| ObjectiveInvestigate the mechanism of Elf5 on TGF-β-EMT axis in prostate cancer.MethodsTo determine whether Elf5 was associated with EMT in prostate cancer, we analyzed the expression of Elf5, E-cadherin and N-cadherin by IHC and ImageJ program in 124 prostate cancer samples. To investigate whether Elf5 can regulate EMT in the context of prostate cancer, Elf5 expression was tested by Western Blot in a panel of prostate cancer cell lines firstly, and then Elf5 was silenced by siRNA in LNCaP cells and stably overexpressed by HA-tagged Elf5 cDNAs in 22Rv1 cells and the morphological change was observed in the presence or absence of TGF-β. At the same time, Western Blot was used to determine the E-cadherin and the EMT markers including N-cadherin, Snail1, Snail2 and ZEB1 in the LNCaP and 22Rv1 cells. Then the Sphere Assay and Migration Assay were applied to determine the anchorage-independent proliferation and migration ability in the LNCaP and 22Rv1 cells. To explored the role of Elf5 in regulating TGF-β-induced signal transduction.The TGF-β response elements CAGA12 or PAI-1 luciferase reporters was cotransfected with HA-tagged Elf5 into HEK293 cells and 22Rv1 cells, or with siElf5 into LNCaP cells respectively. These cells were treated with or without TGF-β and luciferase activity were measured. To investigated whether Elf5’s inhibition on TGF-βsignaling was mediated by suppressing SMADs, Western Blot of endogenous p-SMAD2, SMAD2, p-SMAD3, SMAD3 and Elf5 expression in LNCaP cells was conducted and confirmed in 22Rv1 cells. We next asked whether Elf5 might bind to SMAD3, HA-Elf5 was transiently cotransfected with Flag-vector, Flag-SMAD2,Flag-SMAD3 or Flag-SMAD4 into HEK293 cells which didn’t express endogenous SMAD3 and Elf5, then the immunoprecipitation was conducted and also verified in LNCaP cells which expressed endogenous SMAD3 and Elf5. Lastly, p-SMAD3 and TGF-β were also stained by IHC and positive p-SMAD3 was analyzed, and survival analysis was implemented between Elf5 or p-SMAD3 levels with disease outcome.ResultsElf5 can upregulate the E-cadherin and downregulate the N-cadherin,suggesting that Elf5 may repress EMT in prostate cancer tissues. Compared to 22Rv1 cells,significantly higher Elf5 expression was noticed in LNCaP cells. Elf5 results in the failure of mesenchymal morphogenesis, upregulation of EMT markers, spheres formation and migration in the presence of TGF-β in LNCaP and 22Rv1 cells,demonstrating that the effect on EMT of Elf5 was inversely correlated with TGF-β.Elf5 can repress the CAGA12 and PAI-1 reporeter luciferase activity in HEK293,LNCaP and 22Rv1 cells, suggesting it can inhibit TGF-β-induced signal transduction.Elf5 can repress SMAD3 phosphorylation in LNCaP and 22Rv1 cells and coimmunoprecipitated with SMAD3, but not SMAD2 or SMAD4 in HEK293 and LNCaP cells, which indicated that the mechanism of Elf5 blocking TGF-β signaling was binding to SMAD3. High proportion of p-SMAD3 was detected in Elf5-/TGF-βhigh tumors samples, and Elf5 loss is associated with increased metastasis in patients with TGF-β-positive prostate cancer by survival analysis. Elf5 combined with TGF-β status could serve as a promising marker of metastasis in prostate cancer.ConclusionElf5 is inversely correlated with EMT. Furthermore, Elf5 inhibits TGF-β-driven EMT via repressing SMAD3 phosphorylation by binding to it in prostate cancer cells.Moreover, Elf5 can be used as a biomarker of metastasis-free survival in patients with TGF-β-positive prostate cancer. |
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