Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer

Abstract

Myc activation has been implicated in the pathogenesis of hepatoblastoma (HB), a rare embryonal neoplasm derived from liver progenitor cells. Here, microRNA (miR) expression profiling of 65 HBs evidenced differential patterns related to developmental stage and Myc activity. Undifferentiated aggressive HBs overexpressed the miR-371-3 cluster with concomitant down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster, evoking an ES cell expression profile. ChIP and Myc inhibition assays in hepatoma cells demonstrated that both miR clusters are regulated by Myc in an opposite manner. We show that the two miR clusters exert antagonistic effects on cell proliferation and tumorigenicity. Moreover, their combined deregulation cooperated in modulating the hepatic tumor phenotype, implicating stem cell-like regulation of Myc-dependent miRs in poorly differentiated HBs. Importantly, a four-miR signature representative of these clusters efficiently stratified HB patients, and when applied to 241 hepatocellular carcinomas (HCCs), it identified invasive tumors with a poor prognosis. Our data argue that Myc-driven reprogramming of miR expression patterns contributes to the aggressive phenotype of liver tumors originating from hepatic progenitor cells.

Fig. 1.

miR expression in HB. Unsupervised clustering of HB tumor (T), fetal liver (FL), and normal liver (NL) samples according to their miR profile using 150 probe sets with the highest coefficient of variation. Tumor annotations include the main epithelial component (F, fetal; E, embryonal, crowded fetal and/or macrotrabecular) and molecular classification (C1 or C2) based on the 16-gene signature. A cluster comprising fetal livers and C2-type HBs is ringed by a red square.

Fig. 2.

Specific miR profiles in two HB subtypes. (A) Comparative analysis of gene and miR expression in HB molecular subclasses. For each miR differentially expressed between C1 and C2 HBs, genes with negatively correlated expression profile (r < −0.45) were filtered with candidate miR targets defined by four predictive algorithms. Functional modules or pathways significantly associated with miR-related gene lists are shown as a heat map. Dark yellow, P < 0.05; orange, P < 0.01; light red, P < 0.005; dark red, P < 0.001. (B) GSEA analysis of HB miR data with a list of miRs repressed by Myc, visualized as a heat map in which the red to blue color code indicates high to low miR expression. FDR, false discovery rate. (C) Analysis of let-7, LIN28, and LIN28B expression in HB samples by qPCR. Each bar represents juxtaposed normalized data of gene and miR expression. (D) ISH assay on paraffin-embedded HB and liver samples. F, fetal; E, embryonal; s, siderophage foci. (Scale bar, 100 μm.)

Fig. 3.

Expression of the miR-371–3 cluster is regulated by Myc. (A) Schematic representation of the chromosome 19q13 locus spanning the miR-371–3 cluster (adapted from the Genome Browser Web page). *PolII binding according to Ozsolak et al. (18); **PolII binding according to Crosby et al. (19). (B) Quantitative ChIP analysis of Myc binding to different genomic regions in HepaRG (RG), HepG2 (G2), and Huh6 (H6) cell lines treated with siMyc or control siRNA (siSC). The background threshold shown in gray was set as the highest signal in mock samples. Results are presented as the mean ± SD of at least three independent experiments. (C) Comparative tissue ChIP analysis of C1- and C2-type HB.

Fig. 4.

Altered expression of miR-371–3 and miR-100/let-7a-2/miR-125b-1 clusters influences tumor cell behavior in vitro and in vivo. Huh6 cells were used in all experiments. (A) Box plot (10–90%) representing tumor sizes in mice injected with cells treated with miR-371–3 inhibitor or scrambled control (11 mice for each condition). Mean values are shown by internal horizontal bars, and SD by vertical bars. (B) Proliferation assay of cells treated as indicated. (C) Soft agar assay and Western blot analysis of cells treated as indicated. (D) Growth curve of tumors in nude mice injected with cells treated as indicated, using 5 mice for each condition. Bars indicate SD.

Fig. 5.

Classification of HB and HCC with a four-miR signature. (A) Cm1 and Cm2 HB subclasses defined by differential expression of four miRs are significantly associated with clinical parameters. Kaplan–Meier analysis shows significant differences in overall survival probability between patients carrying Cm1- and Cm2-type HBs. (B) Unsupervised hierarchical clustering of 241 HCC samples by using the HB four-miR signature identifies two main tumor classes. (C) Cm1 and Cm2 HCC subclasses present significant differences in clinical annotations and overall patient survival.