microRNAs and the MYC Network: A Major Piece in the Puzzle of Liver Cancer

Cairo S, Wang Y, de Reyniès A, et al. (Oncogenesis and Molecular Virology Unit, Institut Pasteur, 75015 Paris, France). Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer. Proc Natl Acad Sci U S A 2010;107:20471–20476. Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) represent the most prevalent forms of liver cancer in pediatric and adult patients, respectively. They differ in many aspects, including etiology, risk factors, and clinical management. Typically, HBs arise in absence of viral infection and liver disease, whereas HCC usually develops in a context of liver damage mostly related to hepatitis C and B virus infection or alcohol abuse. Curative treatments (eg, surgical resection, liver transplantation, local ablation) represent the main therapeutic options for these patients and sorafenib is the sole systemic agent approved for advanced HCC (N Engl J Med 2008;359:378–390). Many efforts have been made to understand the molecular pathogenesis of these diseases and several pathways, such as β-catenin/Wnt signaling, have been reported as dysregulated in both malignancies. The transcription factor MYC is often overexpressed in both tumor types and acts as a master promoter of cell proliferation and survival. MYC oncoproteins regulate the expression of multiple genes related to cell cycle such as cyclin-dependent kinases (eg, CDK2, CDK6) and numerous cyclins (eg, CCNA, CCND1, and CCNE). Furthermore, in the recent years it has been shown that MYC can promote tumorigenesis by controlling the expression of microRNAs (miRNAs), small endogenous regulatory molecules (Nat Genet 2008;40:43–50). MiRNAs control a wide variety of cellular processes, including self-renewal, differentiation, and division of cells by translational repression and/or mRNA cleavage. In liver cancer, there is compelling evidence suggesting that miRNAs are important players with pro-oncogenic functions (so called "oncomiRs"), such as miR-21 (Gastroenterology 2007;133:647–658) or having tumor suppressor activities, for example, the let-7 family and miR-26 (Nat Genet 2009;41:843–848; N Engl J Med 2009;361:1437–1447). Different molecular mechanisms are involved in miRNA dysregulation in cancer cells, including copy number changes (amplification/deletion), chromosomal aberrations (eg, translocation), epigenetic modulation, and transcriptional activation or repression. In the study by Cairo et al, the authors performed miRNA expression profiling of 65 HB human samples and identified different miRNAs patterns in the 2 previously reported molecular subtypes named C1 and C2, respectively (Cancer Cell 2008;14:471–484). The C1 class included samples with milder phenotype, whereas the C2 cluster consisted of undifferentiated and highly invasive HBs with poor survival. Supervised clustering analysis showed that C2 subtype was characterized by up-regulation of the miR-371-3 cluster, which is normally expressed in embryonic stem cells, and down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster, which is known to inhibit proliferation and promote cellular differentiation. Furthermore, the authors demonstrated that the stemness-associated miR-371-3 cluster was directly regulated by MYC in a positive manner using siRNA strategy and ChIP assay on HB cell lines. In human samples, MYC and miR-371-3 expression were positively correlated. Functional studies showed that the artificial expression of the miR-371-3 cluster and silencing of the miR-100/let-7a-2/miR-125b-1 cluster inhibited proliferation in vitro as well as tumorigenicity in vivo and these effects were enhanced when simultaneous modulation of the 2 miRNA clusters was achieved. Finally, the authors identified a MYC-dependent miRNA signature consisting of 4 miRNAs (ie, miR-100, let-7a, miR-371 and miR-373), able to detect highly invasive HBs and more undifferentiated tumors (HB Cm2). Of important note, the signature was applied to a large cohort of human HCCs and significantly classified samples in 2 groups resembling HB classes (Cm1 and Cm2) with distinct metastatic phenotypes and clinical behavior. Similarly to the findings reported in HB, HCC Cm2 subgroup was associated with poor differentiation degree, invasive phenotype, and poor outcome. Recently, the prognostic relevance of miRNA expression has been elucidated in numerous cancer types, including HCC (N Engl J Med 2009;361:1437–1447). In the paper discussed herein, Cairo et al identified a MYC-dependent miRNA signature composed of only 4 miRNAs, which efficiently identified poorly differentiated tumor subtypes in both HB and HCC. This study has doubtless important clinical and biological implications. From a clinical perspective, the simple detection of 4 miRNAs in HCC samples would provide important information related to the metastatic potential and outcome of patients. Therefore, it would be possible to simply detect the expression levels of a few miRNAs to refine the prognosis of each patient. As with other prognostic biomarkers in liver cancer, this novel information requires external validation prior to being accepted for decision-making algorithms (Clin Cancer Res 2010;16:4688–4694). From a biological point of view, the study highlights the central role of MYC in a molecular circuitry which involves miRNAs implicated in cell renewal and stemness features of HCC cells. Importantly, the signature, which was generated on HB samples, efficiently classified HCC in 2 similar groups, generating a molecular link between these 2 different forms of liver cancer. Therefore, the next logical step would be to validate the prognostic relevance of this signature in order to complement current HCC outcome prediction algorithms (Clin Can Res 2010;16:4688–4694). In addition, it would also be interesting to test this signature in other liver malignancies with different origin and clinical behavior (eg, fibrolamellar HCC and cholangiocarcinoma) and, in a potential wider scenario, to apply the signature to other cancer types. Indeed, a previous study showed that low expression of let-7a-2 (which is included in the signature reported by Cairo et al) was associated with poor outcomes in patients with lung cancer (Cancer Cell 2006;9:189–198), suggesting that the same miRNAs can have similar functions and prognostic implications in different tumors. Although miRNAs are frequently dysregulated in cancer, few studies have focused on the molecular events triggering their aberrant expression in tumor cells. The study by Cairo et al demonstrated that MYC simultaneously regulates the expression of 2 important miRNA clusters: miR-371-3 and miR-100/let-7a-2/miR-125b-1, which control cell differentiation and proliferation in opposite ways. The relevant role of MYC has been long established in the pathogenesis of liver cancer. Its overexpression into hepatocytes led to the development of HCC and its transient inactivation sufficed to induce regression of invasive liver cancer (Nature 2004;431:1112–1117). MYC is frequently overexpressed in HCC owing to different molecular mechanisms, such as genomic amplification (gains of chromosome 8q) or transactivation by HBx in hepatitis B virus–induced hepatocarcinogenesis. Previous studies showed that HCCs harboring a MYC activation signature were characterized by enrichment of proliferative pathways (eg, AKT signaling) and a stemness-related EpCAM-signature, which supports a link between MYC overexpression and stem cell–like features (Cancer Res 2009;69:7385–7392). This connection is further enhanced by recent studies showing that MYC activates the expression of LIN28 (EMBO J 2009;28:347–358), which maintains cells in an undifferentiated state by post-transcriptional repression of let-7 miRNAs. Accordingly, LIN28 was overexpressed in a subset of HCCs showing coordinate repression of let-7 and enrichment of MYC-targets (Nat Genet 2009;41:843–848). The complexity of this network is further enhanced by the evidence that let-7 inhibits MYC expression in a negative feedback loop. In summary, when MYC is overexpressed, tumor cells are maintained in a more undifferentiated state owing to up-regulation of LIN28, miR-371-3 cluster, and repression of let-7 family. Given the massive relevance of this network in the pathogenesis of HCC, strategies targeting different levels of this molecular cascade could prove to be therapeutically successful. Recently, small-molecule inhibitors that interfere with the MYC/MAX heterodimerization have also been developed to block MYC-mediated transactivation (Anticancer Drugs 2007;18:161–170). The MYC-specific inhibitor 10058-F4 showed significant efficacy in vitro, although less encouraging results were obtained in vivo, probably owing to rapid metabolism resulting in low concentrations in tumors (Cancer Chemother Pharmacol 2009;63:615–625). Other small-molecule compounds with improved pharmacokinetic profiles have been developed. Among these, CX-3453 (Quarfloxin), which acts by decreasing MYC and VEGF mRNA levels, is currently in Phase II clinical trials for neuroendocrine tumors. Nevertheless, the preclinical use of MYC-specific inhibitors caused hepatic hypertrophia and structural damage in the architecture of the liver parenchyma, which might prevent its use in patients with impaired liver function. Nevertheless, the study by Cairo et al suggests that the effects induced by MYC activation can be blocked at different levels by modulating downstream effectors of MYC activity, such as members of the miR-371-3 and/or the miR-100/let-7a-2/miR-125b-1 clusters. As a consequence, they likely represent foreseeable targets for new therapies for the treatment of liver cancer. Strategies modulating miRNA expression are currently under preclinical investigation and an increasing number of oncomiRs (eg, miR-21) and tumor suppressor miRNAs (eg, let-7) have been discovered in HCC, thereby expanding the spectrum of potential targets for the treatment of this disease. The study commented herein highlighted the oncogenic properties of miR-371-3 cluster in liver cancer, similar to the findings reported in human testicular germ cell tumors and thyroid carcinomas (Cell 2006;124:1169–1181; PlosOne 2010;5:e9485), supporting the evidence that strategies inhibiting these specific miRNAs could be successful as antitumoral therapies. Preclinical studies with antagomirs targeting oncogenic miRNAs showed the efficacy and safety of these molecules (Nat Biotechnol 2010;28:341–7), although studies in liver cancer models are still needed. In parallel, the artificial replacement of members of the miR-100/let-7a/miR-125b-1 cluster could be beneficial for the treatment of liver cancer, as suggested by Kota et al (Cell 2009;137:1005–1017). In conclusion, this study further strengthens the key role of miRNAs as prognostic indicators and potential targets for new therapies in liver cancer. Further efforts are undoubtedly required to validate prognostic signatures reported so far and test the efficacy of novel miRNA-based therapeutic approaches in preclinical and early clinical studies. A deeper knowledge of the biological relevance of these small molecules would certainly add an important piece of information into the complex puzzle of liver cancer biology.