Ets-1 and Runx2 Regulate Transcription of a Metastatic Gene, Osteopontin, in Murine Colorectal Cancer Cells

Abstract

Osteopontin (OPN) is a sialic acid-rich phosphoprotein secreted by a wide variety of cancers. We have shown previously that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. Although a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis-and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC (young adult mouse colon) epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nucleotide (nt) ؊107 to ؊174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared with YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt ؊101 to ؊123 and nt ؊121 to ؊145, which specifically bind CT26 nuclear proteins. Competition with unlabeled mutant oligonucleotides revealed that the regions nt ؊115 to ؊118 and nt ؊129 to ؊134 were essential for protein binding. Subsequent supershift and chromatin immunoprecipitation assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in a >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein.Colorectal cancer continues to be the second leading cause of cancer-related deaths in the United States (1, 2). Many patients with advanced or recurrent disease develop distant metastases and fail locoregional therapy. The molecular mechanisms underlying tumor metastasis are not completely understood, but recent evidence implicates osteopontin (OPN) 2 as a key regulator of cancer cell metastasis.OPN, an extracellular matrix protein secreted by a wide variety of cancers, functionally enables tumor progression (3-8).The secreted phosphoprotein binds the ␣ v ␤ integrin and CD44 families of receptors to propagate cellular signals (8 -14). In colorectal cancer, gene profiling studies have identified a positive correlation between advanced or metastatic colon tumors and abundant OPN expression (15, 16). We have previously shown that stable, RNAi-mediated down-regulation of OPN expression in CT26 colon adenocarcinoma cells results in suppression of matrix metalloproteinase-2 (MMP-2) expression, decreased in vitro tumor cell motility and invasiveness, and attenuation of functional in vivo hepatic metastasis (17). Together, these data suggest that OPN is necessary for CT26 colorectal metastasis. Subsequent studies in our laboratory have confirmed that OPN protein expression is highly up-regulated in meta...

Abnormal Expression of the ERG Transcription Factor in Prostate Cancer Cells Activates Osteopontin

“…Another potential inverted EBS was identified at nt À108 to À111 (EBS2), but was found only in the mouse and rhesus macaque genomes. The computer search for transcription factor binding sites (Transfac analysis) also disclosed Runx2, SBF-1, C/EBP, and AP-1 sites, which have already been reported (30).…”

“…Two members of the ETS family, PEA3 and Ets-1, have been shown to be partially responsible for the overexpression of OPN in human breast cancer and in murine colorectal cancer cells, respectively (29,30). In this study, we investigated whether ERG overexpression stimulates OPN expression in PCa cells.…”

A bioinformatical and functional approach to identify novel strategies for chemoprevention of colorectal cancer

“…Moreover, we revealed potentially new interesting pathways, of which the biological relevance for colon adenomas remains to be elucidated. Although the importance of these routes is likely to be the case in colon carcinomas, there is not enough evidence to speculate about implications and targeting of these routes in adenomas (Table 2, for example, the Skp2, the p27 and the Ets pathway) (Timmerbeul et al, 2006;Wai et al, 2006;Fujita et al, 2008).…”

Angiotensin II-Induced Osteopontin Expression in Vascular Smooth Muscle Cells Involves Gq/11, Ras, ERK, Src and Ets-1

“…In the present study, siRNA against Ets-1 significantly suppressed Ang II-induced OPN expression, indicating that Ets-1 plays a role in Ang IIinduced OPN transcription. Indeed, the promoter of the OPN gene has Ets-1 binding sites, and transcriptional regulation of the OPN gene by Ets-1 has been demonstrated in another cell type (23,40). Importantly, Ets-1 has recently been shown to play a critical role in Ang II-mediated vascular remodeling and inflammation (41).…”