Abstract 2996: Overexpression of potential oncogenic microRNAs defines a new molecular subclass of hepatocellular carcinoma
Authors: Toffanin Sara , Hoshida Yujin , Lachenmayer Anja , Cabellos Laia , Lu Jun , Villanueva Augusto , Minguez Beatriz , Savic Radoslav , Chiang Derek Y. , Roayaie Sasan , Bruix Jordi , Schwartz Myron E. , Mazzaferro Vincenzo , Waxman Samuel , Friedman Scott L. , Golub Todd R. , Llovet Josep M. 
Published: February 2011

Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third cause of cancer-related death worldwide. Its incidence is increasing mainly due to hepatitis C virus (HCV) infection. A molecular classification of HCC is still lacking. microRNAs (miRNAs) are small non-coding RNA involved in HCC pathogenesis. Their expression profiling represents a powerful tool to classify cancers.

Objectives: (1) To provide a miRNA-based molecular classification of HCC and, (2) To investigate the function of potential oncogenic miRNAs in HCC models.

Methods: Expression of 358 miRNAs was analyzed in 89 HCV-related HCCs using a bead-based miRNA expression profiling method. Integrative analysis including miRNA profiling, gene expression (Affymetrix U133 2.0®), DNA changes (Affymetrix STY Mapping Array®), IHC (p-Akt, p-IGF-IR, p-S6, p-EGFR, β-catenin) and mutation analysis (β-catenin) was performed. Expression of selected miRNA was validated in a validation set (n=167) by qRT-PCR. Methylation-specific PCR, FISH and SNP-array analyses were performed to identify mechanisms of miRNA deregulation. The function of miRNAs of interest was investigated in vitro by analyzing cell proliferation (thymidine incorporation), migration and invasion (trans-well migration and invasion assays, wound healing assay). Tumor development and growth following direct injection of luciferase-expressing cells stably transfected with specific miRNAs into the liver of nude mice were monitored to investigate their function in vivo. The bioluminescent signal emitted by luciferase-expressing cells was used as indicator of tumor growth.

Results: Three classes of HCC patients were identified and defined by activation of different pathways: Wnt signalling (32/89, 36%), IFN-related genes (29/89, 33%) and proliferative cascades (IGF, Akt/mTOR) (28/89, 31%). A subgroup within the proliferative class (8/89, 9% overexpressed a cluster of miRNAs on 19q13.42 (median fold change: 8.8). Hypomethylation of CpG island upstream the miRNA cluster (2/8, 25%) and copy number gains (1/8, 12.5%) were detected. Their overexpression was confirmed in a validation set (17/167, 10.2%). Members of the cluster family significantly increased proliferation (p&lt;0.05), migration (p&lt;0.02) and invasive capability (p&lt;0.05) in vitro and promoted tumor development in vivo (detectable liver tumor in 4/6 mice compared with 0/6 of the controls after 4 weeks following injection). One mouse of the active arm was sacrificed at week 5 showing large invasive HCC at pathological study, while the others are still alive.

Conclusions: Overexpression of 19q miRNA family and activation of proliferative pathways defined a subclass of HCC. Members of 19q miRNA family increased cell proliferation, migration and invasion in vitro and promoted tumor development in vivo suggesting their role as novel potential oncogenic drivers in HCC.

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 2996.