Introduction: Recent data implicate mTOR signaling in human hepatocellular carcinoma (HCC). However, the specific contribution of mTOR complex components as either potential oncogenes or targets for therapies is unknown. Aims: (1) To evaluate the role of mTOR complex 2 (RICTOR) in human hepatocarcinogenesis and (2) to assess the impact of selective RICTOR blockade in cellular models of HCC. Methods: DNA copy number changes (Affymetrix 238K Sty-arrays) and mRNA levels of RICTOR (Affymetrix U133 Plus 2.0 arrays and qRTPCR using Taqman Probes) were analyzed in 102 human HCV-related HCC (fresh frozen samples). RICTOR over-expression was correlated with both signaling pathway activation (Wnt-ßCatenin, IGF/AKT/MTOR and RAS/MAPK), and with the existing molecular classification of HCC (Chiang et al. Cancer Res 2008). Data were validated in a separate cohort of 164 HCC patients (paraffin-embedded samples), profiled using DASL (Illumina). Molecular data were correlated with clinico-pathological features and patient prognosis using Kaplan-Meier and Cox Proportional Hazard Test. For in vitro studies, Huh-7 cells were transfected with siRNA-RICTOR, and western blots done to evaluate downstream signals (p-AKT, p-RPS6 and p-ERK). Cell viability and proliferation were assessed by MTT and [H3]-Thymidine incorporation assay respectively, whereas FACS was used to evaluate cell cycle. Results: 25% (25/100) of HCV-related HCC samples had DNA gains in the RICTOR locus. Among them, 12% (11/87) had high mRNA levels of RICTOR (P=0.03). These patients had significantly worse survival (P=0.001) and higher rates of early recurrence (P<0.001). Similarly, patients in the validation set with highest mRNA levels of RICTOR (11.6%, 19/163) had significantly higher rates of early recurrence (P=0.01). In cultured cells after 48 hours of transfection with siRNA-RICTOR, cell viability was decreased by 20% (P=0.03) and proliferation by 30% (P=0.03), in addition to a mild but significant G1 cell cycle arrest (p=0.005), without evidence of increased apoptosis. AKT phosphorylation was reduced by siRNA-RICTOR, without interfering with MTOR complex 1 activation (phospho-RPS6). Conclusions: Over-expression of RICTOR identifies a subgroup of HCC patients (10%) with poor prognosis. Selective blockade of RICTOR has anti-neoplastic effects in cellular models of HCC, and thus further evaluation of RICTOR as a potential oncogene in animal models is warranted.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 444.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009