Hippo Tumor Supressor Pathway: Novel Implications for the Treatment of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains the main cause of death in cirrhotic patients and the third most common cause of cancer related death worldwide (CA Cancer J Clin 2005;55(2):74-108). Curative treatment options are restricted to less than 30-40% of patients due to disease progression at diagnosis (Lancet 2003; 362:1907-1917), demanding better surveillance programs for early tumor detection and the development of both chemopreventive treatments and novel treatment strategies. A multikinase inhibitor sorafenib greatly succeeded in the treatment of advanced HCC and became the only FDA-approved therapy, clearly demonstrating that molecular targeted therapy is a realistic treatment option in this cancer (N Engl J Med 2008;4:378-390). This success has facilitated many preclinical and clinical trials investigating novel small molecules and monoclonal antibodies for the treatment of HCC (Hepatology 2008;48:1312-27). In the future, more precise and comprehensive characterization of molecular classes, genetic and epigenetic changes, and altered cell signaling pathways should be taken into account to choose the right treatment scheme for each patient, leading to a much more personalized medicine in HCC (Semin Liver Dis 2010;30(1):35-51). Wnt-β-catenin, p53, IGF, Ras-MAPKK, PI3/Akt/mTor and c-Met are well studied signaling pathways involved in the development and progression of HCC. Recently, genetic and biological studies confirmed the importance of the novel Hippo tumor-suppressor pathway in regulating cell proliferation, apoptosis, organ size and tumorigenesis in mammals (Cancer Cell 2008;13:188-192). This relatively new pathway was first described in Drosophila in 1995 by identifying the tumor suppressor Warts (WTS), followed by the establishment of several other core Hippo pathway components (Fig. 1) (Curr Opin Cell Biol 2008;20(6):638-46). In mammalian cells NF2, FRMD6 and FAT4 are upstream regulators of the MST1/2 kinases that physiologically form a complex with WW45 upon activation to then phosphorylate and activate the downstream kinases LATS1/2. This core complex together with MOB1 phosphorylates and inhibits the transcription co-activators YAP1 and TAZ by causing their cytoplasmatic translocation (Nat Rev Mol Cell Biol 2007;8(8):613-21; Nat Rev Cancer 2007;7(3):182-91). Many of these Hippo pathway members have been implicated in the genesis of cancer. NF2 is mutated or silenced in familial (neurofibromatosis type 2) and sporadic human tumors, MOB1 deletion has been shown in human melanoma and mouse mammary gland carcinoma. LATS1 deficient mice develop ovarian tumors and soft tissue sarcomas and both LATS1 and 2, have been shown to be silenced by methylation in an aggressive subtype of breast cancers. Furthermore, LATS2 seems to be regulated by two micro-RNAs (miRNA), commonly overexpressed in testicular cancer (Nat Rev Cancer 2007;7(3):182-91). Contrary to these tumor suppressors, YAP1 is a candidate oncogene due to their function as growth promoters and induction of EMT, cell migration and invasion, respectively. The amplification of YAP1 in human cancers including liver cancer was first described in 2006 (PNAS 2006;103:12405-12410; Cell 2006;125:1253-1267) and its overexpression in mouse liver induced a dramatic increase in organ size and HCC formation (Cell 2007;130:1120-1133; Curr Biol 2007;17:2054-2060). Prostate, colon, breast, ovarian, lung cancer and medulloblastoma show increased expression of YAP1 and thereby support its oncogenic function. Hypermethylation and silencing of two candidate MST1 regulators RASSF1A and NORE1A/B as well as decreased activity of MST1 was detected in human HCC samples, suggesting an important role in the apoptotic cascade (Gastroenterology 2006;130(4):1117-1128), but only one clinical study of 177 HCC samples detected YAP1 expression in 62% of the cases, associated with poorer tumor differentiation and high serum α-fetoprotein (AFP) levels (Cancer 2009;115:4576-4585). We will summarize and comment herein the recent work of Zhou et al. who confirmed the role of Mst1/2 kinases as tumor suppressors by showing that combined Mst1/2 deficiency leads to loss of the inactivating phosphorylation of YAP1, massive liver overgrowth and the development of HCC in vivo (Cancer Cell;16:425-438). The authors generated germline and liver specific conditional Mst1/2 double knock-out mice to assess the function of the pathway. Germline Mst1−/−Mst2−/− were embryonic lethal, while Mst1−/−Mst2+/− mice developed HCCs due to Mst2 loss of heterozygosity in the tumors as early as at 7 months of age. Interestingly, the authors observed high levels of a cleaved, activated MST1/2 in form of a previously described 34 kDA fragment in normal livers from wild type mice, whereas in spleen only full-length MST1/2 were detectable. This suggests a tissue-specific mechanism of MST1/2 regulation in vivo and a constitutive activation of both kinases in quiescent adult liver. In order to explore simultaneous deficiency of Mst1/2 in the liver, the authors next introduced a Mst2 conditional knockout allele onto the Mst1 and 2 germline null background and detected massive organ overgrowth, increased hepatocyte proliferation and multifocal HCCs. Mst1/2 null livers were protected from apoptosis by injection of the proapoptotic anti-Fas antibody. In a hepatocyte specific Albumin-Cre model, Mst1−/−Mst2F/F mice also developed massive liver overgrowth and HCCs at 3 months of age, suggesting that MST1/2 suppress the development of HCC. To address the influence of Mst1/2 loss on contact inhibition of proliferation, an in vitro model of mouse embryonic fibroblasts (MEF) was used, showing that MST1/2 are not required for LATS1/2 and YAP1 phosphorylation in response to cell density (Fig.1). Mst1 restoration in a HCC cell line derived from the mouse model was able to inhibit the tumorigenic phenotype, leading to MOB1 and YAP1 phosphorylation in the absence of altered LATS1/2 phosphorylation, decreased YAP1 nuclear residence, increased fraction of HCC cells in G1, increased early and late apoptotic cells, decreased c-myc expression, reduced cell proliferation and colony formation. These results were also confirmed when Yap1 depletion in vitro completely restored the phenotype, underlining its critical role as an oncogene in HCC development. Finally, MST1/2 and YAP1 signaling components were examined in 21 matched human HCC and normal liver samples, showing a loss of negative regulation of YAP1, most likely due to decreased MST1/2 activity.