SPINK1Promotes Epithelial-mesenchymal Transition Through EGFR Pathway In Prostate Cancer

Several studies have suggested overexpression of serine protease inhibitor Kazal type1(SPINKl) defines an aggressive molecular subtype of ETS fusion-negative prostate cancer (PCa) patients in western countries. However, how SPINK1contributes to PCa invasion and metastasis is largely unknown. Here we showed that SPINK1and EGFR were significantly co-overexpressed in a cohort of Chinese PCa patients (P=0.003). We further confirmed that SPINK1is an unfavorable prognostic factor in Chinese PCas (P=0.025). SPINK1induced epithelial-to-mesenchymal transition (EMT) in benign prostate RWPE cells, manifested by acquisition of mesenchymal morphology and markers as well as migration and invasion capabilities. Knockdown of SPINK1in22RV1PCa cells results in upregulation of E-cadherin and downregulation of vimentin. Our data further indicated that SPINK1-induced EMT is mediated by epidermal growth factor receptor (EGFR), in which MAPK/MEK/ERK pathway is mainly involved. Connective tissue growth factor (CTGF) might be an important downstream molecule of SPINKl-EGFR axis. In all, our results suggested that SPINK1promotes EMT through EGFR signaling pathway in PCa.Part I Incidence of SPINKl overexpression in prostate cancer tissues and the association of SPINKl overexpression with other moleculesObject:1. To study incidence of SPINK1overexpression in PCa tissues and the relation of SPINK1overexpression with poor prognosis. 2. To identify the association of SPINK1overexpression with other molecular markers.Results:1. In Chinese PCa, frequency of SPINK1overexpression is approximately7.6%. SPINK1overexpression was significantly associated with poor prognosis in prostate cancer.2. SPINK1overexpression exists exclusively with TMPRSS2/ERG gene fusion in Chinese PCa, which is the same as in PCa of western countries.3. EGFR overexpression occurred more frequently in SPINK1-positive cases compared to those of SPINK1-negative cases.4. SPINK1-positive cases tend to show reduced expression of E-Cadherin and increased expression of vimentin, although both of them did not reach statistical significance.Conclusions:In consistence with previous relative reports, SPINK1overexpression is exclusively present with ERG rearrangement in our cohort and SPINK1overexpression was significantly linked to overall survival of PCa patients. For the first time, we demonstrated that SPINK1and EGFR were significantly co-overexpressed. Lower expression of E-Cadherin and higher expression of vimentin in SPINK1positive cases suggest the association of SPINK1with EMT in PCa indirectly.Part Ⅱ SPINK1promotes EMT in vitroObject:1. To identify whether SPINK1can induce PCa cells switch from epithelial phenotype to mesenchymal phenotype.2. To study the regulation effect of SPINK1on expression of epithelial and mesenchymal protein markers in PCa cells.3. To confirm promotive effect of SPINK1on invasive and migratory capacity of PCa cells. Results:1. After treatment with rSPINKl, RWPE cells lost epithelial morphology and acquired mesenchymal morphology. The similar morphologic changes were also observed in LnCap and VCap cells after rSPINK1.2. Compared with the controls, decreased level of E-Cadherin and increased level of vimentin were identified in rSPINKl-treated RWPE, LnCap and VCap cells by western blotting. By contrast, knockdown of SPINK1significantly up-regulated expression of E-Cadherin and down-regulated expression of vimentin in22RV1cells. We observed the same alteration tendency of E-Cadherin and vimentin using laser scanning confocal microscope after immunofuorescent staining.3. Wound healing and transwell assays further demonstrated rSPINK1significantly increased the migratory and invasive capacity of RWPE cells while knockdown of SPINK1resulted in reduced migratory and invasive capacity of22RV1cells.Conclusions:For the first time, we provided substantial evidence to show that SPINK1could promote EMT in PCa.Part Ⅲ Study on mechanism of SPINKl-induced EMTObject:1. To identify whether SPINK1induced EMT through EGFR pathway and which of the three main downstream pathways of EGFR is involved in SPINKl-induced EMT.2. To search the key molecule provoking EMT which is regulated by SPINK1through EGFR pathway.Results:1. rSPINKl could phosphorylate EGFR, STAT3, Akt and ERK in RWPE cells. By contrast, knockdown of SPINK1downregulated phosphorylation level of EGFR, STAT3, Akt and ERK in22RV1cells.2. Western blot showed that alteration of EMT markers (E-Cadherin and vimentin) induced by rSPINK1was completely inhibited by the addition of AG1478and U0126, while AG490and LY294002could inhibit the alteration induced by rSPINK1to a lesser extent. 3. Wound healing assay showed that all these four inhibitors could efficiently reverse migratory capacity induced by rSPINKl in RWPE cells. Transwell assay showed that only AG1478and U0126could reverse invasive capacity induced by rSPINKl.4. We did not find alteration of snail, slug, zeb1, twist or (3-catenin at mRNA or protein level in RWPE cells after rSPINKl treatment, neither nor in22RV1cells transfected with SPINK1siRNA compared with their controls.5.Expression of CTGF is upregulated in RWPE cells treated with rSPINK1.Meanwhile, expression of CTGF is reduced in22RV1cells transfected with SPINK1SiRNA.6. Knockdown of CTGF in RWPE cells could partially reverse the change of E-Cadherin and vimentin as well as the enhanced migratory and invasive capacity induced by rSPINKl.7. AG1478and U0126could totally block the upregulation of CTGF in RWPE cells induced by rSPINK1while AG1478and LY294002could not.Conclusions:1.SPINK1induced EMT mainly through EGFR/MEK/MAPK/ERK signaling pathway in PCa cells.2. CTGF may play an important role in EMT induced by SPINK1as a target molecule of EGFR/MEK/MAPK/ERK signaling pathway.