- Email: [email protected]
Rho GTPase and Wnt Signaling Pathways in Hepatocarcinogenesis
March 2008
18. Moris GP, Beck PL, Herridge MJ, et al. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 2003;125:1750 –1761. 19. Lawrance IC, Wu F, Leite AZ, et al. A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B. Gastroenterology 2003;125:1750 – 1761. 20. Vallance BA, Gunawan MI, Hewlett B, et al. TGF-beta1 gene transfer to the mouse colon leads to intestinal fibrosis. Am J Physiol Gastrointest Liver Physiol 2005;289:G116 – G128. 21. Motomura Y, Khan WI, El-Sharkawy RT, et al. Induction of a fibrogenic response in mouse colon by overexpression of monocyte chemoattractant protein-1. Gut 2006;55:662– 670.
EDITORIALS
875
22. Grassl GA, Valdez Y, Bergstrom KSB, et al. Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis. Gastroenterology 2008;134:768 –780.
Address requests for reprints to: Beth A. McCormick, PhD, Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, 114 16th Street (114-3503), Charlestown, Massachusetts 02129. e-mail: [email protected]. harvard.edu; fax: (617) 726-4172. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2008.01.019
Rho GTPase and Wnt Signaling Pathways in Hepatocarcinogenesis
See “Continuous cell injury promotes hepatic tumorigenesis in Cdc42-deficient mouse liver,” by van Hengel J, D’Hooge P, Hooghe B, et al on page 781.
R
ho GTPase and Wnt/-catenin signaling pathways are interconnected and both pathways are frequently activated in human hepatocarcinogenesis. Rho GTPases and the Wnt/-catenin pathway regulate multiple cellular processes, including cell polarity, proliferation, differentiation, and apoptosis. A functional role of Wnt/-catenin activation in hepatocarcinogenesis has been demonstrated in genetic mouse models. In this issue of GASTROENTEROLOGY, van Hengel et al1 provide first experimental evidence that hepatic deletion of the gene encoding the Rho GTPase—Cdc42—induces chronic liver damage and hepatocarcinogenesis in mice. Wnt/-catenin-signaling. Activation of this pathway occurs in 20%–90% of human hepatocellular carcinomas (HCC).2 Different mechanisms induce Wnt/catenin signaling during hepatocarcinogenesis, including (1) activating mutations of -catenin3,4; (2) inactivation of negative regulators of -catenin like Axin-1, Axin-2,5,6 GSK-3,7 and Prickle-1;8 and (3) overexpression of the Wnt receptor, frizzled-7.9 Genetic mouse models have provided experimental evidence that deletion of adenomatosis polyposis coli (a negative regulator of -catenin) can induce hepatocarcinogenesis.10 However, hepatic expression of -catenin overactive mutants by itself is not sufficient to induce fully malignant tumors in the mouse liver,11,12 but cooperates with other oncogenic stimuli, such as H-ras activation, to promote hepatocarcinogenesis in mice.13
Activation of Wnt-signaling results in nuclear localization of -catenin, thereby inducing cell proliferation via activation of cyclin-D and myc, among other targets. However, Wnt signaling has additional effects that could contribute to cancer formation, including the activation of genes involved in glutamine metabolism like glutamine synthetase14 and the control of cytoskeleton organization, cell-to-cell adhesion, cell polarity, and cell motility.15 These latter effects can also involve Rho GTPase signaling.16,17 Rho family GTPases. Rho GTPases are a subfamily of small GTPases controlling cell polarity, motility, differentiation, apoptosis, and proliferation. The activation of Rho GTPases has been implicated in various types of human cancer.18 There is growing evidence that activation of Rho GTPases by guanine nucleotide exchange factors occurs in human hepatocarcinogenesis. GTPaseactivating proteins (GAPs) inactivate Rho GTPases. Recent studies have identified 2 GAP genes that are frequently deleted in human liver cancer (DLC-1 and DLC2). Both gene products display GAP activity against RhoA and Cdc42 (2 members of the Rho family GTPases) and suppress cell proliferation upon reintroduction in hepatoma cells.18,19 DLC-2 is deleted in 30%–50% of human HCC20 and DLC-1 in 44%–50% of human HCC.21 In addition, p21-activated protein kinase (Pak1), one of the main downstream effectors of Cdc42, is overexpressed in 75% of human HCC.22 The overexpression of Pak1 in human HCC correlates with activation of c-Jun NH2terminal kinase (JNK), suggesting that induction of JNK by Cdc4223 could contribute to hepatocarcinogenesis. Experiments in mouse models have established that c-Jun promotes hepatocyte survival in the context of chronic hepatitis and early HCC formation.24,25
876
EDITORIALS
GASTROENTEROLOGY Vol. 134, No. 3
Interaction Between Rho GTPases and Wnt/Catenin Signaling Pathways. In Drosophila, Rho GTPases
are downstream mediators of the Wnt signaling pathway controlling cell polarity.26 There is ample evidence that these 2 signaling pathways are also interconnected in mammalian cells: (1) Wnt-signaling activates Wrch1 (a Wnt-1 responsive Cdc42 homolog).27 Dysregulation of Wrch1 has been observed in various cancer cell lines, but its role in hepatocarcinogenesis has not been analyzed. (2) Cdc42 impairs -catenin turnover and this regulation is necessary for progenitor cell differentiation in skin.28 In addition, Cdc42-dependent phosphorylation of GSK-3 induces the interaction of adenomatosis polyposis coli with microtubules, thus controlling microtubule network formation and cell polarity.29 (3) Wnt-signaling and Rho GTPases are also interconnected by a family of proteins called the IQGAPs.30 IQGAP1 and IQGAP2 impair the intrinsic GTPase activity of Cdc42, thus stabilizing Cdc42 in its activated form.30 In addition, IQGAP1 can impair complex formation between -catenin and E-cadherin thus affecting cell-to-cell adhesion.30 Interestingly, deletion of the gene encoding IQGAP2 activates Wnt/-catenin signaling in mouse liver and induces hepatocarcinogenesis in an IQGAP1-dependent manner.31 (4) Adenomatosis polyposis coli induces degradation of -catenin. The deletion of adenomatosis polyposis coli is associated with activation of -catenin signaling and frequently occurs in intestinal polyps.15 However, adenomatosis polyposis coli also controls Rho GTPases. It has been shown that adenomatosis polyposis coli forms a complex with IQGAP1.15,30 Although a rare event in human HCC, hepatic deletion of the adenomatosis polyposis coli gene induces -catenin signaling and HCC in mice.10 It is not known whether the effects of adenomatosis polyposis coli on Rho GTPases contribute to hepatocarcinogenesis. In summary, Rho GTPases and Wnt/-catenin are highly interconnected pathways that can influence hepatocarcinogenesis (Figure 1). In agreement with this model, there is experimental evidence that deletion of PTEN-encoding gene (phosphatase and tensin homologue), an inhibition of PTEN expression occurs in 40%– 70% of human HCC,32,33 causes activation of both Cdc4234 and Wnt/-catenin signaling.35 The role of each of these 2 pathways as well as the functional relevance of the crosstalk between the 2 pathways during hepatocarcinogenesis has yet to be identified. In contrast to the in vivo studies on Wnt/-catenin signaling, there are few experimental data analyzing the role of Rho GTPases in mouse models of hepatocarcinogenesis. van Hengel et al1 provide the first experimental evidence that the deletion of the Cdc42-encoding gene is sufficient to induce hepatocarcinogensis in mice. At first glance, these findings appear to be surprising, because activation of Cdc42 signaling has been associated with hepatocarcinogenesis in humans.18 –22 van Hengel et al1
Figure 1. The figure depicts main components of the Wnt/-catenin (white) and Rho GTPase (black) signaling pathways. The 2 pathways are interconnected at multiple levels (grey).
show that hepatic deletion of Cdc42 induces chronic liver damage, jaundice, and fibrosis. It is possible that chronic liver damage and toxic effects of bile acids induce cancer formation in Cdc42-deficient mice. The mice develop small dysplastic foci at 2 months and macroscopic HCC at 8 months of age, indicating that tumor formation in Cdc42-deficient mice occurs early compared with other mouse models of chronic or acute liver injury.36,37 These data suggest that deletion of the gene encoding Cdc42 induces direct molecular effects that promote carcinogenesis. It is possible that alterations in cell adhesion promote carcinogenesis in response to Cdc42 deletion. It is known that Cdc42 can counteract the inhibitory effect of IQGAP1 on cell adhesion.30 In contrast, genetic deletion of Cdc42 lowers intracellular -catenin, thereby impairing the formation of -catenin/E-cadherin complexes and cell adhesion.28 A recent study on hepatocarcinogenesis in IQGAP2-deficient mice has shown that IQGAP1dependent effects on cell adhesion may contribute to the induction of HCC.31 van Hengel et al1 did not detect severe disturbances in tight junctions, but Cdc42-deficient hepatocytes showed an impaired organization of the cytoskeleton, possibly contributing to the tumor phenotype. Given the interconnection between Rho GTPases and Wnt/-catenin signaling, it is of particular interest to investigate whether alterations in the Wnt/-catenin pathway contributed to hepatocarcinogenesis in the Cdc42-deficient mouse liver. van Hengel et al1 provide experimental evidence that -catenin signaling was not activated in hepatic tumors from Cdc42-deficient mice. These findings agree with recent reports that -catenin degradation is increased in response to Cdc42 deletion.28 The data suggest that Cdc42 deletion can induce tumor-
March 2008
igenesis, independent of -catenin signaling. Previous studies on the Wnt/-catenin signaling pathway in mouse models have shown that deletion of adenomatosis polyposis coli induced hepatocarcinogenesis,10 whereas oncogenic -catenin overexpression was not sufficient to induce hepatocarcinogenesis.11,12 These findings indicate that -catenin–independent effects may contribute to hepatocarcinogenesis in adenomatosis polyposis coli mutant mice. Given the known interactions between adenomatosis polyposis coli, IQGAPs, and Rho GTPases, it is tempting to speculate that Rho GTPase signaling can promote hepatocarcinogenesis in the context of Wnt/catenin activation. The findings of van Hengel et al1 provide experimental evidence that disturbances in Rho GTPase signaling promote hepatocarcinogenesis. The study provides a rational basis and valuable tool to analyze molecular mechanisms downstream of Rho GTPases in hepatocarcinogenesis.
ANDRÉ LECHEL KARL LENHARD RUDOLPH Institute of Molecular Medicine and Max-Planck-Research–Group for Stem Cell Aging Ulm, Germany References 1. van Hengel J, D’hooge P, Hooghe B, et al. Continuous cell injury promotes hepatic tumorigenesis in Cdc42-deficient mouse liver. Gastroenterology 2008;134:781–792. 2. Thompson MD, Monga SP. WNT/beta-catenin signaling in liver health and disease. Hepatology 2007;45:1298 –1305. 3. de La Coste A, Romagnolo B, Billuart P, et al. Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci U S A 1998;95: 8847– 8851. 4. Miyoshi Y, Iwao K, Nagasawa Y, et al. Activation of the betacatenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res 1998;58:2524 –2527. 5. Satoh S, Daigo Y, Furukawa Y, et al. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet 2000;24:245–250. 6. Behrens J, Jerchow BA, Würtele M, et al. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 1998;280:596 –599. 7. Ban KC, Singh H, Krishnan R, et al. GSK-3beta phosphorylation and alteration of beta-catenin in hepatocellular carcinoma. Cancer Lett 2003;199:201–208. 8. Chan DW, Chan CY, Yam JW, et al. Prickle-1 negatively regulates Wnt/beta-catenin pathway by promoting Dishevelled ubiquitination/degradation in liver cancer. Gastroenterology 2006;131:1218 –1227. 9. Merle P, de la Monte S, Kim M, et al. Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma. Gastroenterology 2004;127:1110 –1122. 10. Colnot S, Decaens T, Niwa-Kawakita M, et al. Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci U S A 2004;101:17216 –17221. 11. Cadoret A, Ovejero C, Saadi-Kheddouci S, et al. Hepatomegaly in transgenic mice expressing an oncogenic form of beta-catenin. Cancer Res 2001;61:3245–3249.
EDITORIALS
877
12. Harada N, Miyoshi H, Murai N, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res 2002;62: 1971–1977. 13. Harada N, Oshima H, Katoh M, et al. Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations. Cancer Res 2004;64:48 –54. 14. Cadoret A, Ovejero C, Terris B, et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002;21:8293– 8301. 15. Akiyama T, Kawasaki Y. Wnt signalling and the actin cytoskeleton. Oncogene 2006;25:7538 –7544. 16. Schlessinger K, McManus EJ, Hall A. Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity. J Cell Biol 2007;178:355–361. 17. Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer 2002;2:133–142. 18. Leung TH, Ching YP, Yam JW, et al. Deleted in liver cancer 2 (DLC2) suppresses cell transformation by means of inhibition of RhoA activity. Proc Natl Acad Sci U S A 2005;102:15207– 15212. 19. Wong CM, Yam JW, Ching YP, et al. Rho GTPase-activating protein deleted in liver cancer suppresses cell proliferation and invasion in hepatocellular carcinoma. Cancer Res 2005; 65:8861– 8868. 20. Ching YP, Wong CM, Chan SF, et al. Deleted in liver cancer (DLC) 2 encodes a RhoGAP protein with growth suppressor function and is underexpressed in hepatocellular carcinoma. J Biol Chem 2003;278:10824 –10830. 21. Wong CM, Lee JM, Ching YP, et al. Genetic and epigenetic alterations of DLC-1 gene in hepatocellular carcinoma. Cancer Res 2003;63:7646 –7651. 22. Ching YP, Leong VY, Lee MF, et al. P21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation. Cancer Res 2007;67:3601–3608. 23. Coso OA, Chiariello M, Yu JC, et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 1995;81:1137–1146. 24. Eferl R, Ricci R, Kenner L, et al. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 2003;112: 181–192. 25. Hasselblatt P, Rath M, Komnenovic V, et al. Hepatocyte survival in acute hepatitis is due to c-Jun/AP-1-dependent expression of inducible nitric oxide synthase. Proc Natl Acad Sci U S A 2007; 104:17105–17010. 26. Strutt DI, Weber U, Mlodzik M. The role of RhoA in tissue polarity and Frizzled signalling. Nature 1997;387:292–295. 27. Tao W, Pennica D, Xu L, et al. Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1. Genes Dev 2001;15: 1796 –1807. 28. Wu X, Quondamatteo F, Lefever T, et al. Cdc42 controls progenitor cell differentiation and beta-catenin turnover in skin. Genes Dev 2006;20:571–585. 29. Etienne-Manneville S, Hall A. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 2003; 21:753–756. 30. Briggs MW, Sacks DB. IQGAP proteins are integral components of cytoskeletal regulation. EMBO Rep 2003; 4:571–574. 31. Schmidt VA, Chiariello CS, Capilla E, et al. Development of hepatocellular carcinoma in Iqgap2-deficient mice is IQGAP1-dependent. Mol Cell Biol 2008 [Epub ahead of print]. 32. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132: 2557–2576.
878
EDITORIALS
33. Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;133:647– 658. 34. Liliental J, Moon SY, Lesche R, et al. Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. Curr Biol 2000;10:401– 404. 35. He XC, Yin T, Grindley JC, et al. PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nat Genet 2007;39:189 –198. 36. Chisari FV, Klopchin K, Moriyama T, et al. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 1989;59:1145–1156. 37. Sandgren EP, Palmiter RD, Heckel JL, et al. DNA rearrangement causes hepatocarcinogenesis in albumin-plasminogen activator transgenic mice. Proc Natl Acad Sci U S A 1992;89:11523–11527.
GASTROENTEROLOGY Vol. 134, No. 3
Address requests for reprints to: Karl Lenhard Rudolph, MD, Institute of Molecular Medicine and Max-Planck-Research–Group for Stem Cell Aging, Albert-Einstein-Allee 11, 89081 Ulm, Germany; e-mail: [email protected] Supported by the DFG (Ru745-7/1, Ru745-10/1, SFB-738, KFO167) the Deutsche Krebshilfe e.V. (Research Group on Tumor Stem Cells: 106415), the Wilhelm Sander foundation, the Fritz-Thyssen foundation, and the Roggenbuck foundation (to KLR). The authors thank Peter Gierschik for thoughtful comments and discussion. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2008.01.055
To Be or Not to Be: Generation of Hepatocytes From Cells Outside the Liver
See “Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells,” by Campard D, Lysy PA, Najimi M, et al, on page 833.
S
tem cells hold great promise as a source of material for novel, minimally invasive therapies to treat a variety of neurodegenerative disorders, diabetes, heart disease, and numerous other debilitating conditions. It is hoped that stem cells derived from a variety of sources might also produce a plentiful supply of cells with characteristics identical to those of primary human hepatocytes. An unlimited supply of exogenously derived liver cells would facilitate development of cell-based therapies for the treatment of life-threatening liver diseases and could eventually lead to therapies that could improve the lives of other patients with less severe but debilitating liver-based metabolic disorders. The availability of reliable source of highquality liver cells would also facilitate the study of liver diseases and revolutionize the early stages of the drug discovery process, as hepatic biotransformation of drugs can generate metabolites more toxic than the parent drug. The availability of human hepatocytes is limited; most cadaver donor livers are used for transplantation. The quality of human liver cells recovered from less-thanideal donors is often marginal, the cells tolerate cryopreservation inconsistently, and, in culture, they rapidly lose their differentiated functions. Although human fetal liver stem/progenitors seem to have extensive capacity to replicate both in vivo and in culture, ethical issues and limited availability limits their potential for clinical application as well. Although recent studies indicate that
the immunologic barrier to porcine hepatocyte xenografts may not be the limiting factor for cell therapy, formidable infectious and regulatory hurdles remain, and hepatocytes derived from animals have not been useful for the study of either human hepatic drug biotransformation or human hepatitis virus biology. A number of recent publications indicating that liver cells can be generated from non-liver cells have been encouraging. “Hepatocyte-like” cells have, as expected, been derived from embryonic stem cells, but other novel potential sources, associated with fewer ethical concerns, have also been used to generate “hepatocytes.”1–3 These include bone marrow, blood monocytes, umbilical cord, amniotic cells, and even skin fibroblasts. The recent farreaching finding that human skin fibroblasts can be genetically modified to produce inducible pluripotent stem cells— cells that seem to have characteristics nearly identical to human embryonic stem cells—lends credibility to the unlimited extent various differentiated cells might be able to transform into liver cells.4,5 The prospect of generating hepatocytes from patients, using their own skin or blood cells, would enhance the study of specific liver diseases, and might allow correction of liverbased genetic abnormalities by cell therapy without the need for immunosuppressive medications (Figure 1). In this issue of GASTROENTEROLOGY, Campard et al6 provide evidence that hepatocyte-like cells can be derived from the umbilical cord matrix, or Wharton’s jelly. The authors describe a starting cell population that expresses significant amounts of albumin, alpha-fetoprotein (AFP), cytokeratin18, glucose-6-phosphatase (G6P), phosphoenolpyruvate carboxykinase (PEPCK), alpha-1-antitrypsin (AAT), and dipeptidyl peptidase-IV (DPPIV) messenger RNA, and in some cases liver-specific proteins. Because the cells express so
18. Moris GP, Beck PL, Herridge MJ, et al. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 2003;125:1750 –1761. 19. Lawrance IC, Wu F, Leite AZ, et al. A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B. Gastroenterology 2003;125:1750 – 1761. 20. Vallance BA, Gunawan MI, Hewlett B, et al. TGF-beta1 gene transfer to the mouse colon leads to intestinal fibrosis. Am J Physiol Gastrointest Liver Physiol 2005;289:G116 – G128. 21. Motomura Y, Khan WI, El-Sharkawy RT, et al. Induction of a fibrogenic response in mouse colon by overexpression of monocyte chemoattractant protein-1. Gut 2006;55:662– 670.
EDITORIALS
875
22. Grassl GA, Valdez Y, Bergstrom KSB, et al. Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis. Gastroenterology 2008;134:768 –780.
Address requests for reprints to: Beth A. McCormick, PhD, Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, 114 16th Street (114-3503), Charlestown, Massachusetts 02129. e-mail: [email protected]. harvard.edu; fax: (617) 726-4172. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2008.01.019
Rho GTPase and Wnt Signaling Pathways in Hepatocarcinogenesis
See “Continuous cell injury promotes hepatic tumorigenesis in Cdc42-deficient mouse liver,” by van Hengel J, D’Hooge P, Hooghe B, et al on page 781.
R
ho GTPase and Wnt/-catenin signaling pathways are interconnected and both pathways are frequently activated in human hepatocarcinogenesis. Rho GTPases and the Wnt/-catenin pathway regulate multiple cellular processes, including cell polarity, proliferation, differentiation, and apoptosis. A functional role of Wnt/-catenin activation in hepatocarcinogenesis has been demonstrated in genetic mouse models. In this issue of GASTROENTEROLOGY, van Hengel et al1 provide first experimental evidence that hepatic deletion of the gene encoding the Rho GTPase—Cdc42—induces chronic liver damage and hepatocarcinogenesis in mice. Wnt/-catenin-signaling. Activation of this pathway occurs in 20%–90% of human hepatocellular carcinomas (HCC).2 Different mechanisms induce Wnt/catenin signaling during hepatocarcinogenesis, including (1) activating mutations of -catenin3,4; (2) inactivation of negative regulators of -catenin like Axin-1, Axin-2,5,6 GSK-3,7 and Prickle-1;8 and (3) overexpression of the Wnt receptor, frizzled-7.9 Genetic mouse models have provided experimental evidence that deletion of adenomatosis polyposis coli (a negative regulator of -catenin) can induce hepatocarcinogenesis.10 However, hepatic expression of -catenin overactive mutants by itself is not sufficient to induce fully malignant tumors in the mouse liver,11,12 but cooperates with other oncogenic stimuli, such as H-ras activation, to promote hepatocarcinogenesis in mice.13
Activation of Wnt-signaling results in nuclear localization of -catenin, thereby inducing cell proliferation via activation of cyclin-D and myc, among other targets. However, Wnt signaling has additional effects that could contribute to cancer formation, including the activation of genes involved in glutamine metabolism like glutamine synthetase14 and the control of cytoskeleton organization, cell-to-cell adhesion, cell polarity, and cell motility.15 These latter effects can also involve Rho GTPase signaling.16,17 Rho family GTPases. Rho GTPases are a subfamily of small GTPases controlling cell polarity, motility, differentiation, apoptosis, and proliferation. The activation of Rho GTPases has been implicated in various types of human cancer.18 There is growing evidence that activation of Rho GTPases by guanine nucleotide exchange factors occurs in human hepatocarcinogenesis. GTPaseactivating proteins (GAPs) inactivate Rho GTPases. Recent studies have identified 2 GAP genes that are frequently deleted in human liver cancer (DLC-1 and DLC2). Both gene products display GAP activity against RhoA and Cdc42 (2 members of the Rho family GTPases) and suppress cell proliferation upon reintroduction in hepatoma cells.18,19 DLC-2 is deleted in 30%–50% of human HCC20 and DLC-1 in 44%–50% of human HCC.21 In addition, p21-activated protein kinase (Pak1), one of the main downstream effectors of Cdc42, is overexpressed in 75% of human HCC.22 The overexpression of Pak1 in human HCC correlates with activation of c-Jun NH2terminal kinase (JNK), suggesting that induction of JNK by Cdc4223 could contribute to hepatocarcinogenesis. Experiments in mouse models have established that c-Jun promotes hepatocyte survival in the context of chronic hepatitis and early HCC formation.24,25
876
EDITORIALS
GASTROENTEROLOGY Vol. 134, No. 3
Interaction Between Rho GTPases and Wnt/Catenin Signaling Pathways. In Drosophila, Rho GTPases
are downstream mediators of the Wnt signaling pathway controlling cell polarity.26 There is ample evidence that these 2 signaling pathways are also interconnected in mammalian cells: (1) Wnt-signaling activates Wrch1 (a Wnt-1 responsive Cdc42 homolog).27 Dysregulation of Wrch1 has been observed in various cancer cell lines, but its role in hepatocarcinogenesis has not been analyzed. (2) Cdc42 impairs -catenin turnover and this regulation is necessary for progenitor cell differentiation in skin.28 In addition, Cdc42-dependent phosphorylation of GSK-3 induces the interaction of adenomatosis polyposis coli with microtubules, thus controlling microtubule network formation and cell polarity.29 (3) Wnt-signaling and Rho GTPases are also interconnected by a family of proteins called the IQGAPs.30 IQGAP1 and IQGAP2 impair the intrinsic GTPase activity of Cdc42, thus stabilizing Cdc42 in its activated form.30 In addition, IQGAP1 can impair complex formation between -catenin and E-cadherin thus affecting cell-to-cell adhesion.30 Interestingly, deletion of the gene encoding IQGAP2 activates Wnt/-catenin signaling in mouse liver and induces hepatocarcinogenesis in an IQGAP1-dependent manner.31 (4) Adenomatosis polyposis coli induces degradation of -catenin. The deletion of adenomatosis polyposis coli is associated with activation of -catenin signaling and frequently occurs in intestinal polyps.15 However, adenomatosis polyposis coli also controls Rho GTPases. It has been shown that adenomatosis polyposis coli forms a complex with IQGAP1.15,30 Although a rare event in human HCC, hepatic deletion of the adenomatosis polyposis coli gene induces -catenin signaling and HCC in mice.10 It is not known whether the effects of adenomatosis polyposis coli on Rho GTPases contribute to hepatocarcinogenesis. In summary, Rho GTPases and Wnt/-catenin are highly interconnected pathways that can influence hepatocarcinogenesis (Figure 1). In agreement with this model, there is experimental evidence that deletion of PTEN-encoding gene (phosphatase and tensin homologue), an inhibition of PTEN expression occurs in 40%– 70% of human HCC,32,33 causes activation of both Cdc4234 and Wnt/-catenin signaling.35 The role of each of these 2 pathways as well as the functional relevance of the crosstalk between the 2 pathways during hepatocarcinogenesis has yet to be identified. In contrast to the in vivo studies on Wnt/-catenin signaling, there are few experimental data analyzing the role of Rho GTPases in mouse models of hepatocarcinogenesis. van Hengel et al1 provide the first experimental evidence that the deletion of the Cdc42-encoding gene is sufficient to induce hepatocarcinogensis in mice. At first glance, these findings appear to be surprising, because activation of Cdc42 signaling has been associated with hepatocarcinogenesis in humans.18 –22 van Hengel et al1
Figure 1. The figure depicts main components of the Wnt/-catenin (white) and Rho GTPase (black) signaling pathways. The 2 pathways are interconnected at multiple levels (grey).
show that hepatic deletion of Cdc42 induces chronic liver damage, jaundice, and fibrosis. It is possible that chronic liver damage and toxic effects of bile acids induce cancer formation in Cdc42-deficient mice. The mice develop small dysplastic foci at 2 months and macroscopic HCC at 8 months of age, indicating that tumor formation in Cdc42-deficient mice occurs early compared with other mouse models of chronic or acute liver injury.36,37 These data suggest that deletion of the gene encoding Cdc42 induces direct molecular effects that promote carcinogenesis. It is possible that alterations in cell adhesion promote carcinogenesis in response to Cdc42 deletion. It is known that Cdc42 can counteract the inhibitory effect of IQGAP1 on cell adhesion.30 In contrast, genetic deletion of Cdc42 lowers intracellular -catenin, thereby impairing the formation of -catenin/E-cadherin complexes and cell adhesion.28 A recent study on hepatocarcinogenesis in IQGAP2-deficient mice has shown that IQGAP1dependent effects on cell adhesion may contribute to the induction of HCC.31 van Hengel et al1 did not detect severe disturbances in tight junctions, but Cdc42-deficient hepatocytes showed an impaired organization of the cytoskeleton, possibly contributing to the tumor phenotype. Given the interconnection between Rho GTPases and Wnt/-catenin signaling, it is of particular interest to investigate whether alterations in the Wnt/-catenin pathway contributed to hepatocarcinogenesis in the Cdc42-deficient mouse liver. van Hengel et al1 provide experimental evidence that -catenin signaling was not activated in hepatic tumors from Cdc42-deficient mice. These findings agree with recent reports that -catenin degradation is increased in response to Cdc42 deletion.28 The data suggest that Cdc42 deletion can induce tumor-
March 2008
igenesis, independent of -catenin signaling. Previous studies on the Wnt/-catenin signaling pathway in mouse models have shown that deletion of adenomatosis polyposis coli induced hepatocarcinogenesis,10 whereas oncogenic -catenin overexpression was not sufficient to induce hepatocarcinogenesis.11,12 These findings indicate that -catenin–independent effects may contribute to hepatocarcinogenesis in adenomatosis polyposis coli mutant mice. Given the known interactions between adenomatosis polyposis coli, IQGAPs, and Rho GTPases, it is tempting to speculate that Rho GTPase signaling can promote hepatocarcinogenesis in the context of Wnt/catenin activation. The findings of van Hengel et al1 provide experimental evidence that disturbances in Rho GTPase signaling promote hepatocarcinogenesis. The study provides a rational basis and valuable tool to analyze molecular mechanisms downstream of Rho GTPases in hepatocarcinogenesis.
ANDRÉ LECHEL KARL LENHARD RUDOLPH Institute of Molecular Medicine and Max-Planck-Research–Group for Stem Cell Aging Ulm, Germany References 1. van Hengel J, D’hooge P, Hooghe B, et al. Continuous cell injury promotes hepatic tumorigenesis in Cdc42-deficient mouse liver. Gastroenterology 2008;134:781–792. 2. Thompson MD, Monga SP. WNT/beta-catenin signaling in liver health and disease. Hepatology 2007;45:1298 –1305. 3. de La Coste A, Romagnolo B, Billuart P, et al. Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci U S A 1998;95: 8847– 8851. 4. Miyoshi Y, Iwao K, Nagasawa Y, et al. Activation of the betacatenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res 1998;58:2524 –2527. 5. Satoh S, Daigo Y, Furukawa Y, et al. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet 2000;24:245–250. 6. Behrens J, Jerchow BA, Würtele M, et al. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 1998;280:596 –599. 7. Ban KC, Singh H, Krishnan R, et al. GSK-3beta phosphorylation and alteration of beta-catenin in hepatocellular carcinoma. Cancer Lett 2003;199:201–208. 8. Chan DW, Chan CY, Yam JW, et al. Prickle-1 negatively regulates Wnt/beta-catenin pathway by promoting Dishevelled ubiquitination/degradation in liver cancer. Gastroenterology 2006;131:1218 –1227. 9. Merle P, de la Monte S, Kim M, et al. Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma. Gastroenterology 2004;127:1110 –1122. 10. Colnot S, Decaens T, Niwa-Kawakita M, et al. Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci U S A 2004;101:17216 –17221. 11. Cadoret A, Ovejero C, Saadi-Kheddouci S, et al. Hepatomegaly in transgenic mice expressing an oncogenic form of beta-catenin. Cancer Res 2001;61:3245–3249.
EDITORIALS
877
12. Harada N, Miyoshi H, Murai N, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res 2002;62: 1971–1977. 13. Harada N, Oshima H, Katoh M, et al. Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations. Cancer Res 2004;64:48 –54. 14. Cadoret A, Ovejero C, Terris B, et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002;21:8293– 8301. 15. Akiyama T, Kawasaki Y. Wnt signalling and the actin cytoskeleton. Oncogene 2006;25:7538 –7544. 16. Schlessinger K, McManus EJ, Hall A. Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity. J Cell Biol 2007;178:355–361. 17. Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer 2002;2:133–142. 18. Leung TH, Ching YP, Yam JW, et al. Deleted in liver cancer 2 (DLC2) suppresses cell transformation by means of inhibition of RhoA activity. Proc Natl Acad Sci U S A 2005;102:15207– 15212. 19. Wong CM, Yam JW, Ching YP, et al. Rho GTPase-activating protein deleted in liver cancer suppresses cell proliferation and invasion in hepatocellular carcinoma. Cancer Res 2005; 65:8861– 8868. 20. Ching YP, Wong CM, Chan SF, et al. Deleted in liver cancer (DLC) 2 encodes a RhoGAP protein with growth suppressor function and is underexpressed in hepatocellular carcinoma. J Biol Chem 2003;278:10824 –10830. 21. Wong CM, Lee JM, Ching YP, et al. Genetic and epigenetic alterations of DLC-1 gene in hepatocellular carcinoma. Cancer Res 2003;63:7646 –7651. 22. Ching YP, Leong VY, Lee MF, et al. P21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation. Cancer Res 2007;67:3601–3608. 23. Coso OA, Chiariello M, Yu JC, et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 1995;81:1137–1146. 24. Eferl R, Ricci R, Kenner L, et al. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 2003;112: 181–192. 25. Hasselblatt P, Rath M, Komnenovic V, et al. Hepatocyte survival in acute hepatitis is due to c-Jun/AP-1-dependent expression of inducible nitric oxide synthase. Proc Natl Acad Sci U S A 2007; 104:17105–17010. 26. Strutt DI, Weber U, Mlodzik M. The role of RhoA in tissue polarity and Frizzled signalling. Nature 1997;387:292–295. 27. Tao W, Pennica D, Xu L, et al. Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1. Genes Dev 2001;15: 1796 –1807. 28. Wu X, Quondamatteo F, Lefever T, et al. Cdc42 controls progenitor cell differentiation and beta-catenin turnover in skin. Genes Dev 2006;20:571–585. 29. Etienne-Manneville S, Hall A. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 2003; 21:753–756. 30. Briggs MW, Sacks DB. IQGAP proteins are integral components of cytoskeletal regulation. EMBO Rep 2003; 4:571–574. 31. Schmidt VA, Chiariello CS, Capilla E, et al. Development of hepatocellular carcinoma in Iqgap2-deficient mice is IQGAP1-dependent. Mol Cell Biol 2008 [Epub ahead of print]. 32. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132: 2557–2576.
878
EDITORIALS
33. Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;133:647– 658. 34. Liliental J, Moon SY, Lesche R, et al. Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. Curr Biol 2000;10:401– 404. 35. He XC, Yin T, Grindley JC, et al. PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nat Genet 2007;39:189 –198. 36. Chisari FV, Klopchin K, Moriyama T, et al. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 1989;59:1145–1156. 37. Sandgren EP, Palmiter RD, Heckel JL, et al. DNA rearrangement causes hepatocarcinogenesis in albumin-plasminogen activator transgenic mice. Proc Natl Acad Sci U S A 1992;89:11523–11527.
GASTROENTEROLOGY Vol. 134, No. 3
Address requests for reprints to: Karl Lenhard Rudolph, MD, Institute of Molecular Medicine and Max-Planck-Research–Group for Stem Cell Aging, Albert-Einstein-Allee 11, 89081 Ulm, Germany; e-mail: [email protected] Supported by the DFG (Ru745-7/1, Ru745-10/1, SFB-738, KFO167) the Deutsche Krebshilfe e.V. (Research Group on Tumor Stem Cells: 106415), the Wilhelm Sander foundation, the Fritz-Thyssen foundation, and the Roggenbuck foundation (to KLR). The authors thank Peter Gierschik for thoughtful comments and discussion. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2008.01.055
To Be or Not to Be: Generation of Hepatocytes From Cells Outside the Liver
See “Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells,” by Campard D, Lysy PA, Najimi M, et al, on page 833.
S
tem cells hold great promise as a source of material for novel, minimally invasive therapies to treat a variety of neurodegenerative disorders, diabetes, heart disease, and numerous other debilitating conditions. It is hoped that stem cells derived from a variety of sources might also produce a plentiful supply of cells with characteristics identical to those of primary human hepatocytes. An unlimited supply of exogenously derived liver cells would facilitate development of cell-based therapies for the treatment of life-threatening liver diseases and could eventually lead to therapies that could improve the lives of other patients with less severe but debilitating liver-based metabolic disorders. The availability of reliable source of highquality liver cells would also facilitate the study of liver diseases and revolutionize the early stages of the drug discovery process, as hepatic biotransformation of drugs can generate metabolites more toxic than the parent drug. The availability of human hepatocytes is limited; most cadaver donor livers are used for transplantation. The quality of human liver cells recovered from less-thanideal donors is often marginal, the cells tolerate cryopreservation inconsistently, and, in culture, they rapidly lose their differentiated functions. Although human fetal liver stem/progenitors seem to have extensive capacity to replicate both in vivo and in culture, ethical issues and limited availability limits their potential for clinical application as well. Although recent studies indicate that
the immunologic barrier to porcine hepatocyte xenografts may not be the limiting factor for cell therapy, formidable infectious and regulatory hurdles remain, and hepatocytes derived from animals have not been useful for the study of either human hepatic drug biotransformation or human hepatitis virus biology. A number of recent publications indicating that liver cells can be generated from non-liver cells have been encouraging. “Hepatocyte-like” cells have, as expected, been derived from embryonic stem cells, but other novel potential sources, associated with fewer ethical concerns, have also been used to generate “hepatocytes.”1–3 These include bone marrow, blood monocytes, umbilical cord, amniotic cells, and even skin fibroblasts. The recent farreaching finding that human skin fibroblasts can be genetically modified to produce inducible pluripotent stem cells— cells that seem to have characteristics nearly identical to human embryonic stem cells—lends credibility to the unlimited extent various differentiated cells might be able to transform into liver cells.4,5 The prospect of generating hepatocytes from patients, using their own skin or blood cells, would enhance the study of specific liver diseases, and might allow correction of liverbased genetic abnormalities by cell therapy without the need for immunosuppressive medications (Figure 1). In this issue of GASTROENTEROLOGY, Campard et al6 provide evidence that hepatocyte-like cells can be derived from the umbilical cord matrix, or Wharton’s jelly. The authors describe a starting cell population that expresses significant amounts of albumin, alpha-fetoprotein (AFP), cytokeratin18, glucose-6-phosphatase (G6P), phosphoenolpyruvate carboxykinase (PEPCK), alpha-1-antitrypsin (AAT), and dipeptidyl peptidase-IV (DPPIV) messenger RNA, and in some cases liver-specific proteins. Because the cells express so