LSD1-mediated Epigenetic Modification Contributes To Ovarian Cancer Cell Migration And Invasion

Objective:Lysine-specific demethylase 1(LSD1) has been implicated in the process of tumor progression at various steps, but its role in epithelial-messenchymal transition(EMT) and the migration of ovarian cancer cells remains obscure. In this study, we demonstrated the effect of LSD1 on ovarian cancer cell migration and the regulatory role of LSD1 in the expression of EMT markers.Methods:In this study, we constructed stable ovarian cells by using lentiviral transfection. Firstly, several related plasmids were extracted by plasmid miniprep kit and then transfected into 293 T cells using Lipofectamine 2000 to generate lentiviral particles. HO8910 cells were infected by lentiviral supernatant containing the lentiviral particles. After the infection(twice), infected cells were selected with 2.0 μg/ml puromycin for one week. The surviving cells were considered as stable clones. The stable clones were further confirmed by western blot analysis and real-time RT-PCR.Transwell assays were used in vitro to study the role of LSD1 regulating the migration and invasion of ovarian cancer cells. Cells were resuspended with DMEM containing Dox or TCP in upper chamber(with or without coated with Matrigel). Then, 10% FBS-containing medium was placed in the lower chamber to act as a chemoattractant. After a 24-h incubation, the non-invading cells remaining on the upper surface were removed, and the cells on the lower surface were fixed with 4% formaldehyde for 30 min, and stained with 0.1% crystal violet for 15 min. At least 5 fields for each chamber were photographed(x 200 magnification) and counted, and the invading cells were counted in each field.Western blot analysis is used to measure the effect of LSD1 on regulating EMT. Protein lysates were extracted from cells cultured with different doses of Dox for 48 h. Equal amounts of soluble proteins were electrophoresed by SDS-PAGE and transferred to 0.45-μm PVDF membranes. The membranes were blocked with 5% nonfat-dry milk for 1 h at room temperature(RT). After incubation with the primary antibodies(EMT related genes) overnight at 4?C and with the corresponding secondary antibodies for 1 h at RT, the immunoblots were developed by ECL method.LSD1-KD-HO8910 cells were fixed with 1% formaldehyde to cross-link proteins. The reaction was stopped by adding 10 × glycine. Cross-linked cells were washed with PBS twice, pelleted and resuspended in SDS lysis buffer at a concentration of 1x107 cells/ml. Aliquots of 400 μl were sonicated with 4-6 sets of 5-sec pulses(32% output) on ice. Then sonicated lysates were centrifuged and divided into 100 μl aliquots for each ChIP assay(1x106 cells/IP), and precleared with protein g-agarose. After incubation with the antibodies overnight at 4?C, immune complexes were collected with protein G-agarose, and then washed with low salt immune complex wash buffer, high salt immune complex wash buffer, and finally TE buffer. The immune complexes were eluted with 20% SDS, and 1 M NaHCO3. The crosslinks were reversed overnight at 65?C, and then the DNA was purified using spin columns, and finally subjected to q RT-PCR.Results:To investigate the contribution of LSD1 to the migration and invasion of ovarian cancer HO8910 cells, we generated stable LSD1-knockdown(LSD1-KD) clones and LSD1-overexpressing(LSD1-OE) clones from the HO8910 cells. Total RNA and proteins were extracted from these stable cells treated with increasing doses of Dox for 24 or 48 h. Our results showed the mRNA and protein expression of the LSD1 gene was decreased in the LSD1-KD cells in a dose-dependent manner, whereas the levels of LSD1 mRNA and protein expression were increased in the LSD1-OE cells. To understand the effect of LSD1 expression on cell migration and invasion, we performed Transwell assays to measure the migratory capacity of these two transfected cell lines. The LSD1-KD cells displayed less migration and invasion in comparison with the control, whereas the LSD1-OE cells had a higher rate of migration and invasion as compared to the control.To further determine the role of LSD1 in cell migration, we utilized a known potent inhibitor, TCP, to suppress the demethylase activity of LSD1 in HO8910 cells. Inhibition of LSD1 decreased the migration activity of the HO8910 cells in a dose-dependent manner. Taken together, these data suggest that LSD1 is essential for cell migration and invasion in HO8910 ovarian cancer cells.As epithelial to mesenchymal transition(EMT) is involved in tumor migration and invasion, we examined the expression of several EMT markers in LSD1-KD and LSD1-OE HO8910 cells. We found that knockdown of LSD1 upregulated the expression of the epithelial marker E-cadherin and downregulated the expression of the mesenchymal markers N-cadherin, Vimentin, and MMP-2. LSD1 knockdown also caused a decrease in the expression of the transcription factor Snail. Furthermore, inhibition of LSD1 induced an increase in E-cadherin expression and a decrease in the expression of N-cadherin, Vimentin, MMP-2, and Snail in a dose-dependent manner. On the contrary, overexpression of LSD1 induced a decrease in E-cadherin expression, with a concomitant increase in the expression of N-cadherin, Vimentin, MMP-2, and Snail in HO8910 cells.Given that knockdown of LSD1 accompanied by upregulation of E-cadherin at the transcriptional level and inhibition of migration of ovarian cancer cells, we speculated that LSD1 could enhance the migration by downregulating E-cadherin expression via demethylation of H3K4me2, a major substrate of LSD1 in ovarian cancer cells. To confirm this speculation, Ch IP assays were performed in LSD1-KD HO8910 cells incubated with anti-H3K4me2 antibody. Quantitative analysis indicated that the enrichment of H3K4me2 at the promoter of the E-cadherin gene was significantly higher in LSD1-KD cells than in control cells. Collectively, our data reveal that the expression of LSD1 causes a decrease in H3K4me2 levels at the E-cadherin promoter, reduces E-cadherin expression, and consequently contributes to the migration of HO8910 cells.Conclusions:Taking all these pieces of evidence together, we are able to show that knockdown of LSD1 impairs the migration and invasion of HO8910 cells by regulating EMT, while overexpression of LSD1 has a converse effect on cell migration. By demethylating H3K4me2 at the E-cadherin promoter, LSD1 downregulates the E-cadherin expression, and contributes to the metastasis of HO8910 cells. Our results suggest that LSD1 may be a potential therapeutic target for metastatic ovarian cancer.