Abstract 2094: Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen.
Authors: Serova Marie , Tijeras-Raballand Annemilaï , Dos Santos Celia , Muller Nelly , Benhadji Karim A. , Paradis Valerie , Faivre Sandrine , Raymond Eric , de Gramont Armand 
Published: August 2014

Background: Therapeutic options for patients with advanced hepatocarcinomas (HCC) are still limited. TGF-β1 and α-fetoprotein (AFP) have been shown to be independent prognostic factors for disease progression and recurrence. LY2157299, a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation, is currently under clinical investigation in HCC patients. Our study aimed at exploring 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 protein levels were detected by Western blot analysis and mRNA expressions by qRT-PCR. Invasion assays were done on matrigel and in OptiCell devices. Tumor samples from HCC patients were surgically resected, cut in 300 μm thick slices using a Tissue Slicer. Each slice was randomly selected and exposed to LY2157299 (1μM and 10μM) and sorafenib (5μM) for 48h. Tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence (IF).

Results: LY2157299 was evaluated in HEPG2, HEP3B and SK-HEP1 cells as well as SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with LY2157299 treatment at micromolar concentrations. Low concentrations of LY2157299 displayed antiproliferative effects in HEPG2 cells when stimulated by TGF-β but not in SK-HEP1, SK-Sora, SK-Suni and HEP3B cells. Interestingly, HEPG2 cells were the only cell line displaying high levels of AFP as along with an epithelial phenotype. LY2157299 yielded potent anti-migratory and anti-invasive properties in invasive SK-HEP1, SK-Suni and SK-Sora cells. Tumor slices from surgically resected tumor samples from 3 patients with advanced HCC, were exposed ex vivo to 1 μM and 10 μM LY2157299 or 5 μM sorafenib for 48h. This method allows evaluating the effects of novel anticancer agents in whole tumors containing cancer cells and stroma cells. LY2157299 but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling as well as AFP levels. IHC analysis of LY2157299 and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. Interestingly these 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 decreased AFP levels, inhibition of proliferation and apoptosis induction. Our data suggest that LY2157299 may be useful for patients with HCC.

Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Celia Dos Santos, Nelly Muller, Karim A. Benhadji, Valerie Paradis, Sandrine Faivre, Eric Raymond, Armand de Gramont. Effects of TGF-beta signaling inhibition with LY2157299 in hepatocarcinoma models and in ex vivo whole tumor tissue samples from patient specimen. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2013-2094