HMGA2-FOXL2 Axis Regulates Metastases And Epithelial-to-Mesenchymal Transition Of Chemoresistant Gastric Cancer

【Background】Gastric cancer is one of the most common malignancies in China with high morbidity and mortality.Chemotherapy plays fundamental roles in management of advanced stage gastric cancer and is effective in reducing the tumor burden.Tumor cells usually show initial response to chemotherapeutics as is marked by rapid tumor shrinkage,but multidrug resistance might occur during the repeated cycles of chemotherapy.Intriguingly,the chemo-resistance could accelerate the tumor progression and local invasion and systemic metastasis may occur soon,suggesting a connection between chemo-resistance and metastasis.Epithelial-mesenchymal transition(EMT),as a critical step in initiating tumor metastasis,drives malignant progression of tumors.Recent studies showed that in addition to promoting metastasis,EMT is also involved in chemoresistance.On one hand,tumor cells with EMT are usually resistant to various chemotherapeutic agents;on the other hand,chemoresistant cancer cells usually exhibit EMT-like characteristics.Therefore,exploring the mechanisms that conferred the EMT phenotype towards chemoresistant gastric cancer cells will be beneficial for targeting chemoresistance and metastasis simultaneously and improving the survival for patients with gastric cancer.【Objectives】 1.To clarify the roles that EMT play on the increased metastatic capability of chemoresistant gastric cancer cells;2.To screen and verify potential pathway that confer the EMT phenotype and metastases of chemoresistant gastric cancer cells;3.To explicit the clinical significance of the expression of these EMT-associated proteins in gastric cancer tissues.【Methods】1.Transwell and tail vein injection metastasis nude mouse model were used to compare the metastatic potential of chemoresistant and parental gastric cancer cells;quantitative real-time PCR(q RT-PCR),western blot and immunofluorescent(IF)staining were employed to detect the expression of EMT markers;c DNA array and bioinformatics analysis were combined to screen pro-EMT candidates in chemoresistant gastric cancer cells;immunohistochemistry(IHC)was used to evaluate the correlation between the expression of the pro-EMT candidates and E-cadherin in gastric cancer tissue.2.Transient or stable loss-of-function and gain-of-function cell models were established using small interfering RNAs(si RNAs)targeting HMGA2 and FOXL2 or HMGA2 and FOXL2 overexpression and small hairpin RNA(sh RNA)vectors.Transwell assay and an orthotopic gastric cancer xenograft nude mouse model were used to evaluate the metastatic potential of corresponding gastric cancer cells both in vitro and in vivo;western blot and q RT-PCR were employed to detect the expression of EMT markers;The expression levels of HMGA2 and FOXL2 after chemotherapeutic exposure were examined using western blot.3.Western blot and q RT-PCR were used to detect HMGA2 or FOXL2 after manipulating their expression;changes in the in vitro and in vivo metastatic capability were determined with the Transwell assay and the orthotopic transplantation nude mouse model.4.Western blot and q RT-PCR were employed to detect alteration of protein and m RNA levels in E2F1 or Rb loss-of-function and gain-of-function cell model;Promoter Luciferase Report Assay was used to examine transcription of FOXL2 by E2F1;immunoprecipitation(IP)together with western blot was used to check protein-protein interactions.5.c DNA array was utilized to screen downstream genes of FOXL2;ITGA2 expression in FOXL2 silenced or overexpressed cells were detected with western blot and q RT-PCR;western blot,q RT-PCR and IF were applied to examine EMT markers expression in ITGA2 loss-of-function and gain-of-function cells;Transwell assay and the orthotopic gastric cancer xenograft model were used to examine the effects of ITGA2 on cell motility.6.HMGA2,FOXL2 and ITGA2 in primary and metastatic gastric cancer tissues were detected using IHC and the staining intensity was compared.The sole or combined prognostic value of HMGA2,FOXL2 and ITGA2 in gastric cancer was evaluated using the Kaplan-Meier survival analysis.【Results】 1.The in vitro and in vivo metastatic assays showed that chemoresistant gastric cancer cells were more invasive than the parental cells;q RT-PCR and western blot results showed that the expression of the epithelial marker E-cadherin was decreased while the expression of the mesenchymal marker Vimentin was increased in the chemoresistant gastric cancer cells,suggesting the development of EMT.423 genes were found to be elevated in chemoresistant gastric cancer cells with a cut-off of 2-fold change.The list was narrowed to 8 by a bioinformatics strategy focusing on transcription related function,and further to 4 after browsed the prognosis information in the Kaplan-Meier Plot database.Finally,HMGA2 and FOXL2 were found to be negatively correlated with E-cadherin expression in gastric cancer tissues.2.Downregulation of HMGA2 and FOXL2 respectively impaired the metastatic capability of chemoresistant gastric cancer cells both in vitro and in vivo,and promoted E-cadherin expression while inhibited Vimentin expression;conversely,the overexpression of either HMGA2 or FOXL2 in the parental gastric cancer cells promoted cell metastasis,inhibited E-cadherin expression and promoted Vimentin expression;interestingly,short time exposure of chemosensitive gastric cancer cells increased the expression of HMGA2 and FOXL2.3.Interfering endogenous HMGA2 expression or ectopic HMGA2 could inhibit or increase FOXL2,suggesting that FOXL2 may be the downstream target of HMGA2;meanwhile,FOXL2 was proved to be essential for the pro-EMT function of HMGA2.4.q RT-PCR results showed that the classical E2F1 target genes expression were elevated in the chemoresistant gastric cancer cells,indicating that the transcription activity of E2F1 was increased in these cells;when E2F1 expression was manipulated,FOXL2 displayed corresponding alterations;Luciferase Reporter Assay suggested that E2F1 could directly bind to the promoter region of FOXL2;IP together with Luciferase assay showed that interaction between HMGA2 and p Rb promoted the transcription of FOXL2 by E2F1.5.ITGA2 was identified as a downstream target of FOXL2 in a c DNA array screening and it was confirmed in FOXL2 silenced or overexpressed cell models;inhibiting ITGA2 expression in chemo-resistant gastric cancer cells led to impaired cell motility and reversed the EMT phenotype,while its overexpression in the parental cells promoted cell motility and EMT;inhibiting ITGA2 expression antagonized the pro-EMT effect of FOXL2.6.HMGA2,FOXL2 and ITGA2 expression were elevated in metastatic gastric cancer tissues compared with the primary cancer tissues;the expression levels of HMGA2,FOXL2 and ITGA2 were also higher in metastatic gastric cancer tissues than those in metastatic-free gastric cancer tissues;survival analyses showed that increased expression any of HMGA2,FOXL2 or ITGA2 is a sign of poor prognosis,and their combination could better distinguish gastric cancer patients with different survival status.【Conclusion】 In the present study,we elucidated a novel pathway that could promote metastasis and EMT of chemoresistant gastric cancer cells.Chemotherapeutics exposure could induce the expression of HMGA2,and HMGA2 then promots FOXL2 expression by facilitating E2F1 transactivity,leading to metastasis and EMT,which is executed by the integrin subunit ITGA2.This study identified the HMGA2–FOXL2–ITGA2 axis as a potential therapeutic target for those gastric cancer cases with metastasis and reduced sensitivities to chemotherapy.