Abrogation of the transactivation activity of p53 by BCCIP down-regulation

The tumor suppression function of p53 is mostly conferred by its transactivation activity, which is inactivated by p53 mutations in approximately 50% of human cancers. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. Identifying the mechanisms by which wild type p53 transactivation activity can be abrogated may provide insights into the molecular etiology of cancers harboring wild type p53. In this report, we show that BCCIP, a BRCA2 and CDKN1A-interacting protein, is required for the transactivation activity of wild type p53. In p53 wild type cells, BCCIP knock down by RNA interference diminishes the transactivation activity of p53 without reducing the p53 protein level, inhibits the binding of p53 to the promoters of p53 target genes p21 and HDM2, and reduces the tetrameric formation of p53. These data demonstrate a critical role of BCCIP in maintaining the transactivation activity of wild type p53 and further suggest down-regulation of BCCIP as a novel mechanism to impair the p53 function in cells harboring wild type p53.     

Noncanonical DNA motifs as transactivation targets by wild type and mutant p53

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0-13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0-13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi-in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical (1/2)-(a single decamer) and (3/4)-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of (1/2)- and (3/4)-site REs greatly expands the p53 master regulatory network.     

The central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated suppression of E2F and cell growth

Retinoblastoma (Rb) protein is a paradigm of tumor suppressors. Inactivation of Rb plays a critical role in the development of human malignancies. MDM2, an oncogene frequently found amplified and overexpressed in a variety of human tumors and cancers, directly interacts and inhibits the p53 tumor suppressor protein. In addition, MDM2 has been shown to stimulate E2F transactivation activity and promote S-phase entry independent of p53, yet the mechanism of which is still not fully understood. In this study, we demonstrate that MDM2 specifically binds to Rb C-pocket and that the central acidic domain of MDM2 is essential for Rb interaction. In addition, we show that overexpression of MDM2 reduces Rb-E2F complexes in vivo. Moreover, the ectopic expression of the wild type MDM2, but not mutant MDM2 defective in Rb interaction, stimulates E2F transactivation activity and inhibits Rb growth suppression function. Taken together, these results suggest that MDM2-mediated inhibition of Rb likely contributes to MDM2 oncogenic activity.     

Activation of c-myc Gene Expression by Tumor-Derived p53 Mutants Requires a Discrete C-Terminal Domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3′ end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

DNA-binding and transactivation activities are essential for TAp63 protein degradation

The p53-related p63 gene encodes six isoforms with differing N and C termini. TAp63 isoforms possess a transactivation domain at the N terminus and are able to transactivate a set of genes, including some targets downstream of p53. Accumulating evidence indicates that TAp63 plays an important role in regulation of cell proliferation, differentiation, and apoptosis, whereas transactivation-inert deltaNp63 functions to inhibit p63 and other p53 family members. Mutations in the p63 gene that abolish p63 DNA-binding and transactivation activities cause human diseases, including ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndrome. In this study, we show that mutant p63 proteins with a single amino acid substitution found in EEC syndrome are DNA binding deficient, transactivation inert, and highly stable. We demonstrate that TAp63 protein expression is tightly controlled by its specific DNA-binding and transactivation activities and that p63 is degraded in a proteasome-dependent, MDM2-independent pathway. In addition, the N-terminal transactivation domain of p63 is indispensable for its protein degradation. Furthermore, the wild-type TAp63gamma can act in trans to promote degradation of mutant TAp63gamma defective in DNA binding, and the TA domain deletion mutant of TAp63gamma inhibits transactivation activity and stabilizes the wild-type TAp63 protein. Taken together, these data suggest a feedback loop for p63 regulation, analogous to the p53-MDM2 feedback loop.     

The transforming and suppressor functions of p53 alleles: effects of mutations that disrupt phosphorylation, oligomerization and nuclear translocation.

Mutant p53 alleles that have a recessive phenotype in human tumors can, in cooperation with an activated H-ras gene, transform rat embryo fibroblasts (REFs). Mutant p53 proteins differ from wild type, and from each other in conformation, localization and transforming potential. Missense mutations in codons 143, 175 and 275 confer strong transforming potential. A serine 135 p53 mutant has an intermediate transforming potential, while the histidine codon 273 allele transforms weakly, if at all. In contrast to the wild type p53 gene, mutant p53 alleles with strong transforming ability cannot suppress the transformation of REFs by other oncogenes. The His273 allele retains partial suppressor function in this assay. The relevance of p53 oligomerization, phosphorylation and nuclear translocation to the transforming potential of mutant p53 and to wild type p53 suppressor function were examined. The inability of mutant p53 polypeptides to form homodimers correlates with loss of transforming function. Monomeric variants of wild type p53 protein, however, retain the ability to suppress focus formation. Phosphorylation of serine residues 315 and 392 is not required for the transforming function of mutant p53, nor is serine 315 required for suppressor function when these alleles are constitutively expressed in REF assays. Nuclear translocation-defective mutant and wild type p53 proteins retain transforming and suppressor function in REF assays.     

p53 regulates epithelial–mesenchymal transition induced by transforming growth factor β

Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial–mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor β (TGFβ) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFβ‐induced EMT in a well‐established mammary epithelial cell model. We found that diploid NMuMG mammary cells bi‐allelically express a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFβ. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFβ‐driven EMT response. Under serum‐free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFβ. J. Cell. Physiol. 228: 801–813, 2013. © 2012 Wiley Periodicals, Inc.     

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.     

WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.     

Wild type and mutant p53 differentially regulate the gene expression of human collagenase-3 (hMMP-13)

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions including arthritis and cancer metastasis. Recently we have shown for the first time that the human MMP-1 gene is a p53 target gene subject to repression by wild type p53 (Sun, Y., Sun, Y. I., Wenger, L., Rutter, J. L., Brinckerhoff, C. E., and Cheung, H. S. (1999) J. Biol. Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down-regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter- and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis.