Inhibition of Single Minded 2 gene expression mediates tumor-selective apoptosis and differentiation in human colon cancer cells

Abstract

at the Down's syndrome critical region, which in triplication is associated with diverse phenotypic characteristics of Down's syndrome (1). Patients with Down's syndrome show various neurological symptoms and a high incidence of leukemia (1, 2). Members of the SIM family include SIM1 and SIM2, which map to 6q16.3-q21 and 21q22.2, respectively (3), and belong to a family of transcription factors containing a basic helix-loop-helix motif, two period homologue (PER)͞ARNT͞SIM (ARNT, aryl hydrocarbon receptor nuclear translocator) domains, and the HIF1␣͞SIM͞TRH domains (4-6). In Drosophila, SIM is a master regulator of fruit-fly neurogenesis, regulating midline gene expression (6, 7). The SIM2 gene exists in two distinct forms, the long and short forms (SIM2-l and SIM2-s), due to alternative splicing (3). A putative cancer-related role of the SIM family of genes is their ability to transcriptionally regulate key metabolic enzymes to inactivate carcinogens (8). Binding of carcinogens to the aryl hydrocarbon receptor (AhR), which is kept sequestered in the cytoplasm by heat-shock protein (HSP) 90 (9), dissociates HSP 90. The ligand-bound AhR is then translocated into the nucleus, where it can dimerize with ARNT (10). This complex binds to the xenobiotic response element, present in the promoters of key oxidative enzymes, and activates gene transcription (8, 11), thus causing inactivation of the carcinogen. The SIM proteins can inhibit the dimerization of the ligandbound AhR͞ARNT complex (12) and hence prevent carcinogen metabolism, leading to cumulative DNA damage and cancer.The growth arrest and DNA damage (GADD) family of genes was originally isolated from UV radiation-treated cells and subsequently grouped according to their coordinate regulation by growth arrest and DNA damage (13). The GADD family members include GADD34, -45␣, -45␤, -45␥, and -153 (14, 15). These are stressresponse genes induced by both genotoxic and nongenotoxic stresses (16)(17)(18). GADD45␣ is the most extensively studied member of the family and is regulated in both a p53-dependent and -independent manner (13,19). GADD45␣-mediated apoptosis may involve activation of JNK and͞or p38 mitogen-activated protein kinase (MAPK) signaling pathways (14,20).We have recently demonstrated that the SIM2 short-form (SIM2-s) gene is specifically expressed in distinct solid tumors, including colon, pancreas, and prostate, but not in the corresponding normal tissues (21-23). Antisense inhibition of SIM2-s expression caused apoptosis in colon and pancreatic cancer-derived cells. In an effort to understand the molecular mechanism underlying the role of SIM2-s in tumor cell growth, we have embarked on mapping the key pathways linked to SIM2-s. We show that apoptosis is tumor cell-selective and requires GADD45␣ function. Further, we demonstrate that key pathways, including caspase, p38 MAPK activities, and WT p53 status, are critical to apoptosis. Our results link apoptosis to induction of tumor cell-selective differentiation. MethodsCell Culture. Isogenic colon ca...

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

“…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. Moreover, consistent with our findings, others have reported, although the finding is not validated, in RKO colon cancer cells an up-regulation of BNIP3 mRNA following antisense knockdown of endogenous SIM2s, and concomitant loss of cell viability (refer to the supplementary data in Aleman et al, 2005). Unlike SIM2s (Halvorsen et al, 2007), there is no understanding of how BNIP3 levels correlate with prostate tumour cell survival or prognosis.…”

“…Thus, the authors propose SIM2s levels as a novel marker of aggressive prostate cancer. Studies by Narayanan and colleagues have strongly linked SIM2s expression with the survival of tumour-derived cell lines of the pancreas and colon, where antisense-mediated knockdown of SIM2s in cell culture and mouse xenograft models results in cell death and a decrease in tumour size (DeYoung et al, 2002(DeYoung et al, , 2003aAleman et al, 2005). Conversely, however, SIM2s has recently been found to be repressed in breast cancer tumours (Kwak et al, 2007).…”

“…The expression status of SIM2s is emerging as an interesting candidate for a solid tumour marker. Select up-regulation of SIM2s has been identified in solid tumours of the pancreas, colon and prostate (DeYoung et al, 2002(DeYoung et al, , 2003bAleman et al, 2005;Halvorsen et al, 2007). Although the focus has mainly been on SIM2s in these tumour types, SIM2l messenger RNA (mRNA) has also been found to be expressed in tumours and tumour-derived cell lines of the pancreas (DeYoung et al, 2003b) and in prostate tumours (Halvorsen et al, 2007), but not in the corresponding normal cell lines or benign tissue samples.…”

“…The loss of BNIP3 in pancreatic and colon tumours, either by hypermethylation or by transcriptional repression, is correlated with inhibition of cell death (Okami et al, 2004;Murai et al, 2005;Bacon et al, 2007;Mahon et al, 2007), whereas up-regulation of BNIP3 sensitizes pancreatic carcinoma cells to hypoxia-induced cell death (Abe et al, 2005). Aleman et al (2005) have shown that cell death resulting from SIM2s antisense treatment of colon cancer RKO cells was apoptotic, being largely dependent on the activities of caspases 9 and 10; however, mechanisms of autophagy have not been investigated. Autophagic and apoptotic cell death programmes are not mutually exclusive, and BNIP3 has been implicated in both mechanisms (for reviews, see Maiuri et al (2007) and Burton and Gibson (2009)).…”

PCAF Acetylates β-Catenin and Improves Its Stability

“…To test this possibility, alkaline phosphatase activity, a marker of colon cancer cell differentiation (Aleman et al, 2005), was examined in LoVo cells. As shown in Figure 7A, knockdown of PCAF significantly induced the LoVo cell differentiation.…”