Targeting Of MTORC2Prevents Cell Migration And Promotes Apoptosis In Breast Cancer

Background:The mammalian target of rapamycin(mTOR) is a serine/threonine kinase at the nexus between oncogenic phosphoinositide3-kinase(PI3K)/Akt signaling and critical downstream pathways that drive cancer cell growth, survival and resistance to therapeutic agents. mTOR kinase exists in two complexes, mTOR interacts with raptor, Lst8, Deptor and PRAS40to form mTORC1, the sensitive target of rapamycin that phosphorylates downstream targets of S6kinase1(p70S6K1) and eukaryotic initiation factor4E binding protein-1(4E-BP1)and control the cap-dependent protein translation. mTOR with rictor, Lst8, Sin1, Deptor and Protor to form mTORC2, which is insensitive to acute rapamycin treatment and phosphorylates Akt at Ser473. Together, these complexes coordinate a variety of processes that include protein translation, autophagy, proliferation, survival and metabolism in response to nutrient, oxygen, energy, stress and growth factor signals, mTOR signaling is dysregulated in50%of all human malignancies and is a major cancer drug target.As the first generation mTOR inhibitor, rapamycin and rapalogs can slow the proliferation of cancer cell lines and have achieved some success in caner treatment. Unfortunately, their overall efficacy as cancer therapeutics has been limited to a few rare cancers, including mantle cell lymphoma, renal cell carcinoma and endometrial cancer. The major drawbacks of rapalogs are1) S6K is exquisitely inhibited, yet the control of4E-BP andmRNA translation is far less sensitive;2) mTORC2activity is not acutely blocked;3) there is a feedback loop between mTORCl and Akt. Treatment with rapalogs resulted in elevated Akt activity through S6K1and insulin receptor substrate1(IRS-1), which serves as a mechanism to enhance cell survival when mTORC1is inhibited.mTOR ATP-competitive inhibitors as the second generation inhibitors have been developed recently that are able to completely suppress both mTORC1/2complex-mediated signaling, thereby suppressing the feedback activation of Akt. Importantly, they have shown marked improvement of anti-tumor activity in vivo and in vitro and the effectiveness of these drugs in cancer treatment is currently being tested in clinical trials. What’s more, a greater understanding the molecular and cellular mechanisms of action of these compounds is needed. Furthermore, recent studies in cancer biology indicate that mTORC2is emerging as a promising therapeutic target because its activity is essential for the transformation and vitality of a number of cancer cell types. It is important to investigate the efficacy of targeting of mTORC2across abroad range of tumor types, which will provide a strong rationale for targeting of mTORC2in breast cancer treatment.Objectives:The role of mTORC2in pathogenesis and progression of breast cancer, and the effectiveness and mechanisms of targeted inhibition of mTORC2in breast cancer are not known. In this study, the effects of targeting of mTORC2in cell proliferation, migration and apoptosis in a variety of breast cancer cell lines and in breast tumor xenograft were examined. Our study will provide rationales for targeting of mTORC2in breast cancer treatment. Methods:A variety of breast cancer cell lines were used in in vitro studies. The effects of mTOR kinase inhibitors PP242and OSI027, or raptor siRNA (mTORC1) and rictor siRNA (mTORC2) in cell proliferation, migration, apoptosis and mTOR signaling activites in breast cancer cell lines were examined.1) effects of PP242, OSI027and rapamycin on cell proliferation in seven breast cancer cell lines were determined by MTT and clone formation assay;2) effects of raptor or rictor siRNA, and PP242, OSI027or rapamycin treatment on cell migration were detected by wound healing assay;3) effects of raptor or rictor siRNA, and PP242, OSI027or rapamycin treatment on cell apoptosis were examined by Prodium Iodide (PI) staining and cleaved-PARP levels;4) the synergism effects of raptor or rictor siRNA, and PP242, OSI027or rapamycin treatment combined with low dose of cisplatin (200nM) were evaluated by Combination Index (CI) analysis. For in vivo studies, the effects of rapamycin or PP242administration in tumor growth, mTOR signaling and cell apoptosis were observed in breast tumor xenograft.Results:1Targeted inhibition of mTORC1/2signaling by mTOR kinase inhibitors PP242and OSI027effectively suppresses breast cancer cell proliferationWe examined the distinct effects of targeting of mTORC1by rapamycin and targeting of mTORCl/2by two active-site mTOR inhibitors PP242and OSI027on mTORCl/2outputs in breast cancer MCF-7, T47D, MDA-MB-231and Bcap-37cell lines. PP242and OSI-027dose-dependently (50-500nM) suppressed phosphorylation of Akt (S473), a mTORC2phosphorylation site in all tested cell lines, whereas rapamycin treatment caused an increase in phsophorylation of Akt (S473). Although all drugs effectively suppressed phosphorylation of S6(S235/236), only mTOR kinase inhibitors PP242effectively decreased the phosphorylation of4E-BP1(T37/46) in these cells. It is indicated that mTOR kinase inhibitor profoundly diminish mTORC1and mTORC2signaling, whereas rapamycin suppresses mTORC1and enhances phosphorylation of Akt (S473) in some breast cancer cell lines.Secondly, the anti-proliferative activities of PP242and OSI027compared with rapamycin in seven breast cancer cell lines were tested. We found that targeting of mTORC1or dual targeting of mTORC1/2effectively suppressed proliferation in the tested breast cancer cells. Furthermore, PP242and OSI027inhibited clone formation.2Targeting of mTORC2but not mTORC1promotes apoptosis in breast cancer cellsAkt represents an important intracellular survival signaling under a variety of conditions. Rapamycin induced feedback activation of Akt in MCF-7, MDA-MB-231and Bcap-37breast cancer cells. Accordingly, rapamycin did not exhibit any pro-apoptotic activity in these cells. The Akt activation induced by loss of feedback inhibition could be prevented by mTOR kinase inhibitors PP242and OSI027. In consistent with the inhibitory activities of PP242and OSI027in Akt (S473) phosphorylation, these drugs significantly enhanced cleavage of poly (ADP-ribose) polymerase (PARP) and number of apoptotic cells in serum-starved MCF-7, MDA-MB-231and Bcap-37cell lines.Furthermore, raptor, rictor or mTOR siRNA markedly decreased protein levels of raptor, rictor or mTOR and reduced the phosphorylation of their outputs S6(S235/236) and Akt (S473) respectively in MCF-7and MDA-MB-231cells. Knockdown of rictor and mTOR, but not raptor promoted cleavage of PARP and increased the apoptotic cells in serum-starved MDA-MB-231cells. Taken together, it is suggested that targeting of mTORC2but not mTORC1promotes apoptosis in breast cancer cells. 3Inhibition of mTORC2potentiates cisplatin-induced apoptosis in breast cancer cellsCisplatin, a chemotherapeutic agent that is used for many cancers very common, could induce apoptosis in a variety of cancer cell lines. Drug combination index (CI) analysis is a generalized method for analyzing the effects of multiple drugs and for determining summation, synergism and antagonism. The CI method helps answer whether there are synergism with two drugs, how much the two drugs synergism and synergism at what dose levels.We examined the interactions of mTOR kinase inhibitors and low dose of cisplatin. PP242or OSI027was applied at low concentrations (20-400nM) and low dose cisplatin (200nM) in MCF-7, MDA-MB-231and Bcap-37cells. When the CI values were less than1.0, indicating synergistic interactions between PP242or OSI027. On the contrary, rapamycin did not show any synergistic interactions with cisplatin, as the CI values were higher than1.0. Furthermore, PP242or OSI027, but not rapamycin notably enhanced cisplatin-induced cleavage of PARP. Most importantly, rictor or mTOR knockdown, but not raptor knockdown, markedly enhanced cisplatin-induced cleavage of PARP and the number of apoptotic cells.Taken together, our results indicate that targeted inhibition of mTORC2but not mTORC1markedly potentiates cisplatin-induced apoptosis in breast cancer cells.4Targeted inhibition of mTORC2prevents breast cancer cell migrationMetastasis is the major cause of mortality and morbidity among breast cancer patients. Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. This requires migration of cancer cells. To investigate different roles of mTORC1and mTORC2in breast cancer cell migration, effects of targeted inhibition of mTORC1or mTORC2on cell migration were examined by using a wound healing assay in a serum-free medium. PP242-or OSI027-treated and mTOR or rictor siRNA transfected MCF-7cells filled the gap more slowly than vehicle control-or rapmycin-treated or raptor siRNA transfected MCF-7cells did, suggesting that inhibition of mTORC2but not mTORCl prevented cell migration. These results were repeated in MDA-MB-231cells, further confirmed the critical role of mTORC2in breast cancer cell migration.5In vivo administration of PP242but not rapamycin as a single agent effectively prevents breast tumor growth and induces apoptosis in xenograftTo compare the in vivo efficacy of inhibition of mTORC1with dual inhibition of mTORCl/2, nude mice bearing MDA-MB-231breast tumor xenografts were dosed with rapamycin or PP242. The PP242therapy as single agent markedly inhibited breast tumor growth (tumor areas and tumor weight) while rapamycin was ineffective to suppress the tumor growth. Most importantly, in vivo administration of PP242induced significant apoptosis in the breast tumor xenograft. But rapamycin treatment failed to induce significant apoptosis in this model. In consistent with in vitro results, rapamycin suppressed mTORCl (P-S6) but not mTORC2(P-Akt). Our results indicate that dual targeting of mTORC1/2but not mTORC1effectively suppresses breast tumor growth and induces apoptosis in vivo.Conclusion:mTOR kinase inhititors inhibit both mTORC1and mTORC2, rapamycin only inhibits mTORC1but activated Akt in breast cancer cells. Targeting of mTORC2but not mTORC1induced breast cancer cells apoptosis and prevented cell migration, and enhanced pro-apoptosis combined with cisplatin. Dual targeting of mTORC1/2but not mTORC1effectively suppresses breast tumor growth and induces apoptosis in vivo. It is suggested that mTORC2plays a critical role in breast cancer progression. Our data suggested that agents that inhibition of mTORC2may have advantages over selective mTORC1inhibitors in the treatment of breast cancers. Given that mTOR kinase inhibitors are in clinical trials, this study provides a strong rationale for testing the use of mTOR kinase inhibitors or combination of mTOR kinase inhibitors and cisplatin in the clinic.