The Regulatory Effects Of LSD1and Its Specific Inhibitors In The Development Of Gastric Cancer In Vitro And In Vivo And The Underlying Mechanisms

Epigenetics is the study of heritable changes in gene activity that are not caused bychanges in the DNA sequence, inculding DNA methylation, histone modification,chromatin remodeling, gene silencing and RNA editing. Among them, modificationof histone could regulate the chromatin structure and upregulate or downregulate theexpression of genes. LSD1(Histone lysine specific demethylase1) was the firstidentified histone demethylase, which can demethylate mono-, di-methylation ofH3K4and H3K9as well as other non-histone substrates, which result in theregulation of their activity. LSD1is overexpressed in numbers of tumors,downregulate the LSD1expression with RNAi or inhibit LSD1activity could inhibitthe cancer progression. Hence, obtaining selective and potent LSD1inhibitor is apromising way and hot point for cancer treatment.As a homology protein of MAO-A/B(Monoamine oxidase A/B) with FAD as cofactor,LSD1could react against its substrate and remove the methyl group of lysine at thespecific site of histone. Hence, some of the MAO inhibitors could also be applied toinhibit LSD1activity, such as2-PCPA(Tranylcypromine). In2010, it was reportedthat “Click chemistry” was applied for the synthesis of MAO-A inhibitors, hence, wesuppose that1,2,3-triazole containing MAO-A inhibitor could also inhibit LSD1activity. Meanwhile, dithiocarbamate attracted our eyes due to its characters of lowtoxicity, anti-tumor, antibacterial activities of lead compounds. Hence, based on ourexisted compound library, one family of1,2,3-triazole-dithiocarbamate derivateswere designed and synthesized, and their LSD1inhibitory effect were evaluated inorder to have the potent and selective LSD1inhibitor. The in vitro, in vivo anti-cancereffect, cancer migration and invasion inhibitory effect and the mechanism were alsoinvestigated as well as the mechanism study. The detailed work was listed as below:1) Establishment of LSD1inhibitor screening model, design and synthesis of LSD1inhibitor;With molecular biology method, procaryotic expression vector of LSD1wascontructed, the LSD1recombinant was induced and purified. Meanwhile, LSD2, another homology protein of LSD1, was also produced for the selective study. As onemolecular hydrogen peroxide can be produced during the demethylation of LSD1andLSD2, the fluorence probe Amplex Red was applied to quantify the amount ofhydrogen peroxide, which indicated the activity of LSD1and LSD2as well as theinhibitory effect of our candidate compounds to establish the screening model.Meanwhile,691,2,3-triazole dithiocarbamate hybrids were designed and synthesized,their activities against LSD1were also evaluated. With the aid of SAR(Sturecture-Activity Relation) study, one group novel LSD1inhibitor was identified.The most potent one is compound30，could inhibit LSD1activity with IC50=2.11μM.Hence, the following investigation was performed with compound30mainly.2) Mechanism study of LSD1inhibitor and its selectivity study in recombinantlevel;The recombinant level selective study indicated that compound30could specificallyinactivate LSD1with weak inhibitory against LSD2and hardly inhibition againstMAO-A/B. With dilution assay and dialysis experiment, we found that compound30could form non-covalent bond with LSD1and reversible inhibit it. With enzymekinetic study, moledular docking and molecular dynamic study, we found thatcompound30could penetrate into the cavity of FAD in LSD1by forming severlhydrogen bonds with amino acid of LSD1, which means compound30may be acompetitive inhibitor agsinst LSD1cofactor FAD. On the other hand, compound30may also also bind to an allosteric sites of LSD1, resulting in an apparent change inbinding affinity of another ligand at a distinctly different site, but specific mechanismon how does compound30act on LSD1still needs to be clarified in further structureanalysis.3) Inhibitory effect of compound30against LSD1in cell level, and its in vitro andin vivo anti-cancer effect;In gastric cancer cell line MGC-803, we found the LSD1inhibitor compound30could induce the expression of LSD1substrate H3K4me1、H3K4me2and H3K9me2,and specifically inhibit MGC-803and HGC-27, LSD1overexpressed gastric cancercell line, proliferation. Meanwhile, almost no proliferation inhibitory effect on normalgastric epithelial cell line GES-1and gastric cancer cell line SGC07901can be observed as their LSD1expression level is lower. Compound30could also inducecell apoptosis and arrest the cell cycle at G2/M. Our further experiment indicated thatcompound30was efficacious in inhibiting the growth of LSD1overexpressinggastric tumor in vivo but no obvious global toxicity.4) Inhibitory effect of LSD1inhibitor against cell migration, invasion and themechanism sutdy;Wound healing assay and transwell experiment indicated that compound30couldinhibit the cell migration and invasion. Further immunoprecipitation experimentindicated that compound30could interrupt the interaction between LSD1and snai1,which result in the activation of E-Cadherin promoter, and induce the overexpressionof epithelial cell biomarker E-Cadherin. On the other hand, compound30could alsoinhibit the mesenchymal cell biomarker N-Cadherin expression. Elsia experiment alsoindicate that compoud30can inhibit TGF β1expression in MGC-803cell. Hence, wesuppose that compound30could inhibit LSD1activity as well as the amount ofTGFβ1to inhibit EMT(epithelial-mesenchymal transition), which resulted in theinhibition of cell migration and invasion.5) Regulation and the mechanism study of TGF β1on LSD1in MGC-803andGES-1；Transforming growth factor beta (TGF β) is a group of peptides with similar structurebut different function. TGF β1is the most abundant one and plays a key role incancer development and progression. In benign tissues, TGF β1acts as anti-cancerfactor, which could inhibit the cancer cell growth; but in cancer tissues, TGF β1actsas oncogene in the late stage of cancer and promotes the cancer progression. In thisproject, interaction between TGF β1and LSD1was investigated. In MGC-803,different dosages of TGF β1could upregulate LSD1time dependently. ERK inhibitortangeretin, NF-κB inhibitor bay11-7085, p300inhibitor C646combined with TGF β1separately could inhibit TGF β1induced LSD1expression. Hence, we suppose thatTGF β1induced LSD1overexpression was mediated by ERK, NF-κB and p300. Asp300could act at LSD1promoter, our further dual luciferase reporter experimentindicated that ERK, NF-κB and p300mediated LSD1promoter activation by TGF β1.Hence, we suppose that TGF β1could activate ERK by phosphorylation, then NF-κB was activated by ERK and its subunit p65was tranlocated into nucleus andphosphorylated, phosphorylation of p65could recruit p300to LSD1promoter, whichresulted in the activation of LSD1promoter and upregulated LSD1expression. Onthe other hand, TGF β1could inactivate ERK, NF-κB and dephosphorylate p65withno obvious change on LSD1expression.Based on the stated work,691,2,3-triazole-dithiocarbamate derivates weresynthesized, and evaluated on their LSD1inhibitory effect. The most potent one,compound30, could selective and reversible inhibit LSD1and may competitiveinhibit LSD1against cofactor FAD. In cell level, compound30selectively inhibitedLSD1overexpressed gastric cell lines proliferation, and prevent cell migration andinvasion by suppressing EMT. Our further research revealed that in gastric cancer cellline MGC-803, different dosages of TGF β1can induce LSD1overexpression timedependently by activating ERK, NF-κB and phosphorylation of p65, which lead tothe recruitment of p300to the promoter of LSD1, and result in the overexpression ofLSD1. On the other hand, TGF β1can inactive ERK, NF-κB and dephosphorylationof p65, but there is no expression change of LSD1with TGF β1treatment.This research identified novel selective LSD1inhibitor with dual role on cellproliferation and cell migration inhibitory effect. Meanwhile, pleiotropic mechanismof TGF β1on LSD1in gastric cancer cell line MGC-803and normal gastric epithelialcell line GES-1was revealed. All these above give a strong support for the LSD1targeted anti-cancer drug discovery and mechanism for the malignant proliferation.