7161

TP73

P73;tumor protein p73;TP73;tumor protein p73

p53-like transcription factor|p53-related protein

1p36.3

protein-coding

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic hit or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.

The p53 family includes three members that share significant sequence homology, yet exhibit fundamentally different functions in tumorigenesis. Whereas p53 displays all characteristics of a classical tumor suppressor, its homologues p63 and p73 do not. We have previously shown, that NH(2)-terminally truncated isoforms of p73 (Delta TA-p73), which act as dominant-negative inhibitors of p53 are frequently overexpressed in cancer cells. Here we provide evidence that Delta TA-p73 isoforms also affect the retinoblastoma protein (RB) tumor suppressor pathway independent of p53. Delta TA-p73 isoforms inactivate RB by increased phosphorylation, resulting in enhanced E2F activity and proliferation of fibroblasts. By inactivating the two major tumor suppressor pathways in human cells they act functionally analogous to several viral oncoproteins. These findings provide an explanation for the fundamentally different functions of p53 and p73 in tumorigenesis.

p73 is a p53 homolog, as they are similar structurally and functionally. Unlike p53, p73 is not inactivated by the products of viral oncogenes such as SV40 T antigen and human papilloma virus E6. Here we show that the product of adenoviral oncogene E1A inhibits the transcriptional activation by both p73alpha and p73beta. Electrophoretic mobility shift assays revealed that E1A does not inhibit the sequence-specific DNA binding by p73. Transcriptional activation by a fusion protein containing the Gal4 DNA-binding domain and either of the activation domains of p73 was inhibited by wild-type (WT) E1A, but not by the N-terminal deletion mutant E1A(Delta2-36). E1A(Delta2-36), which does not bind to the p300/CBP family of coactivators, failed to inhibit p73-mediated transcription, whereas E1A(DeltaCR2), a deletion mutant that does not bind to the pRb family of proteins, inhibited p73-mediated transcription as efficiently as WT E1A. Consistent with these observations, growth arrest induced by p73 expressed from a recombinant adenovirus was abrogated by WT E1A, which correlated with inhibition of p73-mediated induction of p21(WAF1/CIP1) by E1A. However, p73 was able to induce p21(WAF1/CIP1) and to mediate growth arrest in the presence of E1A(Delta2-36). Furthermore, the expression of either wild-type E1A or E1A(Delta2-36) resulted in the stabilization of endogenous p73. However, p73 stabilized in response to the expression of E1A(Delta2-36), but not WT E1A, was able to activate the expression of p21(WAF1/CIP1). These results suggest that the transcriptional activation function of p73 is specifically targeted by E1A through a mechanism involving p300/CBP proteins during the process of transformation and that p73 may have a role to play as a tumor suppressor.

The p73 protein is a member of the p53 family and, like p53, can induce cell-cycle arrest and apoptosis in response to DNA damage. Because the loss of p53 function is responsible for the progression of well-differentiated thyroid cancer to more aggressive phenotypes, we hypothesized that p73 might also be involved in thyroid carcinogenesis. We find that normal thyrocites do not express p73, whereas most thyroid malignancies are positive for p73 expression. However, the p73 protein of thyroid cancer cells is unresponsive to DNA-damaging agents, failing to elicit a block of the cell cycle or an apoptotic response. Notably, overexpression of transcriptionally active p73 in thyroid cancer lines can arrest the cell cycle but is still unable to induce cell death. The loss of p73 biological activity in neoplastic thyroid cells is partly explained by its interaction with transcriptionally inactive variants of p73 (DeltaNp73) and with mutant p53. Our findings suggest that the functional impairment of p73 could be involved in the development of thyroid malignancies, defining p73 as a potential therapeutic target for thyroid cancer.

PURPOSE: To characterize the expression profile of p73 in human normal tissues by immunohistochemistry (IHC) and to analyze the correlation between p73 expression and bladder cancer progression. EXPERIMENTAL DESIGN: CJDp73 was characterized for p73alpha detection in Western blot and IHC through its application to isoform-transfected 293 cells. Normal tissues were analyzed by IHC with the CJDp73 antiserum. Transitional cell carcinoma (TCC)-derived cell lines were subjected to reverse transcription-PCR and Western blot. TCC tissue microarrays were analyzed for p73alpha expression by IHC, and the results were statistically analyzed. RESULTS: p73 immunostaining was nuclear and restricted to epithelial cells of certain organs such as squamous epithelium of the epidermis and transitional epithelium of the bladder. The expression was also observed in certain specialized glandular epithelia such as acinar cells of breast and parotid gland. Four of seven TCC-derived cell lines had low to undetectable p73alpha protein levels. We found undetectable or low p73alpha expression in 104 of 154 (68%) TCC cases, this phenotype being more frequently observed in invasive tumors when compared with superficial lesions. This association was statistically significant (P < 0.0001). We also observed a significant association between p53, p63, and p73alpha alterations with bladder cancer progression (P < 0.0001). CONCLUSIONS: p73alpha plays a tumor suppressor role in bladder cancer, and its inactivation occurs through an epigenetic mechanism, most probably involving protein degradation.

A novel gene, p73, encoding a protein with significant homology to p53 and showing functional similarities to p53, was identified at chromosome 1p36, at which tumor suppressor gene of hepatocellular carcinoma (HCC) is supposed to be. Involvement of p73 in hepatocarcinogenesis is controversial and clinical value of p73 alterations remains obscure. We investigated allelic status of p73 in 63 patients with HCC. Loss of heterozygosity (LOH) in p73 was analyzed by PCR-RFLP analysis. The results were compared with LOH on chromosome 1p surrounding p73 locus, mutations of p53 and p73, and clinicopathologic characteristics. LOH on p73 was observed in 33% of informative tumors. LOH in p73 was not always observed between the regions with LOH on chromosome 1p examined despite the significant association of LOH in p73 with LOH on chromosome 1p. No mutations were detected in p73. Tumors with LOH in p73 were more frequently detected in liver without cirrhosis than that with cirrhosis. There was no significant statistic association between the presence of LOH in p73 and six different clinicopathologic characteristics such as age, sex, histological type, T stage, tumor diameter, and virus status. Disease-free survival rates of the patients with LOH in p73 were significantly poorer than those without LOH in p73. Multivariate analysis indicated that presence of LOH in p73 was independent prognostic factor in patients with HCC. These findings suggested that p73 might play some role in tumor progression of HCC even though p73 should not be considered a candidate gene on chromosome 1p of HCC and does not function as a tumor suppressor gene like p53. Identifying the patients with LOH of p73 in tumors could be useful to predict early recurrence and to stratify the patients who need adjuvant therapy after operation.

The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. Wwox protein contains two WW domains that are generally known to mediate protein-protein interaction. Here we show that Wwox physically interacts via its first WW domain with the p53 homolog, p73. The tyrosine kinase, Src, phosphorylates Wwox at tyrosine 33 in the first WW domain and enhances its binding to p73. Our results further demonstrate that Wwox expression triggers redistribution of nuclear p73 to the cytoplasm and, hence, suppresses its transcriptional activity. In addition, we show that cytoplasmic p73 contributes to the proapoptotic activity of Wwox. Our findings reveal a functional cross-talk between p73 and Wwox tumor suppressor protein.

Elucidating the mechanisms that regulate the life versus death of mammalian neurons is important not only for our understanding of the normal biology of the nervous system but also for our efforts to devise approaches to maintain neuronal survival in the face of traumatic injury or neurodegenerative disorders. Here, we review the emerging evidence that a key survival/death checkpoint in both peripheral and central neurons involves the p53 tumor suppressor and its newly discovered family members, p73 and p63. The full-length isoforms of these proteins function as proapoptotic proteins, whereas naturally occurring N-terminal truncated variants of p73 and p63 act as prosurvival proteins, at least partially by antagonizing the full-length family members. The authors propose that together, these isoforms comprise an upstream rheostat that sums different environmental cues to ultimately determine neuronal survival during development, during neuronal maintenance in adult animals, and even following traumatic injury.

Reactivation of telomerase is a feature in many cancer cells. Telomerase activation inhibits telomere shortening, thereby preventing cell cycle arrest and apoptosis activated by shortened telomeres or chromosomal rearrangements. The tumor-suppressor gene product, p53, was previously shown to transcriptionally suppress the activation of the catalytic subunit of telomerase (hTERT). Here we have evaluated the role of p73 in hTERT regulation. We found that ectoptic expression of p73beta, in contrast to p73alpha or p53, in p53 null H1299 cells does not lead to suppression of hTERT transcription. However co-expression of p73alpha or p73beta together with p53 abolished p53-mediated hTERT suppression. This phenomenon was found to be dependent on the DNA binding ability of p73. We also show that p53-mediated suppression of hTERT transcription requires a minimum threshold level of p53, and p73 abrogates p53-mediated suppression by reducing p53 levels through the activation of HDM2. Moreover, p53-mediated hTERT suppression was not relieved by p73beta in cells depleted of HDM2 through small interfering RNA-mediated gene silencing. In addition, knockdown of HDM2 in MCF7 cells, which express moderately high levels of p73 and p53, resulted in the reduction of endogenous hTERT levels. Finally, knockdown of p73 in MCF7 cells resulted in increased p53 protein levels and a concomitant decrease in hTERT levels. Together, our data indicate a plausible way by which p73, through HDM2, can oppose p53 tumor suppressor function, thereby possibly contributing to tumorigenesis.

RASSF1A is a tumor suppressor gene that is epigenetically silenced in a wide variety of sporadic human malignancies. expression of alternative RASSF1 isoforms cannot substitute for RASSF1A-promoted cell-cycle arrest and apoptosis. Apoptosis can be driven by either activating Bax or by activation of MST kinases. The Raf1 proto-oncogene binds to MST2, preventing its activation and proapoptotic signaling. Here we show that key steps in RASSF1A-induced apoptosis are the disruption of the inhibitory Raf1-MST2 complex by RASSF1A and the concomitant enhancement of MST2 interaction with its substrate, LATS1. Subsequently, RASSF1A-activated LATS1 phosphorylates and releases the transcriptional regulator YAP1, allowing YAP1 to translocate to the nucleus and associate with p73, resulting in transcription of the proapoptotic target gene puma. Our results describe an MST2-dependent effector pathway for RASSF1A proapoptotic signaling and indicate that silencing of RASSF1A in tumors removes a proapoptotic signal emanating from p73.

The Trp53 gene family member Trp73 encodes two major groups of protein isoforms, TAp73 and DeltaNp73, with opposing pro- and anti-apoptotic functions; consequently, their relative ratio regulates cell fate. However, the precise roles of p73 isoforms in cellular events such as tumor initiation, embryonic development, and cell death remain unclear. To determine which aspects of p73 function are attributable to the TAp73 isoforms, we generated and characterized mice in which exons encoding the TAp73 isoforms were specifically deleted to create a TAp73-deficient (TAp73(-/-)) mouse. Here we show that mice specifically lacking in TAp73 isoforms develop a phenotype intermediate between the phenotypes of Trp73(-/-) and Trp53(-/-) mice with respect to incidence of spontaneous and carcinogen-induced tumors, infertility, and aging, as well as hippocampal dysgenesis. In addition, cells from TAp73(-/-) mice exhibit genomic instability associated with enhanced aneuploidy, which may account for the increased incidence of spontaneous tumors observed in these mutants. Hence, TAp73 isoforms exert tumor-suppressive functions and indicate an emerging role for Trp73 in the maintenance of genomic stability.

Netrins and their receptors deleted in colon cancer (DCC), neogenin, UNC5, and integrins are involved in axon guidance, epithelial morphogenesis, vascular pattering, cancer cell survival, invasion, tumor angiogenesis, and metastasis. Here, we considered the possible contribution of the p53-related apoptosis mediators p63 and p73 in the mechanisms underlying the antagonism between netrin-1 and DCC at the cell death control. We have showed that ectopic expression and external addition of netrin-1 in HeLa and HEK-293 cells with inactive p53 lead to impaired cell viability and induction of apoptosis. These responses were associated with up-regulation of the proapoptotic protein TAp73alpha, decreased Bcl-2/Bax ratio, and caspase-3 cleavage, with no change in protein levels of the antiapoptotic NH(2)-terminal-truncated DeltaNp73alpha isoform, p73 adapter Yap-1 and p73 E3 ubiquitin ligase Itch, and p63, as well as the transcripts encoding p63, TAp73alpha, and DeltaNp73alpha. However, the proteasome inhibitor MG132 potentiated, while DCC counteracted, netrin-1-induced TAp73alpha. Consistently, netrin-1 expression correlated with stabilization of the TAp73alpha protein and lower levels of TAp73alpha ubiquitination that was conversely enhanced by DCC, in a netrin-dependent manner. Our data indicate that netrin-1 selectively up-regulates TAp73alpha by preventing its ubiquitination and degradation. Targeted repression of p73alpha by shRNA reversed TAp73alpha and the apoptosis induced by netrin-1, and exacerbated the growth of HeLa tumor xenografts. Apoptosis induced by cisplatin was markedly enhanced in netrin-1 or DCC-expressing cells. Collectively, our data reveal that the transcriptionally active TAp73alpha tumor suppressor is implicated in the apoptosis induced by netrin-1 in a p53-independent and DCC/ubiquitin-proteasome dependent manner.

Induction of apoptosis by the tumor suppressor p53 is known to protect from Myc-driven lymphomagenesis. The p53 family member p73 is also a proapoptotic protein, which is activated in response to oncogenes like Myc. Here, we have investigated whether p73 provides a similar protection from Myc-driven lymphomas as p53. Confirming previous studies, the inactivation of a single p53 allele (p53+/-) strongly reduced the median survival of Emu-Myc transgenic mice from 103 to 39 days and was invariably associated with a loss of the wild-type p53 allele. In contrast, mutational inactivation of a p73 allele (p73+/-) reduced the median survival by only 12 days. Lymphomas that developed in the p73+/- background showed no loss of heterozygosity (LOH). Furthermore, gene expression profiling of p73+/+, p73+/- and p73-/- lymphomas indicated that p73+/- lymphomas retained p73 transcriptional activity. Subtle gene expression differences between p73+/+ and p73+/- lymphomas, however, suggest a haploinsufficient phenotype on some p73 target genes. This might help to explain why p73+/- animals succumbed to disease slightly earlier than their p73+/+ littermates (log-rank test p<0.0395) and why p73 often shows monoallelic inactivation in human lymphomas. Together these data demonstrate that in Myc-driven lymphomagenesis p73 has weak tumor suppressor activity compared with p53.

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.

Polo-like kinase 1 (Plk1) is overexpressed in tumor tissues and its expression level is tightly associated with the malignancy of tumors and prognosis of tumor patients. Thus, Plk1 is considered as one of the most attractive molecular targets for anticancer therapy. Recently, several small molecule inhibitors of Plk1 have been identified and characterized, and the first generation of Plk1 inhibitors has been investigated in clinical trials. However, the long-term effect of the downregulation of Plk1 on tumor cells has not yet been studied. In this work we have investigated the phenotype of HeLa cells, in which Plk1 is continuously downregulated by constitutive expression of shRNA. The data demonstrate that the long-term suppression of Plk1 increases the levels of cyclindependent kinase inhibitor p21(WAF1/CIP), which is partially induced by the elevated tumor suppressor p73 in p53-inactivated HeLa cells. The increased kinase inhibitor p21(WAF1/CIP1) localizes in both cyctoplasm as well as in nucleus and interacts directly with Cdk1/cyclin B1. Moreover, the knockdown of Plk1 leads to a decreased oncoprotein MDM2 and an elevated pro-apoptotic protein Bax in HeLa cells. Importantly, HeLa cells with reduced level of Plk1, which induces an increase of p21, p73 and Bax, are more sensitive to some chemotherapeutic agents, such as cisplatin.

tumor suppressor p73 plays an important role in the regulation of DNA damage response. E2F-1 acts as a transcriptional regulator for p73. In the present study, we have found that acetylation of E2F-1 has a critical role in the E2F-1-mediated transactivation of p73. In response to adriamycin (ADR), p73 was stabilized in HeLa cells and the expression levels of its target genes increased in association with an induction of apoptosis. Of note, E2F-1 and several its target genes were transactivated in response to ADR, whereas p73 mRNA level remained unchanged. Immunoprecipitation analysis revealed that ADR has a marginal effect on acetylation status of E2F-1. Intriguingly, acetylation level of E2F-1 remarkably increased in the presence of trichostatin A (TSA) and thereby inducing the expression level of p73 mRNA. Taken together, our present findings suggest that acetylation status of E2F-1 contributes to the selective activation of its target genes.

The expression of tumor suppressor p73 is regulated at mRNA and protein levels. It has been shown that E2F1 acts as a transcriptional activator for p73. In this study, we have found that deregulated expression of E2F1 increases the mRNA level of p73, however, E2F1 promotes the degradation of p73. Immunoprecipitation experiments demonstrated that E2F1 forms a complex with p73 and inhibits the transcriptional activity of p73. Enforced expression of E2F1 induces degradation of p73 in a proteasome-independent manner. Additionally, the deletion analysis showed that E2F1(1-117) has an undetectable effect on p73, whereas E2F1(1-285) and E2F1(1-414) have an ability to promote degradation of p73 and inhibition of p73 transcriptional activity, suggesting that the region of E2F1 between amino acid residues 118 and 285 has a critical role in the regulation of p73. Taken together, our present study indicates that E2F1 has a dual role in the regulation of p73.

Aberrant DNA methylation is considered an important epigenetic mechanism for gene inactivation. Monoclonal gammopathy of undetermined significance (MGUS) is believed to be a precursor of multiple myeloma (MM). We have analyzed methylation status of p15 INK4B , p16 INK4A , ARF, SOCS-1, p27 KIP1 , RASSF1A, and TP73 genes in bone marrow DNA samples from 21 MGUS and 44 MM patients, in order to determine the role of aberrant promoter methylation as one of the steps involved in the progression of MGUS to MM. Methylation specific polymerase chain reaction assay followed by DNA sequencing of the resulting product was performed. SOCS-1 gene methylation was significantly more frequent in MM (52%) than in MGUS (14%; p=0,006). Methylation frequencies of TP73, ARF, p15 INK4B , p16 INK4A , and RASSF1A were comparable in MGUS: 33%, 29%, 29%, 5%, and 0%, to that observed in MM: 45%, 29%, 32%, 7%, and 2%. All patients lacked methylation at p27 KIP1 gene. In both entities, a concurrent methylation of p15 INK4B and TP73 was observed. The mean methylation index of MGUS was lower (0.16) than that of MM (0.24; p<0.05). Correlations with clinicopathologic characteristics showed a higher mean age in MGUS patients with SOCS-1 methylated (p<0.001); meanwhile in MM, methylation of p15 INK4B was more frequent in males (p=0.009) and IgG isotype (p=0.038). Our findings suggest methylation of TP73, ARF, p15 INK4B , and p16 INK4A as early events in the pathogenesis and development of plasma cell disorders; meanwhile, SOCS-1 methylation would be an important step in the clonal evolution from MGUS to MM.

The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and DeltaN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas DeltaNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.

Mitochondrial dysfunction and synaptic damage are critical early features of Alzheimers disease (AD) associated with amyloid beta (Abeta) and tau. We previously reported that the scaffolding protein RanBP9, which is overall increased in AD, simultaneously promotes Abeta generation and focal adhesion disruption by accelerating the endocytosis of APP and beta1-integrin, respectively. Moreover, RanBP9 induces neurodegeneration in vitro and in vivo and mediates Abeta-induced neurotoxicity. However, little is known regarding the mechanisms underlying such neurotoxic processes. Here, we show that RanBP9 induces the loss of mitochondrial membrane potential and increase in mitochondrial superoxides associated with decrease in Bcl-2, increase in Bax protein and oligomerization, fragmentation of mitochondria, and cytochrome c release. RanBP9-induced neurotoxic changes are significantly prevented by the mitochondrial fission inhibitor Mdivi-1 and by classical inhibitors of the mitochondrial apoptosis, XIAP, Bcl-2, and Bcl-xl. RanBP9 physically interacts with the tumor suppressor p73 and increases endogenous p73alpha levels at both transcriptional and post-translational levels;moreover, the knockdown of endogenous p73 by siRNA effectively blocks RanBP9 and Abeta1-42-induced mitochondria-mediated cell death. Conversely, siRNA knockdown of endogenous RanBP9 also suppresses p73-induced apoptosis, suggesting that RanBP9 and p73 have cooperative roles in inducing cell death. Taken together, these finding implicate the RanBP9/p73 complex in mitochondria-mediated apoptosis in addition to its role in enhancing Abeta generation.