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Although frequently associated with tumor progression, inflammatory cytokines initially restrain transformation by inducing senescence, a key tumor-suppressive barrier. Here, we demonstrate that the inflammatory cytokine, oncostatin M, activates a mesenchymal/stem cell (SC) program that engages cytokine-induced senescence (CIS) in normal human epithelial cells. CIS is driven by Snail induction and requires cooperation between STAT3 and the TGFβ effector, SMAD3. Importantly, as cells escape CIS, they retain the mesenchymal/SC program and are thereby bestowed with a set of cancer SC (CSC) traits. Of therapeutic importance, cells that escape CIS can be induced back into senescence by CDK4/6 inhibition, confirming that the mechanisms allowing cells to escape senescence are targetable and reversible. Moreover, by combining CDK4/6 inhibition with a senolytic therapy, mesenchymal/CSCs can be efficiently killed. Our studies provide insight into how the CIS barriers that prevent tumorigenesis can be exploited as potential therapies for highly aggressive cancers. IMPLICATIONS: These studies reveal how a normal cell's arduous escape from senescence can bestow aggressive features early in the transformation process, and how this persistent mesenchymal/SC program can create a novel potential targetability following tumor development., (©2020 American Association for Cancer Research.)

Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK) activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1) occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC) transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

Background: Highly aggressive, metastatic and therapeutically resistant triple-negative breast cancers (TNBCs) are often enriched for cancer stem cells (CSC). Cytokines within the breast tumor microenvironment (TME) influence the CSC state by regulating tumor cell differentiation programs. Two prevalent breast TME cytokines are oncostatin-M (OSM) and interferon-β (IFN-β). OSM is a member of the IL-6 family of cytokines and can drive the de-differentiation of TNBC cells to a highly aggressive CSC state. Conversely, IFN-β induces the differentiation of TNBC, resulting in the repression of CSC properties. Here, we assess how these breast TME cytokines influence CSC plasticity and clinical outcome.Methods: Using transformed human mammary epithelial cell (HMEC) and TNBC cell models, we assessed the CSC markers and properties following exposure to OSM and/or IFN-β. CSC markers included CD24, CD44, and SNAIL; CSC properties included tumor sphere formation, migratory capacity, and tumor initiation.Results: There are three major findings from our study. First, exposure of purified, non-CSC to IFN-β prevents OSM-mediated CD44 and SNAIL expression and represses tumor sphere formation and migratory capacity. Second, during OSM-induced de-differentiation, OSM represses endogenous IFN-β mRNA expression and autocrine/paracrine IFN-β signaling. Restoring IFN-β signaling to OSM-driven CSC re-engages IFN-β-mediated differentiation by repressing OSM/STAT3/SMAD3-mediated SNAIL expression, tumor initiation, and growth. Finally, the therapeutic use of IFN-β to treat OSM-driven tumors significantly suppresses tumor growth.Conclusions: Our findings suggest that the levels of IFN-β and OSM in TNBC dictate the abundance of cells with a CSC phenotype. Indeed, TNBCs with elevated IFN-β signaling have repressed CSC properties and a better clinical outcome. Conversely, TNBCs with elevated OSM signaling have a worse clinical outcome. Likewise, since OSM suppresses IFN-β expression and signaling, our studies suggest that strategies to limit OSM signaling or activate IFN-β signaling will disengage the de-differentiation programs responsible for the aggressiveness of TNBCs. [ABSTRACT FROM AUTHOR]

Cytokines play an important role in creating an inflammatory microenvironment, which is now considered a hallmark of cancer. Although tumor cells can exploit cytokine signaling to promote growth, invasion, and metastasis, the response of normal and premalignant epithelial cells to cytokines present in a developing tumor microenvironment remains unclear. Oncostatin M (OSM), an IL-6 family cytokine responsible for STAT3 activation, has been implicated in cancer development, progression, invasion, and metastasis. Paradoxically, OSM can also suppress the growth of normal cells and certain tumor-derived cell lines. Using isogenic human mammary epithelial cells (HMEC) at different stages of neoplastic transformation, we found that OSM signaling suppressed c-MYC expression and engaged a p16- and p53-independent growth arrest that required STAT3 activity. Inhibition of STAT3 activation by expressing a dominant-negative STAT3 protein or a STAT3-shRNA prevented the OSM-mediated arrest. In addition, expression of c-MYC from a constitutive promoter also abrogated the STAT3-mediated arrest, and strikingly, cooperated with OSM to promote anchorage-independent growth (AIG), a property associated with malignant transformation. Cooperative transformation by c-MYC and OSM required PI3K and AKT signaling, showing the importance of multiple signaling pathways downstream of the OSM receptor in defining the cellular response to cytokines. These ?ndings identify c-MYC as an important molecular switch that alters the cellular response to OSM-mediated signaling from tumor suppressive to tumor promoting. [ABSTRACT FROM AUTHOR]

Aberrant regulation of growth signaling is a hallmark of cancer development that often occurs through the constitutive activation of growth factor receptors or their downstream effectors. Using validation-based insertional mutagenesis (VBIM), we identified family with sequence similarity 83, member B (FAM83B), based on its ability to substitute for RAS in the transformation of immortalized human mammary epithelial cells (HMECs). We found that FAM83B coprecipitated with a downstream effector of RAS, CRAF. Binding of FAM83B with CRAF disrupted CRAF/14-3-3 interactions and increased CRAF membrane localization, resulting in elevated MAPK and mammalian target of rapamycin (mTOR) signaling. Ablation of FAM83B inhibited the proliferation and malignant phenotype of tumor-derived cells or RAS-transformed HMECs, implicating FAM83B as a key intermediary in EGFR/RAS/MAPK signaling. Analysis of human tumor specimens revealed that FAM83B expression was significantly elevated in cancer and was associated with specific cancer subtypes, increased tumor grade, and decreased overall survival. Cumulatively, these results suggest that FAM83B is an oncogene and potentially represents a new target for therapeutic intervention. [ABSTRACT FROM AUTHOR]