M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis.

Oncostatin M (OSM), an inflammatory cytokine belonging to the interleukin-6 (IL-6) superfamily, plays a vital role in multitude of physiological and pathological processes. Its role in breast tumor progression and metastasis to distant organs is well documented. Recent reports implicate OSM in macrophage M2 polarization, a key pro-tumoral phenomenon. M2 polarization of macrophages is believed to promote tumor progression by potentiating metastasis and angiogenesis. In the current study, we delineated the mechanism underlying OSM induced macrophage M2 polarization. The findings revealed that OSM skews macrophages towards an M2 polarized phenotype via mTOR signaling complex 2 (mTORC2). mTORC2 relays signals through two effector kinases i.e. PKC-α and Akt. Our results indicated that mTORC2 mediated M2 polarization of macrophages is not dependent on PKC-α and is primarily affected via Akt, particularly Akt1. In vivo studies conducted on 4T1/BALB/c mouse orthotropic model of breast cancer further corroborated these observations wherein i.v. reintroduction of mTORC2 abrogated monocytes into orthotropic mouse model resulted in diminished acquisition of M2 specific attributes by tumor associated macrophages. Metastasis to distant organs like lung, liver and bone was reduced as evident by decrease in formation of focal metastatic lesions in mTORC2 abrogated monocytes mice. Our study pinpoints key role of mTORC2-Akt1 axis in OSM induced macrophage polarization and suggests for possible usage of Oncostatin-M blockade and/or selective mTORC2 inhibition as a potential anti-cancer strategy particularly with reference to metastasis of breast cancer to distant organs such as lung, liver and bone.

Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling.

BACKGROUND: The mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking. METHODS: Protein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion. RESULTS: Here, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling. CONCLUSIONS: Our study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.

Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1ß dependent upregulation of MYO3A gene in breast cancer cells.

The potential of a tumor cell to metastasize profoundly depends on its microenvironment, or "niche" interactions with local components. Tumor-associated-macrophages (TAMs) are the most abundant subpopulation of tumor stroma and represent a key component of tumor microenvironment. The dynamic interaction of cancer cells with neighboring TAMs actively drive cancer progression and metastatic transformation through intercellular signaling networks that need better elucidation. Thus, current study was planned for discerning paracrine communication networks operational between TAMs, and breast cancer cells with special reference to cancer cell invasion and dissemination to distant sites. Here, we report role of MIP-1ß in enhancing invasive potential of metastatic breast cancer MDA-MB-231 and MDA-MB-468 cells. In addition, the poorly metastatic MCF-7 cells were also rendered invasive by MIP-1ß. The MIP-1ß-driven cancer cell invasion was dependent on upregulated expression levels of MYO3A gene, which encodes an unconventional myosin super-family protein harboring a kinase domain. Ex ovo study employing Chick-embryo-model and in vivo Syngenic 4T1/BALB/c mice-model further corroborated aforementioned in vitro findings, thereby substantiating their physiological relevance. Concordantly, human breast cancer specimen exhibited significant association between mRNA expression levels of MIP-1ß and MYO3A. Both, MIP-1ß and MYO3A exhibited positive correlation with MMP9, an established molecular determinant of cancer cell invasion. Higher expression of these genes correlated with poor survival of breast cancer patients. Collectively, these results point toward so far undisclosed MIP-1ß/MYO3A axis being operational during metastasis, wherein macrophage-derived MIP-1ß potentiated cancer cell invasion and metastasis via up regulation of MYO3A gene within cancer cells. Our study exposes opportunities for devising potential anti-metastatic strategies for efficient clinical management of breast cancer.