Expression of P53 and isoforms in bening and malignant lesions of the head and neck.

BACKGROUND: P53, a crucial suppressor of tumor formation, generates multiple isoforms, whose role in disease is still being defined. METHODS: By immunohistochemistry, we studied the expression of P53 protein and relative isoforms in benign papillomas (PA, n=9), inverted papilloma (IPA, n=10) and squamous cell carcinomas (SCC, n=21). RESULTS: In all lesions, P53 isoforms were significantly more expressed than P53. Immunoexpression of P53 matched with P53 isoforms in IPA as well as in SCC. Simultaneous immunoexpression of P53 and related isoforms was double in SCC compared to IPA (10% vs 24%), while expression of P53 isoforms was strongly reduced (70% vs 43%). IPA showed the highest percentage of both reactive cases and immunostained cells expressing P53 isoforms. CONCLUSIONS: We found the higher expression of P53 isoforms in IPA and SCC compared to PA, suggesting their role in local aggressiveness and malignant proliferation in head-neck lesions.

The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer.

Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer. We show that 3-OST3A is epigenetically repressed in all breast cancer cell lines of a panel representative of distinct molecular subgroups, except in human epidermal growth factor receptor 2-positive (HER2+) sloan-kettering breast cancer (SKBR3) cells. Epigenetic mechanisms involved both DNA methylation and histone modifications, producing different repressive chromatin environments depending on the cell molecular signature. Gain and loss of function experiments by cDNA and siRNA transfection revealed profound effects of 3-OST3A expression on cell behavior including apoptosis, proliferation, response to trastuzumab in vitro and tumor growth in xenografted mice. 3-OST3A exerted dual activities acting as tumor-suppressor in lumA-michigan cancer foundation (MCF)-7 and triple negative-MD Anderson (MDA) metastatic breast (MB)-231 cells, or as an oncogenic factor in HER2+-SKBR3 cells. Mechanistically, fluorescence-resonance energy transfer-fluorescence-lifetime imaging microscopy experiments indicated that the effects of 3-OST3A in MCF-7 cells were mediated by altered interactions between HS and fibroblast growth factor-7 (FGF-7). Further, this interplay between HS and FGF-7 modulated downstream ERK, AKT and p38 cascades, suggesting that altering 3-O-sulfation affects FGFR2IIIb-mediated signaling. Corroborating our cellular data, a clinical study conducted in a cohort of breast cancer patients uncovered that, in HER2+ patients, high level expression of 3-OST3A in tumors was associated with reduced relapse-free survival. Our findings define 3-OST3A as a novel regulator of breast cancer pathogenicity, displaying tumor-suppressive or oncogenic activities in a cell- and tumor-dependent context, and demonstrate the clinical value of the HS-O-sulfotransferase 3-OST3A as a prognostic marker in HER2+ patients.

Modulation of p53ß and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response.

In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53ß and p53γ through alternative splicing of exons 9ß and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53ß and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9ß/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9ß/9γ, we demonstrate that cell growth can be driven by modulating p53ß and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53ß and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53ß enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53ß and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53ß and p53γ promote apoptosis in MCF7 cells, p53ß and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53ß and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53ß and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers.

p53 isoforms change p53 paradigm.

Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

The p53 isoform, Δ133p53α, stimulates angiogenesis and tumour progression.

The tumour suppressor p53, involved in DNA repair, cell cycle arrest and apoptosis, also inhibits blood vessel formation, that is, angiogenesis, a process strongly contributing to tumour development. The p53 gene expresses 12 different proteins (isoforms), including TAp53 (p53 (or p53α), p53ß and p53γ) and Δ133p53 isoforms (Δ133p53α, Δ133p53ß and Δ133p53γ). The Δ133p53α isoform was shown to modulate p53 transcriptional activity and is overexpressed in various human tumours. However, its role in tumour progression is still unexplored. In the present study, we examined the involvement of Δ133p53 isoforms in tumoural angiogenesis and tumour growth in the highly angiogenic human glioblastoma U87. Our data show that conditioned media from U87 cells depleted for Δ133p53 isoforms block endothelial cell migration and tubulogenesis without affecting endothelial cell proliferation in vitro. The Δ133p53 depletion in U2OS osteosarcoma cells resulted in a similar angiogenesis blockade. Furthermore, using conditioned media from U87 cells ectopically expressing each Δ133p53 isoform, we determined that Δ133p53α and Δ133p53γ but not Δ133p53ß, stimulate angiogenesis. Our in vivo data using the chicken chorio-allantoic membrane and mice xenografts establish that angiogenesis and growth of glioblastoma U87 tumours are inhibited upon depletion of Δ133p53 isoforms. By TaqMan low-density array, we show that alteration of expression ratio of Δ133p53 and TAp53 isoforms differentially regulates angiogenic gene expression with Δ133p53 isoforms inducing pro-angiogenic gene expression and repressing anti-angiogenic gene expression.

Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation.

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

p53 is activated in response to disruption of the pre-mRNA splicing machinery.

In this study, we show that interfering with the splicing machinery results in activation of the tumour-suppressor p53. The spliceosome was targeted by small interfering RNA-mediated knockdown of proteins associated with different small nuclear ribonucleoprotein complexes and by using the small-molecule splicing modulator TG003. These interventions cause: the accumulation of p53, an increase in p53 transcriptional activity and can result in p53-dependent G(1) cell cycle arrest. Mdm2 and MdmX are two key repressors of p53. We show that a decrease in MdmX protein level contributes to p53 activation in response to targeting the spliceosome. Interfering with the spliceosome also causes an increase in the rate of degradation of Mdm2. Alterations in splicing are linked with tumour development. There are frequently global changes in splicing in cancer. Our study suggests that p53 activation could participate in protection against potential tumour-promoting defects in the spliceosome. A number of known p53-activating agents affect the splicing machinery and this could contribute to their ability to upregulate p53. Preclinical studies indicate that tumours can be more sensitive than normal cells to small-molecule spliceosome inhibitors. Activation of p53 could influence the selective anti-tumour activity of this therapeutic approach.

Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia.

The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms ß and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication.

Diverse p63 and p73 isoforms regulate Δ133p53 expression through modulation of the internal TP53 promoter activity.

In response to stress, p53 binds and transactivates the internal TP53 promoter, thus regulating the expression of its own isoform, Δ133p53α. Here, we report that, in addition to p53, at least four p63/p73 isoforms regulate Δ133p53 expression at transcriptional level: p63ß, ΔNp63α, ΔNp63ß and ΔNp73γ. This regulation occurs through direct DNA-binding to the internal TP53 promoter as demonstrated by chromatin immunoprecipitation and the use of DNA-binding mutant p63. The promoter regions involved in the p63/p73-mediated transactivation were identified using deleted, mutant and polymorphic luciferase reporter constructs. In addition, we observed that transient expression of p53 family members modulates endogenous Δ133p53α expression at both mRNA and protein levels. We also report concomitant variation of p63 and Δ133p53 expression during keratinocyte differentiation of HaCat cells and induced pluripotent stem cells derived from mutated p63 ectodermal dysplasia patients. Finally, proliferation assays indicated that Δ133p53α isoform regulates the anti-proliferative activities of p63ß, ΔNp63α, ΔNp63ß and ΔNp73γ. Overall, this study shows a strong interplay between p53, p63 and p73 isoforms to orchestrate cell fate outcome.

Biological functions of p53 isoforms through evolution: lessons from animal and cellular models.

The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of 'p53 isoforms', including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.