Gene regulatory and pro-tumourigenic mechanisms of the bHLH-PAS transcription factor SIM2s.

Abstract

Single-minded 2 (SIM2), a class I basic Helix-Loop-Helix/PAS (bHLH/PAS) transcription factor, is essential for early development, and the short isoform (SIM2s) is selectively up-regulated in pancreatic and prostate tumours. Mechanistic roll(s) for SIM2 that are essential for development and in these cancers is unknown, largely due to the fact that few bona fide target genes have been described for SIM2. SIM2 must heterodimerise with the obligate class II partner factor ARNT to regulate transcription. Surprisingly, these studies reveal SIM2 plays a role in the regulation of the ARNT homologues, ARNT1 and ARNT2. Two nonexclusive mechanisms were identified; enhanced protein stabilisation, and the specific increased transcription of ARNT2. The regulation of ARNT by a class I family member was found to be unique to the SIM homologues. These findings suggest novel insights into how elevated levels of SIM2s in tumours may confer increased transcriptional activities and/or increase the availability of the essential partner factor for other class I family members to promote their respective activities and functions in developmental and/or tumourigeneic processes. Furthermore, microarray studies in prostate DU145 cells identified the pro-cell death gene, BNIP3 (Bcl-2/adenovirus E1B 19kDa interacting protein 3), as a novel target of SIM2s mediated repression. Further validation showed BNIP3 repression in several prostate and pancreatic carcinoma derived cell lines with ectopic expression of human SIM2s via SIM2s activities at the proximal promoter hypoxia response element (HRE), the site through which bHLH/PAS family member, Hypoxia-Inducible Factor 1α (HIF1α), induces BNIP3. SIM2s attenuates BNIP3 hypoxic induction via the HRE, and increased hypoxic induction of BNIP3 occurs with siRNA knockdown of endogenous SIM2s in prostate PC3AR+ cells. BNIP3 is implicated in hypoxia-induced cell-death processes. PC3AR+ cells expressing ectopic SIM2s have enhanced survival upon treatment with hypoxia mimetics, DP and DMOG. LC3-II protein levels fail to induce in PC3AR+/SIM2s DMOG and hypoxia treated cells, suggesting SIM2s may attenuate autophagic cell-death processes, perhaps via BNIP3 repression. These data show, for the first time, SIM2s cross-talk on an endogenous HRE. SIM2s functional interference with HIF1α activities on BNIP3 may indicate a novel role for SIM2s in promoting tumourigenesis. Moreover, SIM2 expression has previously been implicated in the Hedgehog (Hh) signalling pathway during mouse brain development. The Hh-pathway is known to promote pancreatic and prostate tumour growth, and these studies indicate that SIM2s is indeed implicated in promoting and/or maintaining Hh-signalling in cell lines of these cancer types. Likewise, aberrant Androgen Receptor (AR)-signalling is implicated in prostate tumour development, and androgen-independent AR activity is a hallmark of aggressive prostate cancer. Unexpectedly, SIM2s expression was found to up-regulate endogenous AR protein levels in prostate carcinoma PC3AR+ cells. Furthermore, SIM2s expression is associated with androgen-dependent wtAR-transcriptional responsiveness in these cells, and SIM2s co-immunoprecipitates with endogenous AR in a hormone independent manner. Together these data suggest, for the first time, that SIM2s may function as a coactivator, and concomitant with enhancing AR levels, aid AR-signalling in prostate cancer cells. In summary, these studies sought to identify molecular mechanisms by which aberrant levels of SIM2s expression in solid tumours of the prostate and pancreas may promote tumour development. Several novel mechanisms for SIM2s activities were identified which implicate SIM2s in tumour processes. Namely SIM2s was found to be implicated in: 1) promoting pro-tumourigeneic Hh and AR signalling pathways 2) regulation of the common partner factor ARNT, and 3) attenuation of hypoxically-induced cell-death processes in tumour cells via the direct transcriptional repression of the novel SIM2s target gene, BNIP3.