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Comparative Study
. 2008 Dec;135(6):1972-83, 1983.e1-11.
doi: 10.1053/j.gastro.2008.08.008. Epub 2008 Aug 20.

Pivotal role of mTOR signaling in hepatocellular carcinoma

Augusto Villanueva  1 , Derek Y Chiang, Pippa Newell, Judit Peix, Swan Thung, Clara Alsinet, Victoria Tovar, Sasan Roayaie, Beatriz Minguez, Manel Sole, Carlo Battiston, Stijn Van Laarhoven, Maria I Fiel, Analisa Di Feo, Yujin Hoshida, Steven Yea, Sara Toffanin, Alex Ramos, John A Martignetti, Vincenzo Mazzaferro, Jordi Bruix, Samuel Waxman, Myron Schwartz, Matthew Meyerson, Scott L Friedman, Josep M Llovet
Affiliations
Comparative Study

Pivotal role of mTOR signaling in hepatocellular carcinoma

Augusto Villanueva et al. Gastroenterology. 2008 Dec.

Abstract

Background & aims: The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway.

Methods: The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model.

Results: Aberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade.

Conclusions: MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.

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Figures

Figure 1
Figure 1
Flow chart summarizing the human samples analyzed in the study of mTOR pathway in HCC (n=351). An exploratory set (n=77) intended to find out the expression pattern of key genes of the mTOR pathway in the whole hepatocarcinogeneis process whereas the replication set (n=78) was focused on HCC samples. A clinical training set originated from both exploratory and replication sets was use to generate hypotheses on clinical correlations of mTOR pathway activation (n=82). These hypotheses were validated in an independent clinical cohort (n=196).
Figure 2
Figure 2
Dot-plot of the mRNA levels of 6 genes analyzed in the replication set (very early/early HCC (n=19) and advanced/very advanced HCC (n=59)): IGF2, EGF, IGFBP3, PTEN, RAPTOR, mTOR. Each dot represents the fold-change in expression of each sample normalized to 1 (mean expression in normal liver).
Figure 3
Figure 3
Kaplan-Meier curves showing the correlation between p-RPS6 staining and recurrence (A)/early recurrence (B) in the validation set.
Figure 4
Figure 4
A: Histogram for the copy numbers in 0.5 Mb windows of RICTOR. Red bars represent patients with gains in RICTOR (DNA copy number cutoff=2.27). B: Dot-plot of the mRNA levels of RICTOR in the replication set. C–D: Kaplan-Meier plots of recurrence (C) and early recurrence (D) after clustering patients according to copy number changes of RICTOR. E–F: Downregulation of RICTOR in cell lines transfected with siRNA (Western blot) (E), and significant decrease in cell viability measured with MTT assay in Huh7 cells following downregulation of RICTOR. Graphic shows percentage of viable siRNA-RICTOR transfected cells relative to control siRNA for each time point and cell line analyzed (F).
Figure 5
Figure 5
Cell viability (A–C) and proliferation studies (D) in liver cancer cell lines treated with EGFR inhibitor (AEE788) and mTOR inhibitor (RAD001, everolimus). Results are presented as % of viable cells or % of 3H-Thymidine incorporation (counts per million) in compare to control. Cell cycle profiles (E) and PARP cleavage immunoblotting in Huh7 cells incubated with the compounds (F).
Figure 6
Figure 6
Results of the immunoblotting for total and phosphorylated forms of key proteins of the mTOR pathway (A). Luciferase activity linked to a c-fos reporter in Huh7 cells after incubation with everolimus and EGFR inhibitor (B). Tumor volume and mice survival in xenografts treated with EGFR inhibitor and everolimus (C–D).
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