Abstract 3913: TGF-β signaling mediated suppression ofstat3transcription in hepatocellular cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC At least 40% of hepatocellular cancers (HCC) are clonal, suggesting that HCC may develop from malignant transformation of liver progenitor/stem cells. Several signaling pathways, including IL-6/STAT3 and TGF-β are known to be involved in stem cell renewal, differentiation and survival, and are commonly deregulated in HCC. We have previously demonstrated that human and mouse HCC tissues with aberrant TGF-β signaling show increased expression of STAT3. Moreover, down-regulation of the IL-6/STAT3 pathway in a TGF-β disrupted mouse model (β2SP+/−) by crossing with itih4—/— (Inter-alpha-trypsin inhibitor-heavy chain-4) mice resulted in a significant decrease in the incidence of HCC. Aims: This led us to hypothesize that the TGF-β signaling pathway is a strong candidate pathway for the transition from progenitor to differentiated cells and its disruption may activate the stem cell renewal IL-6/STAT3 pathway. We proceeded to investigate the role of key TGF-β signaling components, including the key Smad3/4 adaptor protein β2SP and Smad3, in the regulation of STAT3 expression. Methods and Results: First, using early passage mouse embryonic fibroblasts (MEFs) from wild type and β2SP−/− embryos, we demonstrate a four-fold increase in STAT3 mRNA by RT-PCR (p<0.001). Moreover, we demonstrate that overexpression of β2SP by transfection or inhibition of β2SP expression by siRNA in several HCC cell lines results in STAT3 suppression or induction, respectively, suggesting that β2SP and the TGF-β signaling pathway regulate STAT3 transcription. Then, using ChiP assay, we demonstrate that β2SP and Smad3 are bound to the STAT3 promoter only following TGF-β stimulation. To then further define the molecular mechanism of TGF-β-mediated STAT3 transcriptional regulation, we then used mutational analysis and demonstrate two transcription factor binding sites within the STAT3 promoter, the cAMP-responsive element (CRE) and STAT3 binding (SBE) sites, are essential for TGF-β-mediated regulation of the STAT3 transcription. Subsequent, electrophoresis mobility shift assays (EMSA) demonstrate that the CRE-binding protein ATF-2, Smad3, and β2SP proteins are major components of the TGF-β-mediated STAT3 transcriptional suppressor complex. Conclusion: These experiments demonstrate a clear link between the TGF-β and IL-6/STAT3 signaling pathways. TGF-β suppression of STAT3 transcription is mediated by a complex including [[Unsupported Character - ]]β2SP, Smad3 and ATF-2 at the CRE site of the STAT3 promoter. Inactivation of TGF-β signaling via disruption of β2SP decreases STAT3 suppression and suggests a potential mechanism for malignant transformation in TGF-β deficient progenitor/stem cells. STAT3 may also present an important target for new therapeutics in transformed cancer progenitor/stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3913.