The Mechanism Of TGF-β Transactivated EGFR Signal Pathway Induced Invasion And Metastasis In Breast Cancer Cells

Objectives:Breast cancer is currently recognized as a common tumor which threatens women’s health.But the detailed mechanism remains elusive.Many researches show that the signal pathway of EGF receptor(EGFR)and Transforming growth factor-beta(TGF-β)play a crucial role in tumor progression.TGF-β exerts a tumor-suppressive role in early stages of breast cancer,but it promotes cancer aggressive in advanced stages.This phenomenon is known as “TGF-β paradox” that has been widely investigated for decades.However,the underlying mechanism by which TGF-β shifts its role from a tumor suppressor to a cancer promoter remains undefined.Methods:1.Immunohistochemistry was used to explore the expression and clinical pathological parameter of TGF-β and relationship between TGF-β and EGFR expression and prognosis.2.QRT-PCR assay and Western blot were used to analyzed TGF-β-induced the m RNA and protein levels of EGFR,the m RNA levels of EGF、TGF-α and the phosphorylation of EGFR,p-Smad3,p-ERK 1/2.3.Inhibition of EGFR by si RNA and molecular inhibitor Erlotinib in MDA-MB-231 and T47 D cell lines,then Transwell assay and in vivo animal assay were used to explore TGF-β-induced cell migration,invasion and metastasis ability in vitro and in vivo.4.Bioinformatics method was used to analyze EGFR promoter region,confirm the transcription factor and the putative binding sites of EGFR promoter,and construct vector: the first vector p GL3-E1 containing Smad3-and Sp1-binding sites;the second one p GL3-E2 comprising the Sp1-binding sites;the third vector p GL3-E3 composed of Smad3-binding sites.5.Chromatin Immunoprecipitation and Dual-luciferase reporter assay were used to explore the regulation mechanism of EGFR promoter with TGF-β treatment.6.PCR-mediated site-directed mutagenesis was used to construct EGFR promoter mutation plasmids.Then dual-luciferase reporter assay was used to confirm the activity of mutated EGFR promoter.7.Knockdown Smad3 and(or)Sp1 by si RNA.Dual luciferase report assay was performed to confirm the effect of Smad3-and(or)Sp1-binding sites in TGF-β-induced EGFR promoter luciferase activity.8.QRT-PCR assay and Western blot were used to explore TGF-β-induced m RNA and protein levels of EGFR using Sp1 si RNA or inhibitor.9.QRT-PCR assay and Western blot were used to explore the crucial role of Erk/Sp1 in TGF-β-induced EGFR upregulation.10.QRT-PCR assay and Western blot were proformed to consider the role of Smad3 in TGF-β-mediated EGFR upregulation.11.Inhibition the expression of Smad3 and(or)Sp1,then q RT-PCR assay and Western blot were used to examine EGFR expression in breast cancer cells which were treated by TGF-β.12.Transwell-based assay was performed to further confirm that the effect of Smad3 and Sp1 in TGF-β-induced breast cancer cell migration and invasion ability.Results:1.Elevated expression of TGF-β induces poor clinical pathological parameters and prognosis,TGF-β is positively correlated with EGFR expression in breast cancer tissues and increased TGF-β and EGFR levels are associated with the poor prognosis of patients with breast cancer.2.The m RNA and protein expression level of EGFR in two breast cancer cells are significantly increased after the TGF-β treatment.But the m RNA level of EGF has no significant enhancement.The phosphorylation of EGFR,ERK1/2 and Smad3 are also upregulated in the TGF-β-treated breast cancer cells.3.EGFR inhibition significantly prevents the TGF-β-induced enhancement of the migration,invasion and metastasis ability in vitro and in vivo.4.A panel of firefly luciferase reporter plasmids with the EGFR promoter region was success constructed.5.Sp1 and Smad3 bind to the EGFR promoter region and regulate the transcriptional activity of EGFR,and the role of Sp1 might be more essential than that of Smad3 in the TGF-β-induced transcriptional activation of the EGFR promoter.6.The luciferase activities of p GL3-E1/E2/E3 mutants are significantly weaker than those of their corresponding wild-type plasmids.7.The knockdown of Sp1 significantly decreases the basal and TGF-β-induced luciferase activity in p GL3-E1-and p GL3-E2-transfected cells but has no effect in p GL3-E3-transfected cells.The inhibition of the promoter activation in Sp1-silenced cells is stronger than that in Smad3-knockdown cells.The double knockdown of Sp1 and Smad3 in p GL3-E1-transfected cells shows the strongest inhibition of luciferase activity among the other cases.8.Inhibition of Sp1 weakens TGF-β-induced upregulation of EGFR in breast cancer cells.9.Erk/Sp1 signaling pathway is implicated in the TGF-β-induced EGFR upregulation.10.Smad3 knockdown markedly suppresses the TGF-β-triggered upregulation of the m RNA and protein expression of EGFR in two breast cancer cells.11.Double knockdown of Sp1 and Smad3 significantly affects the TGF-β-induced increase in EGFR expression.In addition,knockdown of Sp1 showed much stronger inhibitory effect on TGF-β induced EGFR upregulation than that in Smad3 knockdown cells.12.Knockdown of Sp1 or Smad3 by si RNAs prevents TGF-β-induced migration and invasion ability of breast cancer cells.Conclusion:In this study,we demonstrated that elevated expression of TGF-β induces poor clinical pathological parameters and prognosis,TGF-β is positively correlated with EGFR expression in breast cancer tissues and increased TGF-β and EGFR levels are associated with the poor prognosis of patients.We also identified that TGF-β promotes the migration and invasion abilities of breast cancer cells,along with the increase in EGFR expression.EGFR is also essential for TGF-β-induced enhancement of these abilities of breast cancer cells.Canonical Smad3 signaling and ERK/Sp1 signaling pathways mediate TGF-β-induced EGFR upregulation.Hence,our study provided a novel mechanism of TGF-β transactivated EGFR signaling pathway induced invasion and metastasis in breast cancer cells.