Abstract B175: TGF-β-RI kinase inhibitors LY2157299 and LY364947 decrease motility and invasion in hepatocarcinoma (HCC) cells developing resistance to VEGFR/PDGFR kinase inhibitors.

Abstract

Abstract Background: Activity of sorafenib in patients with advanced stages of HCC often decreases over time due to acquired resistance. TGF-β1 is frequently overexpressed in HCC and correlates with poor survival prognosis. TGF-β1 signalling is involved in cellular invasion, mesenchymal transition and may play a role in tumor progression after antiangiogenic therapies. LY2157299 and LY364947 are specific ATP-mimetic inhibitors of TGF-β receptor (TβR)-I activation, LY2157299 having recently entered clinical trials in HCC patients. Our study explores the cellular effects of these TGF-β inhibitors in HCC cells developing resistance to tyrosine kinase inhibitors (TKi). Materials and Methods: TGF-β inhibitors were tested in HEPG2, SK-HEP1 cells and the derived-counterpart SK-suni cells selected by stepwise exposure to the multikinase inhibitor sunitinib. The effects on cellular proliferation, motility, and invasion of LY2157299 and LY364947 were evaluated in human HCC cells by MTT, wound-healing and matrigel assay, respectively. Baseline and phosphorylated (p-) protein levels were studied by Western blot analysis and mRNA expression by qRT-PCR. Results: Protein- and mRNA-expression of TGF-β1, TGF-β2, and TβR-I was detectable in HEPG2, SK-HEP1 and SK-Suni cells. Exogenous stimulation of the three HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 as well as p-ERK1/2, p-AKTser473, and p-S6. In TGF-β-stimulated HEPG2, SK-HEP1 and SK-Suni cells, LY2157299 and LY364947 inhibited p-Smad2 and p-Smad3 at μmolar concentrations. LY364947 also inhibited TGF-β-induced downstream p-AKT473 and p-ERK1/2 signaling in SK-HEP1 cells. LY364947 displayed moderate antiproliferative effects at concentrations up to 20μM (with and without exogenous TGF-β stimulation) after 72h exposure in our cell lines. In contrast, antiproliferative effects of LY2159299 were observed only in presence of TGF-β in HEPG2 cells. Interestingly, 1 and 10μM LY2159299 and LY364947 decreased spontaneous TGF-β-independent cell motility in SK-HEP1 and SK-Suni cells. Both inhibitors at 10 μM decreased TGF-β-independent invasion in SK-HEP1 and SK-Suni. However, TGF-β induced invasion was almost completely abrogated in sunitinib-tolerant cell line after LY2157299 treatment. LY2157299 inhibited also invasion in the less invasive HEPG2 cells. Conclusion: Blockage of TGF-β/TβR-I activation using LY2157299 and LY364947 inhibits TGF-β-dependent cell signaling and reduces cell motility and invasion in parental and TKi-tolerant HCC cells. The antimetastatic potential of LY2157299 deserves further investigations (either as single agents and/or in combination) in clinical trials for patients with HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B175.