent amine oxidase family, LSD1specifically catalyzed the demethylation of mono-and dimethylated histone H3lysine4(H3K4) and H3lysine9(H3K9) through a redox process. Over-expression of LSD1promotes proliferation, migration and invasion of various cancer cells such as prostate cancer, nonsmall-cell lung cancer, breast carcinoma et al. But whether LSD1correlates with metastasis of colon cancer remains to be confirmed.Aims:The aim of this study was to investigate LSD1, E-cadherin, and N-cadherin expression in colon cancer specimens and their clinical significance. And to investigate the influence of inhibition of LSD1expression on proliferation, invasion and apoptosis of colon cancer, and the related mechanismsMethods:(1) The expression of LSD1, E-cadherin, and N-cadherin in colon cancer specimens was determined by immunohistochemistry, and the relationship between the expression of the respective molecules and clinicopathological characteristics was analyzed.(2) The invasive ability of five colon cancer cell lines including Lovo、SW620、HT-29、 HCT-8、HCT-116and control cell HEK293was detected by Transwell Invasion Assay, and the expression of LSD1, N-cadherin and E-cadherin in those six cells were detected by Real-time RT-PCR and western blot. Then the correlation of LSD1, N-cadherin and E-cadherin with invasiveness of colon cancer cells were evaluated.(3) The influence of inhibition of LSD1expression on proliferation, invasion and apoptosis of colon cancer cells were detected by Transwell Invasion Assay, Cell Proliferation Assay and Apoptosis Assay.(4) The enrichment of LSD1at the proximal promoter of E-cadherin in LSD1-silenced SW620cells and Tranylcypromine treated or untreated SW620cells were detected by ChIP assay, and the H3K4me2at the proximal promoter of E-cadherin in the three cells were detected by Western blot.Results:(1) The positive expression rates of LSD1, E-cadherin, and N-cadherin in colon cancer specimens were66.7%(72/108),85.2%(92/108), and41.7%(45/108), respectively. LSD1was significantly more highly expressed in colon cancer specimens classified as high TNM stage lesions and with distant metastasis (P<0.05). However, E-cadherin expression was significantly downregulated in colon cancer specimens classified as high TNM stage lesions and with distant metastasis (P<0.05), whereas the expression of N-cadherin did not differ significantly according to clinical and pathological characteristics (P<0.05). Correlation analysis revealed that LSD1expression was negatively correlated with E-cadherin expression (rs=-0.318,P=0.001), but not evidently correlated with N-cadherin expression (rs=0.182, P=0.06). Colon cancer specimens with positive LSD1expression and negative E-cadherin expression were correlated with significantly lower overall survival.(2) The mRNA and protein levels of LSD1and N-cadherin in SW620cell line were significantly higher than those in the Lovo, HT-29, HCT-8, HCT-116and·HEK293(control) cell lines. Howerver, the levels of E-cadherin in SW620cell line were evidently lower than the other five cell lines (P<0.05). Correlation analysis indicates that the LSD1and N-cadherin expression correlated possitively with the invasiveness of colon cancer cells, whereas the E-cadherin exhibited a negative correlation with the invasiveness of colon cancer cells.(3) Three independent siRNAs targeting LSD1(siRNA-1554、siRNA-705、 siRNA-1973) were transfected to SW620cells to detect gene-silencing efficiency, and the result showed that the knockdown effect of siRNA-705were better than the other two siRNAs at both mRNA and protein levels. Using siRNA-705and Tranylcypromine (2.5mM), we performed Transwell Invasion Assay, Cell Proliferation Assay and Apoptosis Assay in SW620cell lines, and found significant suppression of invasion and growth, and induced cell apoptosis by siRNA and Tranylcypromine (P<0.05). Interestingly, up-regulation of E-cadherin and down-regulation of N-cadherin were observed after treated with siRNA-705and Tranylcypromine for72hours.(4) LSD1is present at the proximal promoter of E-cadherin in SW620cells, and quantitative analysis revealed that the enrichment of LSD1at the proximal promoter of E-cadherin was significantly higher in Tranylcypromine treated and untreated SW620cells than LSD1-silenced SW620cells, which was accordance with invasiveness of SW620cells and LSD1-silenced SW620cells, but was not accordance with invasiveness of Tranylcypromine treated cells. Using an antibody specific for dimethyl H3K4(H3K4me2), we detected relatively high levels of H3K4me2at the promoter of the E-cadherin gene in LSD1-silenced SW620cells and Tranylcypromine treated cells, and found a significant decrease of this active mark specifically at the promoter region in SW620cells.Conclusions:(1) LSD1showed a significantly higher expression, in contrast to the significantly lower expression of E-cadherin, in colon cancer specimens classified as high TNM stage lesions and with distant metastasis. Positive expression of LSD1and negative expression of E-cadherin may be predictors of a worse colon cancer prognosis.(2) The LSD1and N-cadherin expression correlated possitively with the invasiveness of colon cancer cells, whereas the E-cadherin exhibited a negative correlation with the invasiveness of colon cancer cells.(3) Inhibition of LSD1impairs proliferation and invasiveness, and induces apoptosis of colon cancer cells in vitro, and leads to up-regulation of E-cadherin and down-regulation of N-cadherin.(4) LSD1can interact directly with the promoter of E-cadherin and inhibit the expression of E-cadherin by catalyzing the demethylation of H3K4me2at this site, which contributes to metastasis of colon cancer.