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Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B’s obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

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-1β 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-1β. The MIP-1β-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-1β and MYO3A. Both, MIP-1β 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-1β/MYO3A axis being operational during metastasis, wherein macrophage-derived MIP-1β 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.

BackgroundThe 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.MethodsProtein 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.ResultsHere, 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.ConclusionsOur 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.

Augmented reactive oxygen species levels consequential to functional alteration of key mitochondrial attributes contribute to carcinogenesis, either directly via oxidative DNA damage infliction or indirectly via activation of oncogenic signaling cascades. We previously reported activation of a key oncogenic signaling cascade via mammalian target of rapamycin (mTOR) signaling complex-2 (mTORC2) owing to estrogen receptor (ER-α)-dependent augmentation of O within the mitochondria of 17-β-estradiol (E2)-stimulated breast cancer cells. Manganese superoxide dismutase (MnSOD) is the principal mitochondrial attribute governing mitochondrial O homeostasis, raising the possibility that its functional alteration could be instrumental in augmenting mitochondrial O levels in breast cancer cells. Here we show ER-dependent transient inhibition of MnSOD catalytic function in breast cancer cells. Catalytic function of MnSOD is tightly regulated at the post-translational level. Post-translational modifications such as phosphorylation, nitration and acetylation represent key regulatory means governing the catalytic function of MnSOD. Acetylation at lysine-68 (K68) inhibits MnSOD catalytic activity and thus represents an important post-translational regulatory mechanism in human cells. Using reciprocal immunoprecipitation and proximity ligation assay, we demonstrate the occurrence of direct physical interaction between ER-α and MnSOD in human breast cancer cells, which in turn was associated with potentiated acetylation of MnSOD at K68. In addition, we also observed diminished interaction of MnSOD with sirtuin-3, the key mitochondrial deacetylase that deacetylates MnSOD at critical K68 and thereby activates it for scavenging O. Consequently, compromised deacetylation of MnSOD at K68 leading to its inhibition and a resultant buildup of O within the mitochondria culminated in the activation of mTORC2. In agreement with this, human breast cancer tissue specimen exhibited a positive correlation between acetyl-MnSOD levels and phospho-Ser mTOR levels. In addition to exposing the crosstalk of ER-α with MnSOD post-translational regulatory mechanisms, these data also unravel a regulatory role of ER/MnSOD interaction as an important control switch for redox regulation of ER-α-responsive oncogenic signaling cascades. Furthermore, our study provides a mechanistic link for ER-α-dependent O potentiation and resultant mTORC2 activation in breast cancer cells.