Mutant p53 mediates survival of breast cancer cells

Background:

p53 is the most commonly mutated tumour-suppressor gene in human cancers. Unlike other tumour-suppressor genes, most p53 cancer mutations are missense mutations within the core domain, leading to the expression of a full-length mutant p53 protein. Accumulating evidence has indicated that p53 cancer mutants not only lose tumour suppression activity but also gain new oncogenic activities to promote tumourigenesis.

Methods:

The endogenous mutant p53 function in human breast cancer cells was studied using RNA interference (RNAi). Gene knockdown was confirmed by quantitative PCR and western blotting. Apoptosis was evaluated by morphological changes of cells, their PARP cleavage and annexin V staining.

Results:

We show that cancer-associated p53 missense mutants are required for the survival of breast cancer cells. Inhibition of endogenous mutant p53 by RNAi led to massive apoptosis in two mutant p53-expressing cell lines, T47D and MDA-MB-468, but not in the wild-type p53-expressing cells, MCF-7 and MCF-10A. Reconstitution of an RNAi-insensitive mutant p53 in MDA-MB-468 cells completely abolished the apoptotic effects after silencing of endogenous mutant p53, suggesting the specific survival effects of mutant p53. The apoptotic effect induced by mutant p53 ablation, however, is independent of p63 or p73 function.

Conclusion:

These findings provide clear evidence of a pro-survival ‘gain-of-function'' property of a subset of p53 cancer mutants in breast cancer cells.     

Induction of genetic instability by gain-of-function p53 cancer mutants

p53 plays critical roles in tumor suppression and the loss of its function is required for cancer progression. In this context, the p53 gene is the most commonly mutated tumor suppressor gene in human cancers. The majority of the p53 gene mutations in human cancers are missense mutations, leading to the expression of the full-length mutant p53 protein in cancer cells. In addition to the loss of tumor suppression activity, p53 cancer mutants gain new oncogenic activities to promote tumorigenesis and drug resistance. Recent studies have identified a novel gain-of-function of p53 cancer mutants in inducing genetic instability by inactivating critical tumor suppressors such as ATM. Genetic instability is a common mechanism by which cancer cells efficiently accumulate genetic mutations to promote their growth, survival and metastatic potential. Therefore, this gain-of-function of p53 cancer mutants could play important roles in tumorigenesis and drug resistance of various types of human cancers. In addition, because many cancer therapies such as radiation therapy suppress or kill cancer cells by activating ATM-dependent responses to DNA double-stranded break damage, elucidation of this gain-of-function of p53 cancer mutants will have important implications on cancer therapy.     

p73 can suppress the proliferation of cells that express mutant p53

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. p73, a member of the p53 family, has been found to exhibit activity similar to that of p53, including the ability to induce growth arrest and apoptosis. p53 and p73 have a high percentage of similarity at several domains, including the DNA binding domain. This domain in p53 is the location of missense mutations in many human cancers. Mutant p53, which cannot suppress cell proliferation, has been found to have a dominant-negative activity that inactivates wild-type p53. To determine the effects of mutant p53 on wild-type p73, we have established cell lines expressing both mutant p53 and wild-type p73 in a dual-inducible system. This system expresses mutant p53 in a tetracycline-repressible system and p73beta in an ecdysone-inducible system in a p53-null lung carcinoma parental cell line. We have found that wild-type p73beta, in the presence of mutant p53, retains the ability to transactivate p21 and suppresses cell growth through induction of both cell cycle arrest and apoptosis. In addition, in cell lines expressing wild-type p53 and wild-type p73beta, we have found that these proteins cooperate to additively transactivate p21 and suppress cell proliferation.     

Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome

The p53 guardian of the genome is inactivated in the majority of cancers, mostly through missense mutations that cause single residue changes in the DNA binding core domain of the protein. Not only do such mutations result in the abrogation of wild-type p53 activity, but the expressed p53 mutant proteins also tend to gain oncogenic functions, such as interference with wild-type p53-independent apoptosis. Because p53 mutants are highly expressed in cancer cells and not in normal cells, their reactivation to wild-type p53 function may eliminate the cancer by apoptosis or another p53-dependent mechanism. Several studies that embarked on this quest for reactivation have succeeded in restoring wildtype p53 activity to several p53 mutants. However, mutants with more extensive structural changes in the DNA binding core domain may be refractory to reactivation to the wild-type p53 phenotype. Therefore, understanding the structure and functions of oncogenic p53 mutants may lead to more potent reactivation modalities or to the ability to eliminate mutant p53 gain of function.     

Reactivation of p53 mutants by prima-1 [corrected] in thyroid cancer cells

Most undifferentiated thyroid carcinomas express p53 mutants and thereafter, are very resistant to chemotherapy. p53 reactivation and induction of massive apoptosis (Prima-1) is a compound restoring the tumor-suppressor activity of p53 mutants. We tested the effect of Prima-1 in thyroid cancer cells harboring p53 mutations. Increasing doses of Prima-1 reduced viability of thyroid cancer cells at a variable extent (range 20-80%). Prima-1 up-regulated p53 target genes (p21(WAF1) , BCL2-associated X protein (Bax), and murine double minute 2 (MDM2)), in BC-PAP and Hth-74 cells (expressing D259Y/K286E and K286E p53 mutants) but had no effect in SW1736 (p53 null) and TPC-1 (expressing wild-type p53) thyroid cancer cells. Prima-1 also increased the cytotoxic effects of either doxorubicin or cisplatin in thyroid cancer cells, including the chemo-resistant 8305C, Hth-74 and BC-PAP cells. Moreover, real-time PCR and Western blot indicated that Prima-1 increases the mRNA of thyroid-specific differentiation markers in thyroid cancer cells. Fluorescence-activated cell sorting analysis revealed that Prima-1 effect on thyroid cancer cells occurs via the enhancement of both cell cycle arrest and apoptosis. Small interfering RNA experiments indicated that Prima-1 effect is mediated by p53 mutants but not by the p53 paralog p73. Moreover, in C-643 thyroid cancer cells, forced to ectopically express wild-type p53, Prima-1 prevented the dominant negative effect of double K248Q/K286E p53 mutant. Finally, co-IP experiments indicated that in Hth-74 cells Prima-1 prevents the ability of p53 mutants to sequestrate the p53 paralog TAp73. These in vitro studies imply that p53 mutant reactivation by small compounds may become a novel anticancer therapy in undifferentiated thyroid carcinomas.     

Novel p53 mutants selected in BRCA-associated tumours which dissociate transformation suppression from other wild-type p53 functions

Inheritance of germ-line mutant alleles of BRCA1 and BRCA2 confers a markedly increased risk of breast cancer and we have previously reported a higher incidence of p53 mutations in these tumours than in grade matched sporadic tumours. We have now characterized these p53 mutants. The results of these studies identify a novel class of p53 mutants previously undescribed in human cancer yet with multiple occurrences in BRCA-associated tumours which retain a profile of p53-dependent activities in terms of transactivation, growth suppression and apoptosis induction which is close or equal to wild-type. However, these mutants fail to suppress transformation and exhibit gain of function transforming activity in rat embryo fibroblasts. These mutants therefore fall into a novel category of p53 mutants which dissociate transformation suppression from other wild-type functions. The rarity of these mutants in human cancer and their multiple occurrence in BRCA-associated breast tumours suggests that these novel p53 mutants are selected during malignant progression in the unique genetic background of BRCA1- and BRCA2-associated tumours.     

Suppression of glucosylceramide synthase restores p53-dependent apoptosis in mutant p53 cancer cells

Tumor suppressor p53 plays an essential role in protecting cells from malignant transformation by inducing cell-cycle arrest and apoptosis. Mutant p53 that is detected in more than 50% of cases of cancers loses its role in suppression of tumors but gains in oncogenic function. Strategies to convert mutant p53 into wild-type p53 have been suggested for cancer prevention and treatment, but they face a variety of challenges. Here, we report an alternative approach that involves suppression of glucosylceramide synthase (GCS), an enzyme that glycosylates ceramide and blunts its proapoptotic activity in cancer cells. Human ovarian cancer cells expressing mutant p53 displayed resistance to apoptosis induced by DNA damage. We found that GCS silencing sensitized these mutant p53 cells to doxorubicin but did not affect the sensitivity of cells with wild-type p53. GCS silencing increased the levels of phosphorylated p53 and p53-responsive genes, including p21(Waf1/Cip1), Bax, and Puma, consistent with a redirection of the mutant p53 cells to apoptosis. Reactivated p53-dependent apoptosis was similarly verified in p53-mutant tumors where GCS was silenced. Inhibition of ceramide synthase with fumonisin B1 prevented p53 reactivation induced by GCS silencing, whereas addition of exogenous C6-ceramide reactivated p53 function in p53-mutant cells. Our findings indicate that restoring active ceramide to cells can resuscitate wild-type p53 function in p53-mutant cells, offering preclinical support for a novel type of mechanism-based therapy in the many human cancers harboring p53 mutations.     

Chaperone-dependent stabilization and degradation of p53 mutants

p53 missense mutant proteins commonly show increased stability compared to wild-type p53, which is thought to depend largely on the inability of mutant p53 to induce the ubiquitin ligase MDM2. However, recent work using mouse models has shown that the accumulation of mutant p53 occurs only in tumour cells, indicating that stabilization requires additional factors. To clarify the stabilization of p53 mutants in tumours, we analysed factors that affect their folding and degradation. Although all missense mutants that we studied are more stable than wild-type p53, the levels correlate with individual structural characteristics, which may be reflected in different gain-of-function properties. In the absence of Hsp90 activity, the less stable unfolded p53 mutants preferentially associate in a complex with Hsp70 and CHIP (carboxy terminus of Hsp70-interacting protein), and we show that CHIP is responsible for ubiquitination and degradation of these mutants. The demonstration of a complex interplay between Hsp90, Hsp70 and CHIP that regulate the stability of different p53 mutant proteins improves our understanding of the pro-tumorigenic effects of increased Hsp90 activity during multi-stage carcinogenesis. Understanding the roles of Hsp90, Hsp70 and CHIP in cancers may also provide an important avenue through which to target p53 to enhance treatment of human cancers.     

Inactive full-length p53 mutants lacking dominant wild-type p53 inhibition highlight loss of heterozygosity as an important aspect of p53 status in human cancers

Over 1000 different mutants of the tumor suppressor protein p53 with one amino acid change in the core domain have been reported in human cancers. In mouse knock-in models, two frequent mutants displayed loss of wild-type (wt) p53 function, inhibition of wt p53 and wt p53-independent gain of function. The remaining mutants have been systematically characterized for loss of wt p53 function, but not other phenotypes. We report the concomitant assessment of loss of function and interference with wt p53 using URA3-based p53 yeast and confirmatory mammalian assays. We studied 76 mutants representing 54% of over 15 000 reported missense core domain mutations. The majority showed the expected complete loss of wt p53 function and dominant p53 inhibition. A few infrequent p53 mutants had wt p53-like activity. Remarkably, one-third showed no interference with wt p53 despite loss of wt p53 function at 37 degrees C. Half of this group consisted of temperature-sensitive p53 mutants, but the other half was surprisingly made up of mutants with complete loss of wt p53 function. Our findings illustrate the diverse behavior of p53 mutants and mechanisms of malignant transformation by p53 mutants. The identification of full-length p53 mutants without dominant inhibition of wt p53 highlights the importance of determining the status of the wt p53 allele in human cancers, in particular in the context of clinical studies. In the case of p53 mutants with no or weak dominant p53 inhibition, presence of the wt allele may indicate a good prognosis cancer, whereas loss of heterozygosity may spell an aggressive, therapy-resistant cancer.     

Development of antibodies against p53 in lung cancer patients appears to be dependent on the type of p53 mutation.

Using immunoblotting techniques we studied the sera from small cell lung cancer and non-small cell lung cancer patients for antibodies directed against p53. We have also characterized the majority of these patients' tumors for p53 mutations. In the sera of 13% of the patients (4 of 40 small cell lung cancer and 2 of 6 non-small cell lung cancer) we found antibodies specific for the p53 tumor suppressor gene product. All of the antibody-positive patients tested had p53 missense mutations and expressed detectable p53 antigen in their tumor cell lines. No anti-p53 antibodies were detected in sera from patients whose tumor had p53 stop, splice/stop, splice, or frameshift mutations (n = 10). Thus, while we find that the ability of lung cancer patients to develop anti-p53 antibodies is correlated with the type of p53 mutation, many patients have tumors with missense p53 mutations and did not develop anti-p53 antibodies. The presence of p53 antibodies was not correlated to stage, prior treatment, sex, or survival. None of these lung cancer patient sera had measurable amounts of p53 antigen. By immunoblotting all six anti-p53 antisera we were able to detect a variety of mutant p53 proteins (including those from antibody-negative patients) and detected wild-type p53 protein. The development of anti-p53 antibodies represents an interesting model system for studying immune responses in cancer patients against mutant oncogene products.     

Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation.

We investigated the immunocytochemical staining and immunoblotting characteristics of 33 different p53 mutant proteins identified in lung cancer cell lines (18 small-cell lung cancer and 15 non-small-cell lung cancer) using monoclonal antibodies pAbs 240, 421 and 1801. The p53 mutants studied were representative of those found in lung cancer and included three deletions, four nonsense, seven splicing and 19 missense lesions. Control cell lines included six B-lymphoblastoid cell lines and two lung cancer cell lines without p53 mutations. Immunocytochemistry demonstrated 16 cell lines (48%) with definite overexpression of p53 protein (the high-expresser group of mutants), while in the remainder of cases either no p53 expression or low levels of p53 protein expression were found (the low-expresser group of mutants). The type of p53 mutation correlated with the expresser group. High expressers all had p53 missense mutations in exons 5-8, and immunocytochemistry identified 16/17 (94%) of these mutants. Several classes of p53 mutations occur in the low-expresser groups: deletions, splicing mutants, nonsense mutants and missense mutations outside of exons 5-8 all resulted in very low or undetectable levels of p53 protein. We conclude that there are low- and high-expression groups of p53 mutants in lung cancer and that the detection of protein expression in tumor cells by immunocytochemistry and immunoblotting is dependent upon the type of mutation of the p53 tumor-suppressor gene.