Outfoxing liver cancer with p19ARF tumor suppressor?

1010 zy zyxwvutsrqponm zyxwvutsrqponm HEPATOLOGY, November 2004 HEPATOLOGY ELSEWHERE 7. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, et al. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikbeta. Science 2001;293:1673-1677. Transcription factors are proteins that possess separate 10. Pickup JC. Inflammation and activated innate immunity in the pathogenfunctions, such as DNA binding and transactivation and/or esis of type 2 diabetes. Diabetes Care 2004;27:813-823. 11. Senn JJ, Klover PJ, Nowak IA, Mooney RA. Interleukin-6 induces cellular transrepression, that are restricted to distinct domains. In insulin resistance in hepatocytes. Diabetes 2002;51:3391-3399. general, the DNA-binding domains are particularly well 12. Christ B, Yazici E, Nath A. Phosphatidylinositol 3-kinase and protein conserved and used for classification of phylogenetic relakinase C contribute to the inhibition by interleukin 6 of phosphoenolpyruvate carboxykinase gene expression in cultured rat hepatocytes. HEI>A'I.OL-tionships. Also, transcription factors that share the same design for DNA interactions tend to display similar functions, OGY 2000;31:46 1-468. 13. Chen RH, Chang MC, Su YH, Tsai YT, Kuo ML. Interleukin-6 inhibits such as the capacity to heterodimerize and transduce intratransforming growth factor-beta-induced apoptosis through the phosphacellular signals. tidylinositol 3-kinasdAkt and signal transducers and activators of tranConsiderable progress has been made in the understandscription 3 pathways. J Biol Chem 1999;274:23013-23019. 14. Kortylewski M, Feld F, Kruger KD, Bahrenberg G, Roth RA, Joost HG, et ing of the network of transcription factors, collectively real. Akt modulates STAT-3-mediated gene expression through a FKHR ferred to as hepatocyte nuclear factors (HNF), that (FOX0 la)-dependent mechanism. J Biol Chem 2003; 278:5242-5249. Comments zyxwvutsrqpo zyxwvuts zyxwvutsrqpo Copyright 02004 by the American Associationfor the Study of Liver Diseases. Published online in Wiley InterScience (www. interscience. wiley.com). DO1 10.1002ihep.20444 Outfoxing liver Cancer With plSARFTumor Suppressor? Kalinicbenko W Major ML, Wang X, Petrovic K Kuecblej, Yoder HM, etal. Foxml b transcription factor is zyxwvutsrq essential for development of hepatocellular carcinomas and is negatively regulated by the ~ 1 tumor 9 suppres~ sor. Genes Dev 2004;18:830-850. (Reprinted with permission of Genes & Development.) Abstract participate in liver development, differentiation, and regeneration.'" The HNFs are composed of several different fmilies of transcription factors that include the forkhead box (Fox) family.3 The Fox family of transcription factors consists of more than 50 mammalian proteins that share homology in the winged helix DNA-binding domain'4.5 (for useful information visit http://www.biology.pomona.edu/ fox.htm1). Although the Fox family is not among the biggest family of transcription factors, it displays an extraordinary functional diversity and is involved in a wide variety of biological processes that include differen~tiation, ~ cellular proliferation, apoptosis, metabolic homeostasis, longevity, and transformation.> The Fox transcription factors that belong to the HNFs are Foxal, 2 and 3 (also referred to as Hnf3a,P,y), Foxfl (hfhhs, Freacl), and Foxmlb ( H f h l l B , Trident). Among the Fox transcription factors that belong to the HNFs, Foxml b is unique in that it is required for efficient regeneration of adult hepatocytes.6 Indeed, when the Foxml b gene was originally cloned, it was realized that expression of the gene strictly correlated with cell cycling independent of cell type.7 Subsequently, a number of studies have confirmed and extended these original observations.3,s-1" Recent studies have also demonstrated that activation of Foxml B protein requires both Cdk-cyclin phosphorylation-dependent recruitment of p300/CBP and binding of activated Cdk-cyclin complexes to the activation domain. ] Selective overexpression of the Foxml B in hepatocytes results in an 8-hour acceleration of hepatocyte entry into S phase and mitosis during liver regeneration.l0.l2The premature entry into S phase and mitosis is associated with early hepatic expression of cyclin D 1 and CIEBPP, and decreased induction of cdk inhibitor p2 1Cip 1 (p2 l ) , as well as early expression of cyclin B 1, cyclin B2, cdc-2 (CdkI), and cdc25B p h o s p h a t a ~ e . * In ~,~3 contrast, liver regeneration in the Alb-Cre Foxmlb -/mouse revealed that Foxmlb deficiency results in signifi- Hepatocellular carcinoma (HCC) is a leading cause of cancerrelated deaths worldwide. Here, we provide evidence that the Forkhead Box (Fox) mlb (Foxmlb or Foxml) transcription factor is essential for the development of HCC. Conditionally deleted Fomnlb mouse hepatocytes fail to proliferate and are highly resistant to developing HCC in response to a Diethylnitrosamine (DEN)IPhenobarbital (PB) liver tumor-induction protocol. The mechanism of resistance to HCC development is associated with nuclear accumulation of the cell cycle inhibitor ~ 2 7 ~ protein P' and reduced expression of the Cdkl-activator Cdc25B phosphatase. We showed that the Foxmlb transcription factor is a novel inhibitory target of the p l F tumor suppressor. Furthermore, we demonstrated that conditional overexpression of Foxmlb protein in osteosarcoma U20S cells greatly enhances anchorage-independent growth of cell colonies on soft agar. A p l y 26-44 peptide containing nine D-Arg to enhance cellular uptake of the peptide was sufficient to significantlyreduce both Foxmlb transcriptional activity and Foxmlb-induced growth of U20S cell colonies on soft agar. These results suggest that this (D-Arg)9-p13ARF26-44 peptide is a potential therapeutic inhibitor of Foxmlb function during cellular transformation. Our studies demonstrate that the Foxmlb transcription factor is required for proliferative expansion during tumor progression and constitutes a potential new target for therapy of human HCC tumors. zyxwvuts zyx zyxwvutsrqpon zy HEPATOLOGY, Vol. 40, No. 5, 2004 HEPATOLOGY ELSEWHERE / , - 1011 ._...Stabili GI Fig. 1. Schematic representation of FoxMlB regulation of cell cycle genes. Modified from Costa et al.3 cant reduction of hepatocyte replication and mitosis.13 This reduction in hepatocyte replication is associated with increased levels of Cdk inhibitor p21 and decreased expression of Cdc25A phosphatase, resulting in decreased Cdk2 activation and delayed progression into S phase. Also, no expression of the Cdc25B phosphatase is detected in the regenerating Alb-Cre Foxmlb -1- livers resulting in delayed activation of the Cdkl-cyclinB complex and attenuated cell cycle progression. Taken together, the data from the Alb-Cre Foxmlb-/- mouse indicate that the critical targets for the Foxmlb are genes coding for proteins that limit Cdkl and 2 activities needed for the normal cell cycle progression (Fig. 1). In an impressive paper, Kalinichenko et al.14 have extended earlier studies and examined the role of Foxmlb in chemically induced liver cancer. The diethylnitrosamine/ phenobarbital (DEN/PB) liver tumor-induction protocol was used to demonstrate that Alb-Cre Fuxmlb-/- mouse hepatocytes fail to undergo proliferation and are highly resistant to developing hepatocellular carcinoma (HCC). The authors provide evidence that the mechanism of resistance to HCC development in Foxmlb-/- hepatocytes involves (1) increased polyploidization; (2) sustained nuclear accumulation of the Cdk inhibitor ~ 2 7 ~ protein P' and diminished expression of the M-phase promoting Cdc25B phosphatase; (3) transient hepatic expression of p l Ftumor suppressor during DENPB tumor initiation; and (4)increased levels of Foxml b protein, which can stimulate anchorage-independent growth (at least in osteosarcoma U20S cells). The authors also provide evidence that the ( D - A r & - p l F 2644 peptide can inhibit Foxmlb transcriptional activity and might be an effect;ve therapeutic inhibitor of Foxmlb function. Given the critical role of Foxmbl transcription factor in promoting efficient cell cycle progression, it is perhaps not surprisingthat loss ofthe Foxrnbl gene in hepatocytes shortly Transcription M after tumor initiation would significantly reduce and/or delay development of liver cancer. Indeed, one would predict that similar reduction in tumor formation would also occur in other tissues in which the Foxmbl gene was selectively deleted in the target cell population. Although Kalinichenko et al.14 provide an impressive data set on the role of Foxmbl in experimental liver cancer, there are still unanswered questions regarding the precise role of Foxmbl in the development of liver cancer. The fact that Alb-Cre-mediated allele is accomplished 2 to 3 deletion of the Foxmbl weeks after the chemical initiation of the carcinogenic process does not provide precise insight into the potential role or importance of Foxmbl in the later stages (it.,promotion and progression) of the process. It is worth noting that Foxmbl -/- mice do develop liver cancer (albeit with much longer latency, so Foxmbl is not essential for the development of HCC), and therefore it is possible that Foxmbl is not important for the later stages of the process, but these issues need to be clarified. The importance of stage-specific deletion has been well illustrated for another transcription factor, c-jun, that is required for efficient cell cycle progression of hepatocytes and is critical for chemicallyinduced (it., DEN/PB) liver cancer.l5 Wagner and colleagues15 used liver-specific inactivation of c-jun at different stages of HCC development in the mouse to demonstrate that the requirement for c-jun was restricted to early stages of tumor development and inactivation of c-jun in advanced tumors did not impair tumor progression. Similar studies with the Foxml b f j mouse would be highly informative. The most provocative finding reported by Costa and colleagues14 is the demonstration that Foxmb 1 transcription factor is a novel inhibitory target for the ~ 1 tu- 9 mor suppressor. The p 19ARF(p 14ARF in humans) is one of 2 tumor suppressors encoded by the InR4a-Arflocus (the ~ ~ " critical other is p 1 The p 19ARFand ~ 1 6 ' "occupy zyxwvut zyxw zyxwvu zy fufi7 zyxwvu zyxw ~ 10 12 zyxwvutsrqponm HEPATOLOGY ELSEWHERE HEPATOLOGY, November 2004 zyxw SNOW S. THORGEIRSSON nodal points in a signaling network that is disrupted in CbieJf Laboratoy of Experimental Carcinogenesis most, if not all, cancer cells. Recent evidence, primarily Centerfor Cancer Research from mouse tumor models, has demonstrated a clear dif~ a ~ i 5 nCancer al Institute, ~ a t i o n Institutes a~ o f Health ference between the activities of and p16'"k4a in MD Betbesda, regulating both tumor onset and responsiveness to drugs.16 The p19ARFbinds directly to and inactivates References Mdm2, a negative regulator of the p53, and triggers a 1. Zaret KS. Regulatory phases of early liver development: paradigms of orp53-dependent transcriptional program that leads either ganogenesis. Nat Rev Genet 2002;3:499-512. to GI phase arrest or to apoptosis.*7The accumu2. Duncan SA. Mechanisms controlling early developmenl:of the liver. Mech Dev 2003;120:19-33. lates in the nucleolus of cells and can transport targets 3. Costa RH, Kalinichenko W, Holterman AX, Wang X. Transcription such as Mdm2 to this compartment, away from p53. In factors in liver development, differentiation and regeneration. HEPATOLaddition, the binding of ~ 1 to Mdm2 9 ~also inhibits ~ OGY 2003;38:1331-1347. 4. Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the the E3 ubiquitin protein ligase activity of Mdm2 for p53, winged helilforkhead transcription factors. Genes Dev 2000;14: 142-146. and thereby prevents p53 degradation. Furthermore, loss 5. Carlsson P,Mahlapuu M. Forkhead transcription factors: key players in of p53 function can abrogate the tumor suppressor funcdevelopment and metabolism. Dev Biol2002;250: 1-23. tion of p l 9ARF,emphasizing the central role of p53 in the 6 . Kower W, Schilham M W , Moerer P, van den Hoff MJ, Dam K, h e r s WH, et al. Uncoupling of S phase and mitosis in cardiomyocytesand hepatocyteslacking p l 9Aw-Mdm2-p53 pathway.17 the winged-helix transcription factor Trident. C u r Biol 199823:1327-1330. 1 26-44 9 se- ~ 7. Korver W, Roose J, Clevers H. The winged-helix transcription factor TriKalinichenko et al.I4 show that the ~ quenceswere sufficient to associatewith and inhibit Foxmlb dent is expressed in cycling cells. Nucleic Acids Res 1997;25:1715-1719. 8. Ye H, Kelly TF, Samadani U, Lim L, Rubio S, Overdier DG, et al. Hepatranscriptional activity, and increased levels of Foxm 1b protocyte nuclear factor 3/fork head homoiog 11 is expressed in proliferating tein can enhance anchorage-independent growth in the huepithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell man osteosarcoma U20S cells. The authors also provide Biol 1997;17: 1626-1641. 9. Luscher-FirzlaffJM, WestendorfJM, Zwicker J , Burkhardt H , Henriksson evidence in the U20S cells that the (D-Arg)9-p19m 26-44 M, Muller R, et al. Interaction of the fork head domain transcription factor peptide might be an effective therapeutic inhibitor of MPP2 with the human papilloma virus 16 E7 protein: enhancement of Foxml b function. Although the data on the interaction betransformation and transactivation. Oncogene 1999; 18:5620-5630. tween Foxm 1b and p 19ARFare solid, the experimental de- 10. Ye H, Holterman AX, Yo0 KW, Franks RR, Costa RH. Premature expression of the winged helix transcription factor HFH-118 in regenerating mouse liver sign and use of the U20S cells do not allow clear-cut accelerates hepatocyteentry into S phase. Mol Cell Biol1999;19:8570-8580. interpretation of the data obtained with the cells. The U2SO 11. Major ML, Lepe R, Costa RH. Forkhead box M1B transcriptional activity cells lack the expression of p 1 F (or more correctly p 14m) requires binding of Cdk-cydin complexes for phosphory1:ition-dependent recruitment of p300lCBP coactivators. Mol Cell Biol2004;24:2649-2661. but are wild type for the p53 pathway.lS Since the p l F 26-44 sequence contains 1 of the 2 binding sites for Mdm2 12. Wang X, Hung NJ, Costa RH. Earlier expression of the transcription factor HFH 11B diminishes induction of p2 lc'p"wM' levels and accelerthat act both independently and cooperatively,19 it is possible ates mouse hepatocyte entry into S-phase following carbon tetrachloride liver injury. HEPATOLOGY 2001;33: 1404-1414. that the ~ 1 26-44 9 sequence ~ could interact with both Foxml b and Mdm2. For example, the membrane-transduc- 13. Wang X, Kiyokawa H, Dennewia MB, Costa RH. The Forkhead Boxmlb muscription factor is essential for hepatocyte DNA replication and mitosis during 26-44 peptide significantly reduces the ing (D-Ar&-pl PF mouse liver regeneration. Proc Natl Acad Sci U S A 2002;99 16881-16886. number and size of wild-type U2SO colonies in soft agar,I4 14. Kalinichenko W, Major ML, Wang X, Petrovic V, Kuechle J , Yoder HM, et al. Foxmlb transcription factor is essential for development of hepato(Fig 9C-E) and this reduction in soft agar colonies appears to cellular carcinomas and is negatively regulated by the p1 9ARFtumor supbe similar to that seen with the GFP-Foxml b U2SO ~el1s.l~ pressor. Genes Dw 2004;18:830-850. (Fig 9F-H) It is therefore possible that the reduction in soft 15. Eferl R, Ricci R, Kenner L, Zenz R, David JP, Rath M, er al. Liver tumor development: c-Jun antagonizes the proapoptotic activity of p53. Cell agar colonies of U2SO cells is, at least in part, due to activa2003; 112:181-1 92. tion of the p53 pathway by the (D-Ar&-p 1PF 26-44 pep1 6 . Lowe SW, Sherr CJ. Tumor suppression by Ink4u-A$ progress and puztide due to the sequestration of the Mdm2 to the nucleoli. It is zles. Curr Opin Genet Dev 2003;13:77-83. important, and experiientally straightforwxd,to clarifjr the rel- 17. Sherr, CJ. The INK4u/ARFnenvork in tumor suppression. Nat Rev Mol Cell Biol 2001;2:731-737. ative contribution of the p53 and Foxmlb "pathways" to the 18. Martelli F, Hamilton T, Silver DP, Sharpless NE, Bardeesy N, Rokas M, et inhibition of soft agargrowth of U2SO cells and hepatocarcinoal. pl9ARF targets certain E2F species for degradation. Proc Natl Acad Sci genesis in the Alb-Cre Foxml b mouse. U S A 2001;98:4455-4460. Despite some of the limitations and questions discussed 13. Weber JD, Kuo ML, Bothner B, DiGiammarino EL, Kriwacki RW, Roussel MF, et al. Cooperative signals governing ARF-mdm2 interaction and ~~ above, this interesting study by Costa and c o l l e a g ~highlights nucleolar localization of the complex. Mol Cell Biol2000;20:2517-2528. the importance of developing new molecular approaches to identii and test novel targets for the treatment of liver cancer. Copyright 0 2004 by the American Association for the Study ofLiver Diseases. This is particularly important in the context of the current lack Published online in Wiky Interscience (www.interscience. wiley.com). DOI IO.I002/hep.20443 of effective treatment of human liver cancer. zyxwvutsrq zyxwvuts zyxwvutsr zyxwvuts zyxwvut