Portal de Periódicos da CAPES

Wild-Type p53: Tumors Can't Stand It

2007; Cell Press; Volume: 128; Issue: 5 Linguagem: Inglês

10.1016/j.cell.2007.02.022

1097-4172

Michael B. Kastan,

Molecular Biology Techniques and Applications

Most malignant tumors disrupt the p53 signaling pathway in order to grow and survive. Although many genes in addition to p53 are mutated in tumors, recent studies by Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1274) Google Scholar and Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar suggest that restoring p53 function alone is sufficient to cause regression of several different tumor types in mice and thus might represent a potent therapeutic strategy to treat certain human cancers. Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar also demonstrate that restoration of p53 activity results in tumor regression but add the sobering caveat that tumors may be able to quickly generate resistance by finding other ways to disrupt the p53 pathway. Most malignant tumors disrupt the p53 signaling pathway in order to grow and survive. Although many genes in addition to p53 are mutated in tumors, recent studies by Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1274) Google Scholar and Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar suggest that restoring p53 function alone is sufficient to cause regression of several different tumor types in mice and thus might represent a potent therapeutic strategy to treat certain human cancers. Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar also demonstrate that restoration of p53 activity results in tumor regression but add the sobering caveat that tumors may be able to quickly generate resistance by finding other ways to disrupt the p53 pathway. The p53 tumor suppressor protein is a potent roadblock to tumor development. People who carry one altered p53 gene in their germline have an extraordinarily high probability of developing a tumor. Indeed, most human cancers contain either mutations in the p53 gene that generate a dysfunctional or absent protein or have altered expression of other gene products that disrupt p53 function (Vousden and Prives, 2005Vousden K.H. Prives C. Cell. 2005; 120: 7-10PubMed Scopus (233) Google Scholar). Consistent with the demonstrated role of p53 in helping cells to respond to DNA damage and other cellular stresses, tumors lacking p53 demonstrate significant genetic instability and often contain markedly abnormal genomes. Given the many genetic changes that are present in tumors, it has been reasonable to wonder whether “fixing” a single change would be sufficient to curb tumor growth or progression. Despite the potent antitumor properties of p53, it is not clear whether its loss simply facilitates the genetic changes that contribute to tumor development or whether tumor growth is dependent on keeping the p53 pathway turned off permanently. Three recent papers, one in Cell (Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar) and two in Nature (Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1274) Google Scholar, Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar), report the same striking conclusion: restoration of p53 function in established tumors (including lymphomas, sarcomas, and hepatocellular carcinomas) causes regression of tumors in vivo and could represent an effective new approach to treating cancer. With the many genetic changes noted in tumors, it has not been clear whether tumors remain dependent on any single change for their continued growth and progression. It is conceivable that any given genetic change may be required for a particular step in the process of malignant transformation but that once the tumor has progressed beyond that particular stage, that genetic alteration would no longer be required to maintain the transformed phenotype. Such a scenario would suggest that targeting single abnormalities in tumors might be insufficient to control tumor growth and progression. However, work in several mouse models suggests that tumors develop a dependence on growth-promoting oncogenic changes, such as activating mutations in the Myc or Ras oncogenes (Felsher and Bishop, 1999Felsher D.W. Bishop J.M. Mol. Cell. 1999; 4: 199-207Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, Chin et al., 1999Chin L. Tam A. Pomerantz J. Wong M. Holash J. Bardeesy N. Shen Q. O'Hagan R. Pantginis J. Zhou H. et al.Nature. 1999; 400: 468-472Crossref PubMed Scopus (735) Google Scholar). This dependence has been termed “oncogene addiction” (Weinstein, 2002Weinstein I.B. Science. 2002; 297: 63-64Crossref PubMed Scopus (1402) Google Scholar), because tumors regress when these activating mutations are experimentally eliminated from the tumors. Mutations in tumors include those that activate growth-promoting genes and those that inactivate gene products that limit growth or enhance cell death. The p53 protein, perhaps the most intensely studied of such “tumor suppressor” gene products, normally aids the cell in responding to a variety of different cellular stresses (Figure 1). Once activated by a stress, p53 either induces cell-cycle arrest or facilitates programmed cell death (apoptosis). It accomplishes this either through its role as a transcription cofactor or perhaps by carrying out other cellular functions. The “choice” between cell death or growth arrest depends on the cell type and cellular environment. Loss of p53 function probably contributes to tumor progression through a combination of increased genetic instability, loss of growth-arresting signals, and inappropriate cell survival. If the primary reason that p53 loss contributes to tumor development is the generation of increased genetic instability, then p53 dysfunction might act as a “hit-and-run” mutation. In other words, p53 dysfunction would contribute to tumor development because it increases the rate of genetic mutations, but p53 dysfunction may not be required for tumor maintenance once the genetic changes that create the transformed phenotype are in place. However, perhaps not unexpectedly, the three new studies all demonstrate that like “oncogene addiction,” tumors remain addicted to the loss of p53 function. Each paper uses a different mouse-tumor model, and each brings unique insights to the process. The laboratory of Tyler Jacks (Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1274) Google Scholar) used an elegant genetic manipulation to make mice in which p53 was not expressed in any tissues because of the insertion of a STOP cassette in front of the gene. However, they could restore p53 function by removal of the STOP cassette following activation of a tamoxifen-inducible Cre recombinase. In this system, the mice develop spontaneous or irradiation-induced tumors with a high frequency because of the lack of p53 expression, and p53 can be restored at will by administration of tamoxifen. One unique aspect of this system is that the absence of p53 expression causes the mice to develop both lymphomas and sarcomas, allowing exploration of the effects of p53 restoration in at least two different tumor types in a single model system. After allowing the tumors to grow to sizes easily detected by MRI imaging, the investigators treated the mice with tamoxifen, thus activating the nuclear activity of the Cre recombinase, which then cuts out the STOP cassette and restores p53 expression throughout the mouse. Restoration of p53 expression caused significant, sometimes complete, regression of both lymphomas and sarcomas. Interestingly, p53 appeared to work by different mechanisms in the two different tumor types. Its re-expression induced apoptotic cell death in the lymphomas, whereas the sarcomas underwent cell-cycle arrest with signs of cellular senescence. Notably, the re-expression of p53 did not cause toxicity in normal mouse tissues, thus demonstrating that the tumor cells contain signals that activate the p53 pathway, leading to its growth suppressive or death-promoting effects selectively in tumor cells. Cell death is an irreversible state, and cellular senescence may also be irreversible. However, the in vivo permanence of the antitumor effects of p53 restoration was not explored in this model, thus leaving open the question of whether this would be an effective therapeutic intervention. A mouse model of hepatocellular carcinoma was used by Scott Lowe's laboratory (Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar) to address very similar questions. In this system, purified embryonic liver cells are transformed by introduction of an activated Hras oncogene, and p53 expression is downregulated using a doxycycline-repressible short hairpin RNA directed against p53. In this scenario, p53 is suppressed in the absence of doxycycline, and expression is restored when doxycycline is added. The investigators also transduced the cells with a fluorescent (GFP) marker and/or luciferase so that they could follow the tumor size in vivo with imaging. Injection of the Hras-expressing, p53-defective cells into immunodeficient (athymic) mice resulted in rapid growth of invasive hepatocarcinomas. Once the tumors were established, the investigators could restore p53 function by the addition of doxycycline. Established tumors became virtually undetectable within 12 days of doxycycline treatment, and even transient re-expression of p53 after four days of doxycycline treatment caused complete tumor regression. Thus, once activated, the p53-induced tumor suppression seemed irreversible. Similar to the observations of Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1274) Google Scholar in the mouse sarcoma model, Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar found that p53 induction in their hepatocarcinoma model led to growth arrest with senescence rather than apoptotic cell death. These observations are consistent with reports that the cell-cycle suppressive properties of p53 are an important part of its tumor-suppressive activities (Liu et al., 2004Liu G. Parant J.M. Lang G. Chau P. Chavez-Reyes A. El Naggar A.K. Multani A. Chang S. Lozano G. Nat. Genet. 2004; 36: 63-68Crossref PubMed Scopus (252) Google Scholar). The irreversible effects of transient p53 induction caused Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar to explore non-cell-autonomous mechanisms of tumor growth arrest, and they provide evidence for a role of the innate immune system. Inhibition of innate immunity through chemical, antibody, or genetic means reduced tumor regression following p53 restoration. The authors do point out that the athymic nude mice used for the initial studies may lack functional B and T cells, but they retain innate immune function. Interestingly, although the innate immune system seemed to be important for regression of the tumor mass, it was not required for the inhibition of tumor cell growth. Demonstration of a role for this component of the immune system in tumor regression following p53 restoration is a unique aspect of this paper compared to the other two, but the importance of this regression in tumor control and tumor shrinkage is not yet clear. The tumor mass might remain but might have been permanently arrested. The suggestion that even transient restoration of p53 may be sufficient for prolonged tumor control, at least in this tumor type, is a potentially important insight from this model. In their recent study in Cell, Gerald Evans and his team (Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar) used the Eμ-myc mouse model, which recapitulates human Burkitt's lymphoma/leukemia (Christophorou et al., 2005Christophorou M.A. Martin-Zanca D. Soucek L. Lawlor E.R. Brown-Swigart L. Verschuren E.W. Evan G.I. Nat. Genet. 2005; 37: 718-726Crossref PubMed Scopus (145) Google Scholar), as the backbone to ask questions about the effects of modulation of the p53 pathway on various aspects of tumor biology. Using this system (Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), they addressed the question of whether restoration of p53 expression in established tumors would lead to tumor regression. Their model system uses a p53 knockin model in which the normal p53 gene is replaced by a chimeric p53 protein containing an estrogen receptor (ER) domain linked to the wild-type p53 sequence. In this situation, the p53 protein is made but is retained in the cytoplasm in a nonfunctional state; addition of 4-hydroxytamoxifen (4-OHT) then drives transport of the p53 protein into the nucleus, where it can exert its tumor-suppressive activities. Eμ-myc mice were generated that contained one wild-type p53 allele and one p53-inducible allele. The mice rapidly develop B cell lymphoid malignancies as they lose their wild-type p53 allele and assume a p53 null state. Adding 4-OHT to the tumor cells from these mice in vitro activated the function of the remaining p53 allele and inhibited cellular growth and survival. Transplantation of these tumor cells into syngeneic mice resulted in rapid tumor growth and 100% death of the mice in less than four weeks; addition of 4-OHT markedly increased tumor cell death and significantly prolonged survival of the mice. Although survival of the tumor-bearing mice was definitely prolonged after a 7 day pulse of 4-OHT treatment, the mice eventually succumbed to the tumors. The tumor cells from all of these 4-OHT-treated relapsing mice had become resistant to p53 through disruption of the p53 signaling pathway, either by loss of p19ARF expression or by deletion of the p53 ER allele. This potentially important observation is unique among these three studies and raises the specter that even if p53 function could be restored to tumors in vivo that any therapeutic benefit might be short lived, because the tumor could find other ways to inactivate the p53 pathway. One limitation of this pessimistic, but perhaps predictable, conclusion is that only a 7 day pulse of p53 activity was used in these experiments. It is possible that persistent restoration of p53 function would not be as susceptible to the generation of resistance. It is worth noting that Xue et al., 2007Xue W. Zender L. Miething C. Dickins R.A. Hernando E. Krizhanovsky V. Cordon-Cardo C. Lowe S.W. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1629) Google Scholar observed permanent inhibition of tumor growth in their hepatocarcinoma model even after only a 4 day activation of p53 function. Given that their tumor system appeared to depend on cellular senescence and the immune system for tumor control, whereas p53 activation in the Eμ-myc system appears to work via induction of apoptotic cell death, there may be tumor-type specificity in the practical effectiveness of this therapeutic strategy. Interestingly, in the Eμ-myc tumor cells that had inactivated the p53 pathway via loss of p19ARF expression, the p53 protein was still functional and could still be induced by irradiation. Thus, addition of a p53-activating stimulus, such as irradiation or chemotherapy, might make the strategy of p53 functional restoration in tumors more therapeutically effective. Studies of all three mouse tumor model systems yielded the same conclusion about the tumor-reducing effects of p53 restoration in tumor masses. Should we be encouraged that all three mouse models suggest that this is a potentially viable therapeutic intervention, or should we be discouraged by the rapid resistance to p53 restoration generated in the Eμ-myc model? Perhaps both responses are warranted, accompanied by appropriate caveats. It is encouraging that p53 restoration by itself appeared to be sufficient to reduce tumor mass or to prolong survival in two different lymphoma models, a sarcoma model, and a hepatocarcinoma model. Interestingly, the mechanism of tumor suppression by p53 activation appeared to vary (promoting either growth arrest/senescence or apoptosis) depending on the tumor cell type. The resistance to this intervention seen in the Eμ-myc model may reflect the transient (7 days) reintroduction of p53 function or may reflect some unique feature of this tumor model. The relatively persistent cessation of tumor growth in the hepatocarcinoma model after just 4 days of p53 restoration may suggest that some tumor types will not easily generate resistance either because of the mechanism of tumor suppression in that cell type, the nature of other genetic alterations in the tumor type, or the influence of the immune system on the tumor suppressive effects (as intriguingly suggested by Xue and colleagues). Simple re-expression of p53 in cells does not seem to be sufficient to activate the p53 pathway (Ventura et al., 2007Ventura A. Kirsch D.G. McLaughlin M.E. Tuveson D.A. Grimm J. Lintault L. Newman J. Reczek E.E. Weissleder R. Jacks T. Nature. 2007; 445: 606-607Crossref PubMed Scopus (1274) Google Scholar, Christophorou et al., 2005Christophorou M.A. Martin-Zanca D. Soucek L. Lawlor E.R. Brown-Swigart L. Verschuren E.W. Evan G.I. Nat. Genet. 2005; 37: 718-726Crossref PubMed Scopus (145) Google Scholar, Christophorou et al., 2006Christophorou M.A. Ringshausen I. Finch A.J. Swigart L.B. Evan G.I. Nature. 2006; 443: 214-217Crossref PubMed Scopus (324) Google Scholar). Rather, the transformed environment of tumor cells appears to be required to activate the restored p53 protein. In fact, it was argued in the Eμ-myc system that p19ARF is the only persistent determinant of p53 activation in the tumor cells (Christophorou et al., 2005Christophorou M.A. Martin-Zanca D. Soucek L. Lawlor E.R. Brown-Swigart L. Verschuren E.W. Evan G.I. Nat. Genet. 2005; 37: 718-726Crossref PubMed Scopus (145) Google Scholar, Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). This total dependence on p19ARF for p53 activation may be a reflection of the unusual nature of the Eμ-myc system and may not be representative of all tumors or reflect the situation in human tumors. In addition to activation of p14ARF, other signals that activate p53 in human tumors (Figure 1) include telomere abnormalities, hypoxia, replicative stress, oxidative stress, and other types of DNA damage (Giaccia and Kastan, 1998Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1143) Google Scholar, Karlseder et al., 1999Karlseder J. Broccoli D. Dai Y. Hardy S. de Lange T. Science. 1999; 283: 1321-1325Crossref PubMed Scopus (881) Google Scholar). In contrast to human cells, and in particular many human tumors, mouse cells have long telomeres. Long telomeres in mouse tumors means that these tumor cells lack one of the signaling pathways to p53. Interestingly, mice lacking p19ARF develop significantly fewer tumors when they also contain “humanized” telomeres (shortened over generations by genetic deletion of telomerase) (Greenberg et al., 1999Greenberg R.A. Chin L. Femino A. Lee K.H. Gottlieb G.J. Singer R.H. Greider C.W. DePinho R.A. Cell. 1999; 97: 515-525Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Similarly, although hypoxia and oxidative stress may be important stimuli for activating the p53 pathway during human tumorigenesis, tumor development in most mouse models is not dependent on these stimuli. An additional difference between these mouse tumor models and human tumors is the lack of dominant-negative p53 alleles. As noted by Martins et al., 2006Martins C.P. Brown-Swigart L. Evan G.I. Cell. 2006; 127: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar even the relapsed tumors in the Eμ-myc mice failed to mutate the p53 gene and instead lost the entire p53 allele. This is not surprising given that these mouse tumors only had one p53 allele to start with. However, p53 is frequently mutated in human tumors, the mutant protein is frequently overexpressed because of a prolonged half-life, and the mutant overexpressed p53 protein has “dominant-inhibitory” and perhaps “gain-of-function” biochemical activities. It is possible that the presence of these mutant p53 proteins in human tumors could negatively affect the outcome of functional p53 restoration depending on how p53 function is restored. Of course, if the mechanism by which p53 function is restored is through conversion of mutant p53 protein to a functional p53 protein (Bykov et al., 2002Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (806) Google Scholar), then this is no longer a concern and, in fact, is a therapeutic benefit. Regardless of which signal in the tumor cells activates p53 following its restoration, the fact that the pathway is activated in tumor cells but not normal cells provides a potentially important therapeutic selectivity. The goal of tumor therapy is to develop interventions that selectively kill tumor cells relative to normal cells. The suggestion that tumor cells, but not normal cells, have a cellular environment that activates the p53 pathway would create a setting of an advantageous therapeutic index. Although genetically malleable mouse models are powerful tools for proof of principle, there are numerous practical challenges before restoring p53 function in vivo in human tumors becomes a reality. However, different approaches to achieve this are already in various stages of development: gene therapy approaches directly introducing wild-type p53 genes into tumors are in clinical trials (Roth, 2006Roth J.A. Expert Opin. Biol. Ther. 2006; 6: 55-61Crossref PubMed Scopus (101) Google Scholar), the small molecule PRIMA-1 has been reported to restore mutant p53 protein to wild-type function in tissue culture (Bykov et al., 2002Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (806) Google Scholar), and the small molecule nutlin can induce p53 protein (as long as the p53 gene itself has not been mutated) in cells by relieving its inhibition by HDM2 (Vassilev et al., 2004Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. et al.Science. 2004; 303: 844-848Crossref PubMed Scopus (3519) Google Scholar). The therapeutic synergy of DNA-damage induction with p53 restoration in the Eμ-myc model further suggests that combinations of standard cytotoxic therapies with any of these approaches might add to their effectiveness. Although, there are many challenges ahead, these three new elegant studies are likely to stimulate new interest in developing approaches for restoring p53 function in vivo in human tumors.