Abstract 4718: Potent preclinical antitumor effects induced by pan-histone deacetylase inhibitor panobinostat in combination with sorafenib in hepatocellular carcinoma
Authors: Sara Toffanin, Anja Lachenmayer, Clara Alsinet, Laia Cabellos, Beatriz Minguez, Hung-Wen Tsai, Yujin Hoshida, Augusto Villanueva, Stephen C. Ward, Swan Thung, Scott L. Friedman, Josep M. Llovet
Journal: Cancer Research (2012)

Abstract Background: Hepatocellular carcinoma (HCC) is a major worldwide health problem, with over 750,000 new cases diagnosed each year. Sorafenib represents the only systemic agent approved for advanced HCC and new therapies are eagerly demanded. Histone Deacetylases (HDAC) are regulators of gene expression frequently overexpressed in cancer. HDAC-inhibitors (HDACi) represent promising agent for the treatment of HCC. Aims: (1) To analyze the expression levels and copy number alterations of 11 HDACs in human HCC and (2) to investigate the effects of the pan-HDACi panobinostat and its combination with sorafenib in experimental models of HCC. Methods: Gene expression and copy number analysis of HDACs were analyzed in 101 HCC samples, matched cirrhotics, and 10 normal livers. Cell viability (MTT assay), proliferation (3[H]-Thymidine incorporation) and cell cycle changes (FACS) were performed on Huh7, HepG2 and Hep3B cells treated with panobinostat and combination with sorafenib. A subcutaneous xenograft model using Huh7 cells was generated to assess the anti-tumor effects in vivo. Mice were randomized into 4 arms: vehicle (n=10), panobinostat (n=14, 15mg/kg), sorafenib (n=12, 30mg/kg), and combination (n=12, panobinostat 7.5 mg/kg). Endpoints were tumor growth, toxicity and survival. Vessel density, proliferation index (Ki-67 staining) and apoptosis (Tunnel assay) were analyzed on tumor sections. Target gene and protein expression levels were investigated by qRT-PCR and western blot on tumor tissues. Results: HDAC2, 4, 11 and HDAC3 and 5 were significantly upregulated in HCCs compared to normal and cirrhotic livers, respectively (p&amp;lt;0.05). Overexpression of HDAC3 and 5 correlated with copy number gains (p&amp;lt;0.05). Panobinostat together with sorafenib inhibited cell viability (26.3-73.4%) and proliferation (0.1-14.2%) of HCC cells at 72h and induced apoptosis (23% Huh7 sub-G1 phase). In vivo, combination therapy significantly inhibited tumor growth at day 20 (227.3 mm3) compared to panobinostat (531.2 mm3) or sorafenib (806.7mm3) alone (p=0.005 and p&amp;lt;0.001, respectively). Median survival improved to 33 days in the combination arm compared to 22 days in panobinostat and sorafenib groups, and 12 days in control mice (p&amp;lt;0.05). Histone 3 acetylation, p21 protein levels and autophagy (increased LC3 II, decreased p62) were significantly induced by this combination. Decrease in vessel density and Ki-67 staining as well as increase in TUNEL staining were reported. Panobinostat induced downregulation of Survivin and UHRF1 target genes and up-regulation of CDH1. Conclusion: HDACs expression is altered in HCC and related to copy gains for HDAC3 and 5. The HDACi panobinostat exhibited strong antitumor effects enhanced by the combination with sorafenib. These findings support the rationale for early clinical studies with this novel combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4718. doi:1538-7445.AM2012-4718