| As the first identified histone lysine demethylase,lysine specific demethylase 1(LSD1)utilizes the flavin adenine dinucleotide(FAD)as a cofactor to specifically demethylates di-and monomethylated histones H3K4 and H3K9.Wnt signaling pathway regulates many cell functions,including proliferation,migration and cell death.And an increasing body of evidence indicates that regulators controlling Wnt/β-catenin pathway are frequently dysregulated in human cancers owing to genetic and epigenetic defects.Until now,several articles have published the cooperation between LSD1 and some key proteins in Wnt/β-catenin pathway to promote EMT,cell proliferation in breast cancer,colon cancer.In the previous study,it was found that abrogation of LSD1 inhibited Wnt3 a secretion in male derived gastric cancer cell MGC-803,while it was not affected in female derived gastric cancer cells.Therefore,the aim of this study is to investigate whether Wnt3 a mediated LSD1 induced gastric cancer cell metastasis in a gender specific manner.1)The correlation analysis between Wnt3 a and LSD1,EMT related proteins in gastric cancer tissuesIn this study,the expression of Wnt3 a,LSD1 and EMT marker proteins(including Snail,N-Cadherin and Vimentin)were detected in 269 gastric cancer tissues by immunohistochemical staining.The correlation analysis results showed that the expression of Wnt3 a was strong significantly correlated with LSD1 expression in male and metastasis specimens while there was no significant correlation between the expression of LSD1 and Wnt3 a in female or in non-metastasis patients.Based on the above findings,we hypothesized that Wnt3 a and LSD1 may affect each other’s expression.To further explore the effect of gender,269 specimens were subject to immunohistochemical analysis to clarify the expression relationship among Wnt3 a,LSD1 and EMT markers in female and male derived gastric cancer tissue specimens.As indicated in results,in female derived gastric cancer tissue specimens,the correlationship between Wnt3 a and LSD1,Snail,N-Cadherin are not significant,suggesting that Wnt3 a may not impact on EMT in female derived gastric cancer cells.On the other hand,all these proteins were positively correlated with Wnt3 a in male derived gastric cancer tissue specimens.As LSD1 was reported to be overexpressed in gastric cancer,LSD1 lowexpressed and overexpressed gastric cancer tissues were selected and subjected to correlationship analysis between Wnt3 a and others.As shown in results,in male derived LSD1 overexpressed gastric cancer tissues,Wnt3 a performed stronger correlationship with LSD1,Snail,N-Cadherin and Vimentin.Therefore,we deem that LSD1 may interregulate with Wnt3 a and then contribute to EMT in male derived LSD1 overexpressed gastric cancer cells in a gender specific manner.2)Wnt3a-mediated LSD1 regulates Wnt/β-catenin pathway in gastric cancer cellsThe aforesaid research work of this subject showed that there was a significant positive correlation between the expression of Wnt3 a and LSD1 in male derived LSD1 overexpressed gastric cancer tissues.Therefore,we believe that there may be the impact of LSD1 on Wnt3 a.In this chapter,we will further investigate the role of LSD1 in the regulation of Wnt3 a and the effect of LSD1 on Wnt/β-catenin pathway in gastric cancer cell lines and gastric mucosal epithelial cells,in order to investigate whether this regulation occurs only in male derived LSD1 overexpressed gastric cancer cells.By Western blot,Elisa and RT-qPCR experiments,we found that when LSD1 was knocked down,Wnt3 a protein secretion in the cell culture medium were significantly decreased in MGC-803 cells,but not in MKN45,SGC-7901 and GES-1 cells while no significant change in Wnt3 a mRNA can be detected when LSD1 was knocked down in these cells.Similarly,amount of Wnt3 a was greatly increased when LSD1 was overexpressed in MGC-803 cells.Subsequent detection of proteins involved in Wnt/β-catenin pathway activation revealed that LSD1 knockdown decreased expression of PORCN and secretion of Wnt3 a as well as nucleus translocation of β-catenin and Snail in MGC-803 cells but not in MKN45 cells.The results of RT-qPCR showed that the mRNA level of Wnt/β-catenin pathway targets c-Myc and CyclinD1 were also decreased in MGC-803 cells,but not in MKN45 cells.These suggested that LSD1 knockdown inactivate the Wnt/β-catenin pathway and prevent nucleus translocation of Snail in MGC-803 cells.MGC-803 cells subcutaneous xenografts detected by immunohistochemical staining showed that deficiency of LSD1 led to low expression of Wnt3 a and β-catenin but up-regulated P-β-catenin in MGC-803 cells,suggesting that knockdown of LSD1 can also down-regulate the Wnt/β-catenin pathway in vivo.All of the above results indicated that LSD1 may regulate Wnt3 a secretion via PORCN and then activate Wnt/β-catenin pathway in male derived gastric cancer cell line MGC-803 cells.Therefore,we speculate that LSD1 may make further effect on EMT by Wnt/β-catenin pathway.3)The ability of LSD1 to mediate invasion and metastasis of MGC-803 by Wnt/β-catenin pathwayAt present,there are many reports that LSD1 is involved in cancer cells metastasis and invasion,the activity loss of LSD1 leads to the reversal of EMT process and then inhibits cells metastasis and invasion.Meanwhile,Wnt3 a is as an inducer of EMT to participate in the occurrence of diseases in a variety of cancers.Therefore,whether Wnt3a-mediated LSD1 promotes invasion and metastasis of gastric cancer cells was further explored in this chapter.By Elisa,Western blot,wound healing and transwell experiment,it was found that LSD1 overexpression promoted Wnt3 a secretion,metastasis and invasion as well as EMT in MGC-803.However,the PORCN inhibitor LGK974 disputes the secretion of Wnt3 a by preventing palmitoylation of Wnt3 a,and also prevents the accelerated invasion and EMT by LSD1 overexpression.Therefore,we believe that blockade of Wnt3 a signaling pathway can inhibit LSD1-induced cell metastasis and EMT in MGC-803,so Wnt3 a plays a very important role in this process.To further validate this conclusion,we established a mouse model of MGC-803 Control and MGC-803 LSD1-KD systemic transfer.After 7 weeks of observation,we found that LSD1 knockdown can inhibit the migration of MGC-803 in mice to some extent,meanwhile,prolong the survival time of the mouse.HE staining experiments further confirmed that LSD1 promoted lung metastasis and liver metastasis of MGC-803 in mice.Subsequent immunohistochemical staining experiments showed that LSD1 lowexpression reduced the expression of Wnt3 a,β-catenin,and the mesenchymal marker proteins,including N-Cadherin,Vimentin,and Snail.The above results suggested that LSD1 promotes metastasis and invasion of MGC-803 by the Wnt/β-catenin pathway.In summary,LSD1 may promote the secretion of Wnt3 a by regulating PORCN,and then activate Wnt/β-catenin pathway,subsequently upregulate Wnt pathway target genes c-Myc,CyclinD1 and Snail,thereby promoting the metastasis and EMT of male derived gastric cancer cell lines.This study explored the mechanism of Wnt3a-mediated LSD1 promoting gastric cancer cells metastasis by Wnt/β-catenin pathway in a gender specific manner,presented a new idea for personalized treatment of gastric cancer. |
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