HDAC9 Affects The Invasion And Migration Of Ovarian Cancer Cells Through The FOXO1/TGFβ Axis

Ovarian cancer is the most lethal gynecologic malignancy.As there are no specific symptoms at the early stage of ovarian cancer,most patients are diagnosed at stage III/IV when the tumor has metastasized throughout the peritoneal cavity.The relative five-year survival rate of patients with late-stage ovarian cancer is less than 30%.Ovarian cancer comprises of a series of tumors subtypes characterized by different tissue origins,histopathological features,clinical manifestations,and molecular events.The management of ovarian cancer patients is shifting from a "one-size-fits-all" to a molecularly-driven,histologically subtype-specific management strategy.Therefore,further studies are needed for identification of the prognosis of ovarian cancer patients and molecular signaling network complexities associated with tumor metastasis.Further in-depth study of the metastatic potential of primary ovarian cancer can promote the development of personalized therapies and the optimization of management of ovarian cancer patients.There are extensive epigenetic alterations in ovarian cancer.As a member of class IIa histone deacetylases,HDAC9(histone deacetylase 9)catalyzes the deacetylation of histones and non-histone substrates,which regulates a variety of key cellular processes,such as cell cycle,immune response,angiogenesis,and apoptosis.In a variety of tumors,HDAC9 functions as an oncogene.In triple-negative breast cancer and non-small cell lung cancer,aberrant HDAC9 expression promotes tumor cell migration and is associated with poor prognosis for patients.However,its roles in ovarian cancer remain unclear.The analysis based on human ovarian cancer tissues from 100 patients revealed a histological subtype-specific effect of HDAC9 on the prognosis of ovarian cancer patients.High expression levels of HDAC9 were associated with a poor prognosis for patients with high-grade serous ovarian cancer.In contrast,high expression levels of HDAC9 were associated with a higher survival rate for patients with non-serous ovarian cancer.HDAC9 may be a promising biomarker for judging the prognosis of ovarian cancer patients.FOXO1(forkhead box O1)has complex roles in the development of malignant tumors,and is considered as focus of attention in tumor research.Compared with normal tissues,the expression level of FOXO1 was significantly increased in epithelial ovarian cancer.And overexpressed FOXO1 is associated with poor prognosis for patients with epithelial ovarian cancer,but the exact mechanism remains unclear.Recent studies have shown that FOXO1 is able to bind to the TGFβ(transforming growth factor β)promoter and promote the expression of TGFβ,which is involved in the regulation of tumor metastasis and chemoresistance,suggesting that FOXO1 may contribute to the malignant progression of ovarian cancer by regulating the expression of TGFβ.The Wnt/β-catenin signaling pathway is a classical tumor metastasis-related pathway.β-catenin is the core transcriptional coactivator of the classical Wnt/β-catenin signaling pathway.After transferring from the cytoplasm to the nucleus,β-catenin promotes the expression of target genes by binding with TCF/LEF.Acetylation can regulate the transcriptional activity of FOXO1 and β-catenin via altering their subcellular localization.However,in ovarian cancer,the effects of HDAC9 on the subcellular localization of FOXO1 and β-catenin is still unclear.We believe that exploring the effects of HDAC9 on the subcellular localization and transcriptional activity of FOXO1 and β-catenin may be useful to elucidate the mechanisms by which HDAC9 affects the prognosis of ovarian cancer patients.In this study,epigenetic regulation was used as point of penetration,basing on clinical patient specimens,ovarian cancer tissue c DNA microarrays and human ovarian cancer cell lines SKOV3 and A2780,to explore the effects of HDAC9 on the transcriptional activity and subcellular localization of FOXO1 and β-catenin,elucidating the regulatory mechanism of HDAC9 on the invasion and migration of ovarian cancer cells.And provide new clues for the treatment of ovarian cancer by targeting HDAC9.Methods1.Immunohistochemical staining was used to detected the expression of HDAC9 in 100 cases of ovarian cancer patients.The correlation between HDAC9 expression and overall survival of ovarian cancer patients was detected by Kaplan-Meier survival analysis.The correlation between HDAC9 expression and the risk of recurrence,and tumor metastasis were analyzed by a two-tailed,unpaired student t test.2.A2780(endometrioid ovarian cancer cell line)and SKOV3 cells(serous ovarian cancer cell line)were transfected with HDAC9-sh RNA plasmids(sh HDAC9-1,2,3)and HDAC9 overexpression constructs(pc DNA3.1-HDAC9).The protein expression of HDAC9 was detected by Western blot.The m RNA expression of HDAC9 was detected by q RT-PCR.3.To explore the effects of HDAC9 on the proliferation and apoptosis of ovarian cancer cells.A2780 and SKOV3 cells were transfected with empty vector(NC),HDAC9-sh RNA plasmids(sh HDAC9-1/2),or pc DNA3.1-HDAC9 for 24 h.Clonogenic assays was used to detect the proliferation of A2780 and SKOV3 cells.Annexin V/PI staining was used to detect apoptosis of A2780 and SKOV3 cells.4.To explore the effect of HDAC9 on the migration and invasion of ovarian cancer cells.A2780 and SKOV3 cells were transfected with NC,sh HDAC9-1/2,or pc DNA3.1-HDAC9 for 24 h.The in vitro wound-healing assay was used to detect the migration of A2780 and SKOV3 cells.Transwell assay was used to detect the invasion of A2780 and SKOV3 cells.The protein level of E-cadherin,N-cadherin,vimentin,MMP2 and MMP9 was detected by Western blot.5.Microarrays from 80 ovarian cancer patients in the GEO database were used to detect the differentially expressed genes between patients with high HDAC9 expression and patients with low HDAC9 expression,and performed KEGG enrichment analysis on these genes.6.To explore the effects of HDAC9 on the subcellular localization of FOXO1 in ovarian cancer cells.A2780 and SKOV3 cells were transfected with NC,sh HDAC9-1/2,or pc DNA3.1-HDAC9 for 24 h.The protein level of FOXO1 was detected by Western blot.Immunofluorescence assay was used to detect the effects of HDAC9 on the subcellular localization of FOXO1 in A2780 and SKOV3 cells.After nucleus isolation,the protein level of FOXO1 was detected by Western blot.7.Microarrays from 80 ovarian cancer patients in the GEO database were used to detect the differentially expressed genes between patients with high FOXO1 expression and patients with low FOXO1 expression,and performed KEGG enrichment analysis on these genes.8.To explore the effects of HDAC9 on TGFβ signaling pathway in ovarian cancer cells.A2780 and SKOV3 cells were transfected with NC,sh HDAC9-1/2,or pc DNA3.1-HDAC9 for 24 h.Western blot was used to detect the effects of HDAC9 on the protein level of TGFβ,SMAD2/3 and P-SMAD2/3.The protein level of snail and slug was detected by Western blot.9.To explore the effects of HDAC9 on the subcellular localization of β-catenin in ovarian cancer cells.A2780 and SKOV3 cells were transfected with NC,sh HDAC9-1/2,or pc DNA3.1-HDAC9 for 24 h.The protein level of β-catenin was detected by Western blot.Immunofluorescence assay was used to detect the effect of HDAC9 on the subcellular localization of β-catenin in A2780 and SKOV3 cells.Western blot was used to detect the effects of HDAC9 on the protein level of ac-lys 49 β-catenin.Results1.According to the histological subtypes,patients were divided into “high-grade serous ovarian cancer group” and “non-serous ovarian cancer group”.High expression levels of HDAC9 were associated with a poor prognosis for patients with high-grade serous ovarian cancer.In contrast,high expression levels of HDAC9 were associated with a higher survival rate for patients with non-serous ovarian cancer.2.The expression of HDAC9 was positively correlated with the risk of relapse in patients with high-grade serous ovarian cancer.3.The expression of HDAC9 was negatively correlated with the risk of relapse and metastasis in patients with non-serous ovarian cancer.4.HDAC9 was successfully knocked down or overexpressed.Consequently,we chose sh HDAC9-1,2 for subsequent experiments.5.Results of clonogenic assays and Annexin V/PI staining have showed that HDAC9 does not significantly affect the proliferation and apoptosis of human serous ovarian cancer SKOV3 cells and human non-serous ovarian cancer A2780 cells.6.Overexpressed HDAC9 can promote the migration and invasion of SKOV3 cells and the expression of N-cadherin,vimentin,MMP2 and MMP9,and inhibit the expression of E-cadherin.Downregulated HDAC9 inhibits the migration and invasion of SKOV3 cells and the expression of N-cadherin,vimentin,MMP2 and MMP9,and promotes the expression of E-cadherin.7.Overexpressed HDAC9 can inhibit the migration and invasion of A2780 cells and the expression of N-cadherin,vimentin,MMP2 and MMP9,and promote the expression of E-cadherin.Downregulated HDAC9 promotes the migration and invasion of A2780 cells and the expression of N-cadherin,vimentin,MMP2 and MMP9,and inhibits the expression of E-cadherin.8.In serous ovarian cancer,the differentially expressed genes between patients with high HDAC9 expression and patients with low HDAC9 expression were enriched in FOXO signaling pathway upon KEGG pathway analysis.However,in non-serous ovarian cancer,the differentially expressed genes between patients with high HDAC9 expression and patients with low HDAC9 expression were not enriched in FOXO signaling pathway.These genes were enriched in Wnt signaling pathway.9.HDAC9 did not control the expression of FOXO1 in A2780 and SKOV3 cells.In SKOV3 cells,downregulated HDAC9 induced FOXO1 translocation to the cytoplasm,and HDAC9 upregulation increased the nuclear accumulation of FOXO1.However,HDAC9 did not control the subcellular localization of FOXO1 in A2780 cells.10.In serous ovarian cancer,the differentially expressed genes between patients with high FOXO1 expression and patients with low FOXO1 expression were enriched in TGFβ signaling pathway upon KEGG pathway analysis.However,in non-serous ovarian cancer,the differentially expressed genes between patients with high FOXO1 expression and patients with low FOXO1 expression were not enriched in TGFβ signaling pathway.11.In SKOV3 cells,overexpressed HDAC9 promoted the expression of TGFβ and increased the protein level of P-SMAD2/3,promoting the expression of snail and slug.Downregulated HDAC9 inhibited the expression of TGFβ and decreased the protein level of P-SMAD2/3,leading to the inhibition of snail and slug expression.12.HDAC9 did not control the expression of TGFβ and the protein level of PSMAD2/3 in A2780 cells.However,downregulated HDAC9 promoted the expression of snail and slug in A2780 cells.Overexpressed HDAC9 lead to the inhibition of snail and slug expression.13.In A2780 cells,overexpressed HDAC9 decreased the acetylation of β-catenin K49 and induced β-catenin translocation to the cytoplasm.In contrast,downregulated HDAC9 increased the nuclear accumulation of β-catenin and the acetylation of β-catenin K49.14.In SKOV3 cells,overexpressed HDAC9 decreased the acetylation of β-catenin K49 and downregulated HDAC9 increased the acetylation of β-catenin K49.However,HDAC9 had no significant effect on the subcellular localization of β-catenin in SKOV3 cells.Conclusion1.HDAC9 expression levels were negatively correlated with the prognosis for patients with high-grade serous ovarian cancer.In contrast,HDAC9 expression levels were positively correlated with the prognosis for patient with non-serous ovarian cancer.HDAC9 may be a potential target for individualized treatment of patients with different histological subtypes of ovarian cancer,and a promising biomarker for judging the prognosis of ovarian cancer patients.2.In serous ovarian cancer cells,overexpressed HDAC9 can increase the nuclear localization of FOXO1 and promote the expression of TGFβ,activating EMT and promoting cell migration and invasion.On the contrary,in non-serous ovarian cancer cells,overexpressed HDAC9 decreased the acetylation of β-catenin K49 and induced β-catenin translocation to the cytoplasm,inactivating EMT and inhibiting cell migration and invasion.