Twist Induces EMT And Cell Motility In Breast Cancer Via Regulating A Downstream Signaling Network

Objective To understand the effects of Twist on EMT, migratoryand invasive ability of human mammary epithelial cells, and its possibledownstream signaling network.Methods (1) Myc-Twist was digested from pcDNA3/myc-Twist andsubcloned into the retroviral vector pBABE-puro to construct arecombinant plasmid (pBABE-myc-Twist). The inserted Twist gene wasconfirmed by restriction enzyme digestion and DNA sequencing.(2) Theplasmid pBABE-myc-Twist and its control were transfected into293T cellsrespectively for packaging of retrovirus. Human mammary epithelial cellsMCF10A were infected with the retroviruses carrying Twist gene or itscontrol. The expression of Twist mRNA and protein in the MCF10A-Twistand MCF10A-Vector cells were determined by the RT-PCR and Westernblotting, respectively.(3) The expressions of EMT markers induced byTwist in MCF10A cells were detected using immunofluorescence staining and Western blotting. Cell proliferation was tested with the use of MTT andFCM. Cell migration and invasion were analyzed by Transwell assay.(4)iTRAQ-based analysis was employed to compare the differential proteinexpressions in MCF10A-Twist cells undergoing EMT againstMCF10A-Vector control cells. The biological processes and signalingpathways in which these significant proteins involved were analyzed by theDAVID v6.7, a on-line analysis database, to construct a downstreampathway network in Twist-induced EMT.(5) The activation of MAPK andPI3K/AKT pathways was detected by Western blotting. The expressions ofEMT markers in MCF10A-Twist cells treated with PD98059or LY294002were determined by the qRT-PCR and Western blotting. Cell migration wasanalyzed by Transwell assay.(6) The Twist siRNA was transfected intobreast cancer cells BT549and Hs578T by lipofectamine. The role ofsiTwist in the expression of EMT markers was determined by the qRT-PCRand Western blotting. The activation of MAPK and PI3K/AKT pathwayswas detected by Western blotting. The effect of siTwist on the migrationcapacity of BT549and Hs578T cells was analyzed by Transwell assay.Results (1) The myc-tagged Twist gene was correctly inserted intothe expression retroviral vector as a recombinant plasmid(pBABE-myc-Twist) identified by restriction analysis and DNA sequencing.(2) The Twist gene was efficiently delivered into human mammaryepithelial cells MCF10A by the retrovirus, resulting in stable expression of Twist mRNA and myc-tagged Twist protein as shown by RT-PCR andWestern blotting, respectively.(3) The expressions of the epithelialbiomarkers E-cadherin and-catenin were downregulated, whereas themesenchymal markers vimentin and fibronectin were upregulated inMCF10A-Twist cells as shown by immunofluorescence and Westernblotting. By using the MTT and FCM analysis, Twist was found to inhibitthe cell proliferation of MCF10A (P<0.05). Twist can also notably promoteinvasive and migratory of MCF10A (P<0.01).(4)194significantlychanging proteins were identified and quantified by iTRAQ-based analysis(P<0.05). These significant proteins analyzed by the DAVID v6.7werefound to play roles in cytoskeleton organization, cell adhesion, cellproliferation, cell migration and EMT process. Moreover, these proteinswere involved in some pathways, such as PI3K/AKT, ECM-receptor,MAPK, WNT and P53signaling, which formed a signaling networkassociated with Twist-induced EMT.(5) The ERK/MAPK and PI3K/AKTpathways were activated obviously in MCF10A-Twist. The expressions ofE-cadherin and-catenin were up-regulated, and the expressions ofvimentin, fibronectin, N-cadherin, MMP2and MMP9were down-regulatedin MCF10A-Twist cells treated with PD98059or LY294002. Inhibiting thetwo pathways also decreased cell migration (P<0.05).(6) Knockdown ofTwist increased the expressions of the epithelial biomarkers, and decreasedthe expressions of mesenchymal markers in BT549and Hs578T cells. Consistent with these findings, silencing of Twist inhibited the activation ofERK/MAPK and PI3K/AKT pathways, and the migration ability of BT549and Hs578T cells (P<0.05).Conclusion Twist induces EMT of MCF10A cells and promotes themigration and invasion of MCF10A cells via a signaling pathway network.