The Study For The Interaction Between Rictor And PKCζ And Its Role In Metastasis Of Breast Cancer Cells

ObjectivesMetastasis accounts for the majority of patients'deaths due to cancer, and thus cut off the metastatic process is of critical importance for cancer treatment, especially in late stage. Breast cancer is one of the principal diseases that are threatening women's health, over 80% of the metastatic cells are present in the lympgatic vessel or the bloodstream and manage to enter a jacent or distant organ eventually. Chemotaxis is one of the basic functions of cells, and directly participates in vital activities of immunological cell homing, neural development, sperm cell movement and ferrtilization, wound healing and so on. The latest researches shows that chemotaxis plays critical role in cancer metastasis, and the main contribution to cancer metastasis arises from the failture of restriction in cancer cell migration. So, cell migration mediation-associated proteins are to be target of cancer metastasis treatment.mTORC2 (mammalian target of rapamycin complex 2) is critical protien kinase enzyme complex that mediates reconstitution of cytoskeleton and cell proliferation, it is consist of mTOR, mLST8, mSinl and Rictor (the rapamycin intensitive companion of mTOR) and so on. mTORC2 is key protien kinase that phosphorylates Akt(Ser473) site. Our previous reseach has found that EGF induces breast cancer cell chemotaxis by the signaling conductioon pathway of PI3K/PDK1/Akt/PKCζ, Akt2 and its downstream substrate PKCζplay important role in invation and metastasis of breast cancer cells. We speculate that mTORC2 can alter PKCζactivity by phosphorylating Akt to regulate breast cancer metastasis. Rictor is the core constituent that defines mTORC2 and is critical protein in the function of mTORC2 as a protein kinase.Here, we take breast cancer cells as an example and Rictor as the target protein to investigate the interatction of Rictor and PKCζand the role of Rictor in cancer cell migration and cancer cell metastasis in vitro and in vivo.Methods1) Immunohistochemistry analysis detected the expression of Rictor in breast infitrating ductal carcinoma;2) Plasmid and StealthTM RNA lipofectamin2000 transfection assays was performed to knockdown the expression of Rictor in breast cancer MDA-MB-231 cells and T47D cells, and validated by Western Blotting and RT-PCR assays. At the same time, detected the effects of Rictor knockdown in breast cancer proliferation by MTT and flow cytometry;3) Chemotaxsis assay, wound healing assay and adhesion assay were utilized to detect the effects of Rictor knockdown in breast cancer MDA-MB-231 cells and T47D cells on capacity of cell migration and investigated the molecular mechanism by Western Blotting;4) Fluorescence microscope technology and co-immunoprecipitation assays were used to examine and observe whether there is interaction between Rictor and PKCζin breast cancer cells. And examined the effects of Rictor knockdown on PKCζactivity by Western Blotting;5) Fluorescence microscope technology and Western Blotting were performed to analyze the effects of Rictor knockdown on breast cancer MDA-MB-231 cells actin polymerization;6) The SCID mice were adopted to investigate the effects of Rictor knockdown in breast tumor transplanted tumor of breast cancer MDA-MB-231 cells on tumor growth and metastasis.Results1) The expression of Rictor is elevated in breast cancer tissues and is positively associated with lymph node metastasis;2) Rictor knockdown inhibited inhibted EGF induced activation of Akt(Ser473) site, and showed noticeable inhibiton on breast cancer cell proliferation;3) Rictor knockdown significantly inhibited the capacities of EGF induced chemotaxis, wound healing and cell adhesion of breast cancer cells;4) Rictor interacted and co-localized with PKCζat the leading edge of migrating cells, and Rictor activated PKCζby phosphorylation;5) Rictor knockdown inhibited EGF induced actin polymerization;6) Rictor knockdown inhibited breast cancer transplanted tumors growth and metastasis on SCID mice in vivo.ConclusionsOur research results displays that Rictor is corelated with lymphnode metastasis of breast cancer. Rictor knockdown can inhibite chemotaxis and migration of cells and prevent metastasis of cancer cells. Rictor interacts with PKCζand regulates breast cancer cells chemotaxis by controling the activity of PKCζ, and then plays a critical role in regulating breast cancer lung metastasis. It hints that Rictor is not only to be new markers in breast cancer diagnosis and prognosis but also to be a new target in breast cancer metastasis treatment.