IBP Regulates Epithelial-to-mesenchymal Transition And Promote Breast Cancer Cell Metastasis

The spread of tumor cells from a primary tumor to a secondary site remains one of themost life-threatening pathological events. Epithelial-to-mesenchymal transition (EMT) is acrucial process for the invasion and metastasis of epithelial tumors. Dissecting the molecularmechanisms that regulate EMT is pivotal for controlling tumor invasiveness and metastasis.However, the molecular mechanisms underlying this transition are poorly understood. IBPfunctions as a guanine nucleotide exchange factor (GEF) for Rho-family GTPases, includingRac1, RhoA, and Cdc42. Loss of IBP in mice leads to the spontaneous development of a SLElike systemic autoimmune disorder characterized by the hypergammaglobulinemia,autoantibody production and accumulation of effecter and memory T cells. IBP is involved inregulation of immunological synapse formation, T cell activation and Th1/Th2cellpolarization. Studies indicate that IBP regulate cell morphology in cooperation with activatedRac1and affect cell differentiation with integrins. Recent report show that IBP mayparticipate in the regulation of post-translational modifications. Previous studies suggest asignificant physiological role for IBP. Most of the studies were limited to the function of IBPin immune system. We, for the first time, report that IBP is correlated with progression ofbreast cancer. Weather IBP regulate Epithelial-to-mesenchymal transition of breast cancercells is still unknown. In this study, we focus on the mechanism that IBP regulateEpithelial-to-mesenchymal transition of breast cancer cells and promote tumor metastasis.Methods and results1. Expression of IBP in breast cancer patient specimens and cell lines.1) Tumors and lymphnodes tissues from109patients were analyzed by IHC. Eachsample was assigned an immunoreactivity score ranging from0to6. Primary tumors andcorresponding lymph node metastases exhibited diffuse cytoplasmic staining for IBP. Pairedcomparisons of immunoreactivity scores between primary and metastatic tumors weresignificant (p <0.001).2) Kaplan–Meier plots of IBP expression in20cases of breast cancer patients. Overall survival rate was performed. Increased IBP expression was found to correlate with shorteroverall survival (p=0.041) of patients.3)9breast cancer cell lines and normal human mammary epithelial cells (HMC) wereanalyzed by western blot. Higher levels of IBP protein were observed in most breast cancercell lines compared to HMC. Higher Metastatic cancer cell lines contained higher levels ofIBP.2. Mechanism of IBP regulate Epithelial-to-mesenchymal of breast cancer1) By Western blot and Immunofluorescence, we found that IBP regulateEpithelial-to-mesenchymal markers (E-cadherin, cytokeratin18, N-cadherin, fibronectin andvimentin).2) We examined breast cancer cell with phase contrast microscope and found that IBPregulates the EMT phenotype of breast cancer cells.3) EGF induced marked EMT morphology in breast cancer cells and upregulated theexpression of the mesenchymal marker fibronectin. More importantly, IBP is upregulated inEGF induced EMT.4) Loss of IBP inactivates EGFR signaling in breast carcinoma cell lines. Activation ofEGFR (1068,1173), p38MAPK, and ERK1/2was analyzed by immunoblotting.5) The mRNA levels of EMT-related transcription factors in breast cancer cell weremeasured by quantitative Real-timePCR. IBP silencing reduces EMT-induced transcriptionfactor Snail, Slug, ZEB1and ZEB2expression.6) Migration and invasion assays indicate that IBP promote Migration and invasion ofbreast cancer cells; Western blot analysis demonstrates that IBP regulate expression of MMPsin breast cancer.7) We investigated IBP regulation of cytoskeleton in breast cancer cells by stainingtubulin and F-actin. IBP overexpression induced formation of pseudopodium; IBP silencingcause disorder of cytoskeleton rearrangement.8) We found that IBP affect activity of Rac1, RhoA, and Cdc42via its GEF activity inbreast cancer cell.3．Silencing of IBP inhibit breast cancer cell tumor formation and metastasis in vivo.1) Subcutaneous tumor formation analysis of human breast cancer cell in nude mouseimply that silencing of IBP inhibit breast cancer cell tumor formation.2) Nude mice metastatic model analysis imply that silencing of IBP inhibit breast cancer cell in vivo.In summery, we defined IBP as an oncogene frequently overexpressed in breast cancercells that promotes EMT and cellular motility. First, we confirmed the increased expression ofIBP in breast cancer, in particular in advanced cancer. Second, we showed that IBP is apositive regulator of EMT in breast cancer cells and plays a role in EGF-induced EMT. Third,we show depletion of IBP attenuated the migration and invasion of breast cancer cellsconcomitant with altered cytoskeletal rearrangement and decreased activation of Rho GTPase.Finally, we confirm that silencing of IBP inhibit breast cancer cell tumor formation andmetastasis in vivo. Our results suggest that IBP serve as a predictive marker for breastcancer with high metastatic potential, and IBP maybe a new therapy target for breast cancer.