The Effect Of P32in EGF Induced Chemotaxis And Metastasis Of Human Breast Cancer

Background and ObjectiveBreast cancer is one of the leading causes of death in women. Metastasis is one of the basic biology characteristics of malignant tumor, and it is the main cause of tumor relapse, treatment failure or even death. Here, we aim to study the relationship between p32and tumor cell chemotaxis and metastasis.Methods1) IHC staining was used to detect the expression of p32in breast cancer and breast lobular hyperplasia tissues, and analyzed the relationship of p32and patients’ pathological parameters.2) Western blotting was used to detect the expression of p32in several breast cancer cell lines, and immunofluoresence and subcellular fractionation were used to detect the distribution of p32.3) MTT assay and soft agar colony formation assay were used to detect the influence of p32downregulation on the proliferation of breast cancer cells.4) Chemotaxis and wound healing assay demonstrated the influence of p32on the chemotaxis ability and migration ability of breast cancer cells.5) Rotenone which is an mitochondrial complex I inhibitor to detected the role of oxidative phosphorylation on cell chemotaxis and migration ability.6) Mass spectrometric analysis was use to identify p32interacting proteins. PKCζ wad found as one of p32interacting proteins.7) We used western blotting assay and immunofluoresence assay to detecte the effect of p32binding on the activity of PKCζ. Then we used rotenone to detected the role of oxidative phosphorylation on the activity of PKCζ.8) We used immunofluoresence and Western blot to examine the influence of p32on polarity and F-actin polymerization.9) We examined the role of p32in vivo.Results1) P32was expression higher in breast cancer tissues and related with TNM stage and distant metastasis.2) P32was overexpression in breast cancer cell lines. P32was mainly distribute at mitochondria, it also can be detected at membrane, cytosal, and nuclears. P32 can translocation to cell membrane after EGF stimulation.3) The proliferation of MDA-MB-231cells was impaired after down-regulation of p32(P<0.05).4) P32depleted breast cancer cells showed decreased chemotaxis and migration ability compared with control cells (P<0.01).5) After using of mitochondrial complex I inhibitor, rotenone, cells chemotaxis and migration ability were no effect.6) PKCζ, BAT2, CHCHD2, Lamin-B receptor were found as p32interacting proteins by mass spectrometric analysis. Immunofluoresence assay showed p32was colocalized with PKCζ at cell membrane after EGF stimulation.7) Knockdown of p32could impair EGF induced PKCζ membrane translocation and phosphoralation of PKCζ. But downregulation of PKCζ did not effect the p32membrane translocation.8) P32(74-174aa) interacted with PKCζ, regulatory domain. P32(74-174aa) fragment can rescue sip32-caused EGF induced PKCζ membrane translocation and cell migration ability.9) P32impaired the cells’ giantin and γ-tubulin reorientation through reduce phosphoralation of GSK-3β.P32impaired cells actin polymerization through regulation the phosphorylation of cofilin.10) P32imparted breast cancer cells metastasis in SCID mice.Conclusions1) P32was overexpression in human breast cancer tissues, and its expression was related with TNM stage and distant metastasis.2) P32regulated breast cancer cell chemotaxis and migration abiltiy through binding with PKCζ regulatory domain and regulated activity of PKCζ, then regulated activity of GSK-3β and cofilin to effected cell polarity and cytoskeleton rearrangement. P32playing an important role in breast cancer cell mobility independent oxidative phosphorylation.3) In vivo, p32depletion could impair breast cancer cells metastasis.