Abstract A228: TGF-beta signaling inhibition using LY2157299 affects proliferation or invasion in hepatocarcinoma cells and patient samples.

Abstract

Abstract Background: To date, the only FDA approved targeted agent in advanced hepatocarcinomas is sorafenib, indicating a critical need for innovative therapeutic options. LY2157299, a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation, is currently under clinical investigation in HCC patients after sorafenib or in patients ineligible for sorafenib. Our study aimed at investigating the effects of LY2157299 in HCC cell lines and patient samples with various AFP expression levels. Materials and Methods: Antiproliferative effects of LY2157299 were evaluated in a panel of human HCC cells by MTT assay. Baseline and phosphorylated (p-) protein levels were assessed by Western blot analysis and mRNA expressions by qRT-PCR. Invasion assays were done on matrigel and in OptiCell systems. Tumor samples from HCC patients were surgically resected and cut in 300 µm thick slices using a Tissue Slicer. Each slice was randomly exposed to LY2157299 (1µM and 10µM) and sorafenib (5µM) for 48h. At the end of treatment, tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence (IF). Results: LY2157299 was evaluated in HEPG2 and HUH-7 AFP-positive and HEP3B and SK-HEP1 AFP negative cells as well as “in lab-established” SK-HEP1-derived cell lines resistant to the tyrosine kinase inhibitors sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of canonical Smad pathway that was potently inhibited with LY2157299 treatment at micromolar concentrations. In contrast, the non-canonical pathways such as MAPK and Akt/mTOR were not activated by TGF-β in most cell lines with the exception of SK-HEP1 cells where LY2157299 restored the basal level of p-ERK1/2, p-AKT and p-S6 kinase phosphorylation. Low concentrations of LY2157299 displayed antiproliferative effects in HEPG2 and HEP3B cells when stimulated by TGF-β but not in other cell lines. LY2157299 yielded potent anti-migratory and anti-invasive properties in invasive SK-HEP1, SK-Suni and SK-Sora cells. Tumor slices from surgically resected advanced HCC tumor from 5 different patients, were exposed ex vivo to LY2157299 or sorafenib for 48h. This method allows the evaluation of novel anticancer agents in whole tumors containing cancer cells and stromal cells. LY2157299 but not sorafenib decreased 2-5 fold the p-Smad2/3. LY2157299 treatment mainly affected PI3K/AKT rather than MAPK signalling pathway. In contrast, sorafenib treatment reduced both PI3K and MAPK pathway activation. IHC analysis of LY2157299 and sorafenib-exposed samples showed a significant decrease (>3 fold) of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3 (3-5 fold). Interestingly, all molecular effects were independent of AFP expression. Conclusion: TGF-β/TβR-I inactivation using LY2157299 inhibits TGF-β-dependent cell signaling in HCC cell lines with either anti-proliferative or anti-invasive effects depending on the model. In tumor samples from patients, inhibition of TGF-β signaling was associated with inhibition of proliferation and apoptosis induction regardless AFP level. Our data suggest that LY2157299 may be useful for patients with HCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A228. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Celia Dos Santos, Karim A. Benhadji, Valerie Paradis, Eric Raymond, Sandrine Faivre, Armand de Gramont. TGF-beta signaling inhibition using LY2157299 affects proliferation or invasion in hepatocarcinoma cells and patient samples. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A228.