P73 regulates cisplatin-induced apoptosis in ovarian cancer cells via a calcium/calpain-dependent mechanism

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca2+]i) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca2+-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca2+]i increase in OV2008 cells was not effected by the elimination of extracellular Ca2+, but this was attenuated by the depletion of internal Ca2+ store, indicating that mobilization of intracellular Ca2+] stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca2+-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca2+/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA

Effects of inducible overexpression of DNp73(alpha) on cancer cell growth and response to treatment in vitro and in vivo

The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73(alpha) expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73(alpha). In vitro the two DNp73(alpha) overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73(alpha) both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73(alpha) does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents. (copyright) 2005 Nature Publishing Group. All rights reserved

Characterization of ΔNp73 expression and regulation in gastric and esophageal tumors

p73 is a member of the p53 protein family. Although the tumor suppressor function of p53 is clearly defined, the role of p73 in tumorigenesis is still a matter of debate. A complex pattern of expression of p73 isoforms makes it difficult to unambiguously interpret the experimental results. Previously, we and others have found that the N-terminally truncated isoform of p73, ΔNp73, has potent anti-apoptotic and oncogenic properties in vitro and in vivo. In the present study, we analyzed, for the first time, the regulation of ΔNp73 in a large number of gastric, gastroesophageal junction and esophageal tumors. We found that expression of ΔNp73 mRNA and protein is increased in these neoplasms. Furthermore, the up-regulation of the ΔNp73 protein is significantly associated with poor patient survival. Oncogenic properties of ΔNp73 were further confirmed by finding that ΔNp73 facilitates anchorage-independent growth of gastric epithelial cells in soft agar. As little is currently known about the regulation of ΔNp73 transcription, we investigated the alternative p73 gene promoter that mediates the ΔNp73 expression. Analyzing the ΔNp73 promoter in silico as well as by using chromatin immunoprecipitation, site-directed mutagenesis and deletion analyses we identified the evolutionary conserved region within the ΔNp73 promoter that contains binding sites for HIC1 protein. We found that HIC1 negatively regulates ΔNp73 transcription in mucosal epithelial cells. This leads to a decrease in ΔNp73 protein levels and may normally control the oncogenic potential of the ΔNp73 isoform

DEC1 Coordinates with HDAC8 to Differentially Regulate TAp73 and ΔNp73 Expression

P73, a member of the p53 family, plays a critical role in neural development and tumorigenesis. Due to the usage of two different promoters, p73 is expressed as two major isoforms, TAp73 and ΔNp73, often with opposing functions. Here, we reported that transcriptional factor DEC1, a target of the p53 family, exerts a distinct control of TAp73 and ΔNp73 expression. In particular, we showed that DEC1 was able to increase TAp73 expression via transcriptional activation of the TAp73 promoter. By contrast, Np73 transcription was inhibited by DEC1 via transcriptional repression of the ΔNp73 promoter. To further explore the underlying mechanism, we showed that DEC1 was unable to increase TAp73 expression in the absence of HDAC8, suggesting that HDAC8 is required for DEC1 to enhance TAp73 expression. Furthermore, we found that DEC1 was able to interact with HDAC8 and recruit HDAC8 to the TAp73, but not the ΔNp73, promoter. Together, our data provide evidence that DEC1 and HDAC8 in differentially regulate TAp73 and ΔNp73 expression, suggesting that this regulation may lay a foundation for a therapeutic strategy to enhance the chemosensitivity of tumor cells

Diverse p63 and p73 isoforms regulate Delta 133p53 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: p63β, ΔNp63α, ΔNp63β 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 p63β, ΔNp63α, ΔNp63β and ΔNp73γ. Overall, this study shows a strong interplay between p53, p63 and p73 isoforms to orchestrate cell fate outcome

Yes-associated protein (YAP) functions as a tumor suppressor in breast

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling