Loss of the Ras Regulator RASAL1: Another Route to Ras Activation in Colorectal Cancer
2008; Elsevier BV; Volume: 136; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2008.11.024
ISSN1528-0012
AutoresAndré Bernards, Jeffrey Settleman,
Tópico(s)Cancer-related Molecular Pathways
ResumoSee “Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression,” by Ohta M, Seto M, Ijichi H, et al, on page 206. See “Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression,” by Ohta M, Seto M, Ijichi H, et al, on page 206. Members of the Ras superfamily of small guanosine triphosphate (GTP) binding proteins function as binary on/off switches to control many biological processes. Ras proteins perform this function by cycling between active GTP bound and inactive guanosine diphosphate (GDP)-bound conformations. In their active state, the Ras proteins are capable of interacting with a diverse set of effector proteins. By modifying the enzymatic activity of these effectors, changing their subcellular localization, or altering their interaction with other proteins, activated Ras proteins elicit specific biological responses. Genes that encode the 3 highly related H-Ras, K-Ras, and N-Ras proteins were among the first identified human oncogenes.1Parada L.F. Tabin C.J. Shih C. et al.Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene.Nature. 1982; 297: 474-478Crossref PubMed Scopus (605) Google Scholar Somatically acquired mutations of these Ras genes are among the most common genetic changes in cancer, and are frequently observed in colorectal, pancreatic, and lung cancers. These mutationally altered Ras proteins are defective in their ability to recover their inactive, GDP-bound state, and instead remain constitutively active, transducing excessive signals that promote cancer cell proliferation and survival. Mutations giving rise to activated K-Ras occur during the advanced adenoma stage in up to 50% of colorectal cancers (CRCs).2Bos J.L. Fearon E.R. Hamilton S.R. et al.Prevalence of ras gene mutations in human colorectal cancers.Nature. 1987; 327: 293-297Crossref PubMed Scopus (1624) Google Scholar, 3Vogelstein B. Fearon E.R. Hamilton S.R. et al.Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (5990) Google Scholar The fact that not all CRCs harbor Ras mutations is potentially interesting, and suggests either that increased Ras signaling is not strictly required for tumor development, or that increased Ras signaling can be achieved by other means. Much evidence favors the latter hypothesis. Thus, among a surprisingly large set of putative and confirmed Ras effector proteins,4Rodriguez-Viciana P. Sabatier C. et al.Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate.Mol Cell Biol. 2004; 24: 4943-4954Crossref PubMed Scopus (263) Google Scholar Raf kinases, phosphatidylinositol 3-kinases (PI3Ks), and Ral GTPase exchange factors are believed to be relevant primarily to Ras-induced mitogenic signal transduction (Figure). Raf kinases act as upstream activators of extracellular signal–regulated kinase (ERK) cascades, and mutations that activate B-Raf are commonly found in melanoma and other cancers.5Davies H. Bignell G.R. Cox C. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8553) Google Scholar In CRC, mutations that activate K-Ras or B-Raf define mutually exclusive subsets of cases.6Rajagopalan H. Bardelli A. Lengauer C. et al.Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status.Nature. 2002; 418: 934Crossref PubMed Scopus (1086) Google Scholar Moreover, up to 30% of CRCs harbor PIK3CA mutations that activate the p110α catalytic subunit of PI3K, another important mitogenic Ras effector.7Samuels Y. Wang Z. Bardelli A. et al.High frequency of mutations of the PIK3CA gene in human cancers.Science. 2004; 304: 554Crossref PubMed Scopus (2991) Google Scholar GDP-bound, inactive Ras is activated by guanine nucleotide exchange factors (GEFs), whereas Ras-GTP inactivation is promoted by so-called GTPase-activating proteins (GAPs). The former proteins promote GTP for GDP exchange on Ras,8Bernards A. Settleman J. GEFs in growth factor signaling.Growth Factors. 2007; 25: 355-361Crossref PubMed Scopus (16) Google Scholar and GAPs stimulate the low intrinsic GTPase activity of Ras.9Bernards A. Settleman J. GAPs in growth factor signalling.Growth Factors. 2005; 23: 143-149Crossref PubMed Scopus (66) Google Scholar Mutations that activate GEFs are uncommon, but mutational activation of the SOS1 RasGEF has been identified in Noonan syndrome,10Roberts A.E. Araki T. Swanson K.D. et al.Germline gain-of-function mutations in SOS1 cause Noonan syndrome.Nat Genet. 2007; 39: 70-74Crossref PubMed Scopus (492) Google Scholar, 11Tartaglia M. Pennacchio L.A. Zhao C. et al.Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome.Nat Genet. 2007; 39: 75-79Crossref PubMed Scopus (474) Google Scholar a genetically heterogeneous disease that can also be caused by gain-of-function mutations in Ras signaling components KRAS, RAF1, or PTPN11.12Aoki Y. Niihori T. Narumi Y. et al.The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders.Hum Mutat. 2008; 29: 992-1006Crossref PubMed Scopus (287) Google Scholar In addition to activation by GEFs, Ras can be activated also by loss of RasGAP activity. The best known example of this scenario involves neurofibromatosis type 1 (NF1), which is among the most common tumor predisposition syndromes and is caused by loss of the NF1 RasGAP neurofibromin.13Cichowski K. Jacks T. NF1 tumor suppressor gene function: narrowing the GAP.Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar Recently, NF1 loss-of-function mutations have also been identified in 23% of glioblastomas,14McLendon R. Friedman A. Bigner D. et al.Comprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; Google Scholar and similar mutations have also been detected in CRC.15Wood L.D. Parsons D.W. Jones S. et al.The genomic landscapes of human breast and colorectal cancers.Science. 2007; 318: 1108-1113Crossref PubMed Scopus (2614) Google Scholar Another cancer-implicated RasGAP is DAB2IP, the gene for which is epigenetically silenced in several cancers, including prostate, breast, lung, liver, and gastrointestinal neoplasms.16Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. Epigenetic regulation of a novel tumor suppressor gene (hDAB2IP) in prostate cancer cell lines.J Biol Chem. 2003; 278: 3121-3130Crossref PubMed Scopus (124) Google Scholar, 17Dote H. Toyooka S. Tsukuda K. et al.Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in breast cancer.Clin Cancer Res. 2004; 10: 2082-2089Crossref PubMed Scopus (102) Google Scholar, 18Dote H. Toyooka S. Tsukuda K. et al.Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in gastrointestinal tumour.Br J Cancer. 2005; 92: 1117-1125Crossref PubMed Scopus (75) Google Scholar, 19Yano M. Toyooka S. Tsukuda K. et al.Aberrant promoter methylation of human DAB2 interactive protein (hDAB2IP) gene in lung cancers.Int J Cancer. 2005; 113: 59-66Crossref PubMed Scopus (98) Google Scholar, 20Qiu G.H. Xie H. Wheelhouse N. et al.Differential expression of hDAB2IPA and hDAB2IPB in normal tissues and promoter methylation of hDAB2IPA in hepatocellular carcinoma.J Hepatol. 2007; 46: 655-663Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar In this issue of Gastroenterology, Ohta et al21Ohta M. Seto M. Ijichi H. et al.Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.Gastroenterology. 2009; 136: 206-216Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar provide evidence that reduced expression of another RasGAP, dubbed RASAL1, plays an important role during CRC progression. Ohta et al21Ohta M. Seto M. Ijichi H. et al.Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.Gastroenterology. 2009; 136: 206-216Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar report a potential role for one of the human RasGAPs in a subset of CRCs. Their effort began with an in silico analysis of human proteins harboring putative RasGAP catalytic domains. This revealed 12 such proteins, representing the majority of the 15 potential RasGAPs detected in a previous survey.22Bernards A. Ras superfamily and interacting proteins database.Methods Enzymol. 2006; 407: 1-9Crossref PubMed Scopus (12) Google Scholar Then, with the goal of identifying potentially dysregulated RasGAPs in CRC, a disease frequently associated with K-Ras mutational activation and augmented Ras pathway signaling, they examined the mRNA expression of each of the RasGAPs. This analysis revealed that the expression of mRNA encoding 1 of these proteins, RASAL1, was significantly reduced in a subset of CRC-derived cell lines demonstrating wild-type K-Ras. The enrichment in this genetically defined subset of tumors is potentially significant, because CRCs expressing mutant K-Ras would be expected to demonstrate constitutive “GAP-refractory” Ras pathway signaling, thereby obviating a role for RasGAP loss in activating Ras-mediated signaling pathways. The reduced RASAL1 expression in this subset of CRC lines was confirmed by immunoblotting to detect the RASAL1 protein. Ohta et al21Ohta M. Seto M. Ijichi H. et al.Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.Gastroenterology. 2009; 136: 206-216Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar were able to confirm that ectopic expression of RASAL1 in transfected cells in culture could promote Ras inactivation, as well as suppression of signaling to the Ras downstream effector, ERK. Moreover, as expected, RASAL1 expression failed to affect the activity of mutationally activated oncogenic K-RasV12. The potential relevance of reduced RASAL1 expression to the activation state of K-Ras in CRCs expressing wild-type K-Ras was further supported by the additional finding that Ras-GTP levels were elevated in CRC lines demonstrating both wild-type K-Ras and reduced RASAL1 expression. Furthermore, RNAi-mediated knockdown of RASAL1 expression in CRC cells with wild-type K-Ras yielded increased Ras-GTP levels, and corresponding cells were found to exhibit enhanced tumor growth potential in mouse xenograft studies. Such findings nicely support a role for RASAL1 as a regulator of wild-type K-Ras in CRC cells. The investigators then turned their attention to primary CRC tumors to verify the cell line findings. An analysis of 152 CRC tumors of various histologic grades revealed that RASAL1 protein expression was reduced in 47% of adenocarcinomas and 17% of advanced adenomas, but no notable expression differences were observed in small adenomas. This finding suggests that reduced RASAL1 expression may be associated with CRC disease progression, with expression diminishing as cells progress from early adenomas to adenocarcinomas. Furthermore, reduced RASAL1 expression was primarily seen in tumors of the distal colon or rectum, suggesting that a particular cell of origin, defined by anatomic distribution, may be especially vulnerable to loss of RASAL1 expression. Indeed, the authors suggest that a seemingly unique role for RASAL1 as a tumor suppressor in CRC, despite the fact that there are many other RasGAPs, is that, unlike many of the other RasGAPs, whose expression is somewhat ubiquitous, RASAL1 expression exhibits clear enrichment in subsets of human tissues, including very high expression in thyroid and adrenal medulla. However, a specific enrichment in colonic epithelia has not been reported thus far. Ohta et al21Ohta M. Seto M. Ijichi H. et al.Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.Gastroenterology. 2009; 136: 206-216Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar also report findings implicating epigenetic regulation in the reduced expression of RASAL1 in a subset of CRCs. Using an in silico analysis, they identified a 5′ CpG island within the RASAL1 gene containing 9 CpG sites, and found that these were indeed methylated in several of the CRC cells, particularly those with wild-type K-Ras. Methylation of such islands is often associated with transcriptional silencing. By treating cells with reduced RASAL1 expression with 5-azacytidine, a demethylating agent, they observed the expected restoration of RASAL1 expression. Based on this observation, the investigators concluded that reduced RASAL1 expression in a subset of wild-type K-Ras–expressing CRCs may reflect epigenetic gene regulation. Moreover, the fact that they did not observe either loss of heterozygosity or homozygous gene loss at the RASAL1 locus in CRC tumors is consistent with a nonmutational loss of RASAL1 as a potential tumor suppressor. There are certainly other examples of epigenetic loss of tumor suppressor function in human cancer, such as CDKN2A (which encodes p16), whose expression is reduced in a large fraction of many tumor types, and MLH1, whose expression is epigenetically suppressed in many CRCs. These overall findings implicate RASAL1 as another example of an epigenetically regulated tumor suppressor, whose expression loss may lead to increased Ras-mediated signaling in CRCs. The Ras signaling pathway is among the cellular signaling pathways that are most frequently dysregulated in human cancers. Although this is typically associated with direct mutational activation of the Ras genes, accumulating evidence supports a likely role for alterations of additional Ras pathway components (see above). The findings by Ohta et al21Ohta M. Seto M. Ijichi H. et al.Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.Gastroenterology. 2009; 136: 206-216Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar further support a role for nonmutational, epigenetic regulation of Ras pathway components in human cancer. Furthermore, the previous studies implicating epigenetic regulation of DAB2IP, another RasGAP, in cancer suggest a theme that should prompt further analysis of expression of the various other RasGAP family proteins in additional human malignancies. Although the analysis of RASAL1 in both CRC cell lines and primary tumors supports an apparent role for this protein in CRC disease progression, formal demonstration of a causative role for RASAL1 loss in cancer progression will undoubtedly require additional studies. For example, it could be informative to determine whether mice with a targeted disruption of this locus (potentially requiring conditional tissue-specific knockout technology) are prone to tumor development. This kind of analysis is generally considered to be the “litmus test” for putative tumor suppressors. Of course, RASAL1 may not perform an analogous role in mice, and it is also possible that additional oncogenic “hits” would be required to reveal its tumor suppressor function in such studies. Future studies will undoubtedly also need to address the mechanisms underlying the epigenetic regulation of RASAL1 expression. By analogy, the epigenetic regulation of DAB2IP seems to be under the control of epigenetic repressive control of EZH2, a histone methyltransferase, and putative oncogene.23Chen H. Tu S.W. Hsieh J.T. Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer.J Biol Chem. 2005; 280: 22437-22444Crossref PubMed Scopus (201) Google Scholar Perhaps a similar regulatory mechanism underlies reduced RASAL1 expression in some CRCs. Overall, these new findings highlight the potential broader significance of epigenetic regulation in cancer development and progression, and are likely to prompt further studies into this emerging aspect of tumorigenesis. Decreased Expression of the RAS-GTPase Activating Protein RASAL1 Is Associated With Colorectal Tumor ProgressionGastroenterologyVol. 136Issue 1PreviewAlthough colorectal cancer (CRC) progression has been associated with alterations in KRAS and RAS signaling, not all CRC cells have KRAS gene mutations. RAS activity is modulated by RAS-GTPase-activating proteins (RASGAPs), so we investigated the role of RASGAPs in CRC progression. Full-Text PDF
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