Haploinsufficiency of Mdm2 and Mdm4 in tumorigenesis and development

The tumor suppressor p53 is inactivated by multiple mechanisms that include mutations of the p53 gene itself and increased levels of the p53 inhibitors MDM2 and MDM4. Mice lacking Mdm2 or Mdm4 exhibit embryo-lethal phenotypes that are completely rescued by concomitant deletion of p53. Here we show that Mdm2 and Mdm4 haploinsufficiency leads to increased p53 activity, exhibited as increased sensitivity to DNA damage and decreased transformation potential. Moreover, in in vivo tumor development, Emu-myc Mdm4+/- mice show a delayed onset of B-cell lymphomas compared to Emu-myc mice. Additionally, Mdm2+/- Mdm4+/- double-heterozygous mice are not viable and exhibit defects in hematopoiesis and cerebellar development. The defects in Mdm2+/- Mdm4+/- mice are corrected by deletion of a single p53 allele. These findings highlight the exquisite sensitivity of p53 to Mdm2 and Mdm4 levels and suggest that some cell types may be more sensitive to therapeutic drugs that inhibit the Mdm-p53 interaction.     

Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.     

Wild type p53 reactivation: From lab bench to clinic

The p53 tumor suppressor is the most frequently inactivated gene in cancer. Several mouse models have demonstrated that the reconstitution of the p53 function suppresses the growth of established tumors. These facts, taken together, promote the idea of p53 reactivation as a strategy to combat cancer. This review will focus on recent advances in the development of small molecules which restore the function of wild type p53 by blocking its inhibitors Mdm2 and MdmX or their upstream regulators and discuss the impact of different p53 functions for tumor prevention and tumor eradication. Finally, the recent progress in p53 research will be analyzed concerning the role of p53 cofactors and cellular environment in the biological response upon p53 reactivation and how this can be applied in clinic.     

Selective inactivation of p53 facilitates mouse epithelial tumor progression without chromosomal instability

We examined the selective pressure for, and the impact of, p53 inactivation during epithelial tumor evolution in a transgenic brain tumor model. In TgT(121) mice, cell-specific inactivation of the pRb pathway in brain choroid plexus epithelium initiates tumorigenesis and induces p53-dependent apoptosis. We previously showed that p53 deficiency accelerates tumor growth due to diminished apoptosis. Here we show that in a p53(+/-) background, slow-growing dysplastic tissue undergoes clonal progression to solid angiogenic tumors in all animals. p53 is inactivated in all progressed tumors, with loss of the wild-type allele occurring in 90% of tumors. Moreover, similar progression occurs in 38% of TgT(121)p53(+/+) mice, also with loss of at least one p53 allele and inactivation of p53. Thus, the selective pressure for p53 inactivation, likely based on its apoptotic function, is high. Yet, in all cases, p53 inactivation correlates with progression beyond apoptosis reduction, from dysplasia to solid vascularized tumors. Hence, p53 suppresses tumor progression in this tissue by multiple mechanisms. Previous studies of fibroblasts and hematopoietic cells show that p53 deficiency can be associated with chromosomal instability, a mechanism that may drive tumor progression. To determine whether genomic gains or losses are present in tumors that progress in the absence of p53, we performed comparative genomic hybridization analysis. Surprisingly, the only detectable chromosomal imbalance was partial or complete loss of chromosome 11, which harbors the p53 gene and is thus the selected event. Flow cytometry confirmed that the majority of tumor cells were diploid. These studies indicate that loss of p53 function is frequent under natural selective pressures and furthermore that p53 loss can facilitate epithelial tumor progression by a mechanism in addition to apoptosis reduction and distinct from chromosomal instability.     

The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation

Although DNA damaging agents have revolutionized chemotherapy against solid tumors, a narrow therapeutic window combined with severe side effects has limited their broader use. Here we show that RAD001 (everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53, but not mutant p53 tumor cells. The use of isogenic tumor cell lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. We further show that RAD001 sensitizes cells to cisplatin by inhibiting p53-induced p21 expression. Unexpectedly, this effect is attributed to a small but significant inhibition of p21 translation combined with its short half-life. These findings provide the molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors.     

Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage.

Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild-type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations.     

Family Feud in Chemosensitvity: p73 and Mutant p53

The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 (“gain of function”) are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.     

Timed Somatic Deletion of p53 in Mice Reveals Age-Associated Differences in Tumor Progression

Inactivating mutations in the p53 tumor suppressor gene occur often in the progression of human cancers. p53 inhibits the outgrowth of nascent cancer cells through anti-proliferative actions (including induction of apoptosis or senescence). To test p53 tumor suppressor functions in a novel experimental context, we somatically deleted both p53 alleles in multiple tissues of mice at various ages. Mice homozygously deleted for p53 at 3 months of age showed a longer tumor latency compared to mice deleted for p53 at 6 and 12 months of age. These results are consistent with a model in which tissues accumulate oncogenically activated cells with age and these are held in check by wildtype p53. We also deleted p53 before, concurrent with, and after treatment of mice with ionizing radiation (IR). The absence or presence of p53 during IR treatment had no effect on radiation-induced lymphoma latency, confirming that the immediate p53 damage response was irrelevant for cancer prevention. Even the presence of wildtype p53 for up to four weeks post-IR provided no protection against early lymphoma incidence, indicating that long term maintenance of functional p53 is critical for preventing the emergence of a cancer. These experiments indicate that sustained p53 anti-oncogenic function acts as a final or near final line of defense preventing progression of oncogenically activated cells to malignant tumors.     

Apoptosis is the essential target of selective pressure against p53, whereas loss of additional p53 functions facilitates carcinoma progression

The high frequency of p53 mutation in human cancers indicates the important role of p53 in suppressing tumorigenesis. It is well established that the p53 regulates multiple, distinct cellular functions such as cell-cycle arrest and apoptosis. Despite intensive studies, little is known about which function is essential, or if multiple pathways are required, for p53-dependent tumor suppression in vivo. Using a mouse brain carcinoma model that shows high selective pressure for p53 inactivation, we found that even partially abolishing p53-dependent apoptosis by Bax inactivation was sufficient to significantly reduce the selective pressure for p53 loss. This finding is consistent with previous reports that apoptosis is the primary p53 function selected against during Eμ-myc-induced mouse lymphoma progression. However, unlike observed in the Eμ-myc-induced lymphoma model, attenuation of apoptosis is not sufficient to phenocopy the aggressive tumor progression associated with complete loss of p53 activity. We conclude that apoptosis is the primary tumor suppressive p53 function and the ablation of additional p53 pleiotropic effects further exacerbates tumor progression.     

Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA). Immunization against a self-antigen is challenging because an antigen-specific immune response likely generates only low affinity antigen-specific CD8⁺ T-cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous TAAs expressed by tumors. The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine. The vaccine employs a xenogeneic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Mice immunized with xenogeneic full length hp53 DNA plasmid intramuscularly followed by electroporation were protected against challenge with murine colon cancer MC38 while those immunized with mp53 DNA were not. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53 DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8⁺ T-cell precursors compared to vaccination with mp53 DNA. Antibody depletion experiments also demonstrated that CD8⁺ T-cells play crucial roles in the antitumor effects. This study showed intramuscular vaccination with xenogeneic p53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against tumors expressing mutated p53 through CD8⁺ T cells.     

An MVA vaccine overcomes tolerance to human p53 in mice and humans

Background The cellular regulatory protein p53 is overexpressed by almost 50% of all malignancies making it an attractive target for a vaccine approach to cancer. A number of immunotherapy approaches targeting p53 have been evaluated successfully in murine models, but translation of these preclinical findings to the clinic has been unsuccessful. Prior studies in our laboratory employing murine models demonstrated that a modified vaccinia virus Ankara (MVA) vaccine expressing murine p53 could stimulate p53 specific immunity. Systemic administration of the MVA vaccine was able to effect the rejection of established tumors. To better understand the immunologic mechanisms that underlie the vaccine function of human p53, we utilized a murine model in which the murine germ line copy of p53 was replaced with a modified human one. These mice, referred to as Hupki, were evaluated as a tolerant model to explore the capacity of MVA expressing human p53 to overcome tolerance and reject human p53-expressing tumors. Results MVAp53 immunization of Hupki mice resulted in the generation of p53-specific CD8⁺ T cells and the rejection of a highly aggressive murine mammary carcinoma cell line 4T1(H-2d) transfected with human p53 (4T1p53). An immunologic correlate of tumor protection was evaluated utilizing an overlapping peptide library spanning the full length of human p53. This reagent was also used in combination with MVAp53 to stimulate p53-specific CD8⁺ T cell responses in cancer patients. Conclusion These studies demonstrate the potential of MVAp53 to overcome tolerance to p53 for cancer immunotherapy.     

Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome

The p53 tumor suppressor gene is commonly altered in human tumors, predominantly through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of-function properties to p53. To ascertain the physiological effects of p53 point mutation, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are models of Li-Fraumeni Syndrome; they developed allele-specific tumor spectra distinct from p53+/- mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including a variety of carcinomas and more frequent endothelial tumors. Dominant effects that varied by allele and function were observed in primary cells derived from p53R270H/+ and p53R172H/+ mice. These results demonstrate that point mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss of p53 function.     

Family feud in chemosensitvity: p73 and mutant p53

The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 ("gain of function") are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.     

Mutant p53 reactivation by small molecules makes its way to the clinic

The TP53 tumor suppressor gene is mutated in many human tumors, including common types of cancer such as colon and ovarian cancer. This illustrates the key role of p53 as trigger of cell cycle arrest or cell death upon oncogenic stress. Most TP53 mutations are missense mutations that result in single amino acid substitutions in p53 and expression of high levels of dysfunctional p53 protein. Restoration of wild type p53 function in such tumor cells will induce robust cell death and allow efficient eradication of the tumor. Therapeutic targeting of mutant p53 in tumors is a rapidly developing field at the forefront of translational cancer research. Various approaches have led to the identification of small molecules that can rescue mutant p53. These include compounds that target specific p53 mutations, including PK083 and PK5174 (Y220C mutant p53) and NSC319726 (R175H mutant p53), as well as PRIMA-1 and its analog APR-246 that affect a wider range of mutant p53 proteins. APR-246 has been tested in a Phase I/II clinical trial with promising results.