TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells

Abstract

Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation, serves as an important tumor-suppressive barrier. We show here that finite lifespan human mammary epithelial cells (HMEC) undergo a p16/ RB-and p53-independent OIS in response to oncogenic RAS that requires TGF-β signaling. Suppression of TGF-β signaling by expression of a dominant-negative TGF-β type II receptor, use of a TGF-β type I receptor inhibitor, or ectopic expression of MYC permitted continued proliferation upon RAS expression. Surprisingly, unlike fibroblasts, shRNA-mediated knockdown of ATM or CHK2 was unable to prevent RAS-mediated OIS, arguing that the DNA damage response is not required for OIS in HMEC. Abrogation of TGF-β signaling not only allowed HMEC lacking p53 to tolerate oncogenic RAS but also conferred the capacity for anchorage-independent growth. Thus, the OIS engaged after dysregulated RAS expression provides an early barrier to malignant progression and is mediated by TGF-β receptor activation in HMEC. Understanding the mechanisms that initiate and maintain OIS in epithelial cells may provide a foundation for future therapies aimed at reengaging this proliferative barrier as a cancer therapy. (ii) constitutive growth signaling, (iii) unlimited replication potential, and (iv) invasive potential (1). Early studies using normal mouse cells indicated that a limited set of genetic manipulations could confer neoplastic potential (2). However, normal human cells have been more difficult to transform to malignancy, indicative of their more stringent tumor-suppressive pathways. Extensive study of cultured human mammary epithelial cells (HMEC) has identified two senescence barriers. One involves the stress-associated induction of the cyclin-dependent kinase inhibitor p16 before attaining critically short telomeres. This stasis barrier can be overcome by inhibiting p16, allowing continued proliferation, which results in agonescence, a proliferative barrier mediated by telomere depletion (3). Additionally, the ability of dysregulated oncogenic signaling to induce senescence in human cells has implicated oncogene-induced senescence (OIS) as an important tumor-suppressive barrier. A number of recent studies have demonstrated the physiological relevance of OIS in human tumorigenesis and in vivo tumor mouse models (4). Additionally, the presence of senescent cells in benign but not advanced tumors argues that OIS serves as an early tumorsuppressive barrier that needs to be dismantled for full oncogenic progression (4). In human fibroblasts, OIS could be bypassed by disabling p16 or molecular components of the DNA damage response (DDR), including ATM, CHK2, or p53, before RAS, MOS, or STAT5 overexpression (5-9). However, OIS in HMEC has been shown to be independent of p53 and the p16-RB pathway after oncogenic RAF-1 expression (10). The contrasting responses between epithelial and fibroblast cells argue that the signaling networks responsible for OIS have tissue specificity. Indeed, fibrob...

The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway

“…Nevertheless, a growing body of evidence supports the involvement of other pathways in the regulation of senescence, and in particular, in that of oncogene‐induced senescence (OIS) (Bianchi‐Smiraglia & Nikiforov, 2012; Christoffersen et al., 2010; Cipriano et al., 2011; Humbert et al., 2010; Lin et al., 2010; Scurr et al., 2010). …”

STAT3-mediated SMAD3 activation underlies Oncostatin M-induced Senescence

“…41,42 Likewise, persistent Oncostatin M (OSM)-mediated activation of STAT3 also induces a p16/p53-independent senescence. 19 Given that RAS-induced OIS requires functional Transforming Growth Factor-b (TGF-b) signaling, we hypothesized that persistent OSM/STAT3-induced senescence would also utilize the TGF-b signaling pathway.…”

“…40,[56][57][58][59] Our lab has previously demonstrated that the expression of MYC from a constitutive promoter prevents OSM-or RAS-induced senescence and alters the response of HMEC to persistent oncogenic stimuli, from growth suppressive to growth promoting. 19,41 Moreover, after dismantling the tumor suppressive senescence barrier, MYC expression cooperates with persistent OSM or RAS signaling to drive transformation. 19,41 Thus, similar to the reported "TGF-b paradox," we hypothesized that once OSM/ STAT3-induced senescence was dismantled by constitutive MYC expression, persistent OSM-induced STAT3/SMAD3 signaling would promote phenotypic traits associated with malignant progression (anchorage-independent growth, epithelial-mesenchymal transition (EMT), and invasive properties).…”

“…19,41 Moreover, after dismantling the tumor suppressive senescence barrier, MYC expression cooperates with persistent OSM or RAS signaling to drive transformation. 19,41 Thus, similar to the reported "TGF-b paradox," we hypothesized that once OSM/ STAT3-induced senescence was dismantled by constitutive MYC expression, persistent OSM-induced STAT3/SMAD3 signaling would promote phenotypic traits associated with malignant progression (anchorage-independent growth, epithelial-mesenchymal transition (EMT), and invasive properties). To test this hypothesis, MYC expressing HMEC (shp53/MYC-HMEC) were treated with either recombinant OSM or TGF-b1 and plated into soft agar to assess anchorage independent growth (AIG), a characteristic associated with cellular transformation.…”

“…19 Given that RAS-induced OIS requires functional Transforming Growth Factor-b (TGF-b) signaling, we hypothesized that persistent OSM/STAT3-induced senescence would also utilize the TGF-b signaling pathway. 41 To test this hypothesis, post-selection shp53-HMEC (lacking p16 expression due to endogenous p16 promoter methylation and p53 due to shRNA-mediated ablation) were infected with retroviruses encoding either a dominant-negative STAT3 (shp53/DN-STAT3-HMEC) or a dominant-negative TGF-b type II receptor (shp53/ DN-TGFbR2-HMEC) to inhibit TGF-b signaling. An empty vector (shp53/vector-HMEC) was used as a control.…”