| Gastric cancer(GC)is a highly aggressive cancer and is the third leading cause of cancer-related deaths worldwide.The world’s GC cases and deaths mainly occur in east Asian countries such as China and Japan.Of note,the majority of worldwide GC deaths annually occur in China,which accounts for 45.0%of worldwide deaths.GC patients are often diagnosed at advanced stages due to the absence of specific symptoms.Despite the availability of new diagnostic techniques and an increase in the early detection rate,poor prognosis and survival for GC patients remains serious health problem.It urgently requires the further study of the molecular mechanism and the clinical characteristics of GC,and provides a theoretical basis for the diagnosis and personalized treatment of GC.In recent years,epigenetics has been widely studied in the development of tumors.Histone lysine-specific demethylase 1(LSD1)is the first reported histone demethylase as a member of amine oxidase superfamily.And it specifically removes mono-or dimethylated histone H3 lysine 4(H3K4)and H3 lysine 9(H3K9)and also demethylates other nonhistone substrate.LSD1 has been reported to be overexpressed in many malignant tumors.Abrogation of LSD1 by RNAi or various kinds of inhibitors has been shown to effectively treat those cancers.TGF β1(Transforming growth factor β1)is a primary inducer of EMT and it promote proliferation and migration of cancer cells.We have previously reported that LSD1 inhibitors can decrease cell migration and invasion in gastric cancer cell line MGC-803 by stimulating the epithelial marker E-cadherin and downregulating the mesenchymal marker N-cadherin,which are downstream of TGF β1.Based on the above,we hypothesized that LSD1 may play a vital role in gastric cancer cell mobility and metastasis by involved in TGF β1 pathway.The detailed work was listed as below:1.Analysis of the expression correlationship between TGF β1 and LSD1 in human gastric cancer tissues.We collected gastric cancer tissue specimens from 235 patients diagnosed with gastric cancer and performed immunohistochemical staining to examine the expression of LSD1 and TGF β1.After staining,the immunostained sections were digitally scanned and analyzed,and the clinicopathological data indicated 64.8%of tumors were accompanied with lymph node metastases.Moreover,we determined the correlationship between LSD1 and TGF β1 expression with clinicopathologic features by Pearson’s correlation,and the expression of TGF β1 was significantly correlated with LSD1 expression in metastasis specimens,while there was no significant correlation between the expression of LSD1 and TGF β1 in non-metastasis patients.Also,the expression of LSD1 is significantly correlated with TGF β1 in the poorly differentiated and moderately differentiated tumors.Based on the above findings,we suppose that LSD1 and TGF(31 affect each other’s expression in specific states in gastric cancer cells.2.Regulation of TGF β1 on LSD1 in gastric cancer cell lines and a benign gastric epithelial cell line.Human gastric cancer cell line,including MGC-803,HGC-27 and SGC-7901,were treated with different concentrations of TGF β1,which significantly increased the expression levels of LSD1.However,TGF β1 did not significantly alter LSD1 expression in the benign gastric epithelial cell,GES-1.Furthermore,LSD1 expression levels in tumor tissues were also significantly increased by TGF β1 in MGC-803 cells in vivo.Thus,TGF β1 has distinct effects on the induction of LSD1 expression in gastric cancer cell lines compared to benign gastric epithelial cells.3.Mechanism study of TGF β1 regulating LSD1 expression;We investigated the possible involvement of the SMAD dependent canonical pathway in TGF(31 induced LSD1 expression.The result indicated that SMAD2/3 phosphorylation was significantly increased by TGF(31,and SMAD3 inhibitor significantly inhibited TGFβ1-induced SMAD3 phosphorylation.However,TGF β1-induced LSD1 overexpression was not abrogated by SMAD3 inhibitor.This finding prompted us to focus on the non-canonical pathway for the induction of LSD1 expression by TGF β1.We evaluated the activation of ERK1/2 and p65 in MGC-803 cells after incubation with TGF β1.And ERK1/2 and p65 can be phosphorylated after incubation with TGFβ1,and promoted the nuclear translocation of p65.When ERK1/2 phosphorylation were abrogated by ERK1/2 siRNA or ERK1/2 inhibitor in MGC-803 cells,overexpression of LSD1 induced by TGF β1 was also blocked.Similarly,cells were incubated with p65 siRNA or NF-κB inhibitor BAY 11-7085,expression of LSD1 was constant,even in the presence of TGF β1.Our further ChIP experiment indicated that TGF β1 significantly enhances p300 binding to the promoter of LSD1,and the TGF β1-induced increase in promoter activity was significantly decreased by preincubating cells with BAY 11-7085,tangeretin and the p300 inhibitor,C646.These results support the concept that the TGF β1-induced overexpression of LSD1 is mediated by the sequential activation of ERK1/2-NF-κB-p300 and LSD1 promoter activation.Following the incubation of GES-1 cells with TGF β1,ERK1/2 was dephosphorylated,nucleus translocation of p65 was also abolished,as opposed to MGC-803 cells,without altering the expression of LSD1.4.Mechanism study of LSD1 regulating TGF β1 expression.We generated a stable LSD1 knock down MGC-803 cell line by using lentivirus vector based on Cas9 technology.ChIP experiment indicated H3K9me2 at the promoter of TGF β1 was significantly accumulated in cells where LSD1 was knocked down in MGC-803 cells,which could contribute to the transcriptional repression and the reduced secretion of TGF β1 in culture media.Significant correlations were found between LSD1 and TGF β1,vimentin,N-Cadherin in gastric adenocarcinoma with metastasis specimens,but not in normal gastric specimens adjacent to the gastric carcinoma.RNA-Sequence analysis for the MGC-803 and LSD1 knockdown MGC-803 cell lines indicated there 318 differential expressed genes,which included the TGF β signaling pathway and multiple signaling pathways by the KEGG annotation.This result further demonstrated LSD1 plays an important role in the regulation of gastric cancer cell metastasis.5.Mechanism study of LSD1 on gastric cancer metastasis.TGF β1 induced cell migration by up-regulating MMP2,N-cadherin,vimentin and downregulating E-cadherin.LSD1 knockdown partially reversed the TGF β1 induced expression of E-cadherin,whereas expression of N-cadherin,MMP2 and vimentin were significantly reduced,which inhibited cell migration.Overall,the above results indicated that LSD1 is a key factor in TGF β1-induced cell migration.Subsequently,we investigated the effect of the loss-of-function of LSD1 on seeding lung metastasis.The data indicated metastasis of lung tumor cells was significantly reduced in animals injected with MGC-803/LSD1 knockdown cells compared with those injected with MGC-803 cells,and LSD1 knockdown improved the median survival of the mice.6.Role of estrogen on gastric cancer cells migration.Based on the role of LSD 1 in nuclear receptor-mediated tumor proliferation and metastasis and gender differences in gastric cancer incidence,in this study,we found estradiol E2 inhibited the migration of gastric cancer cell MGC-803,but has no effect on cell proliferation.Interestingly,E2 can promote the binding of ERa and LSD1 and down-regulate the expression of LSD1,and in addition,E2 increased ubiquitination of LSD 1 in the presence of the proteasome inhibitor MG-132.In conclusion,our data indicate that the expression of LSD1 is positively correlated with TGF β1 in metastasis specimens and the moderately and poorly differentiated gastric cancer tissues.Furthermore,TGF β1 promotes LSD1 expression by sequentially activating ERK1/2-NF-κB-p300,and LSD1 positively regulates TGF β1 expression by increasing the accumulation of H3K9me2 around the TGF β1 promoter in gastric cancer cell lines.The TGF β1-LSD1 amplification loop is critical for EMT in gastric cancer,which resulted in the progression of EMT and promoted cell migration.However,the activation LSD1 is not present in the human gastric mucosa epithelial cell line GES-1 due to TGF β1-induced inactivation of ERK1/2.We further determined the correlationship among LSD1,TGF β1,E-cadherin and vimentin in gastric metastasis specimens,and significant correlations were found between LSD1 and TGF β1 and vimentin.In contrast,the expression between LSD1 and E-Cadherin were significantly negative correlated.Furthermore,when LSD1 expression was abrogated,cell migration and spontaneous lung metastasis were suppressed in vitro and in vivo,and LSD1 knockdown partially inhibited TGF β1-induced cell migration by reversing expression of E-cadherin and N-cadherin.Besides,E2 can promote the binding of ERα and LSD1 and degradation via ubiquitination,which inhibited the migration of gastric cancer cell.The study above first discovered the correlation between the expression of LSD1 and TGF β1 in human gastric cancer tissues and a novel mechanism for metastasis in gastric cancer that was mediated by TGF β1 and LSD1.Meanwhile,the regulatory mechanism of LSD1 in inhibiting migration of gastric cancer cell by E2 was explored.These gave a strong support for targeting LSD 1 to serve as a new strategy for gastric cancer. |
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