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WT1 the Oncogene: A Tale of Death and HtrA

2010; Elsevier BV; Volume: 37; Issue: 2 Linguagem: Inglês

10.1016/j.molcel.2010.01.010

1097-4164

Abdelkader Essafi, Nicholas D. Hastie,

Energy and Environment Impacts

Here, Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar identify WT1 as a novel bona fide substrate of the HtrA2/Omi mitochondrial protease and show that this reaction modulates WT1 antiapoptotic activity under cytotoxic stress. This supports an oncogenic function for WT1, with implications for novel chemotherapeutic avenues. Here, Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar identify WT1 as a novel bona fide substrate of the HtrA2/Omi mitochondrial protease and show that this reaction modulates WT1 antiapoptotic activity under cytotoxic stress. This supports an oncogenic function for WT1, with implications for novel chemotherapeutic avenues. Hanahan and Weinberg identified six hallmarks of most, if not all, cancers (Hanahan and Weinberg, 2000Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (20663) Google Scholar). These involve growth independent of mitogenic signals, insensitivity to antigrowth signals, evasion of apoptosis, indefinite replicative potential, sustainable angiogenesis, and, finally, invasion and metastasis. These hallmarks define genes involved in cancer progression, and oncogenes such as Bcl2 promote these hallmarks, whereas tumor suppressor genes such as p53 oppose them. The balance between the two types of genes determines the difference between normal development and tumorigenesis. However, some genes defy such simplistic definition and have a dichotomous function in cancer development. In this issue of Molecular Cell, Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar explore the mechanisms underlying the function of one such gene, the Wilms' tumor suppressor gene (WT1). The Wt1 gene was first cloned in 1990 as one of the first tumor suppressors, and its inactivation in humans causes the eponymously named childhood kidney tumor (Hohenstein and Hastie, 2006Hohenstein P. Hastie N.D. Hum. Mol. Genet. 2006; 15: R196-R201Crossref PubMed Scopus (282) Google Scholar). The WT1 protein contains four C2H2 zinc finger (ZF) domains at the C terminus and an unstructured N terminus containing the activation and suppression regulatory regions essential for protein-protein interactions. Wt1 epitomizes the complexity of the mammalian genome, encoding for at least 24 different isoforms as a result of a combination of alternative promoters, start codons, and splicing and RNA editing events. There are four most-studied isoforms, which include those encoding or skipping the 51 nt exon 5 and the variants that include or exclude the amino acids (KTS) between ZF3 and ZF4 through a cryptic splice donor site. WT1 binds both DNA and RNA and is hypothesized to regulate gene expression from transcription to translation. Differential binding to DNA and RNA is partly explained by the absence and presence of KTS, respectively (Hohenstein and Hastie, 2006Hohenstein P. Hastie N.D. Hum. Mol. Genet. 2006; 15: R196-R201Crossref PubMed Scopus (282) Google Scholar). Evidence for WT1's role as a tumor suppressor was reinforced by studies in cells grown in culture. Overexpression of WT1 in cancer cells lowered their colony formation rates and slowed their growth in vitro, and cancer cells ectopically expressing WT1 exhibited reduced tumor formation in nude mice (reviewed in Yang et al., 2007Yang L. Han Y. Suarez Saiz F. Saurez Saiz F. Minden M.D. Leukemia. 2007; 21: 868-876PubMed Google Scholar). This was mediated by promoting apoptosis through the induction of BAK and/or the suppression of growth signals. However, recent studies have shown that WT1 is overexpressed or expressed de novo in adult cancers that arise from tissues in which WT1 is not normally expressed. They include colorectal, breast, lung, desmoid, and brain tumors. Moreover, in breast cancer and leukemia, WT1 expression is correlated with poor prognosis (Yang et al., 2007Yang L. Han Y. Suarez Saiz F. Saurez Saiz F. Minden M.D. Leukemia. 2007; 21: 868-876PubMed Google Scholar). WT1 also regulates key oncogenes such as Bcl-2 and c-Myc in a dichotomous manner. WT1 activates the genes in one cell type and represses them in another, thus acting as an oncogene in one context (e.g., adult tumors) and a tumor suppressor in another (e.g., childhood tumors). These and other findings support the notion that Wt1 may function as an oncogene in adult cancers, but the crucial genetic proof remains to be demonstrated. One key question is whether WT1 continues to promote cell survival in adult cancers that are subjected to drug-induced cytotoxic stress and, if so, how this effect may be abrogated. In this issue of Molecular Cell, Roberts and colleagues show that WT1 mediates antiapoptosis signaling downstream of cytotoxic drugs, an effect that is negated by a novel binding partner high temperature requirement protein A2 (HtrA2/Omi), a serine protease (Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). They show that WT1 is a bona fide substrate for HtrA2-mediated proteolysis and delineate the cleavage sites and the transcriptional targets deregulated by the processing (Figure 1). Previously, the authors have shown that the WT1 N terminus harbors a 30 amino acid suppression domain (SD) that interacts with the cosuppressor BASP1 (Carpenter et al., 2004Carpenter B. Hill K.J. Charalambous M. Wagner K.J. Lahiri D. James D.I. Andersen J.S. Schumacher V. Royer-Pokora B. Mann M. et al.Mol. Cell. Biol. 2004; 24: 537-549Crossref PubMed Scopus (95) Google Scholar). In this study, they used SD as bait in a yeast two-hybrid screen to identify HtrA2 as the main target to bind SD, whereas another region of WT1 (245–297) did not capture HtrA2. HtrA2 is a mitochondrial protease that is present at low levels in the nucleus. Its main known biological function is to act as a tumor suppressor taming the activity of prosurvival oncogenes through proteolysis. Its main substrates are inhibitors of apoptosis (IAPs). In cancer cells, HtrA2 is catalytically activated under cytotoxic stress, resulting in the cleavage of target oncoproteins (Vande Walle et al., 2008Vande Walle L. Lamkanfi M. Vandenabeele P. Cell Death Differ. 2008; 15: 453-460Crossref PubMed Scopus (217) Google Scholar). In the current study, the authors showed that WT1 is cleaved at multiple sites by HtrA2 using in vitro translation assays and 32S radiolabeling. To delineate the cleavage sites within WT1, the authors took advantage of the presence of two antibodies that recognize either the N or C terminus of WT1. Combining product fragment size information with a search for conserved HtrA2 preference cleavage sites, they predicted two such cleavage sites in the C terminus of WT1 at Valine-286 and Leucine-320, resulting in fragments of 20 and 35 Kda, respectively. They also identified a cleavage site in the N terminus at Leucine-91 adjacent to SD. They further confirmed their findings in living cells by coexpressing WT1 with wild-type or protease-deficient HtrA2 (HtrA2S306A). As expected, the latter was not able to cleave WT1. However, wild-type HtrA2 cleaved all of the four major isoforms of WT1 (i.e., with or without exon 5 and with and without KTS), suggesting that WT1 is a physiological substrate of HtrA2. The number of HtrA2 substrates is limited. In fact, a recent proteome-wide screen of more than 1000 proteins identified only 15 substrates (Vande Walle et al., 2007Vande Walle L. Van Damme P. Lamkanfi M. Saelens X. Vandekerckhove J. Gevaert K. Vandenabeele P. J. Proteome Res. 2007; 6: 1006-1015Crossref PubMed Scopus (94) Google Scholar), and more recently, only three mitochondrial proteins have been identified as substrates for this protease in a mitoproteome analysis (Johnson and Kaplitt, 2009Johnson F. Kaplitt M.G. PLoS ONE. 2009; 4: e7100Crossref PubMed Scopus (16) Google Scholar). Both would have failed to identify WT1 as a target, as the cells used, HeLa and HEK293, do not express WT1. Cytotoxic stress is a major targeted therapy in the fight against cancer and is induced by drugs such as Etoposide, Staurosporine, and Anisomycin. Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar treated hematopoietic and solid tumor cell lines expressing endogenous WT1 with these drugs, which induced WT1 processing in an HtrA2-dependent manner. This was reversed by inhibiting HtrA2 activity by addition of either the small molecule inhibitor (UCF101) or HtrA2-specific siRNA. This confirmed that HtrA2 is necessary to cleave WT1 under cytotoxic stress. They also found that WT1 is processed equally in the cytoplasm and the nucleus. One of the most surprising findings of Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar is that the loss of HtrA2 leads to WT1 accumulation not only at the protein level, but also at the mRNA level. In HtrA2-deficient mouse embryonic fibroblasts (MEFs) Wt1 mRNA and protein levels are highly upregulated to levels comparable to those found in M15, a murine mesonephric cell line. This points to a negative feedback loop by which WT1 might autoregulate its own expression. Moreover, under normal conditions, HtrA2 controls Wt1 expression directly or indirectly at the transcriptional level and clearly, under cytotoxic stress, processes WT1 at the protein level. The authors further showed that only catalytically active HtrA2 was able to rescue WT1 cleavage in HtrA2-deficient MEFs, suggesting that HtrA2 is sufficient to cleave WT1. Finally, Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar investigated the interplay between WT1, HtrA2, and apoptosis induced downstream of cytotoxic stress. Upon treatment with Staurosporine, cancer cells and HtrA2−/− MEFs undergo apoptosis that is enhanced by WT1 knockdown. The data show that WT1 impedes the proapoptotic effects of cytotoxic drugs, whereas the loss of HtrA2 promotes survival and impairs drug-induced cell death, at least partially through the resulting proteolysis of WT1. The authors provide a potential mechanism by which WT1 may achieve this function. They investigated two known targets of WT1, JunB and c-Myc, which were maintained at low levels by WT1-mediated repression under normal circumstances in U2OS cells. Cytotoxic drug treatment leads to the HtrA2-mediated proteolysis of WT1, culminating in high levels of c-Myc and JunB transcripts. High levels of c-Myc, paradoxically, induce apoptosis in cancer cells (Murphy et al., 2008Murphy D.J. Junttila M.R. Pouyet L. Karnezis A. Shchors K. Bui D.A. Brown-Swigart L. Johnson L. Evan G.I. Cancer Cell. 2008; 14: 447-457Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), and JunB is proapoptotic, as it activates Ink4a and represses Bcl2 and BclXL (Eferl and Wagner, 2003Eferl R. Wagner E.F. Nat. Rev. Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1490) Google Scholar). In summary, Hartkamp et al., 2010Hartkamp J. Carpenter B. Roberts S.G.E. Mol. Cell. 2010; 37 (this issue): 159-171Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar identified a novel bona fide substrate of HtrA2 and confirmed this relationship in vivo. They have provided strong evidence for WT1 as a key mediator of oncogenic signaling downstream of cytotoxic drugs and discovered a novel pathway that regulates WT1-mediated derepression of apoptosis inducers under cytotoxic stress (Figure 1). Future work should concentrate on pinpointing other targets downstream of WT1 that amplify apoptotic signals emanating from cytotoxic drugs. It will also be important to analyze the effect of other types of stresses (e.g., genotoxic stress) on the HtrA2-WT1 interaction and the role of this interaction when WT1 acts as a tumor suppressor, such as in Wilms' tumors that have functional WT1. Furthermore, this study extends the limited list of HtrA2 substrates. Future work may concentrate on understanding the biological role of cleavage-resistant WT1 in normal development and tumorigenesis. In fact, Bcl2, a potent oncogene that promotes survival, was converted into an apoptotic effector by constructing a cleavage-resistant variant (Cheng et al., 1997Cheng E.H. Kirsch D.G. Clem R.J. Ravi R. Kastan M.B. Bedi A. Ueno K. Hardwick J.M. Science. 1997; 278: 1966-1968Crossref PubMed Scopus (975) Google Scholar). Also, the role of HtrA2 in regulating WT1 mRNA and protein levels, independent of WT1-dependent autoregulation, could be investigated by using such cleavage-resistant variants. Last but not least, the current work offers a novel pathway for potential therapeutic intervention in cancer. The authors would like to thank Craig Nicol (MRC Human Genetics Unit) for his assistance in generating the figure. The Wilms' Tumor Suppressor Protein WT1 Is Processed by the Serine Protease HtrA2/OmiHartkamp et al.Molecular CellJanuary 29, 2010In BriefThe Wilms' tumor suppressor protein WT1 functions as a transcriptional regulator of genes controlling growth, apoptosis, and differentiation. It has become clear that WT1 can act as an oncogene in many tumors, primarily through the inhibition of apoptosis. Here, we identify the serine protease HtrA2 as a WT1 binding partner and find that it cleaves WT1 at multiple sites following the treatment of cells with cytotoxic drugs. Ablation of HtrA2 activity either by chemical inhibitor or by siRNA prevents the proteolysis of WT1 under apoptotic conditions. Full-Text PDF Open Access