Loss of Singleminded-2s in the Mouse Mammary Gland Induces an Epithelial-Mesenchymal Transition Associated with Up-Regulation of Slug and Matrix Metalloprotease 2

Abstract

The short splice variant of the basic helix-loop-helix Per-Arnt-Sim transcription factor Singleminded-2, SIM2s, has been implicated in development and is frequently lost or reduced in primary breast tumors. Here, we show that loss of Sim2s causes aberrant mouse mammary gland ductal development with features suggestive of malignant transformation, including increased proliferation, loss of polarity, down-regulation of Ecadherin, and invasion of the surrounding stroma. Additionally, knockdown of SIM2s in MCF-7 breast cancer cells contributed to an epithelial-mesenchymal transition (EMT) and increased tumorigenesis. In both Sim2؊/؊ mammary glands and SIM2s-depleted MCF7 cells, these changes were associated with increased SLUG and MMP2 levels. SIM2s protein was detectable on the SLUG promoter, and overexpression of SIM2s repressed expression from a SLUG-controlled reporter in a dose-dependent manner. To our knowledge, SIM2s is the first protein shown to bind and repress the SLUG promoter, providing a plausible explanation for the development role and breast tumor-suppressive activity of SIM2s. Together, our results suggest that SIM2s is a key regulator of mammary-ductal development and that loss of SIM2s expression is associated with an invasive, EMT-like phenotype.

Signalling pathways involved in endocrine resistance in breast cancer and associations with epithelial to mesenchymal transition (Review)

“…In support of the former, loss of sINGLEMINdEd-2s in the mouse mammary gland induces sNAIL2-mediated EMT, indicating how gene inactivation may circumvent EMT dormancy in the adult mammary gland (219). In support of the latter, there is evidence for the heterogeneous expression and nuclear translocation of sNAIL (220) and sNAIL2 (221) following exposure to hypoxia at the invasive front, highlighting the importance of the microenvironment in providing extracellular signals for initiation of EMT at metastasis.…”

The HIF1α-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element

“…Mechanistic roles for SIM2s and BNIP3 in cancer remain to be fully defined; however, deregulation of both factors are emerging as select solid tumour markers and are associated with poor prognostic outcomes (DeYoung et al, 2002;Halvorsen et al, 2007;Laffin et al, 2008;Burton and Gibson, 2009). As discussed previously, decreased BNIP3 levels and poor prognosis (Okami et al, 2004;Erkan et al, 2005;Mahon et al, 2007) clearly correlate with elevated SIM2s expression (DeYoung et al, 2002(DeYoung et al, , 2003b in pancreatic cancer.…”

“…Conversely, however, SIM2s has recently been found to be repressed in breast cancer tumours (Kwak et al, 2007). In contrast to the colon and pancreatic tumour-derived cell lines tested, loss of SIM2s in MCF-7 breast cancer cells correlates to cell survival through the activation of SLUG-mediated EMT (epithelial-mesenchymal transition) (Kwak et al, 2007;Laffin et al, 2008). Whether changes in SIM2s expression levels are required for tumour growth and/or tumour maintenance, and how SIM2s expression and transcriptional activities fit into the molecular profile of disease progression, is in need of further investigation.…”

“…Cell-based reporter gene assays have shown the murine long isoform to mediate repression of transcription via carboxy-terminal repression domains (Ema et al, 1996b;Moffett et al, 1997;Woods and Whitelaw, 2002). Recently, hSIM2s-mediated repression of the SLUG gene in breast cancer cells has been reported (Laffin et al, 2008). Another repressive mode of SIM2 is by sequestration of ARNT from other bHLH/PAS transcription factors, a mechanism by which SIM2 can interfere with the function of the hypoxia-inducible factor 1a (HIF1a) (Woods and Whitelaw, 2002).…”

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer

“…TGF can also phosphorylate certain cytoplasmic proteins regulating cell polarity and tight junction formation. These include RAS/MAPK (Xue et al, 2003), integrin -1 (Blanco et al, 2002), integrin-linked kinase (Hartwell et al, 2006), p38 MAPK (Mani et al, 2007), RHOA kinase (ROCK) (Moody et al, 2005), PI3K , JAGGED1/NOTCH (Come et al, 2006), SARA (Laffin et al, 2008), NFKB (Lester et al, 2007), PAR6 (Berx et al, 2001;Storci et al, 2008), pAR66A and ERK (Wu et al, 2009). Furthermore, EMT induced by the oncogenic stimulation by RAS and/or RAF activation in mammary, kidney and skin epithelial tissue was found to depend almost completely on TGF-signaling (Moustakas and Heldin, 2009).…”