Final and future frontiers
Authors: Scott L. Friedman
Journal: Journal of Hepatology (2014)

YAP activation is an early event and a potential therapeutic target in liver cancer developmentJournal of HepatologyVol. 61Issue 5PreviewAlthough the growth suppressing Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event. Full-Text PDF Telomerase activated thymidine analogue pro-drug is a new molecule targeting hepatocellular carcinomaJournal of HepatologyVol. 61Issue 5PreviewHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although hepatectomy and transplantation have significantly improved survival, there is no effective chemotherapeutic treatment for HCC and its prognosis remains poor. Sustained activation of telomerase is essential for the growth and progression of HCC, suggesting that telomerase is a rational target for HCC therapy. Therefore, we developed a thymidine analogue pro-drug, acycloguanosyl-5′-thymidyltriphosphate (ACV-TP-T), which is specifically activated by telomerase in HCC cells and investigated its anti-tumour efficacy. Full-Text PDF Open Access In hepatology, hepatocellular carcinoma (HCC), not outer space, is the final frontier (sorry, Star Trek fans, https://www.youtube.com/watch?v=S6R3MiAv9ac), given its extremely heterogeneous molecular pathogenesis, dismal prognosis and limited treatment options [[1]Villanueva A. Hernandez-Gea V. Llovet J.M. Medical therapies for hepatocellular carcinoma: a critical view of the evidence.Nat Rev Gastroenterol Hepatol. 2012; 10: 34-42Crossref PubMed Scopus (255) Google Scholar]. Treatment of this neoplasm remains a huge unmet need with no major successes imminent, in contrast to the recent transformative progress in HCV antivirals, and the heightened prospects for antifibrotic therapies. Two articles, by Perra et al. and Tarocchi et al., in this month’s Journal of Hepatology offer valuable insights into HCC pathogenesis and treatment, respectively, which represent much-needed, albeit only incremental progress in this area. The study by Perra et al. examines the role of Hippo signalling pathway inactivation in experimental and human carcinogenesis. First discovered in Drosophila, the name ‘Hippo’ refers to a phenotype of marked organomegaly in flies associated with loss of Hippo’s normal tumour suppressor function. In mammals, the Hippo pathway contains a highly conserved core kinase cassette [2Yu F.X. Guan K.L. The Hippo pathway: regulators and regulations.Genes Dev. 2013; 27: 355-371Crossref PubMed Scopus (844) Google Scholar, 3Lin J.I. Poon C.L. Harvey K.F. The Hippo size control pathway-ever expanding.Sci Signal. 2013; 6: pe4PubMed Google Scholar, 4Barry E.R. Camargo F.D. The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development.Curr Opin Cell Biol. 2013; 25: 247-253Crossref PubMed Scopus (164) Google Scholar]; when Hippo signalling is active and growth is suppressed, two growth promoting transcriptional co-activators, YAP and Taz, are sequestered and degraded in the cytoplasm following phosphorylation by the kinases MST1/2 and LATS1/2. In absence of this phosphorylation, YAP and Taz are nuclear, where they interact with the molecules TEAD1-4 to promote a range of biologic activities, including cell fate (in part through convergence with Wnt signalling [[4]Barry E.R. Camargo F.D. The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development.Curr Opin Cell Biol. 2013; 25: 247-253Crossref PubMed Scopus (164) Google Scholar]), proliferation, polarity, and, most importantly, organ size control. In liver, the dominant contributions of YAP activation are to organ size [5Kowalik M.A. Saliba C. Pibiri M. Perra A. Ledda-Columbano G.M. Sarotto I. et al.Yes-associated protein regulation of adaptive liver enlargement and hepatocellular carcinoma development in mice.Hepatology. 2011; 53: 2086-2096Crossref PubMed Scopus (69) Google Scholar, 6Piccolo S. Cordenonsi M. Dupont S. Molecular pathways: YAP and TAZ take center stage in organ growth and tumorigenesis.Clin Cancer Res. 2013; 19: 4925-4930Crossref PubMed Scopus (112) Google Scholar, 7Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. et al.Elucidation of a universal size-control mechanism in Drosophila and mammals.Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1715) Google Scholar] and HCC [5Kowalik M.A. Saliba C. Pibiri M. Perra A. Ledda-Columbano G.M. Sarotto I. et al.Yes-associated protein regulation of adaptive liver enlargement and hepatocellular carcinoma development in mice.Hepatology. 2011; 53: 2086-2096Crossref PubMed Scopus (69) Google Scholar, 8Liu A.M. Xu Z. Luk J.M. An update on targeting Hippo-YAP signaling in liver cancer.Expert Opin Ther Targets. 2012; 16: 243-247Crossref PubMed Scopus (27) Google Scholar, 9Moeini A. Cornella H. Villanueva A. Emerging signaling pathways in hepatocellular carcinoma.Liver Cancer. 2012; 1: 83-93Crossref Google Scholar]. In further support of Hippo’s contribution to HCC, loss of MST1/2, which leads to YAP activation, also causes HCC [10Lu L. Li Y. Kim S.M. Bossuyt W. Liu P. Qiu Q. et al.Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver.Proc Natl Acad Sci U S A. 2010; 107: 1437-1442Crossref PubMed Scopus (552) Google Scholar, 11Zhou D. Conrad C. Xia F. Park J.S. Payer B. Yin Y. et al.Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene.Cancer Cell. 2009; 16: 425-438Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar] and Hippo dysregulation accompanies many human HCCs. The study by Perra et al. further implicates disrupted Hippo signalling with YAP activation in HCC pathogenesis, and specifically suggests that YAP activation is an early event in carcinogenesis, based on two animal models and immunohistochemical studies of human HCC. In the animal studies, the investigators used a model originally described in 1976, in which animals are administered a carcinogen (diethylnitrosamine, or DEN) and cell cycle inhibitor (2-acetylaminoflourine), then undergo partial hepatectomy, leading first to massive expansion of oval cells (i.e., rodent liver progenitor cells), then to cancer. They complement this method with another HCC model using a combination of DEN and a methionine-choline deficient diet. The authors describe early accumulation of YAP and its target genes in pre-neoplastic hepatocytes and in oval cells. Increased YAP was associated with downregulation of β-TRCP, an ubiquitin ligase molecule that ordinarily ubiquitinates YAP to hasten its degradation by the proteasome, implying that reduced ubiquitinylation of YAP through reduced β-TRCP leads to its accumulation. A specific role for YAP in inducing carcinogenesis was supported by its attenuation in the presence of an inhibitor of YAP signalling, verteporfin. These findings were validated in culture using human cell lines, and also correlated with immunohistochemical evidence of YAP induction in human HCCs. They also describe YAP accumulation in adenomas, consistent with one recent report [[12]Anakk S. Bhosale M. Schmidt V.A. Johnson R.L. Finegold M.J. Moore D.D. Bile acids activate YAP to promote liver carcinogenesis.Cell Rep. 2013; 5: 1060-1069Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar] but in contrast to another [[13]Tschaharganeh D.F. Chen X. Latzko P. Malz M. Gaida M.M. Felix K. et al.Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma.Gastroenterology. 2013; 144: e1512Abstract Full Text Full Text PDF Scopus (246) Google Scholar]. Hippo/YAP is among the ‘hottest’ intracellular pathways in liver, and the Perra et al. paper adds to the interest in Hippo/YAP, but also further reveals its complexity. The findings complement a lovely study by Yimlamai et al. in Cell, which implicates YAP signalling in liver cell fate determination [[14]Yimlamai D. Christodoulou C. Galli G.G. Yanger K. Pope-Mooney B. Gurung B. et al.Hippo pathway activity influences liver cell fate.Cell. 2014; 157: 1324-1338Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar]. Altogether, they reinforce the growing appreciation for Hippo/YAP as a regulator of organ size, growth regulation, cancer, progenitor cell amplification and cellular identity in liver. On the other hand, the Perra et al. study also reinforces the dearth of adequate animal models for HCC. Moreover, given HCC’s remarkable heterogeneity, no single model will replicate all subtypes of HCC. The models used in this study have uncertain relevance to human disease at best, since they do not include a fibrotic, pre-malignant phase of disease. The study would have benefited greatly from a molecular analysis of the tumours and pre-malignant lesions to determine whether the signalling pathways in these models are also prominent in human disease. Given the complexity and incomplete characterization of Hippo signalling in normal and injured human liver disease, it is premature to contemplate how to exploit new knowledge about this pathway to develop new therapeutics. On the other hand, if YAP signalling is consistently increased in early HCC, this could lead to its use either as a tissue marker of early cancer, or to the discovery of secreted molecules induced by YAP that might form the basis for a new circulating HCC biomarker. In contrast to the Perra et al. study, the article by Tarocchi et al. has more direct translational potential. The investigators utilize a clever approach that exploits the pathologic re-appearance of telomerase activity in HCC, which is absent from normal hepatocytes. Telomerase is an enzyme that restores DNA to the ends of chromosomes, or telomeres. Not only is telomerase induced in HCC, but telomerase re-expression is a more generalized feature of many human cancers [[15]Mocellin S. Pooley K.A. Nitti D. Telomerase and the search for the end of cancer.Trends Mol Med. 2013; 19: 125-133Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar]. Tarocchi et al. used a pro-drug compound previously described in their studies of pancreatic cancer [[16]Polvani S. Calamante M. Foresta V. Ceni E. Mordini A. Quattrone A. et al.Acycloguanosyl 5′-thymidyltriphosphate, a thymidine analogue prodrug activated by telomerase, reduces pancreatic tumor growth in mice.Gastroenterology. 2011; 140: e709Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar], which is activated into the nucleoside analogue acyclovir (ACV) by the enzymatic activity of telomerase. Thus, only cells with active telomerase will activate the substrate pro-drug to induce cellular apoptosis and cell cycle arrest from acyclovir’s actions in telomerase-expressing cells. The authors first validate the activity of the pro-drug compound in one murine and three human hepatic cancer cell lines, then extend their findings in vivo in an HBV transgenic mouse model that develops tumours, as well as to a syngeneic orthotopic murine model in which a human cell line (Hepa1-6) is directly implanted within the liver. The culture studies demonstrate that ectopic modulation of telomerase activity correlates with susceptibility to the acyclovir pro-drug. Importantly, the strategy requires wild type p53 activity in cells, such that Hep3B cells, which are p53 null, were not affected, but p53 re-introduction conferred responsiveness. In the HBV transgenic mouse studies, three times weekly intraperitoneal administration of the ACV pro-drug for four weeks reduced the size of neoplastic foci, associated with decreased α-fetoprotein levels, as well as reduced tumour cell proliferation and increased apoptosis. Interestingly, use of the pro-drug in combination with the clinically approved multi-kinase inhibitor sorafenib offered no additional benefit over the pro-drug alone in this orthotopic tumour model. The findings introduce a new mode of attack for this highly recalcitrant tumour. The appeal of this approach is its built-in targeting mechanism to selectively deliver a novel therapeutic to tumour cells only. While promising, several hurdles remain. First, the requirement for wild type p53 activity makes this approach much less valuable in the ∼1/3 of HCCs with p53 inactivation. Similar to p53 heterogeneity, the expression of telomerase is not always present in HCC, and it is higher in more poorly differentiated tumours, indicating that early tumours might not be as responsive [[17]Kawakami Y. Kitamoto M. Nakanishi T. Yasui W. Tahara E. Nakayama J. et al.Immuno-histochemical detection of human telomerase reverse transcriptase in human liver tissues.Oncogene. 2000; 19: 3888-3893Crossref PubMed Scopus (56) Google Scholar]. Of greater concern, some telomerase activity is present in cirrhosis, which could lead to unwanted targeting of acyclovir to non-malignant hepatocytes [[17]Kawakami Y. Kitamoto M. Nakanishi T. Yasui W. Tahara E. Nakayama J. et al.Immuno-histochemical detection of human telomerase reverse transcriptase in human liver tissues.Oncogene. 2000; 19: 3888-3893Crossref PubMed Scopus (56) Google Scholar]. This issue reflects a broader challenge in cancer, in which heterogeneity may be present not only among tumours from different patients, but even in a single tumour from one patient, a challenge particularly well described in breast cancer [[18]Polyak K. Heterogeneity in breast cancer.J Clin Invest. 2011; 121: 3786-3788Crossref PubMed Scopus (597) Google Scholar]. In addition, since the ACV pro-drug does not consistently kill tumour cells, the therapy would need to be combined with some cytotoxic therapy, otherwise cellular escape from the drug’s effect may emerge. Finally, newer mouse models utilizing patient derived xenografts (PDX) might enhance the efficacy of this approach [[19]Rosfjord E. Lucas J. Li G. Gerber H.P. Advances in patient-derived tumor xenografts: from target identification to predicting clinical response rates in oncology.Biochem Pharmacol. 2014; 91: 135-143Crossref PubMed Scopus (120) Google Scholar]. PDX models implant human tumour tissue directly from the patient into mice to more rapidly identify personalized combination therapies; as yet, however, not all studies have conclusively documented improved outcomes using these personalized avatars. The studies highlighted in this final Focus piece reflect the remarkable arc of progress in basic and translational hepatology over the past 30 years. As this is my last single-author Focus article (Professor Shouval and I will share authorship of the final piece next month), I want to express my gratitude to Professor Samuel and the Journal of Hepatology for this opportunity as a Focus co-Editor. It has been a privilege to document many fine examples in the Journal of Hepatology in which investigators have advanced our field towards the frontiers of the future. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.