DNp73 a matter of cancer: Mechanisms and clinical implications

The p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis, and chemosensitivity of tumors. NH2-terminally truncated p73 (referred to as DNp73) acts as a potent inhibitor of all these tumor suppressor properties, implying that it has oncogenic functions in human tumorigenesis. This was favored by the observation that high DNp73 expression levels in a variety of cancers are associated with adverse clinico-pathological characteristics and the response failure to chemotherapy. The actual challenge is the deciphering of the molecular mechanisms by which DNp73 promotes malignancy and to unravel the regulatory pathways for controlling TP73 isoform expression. This review is focused on recent findings leaving no doubt that N-terminally truncated p73 proteins are operative during oncogenesis, thus underscoring its significance as a marker for disease severity in patients and as target for cancer therapy.     

DNp73 a matter of cancer: mechanisms and clinical implications.

The p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis, and chemosensitivity of tumors. NH2-terminally truncated p73 (referred to as DNp73) acts as a potent inhibitor of all these tumor suppressor properties, implying that it has oncogenic functions in human tumorigenesis. This was favored by the observation that high DNp73 expression levels in a variety of cancers are associated with adverse clinico-pathological characteristics and the response failure to chemotherapy. The actual challenge is the deciphering of the molecular mechanisms by which DNp73 promotes malignancy and to unravel the regulatory pathways for controlling TP73 isoform expression. This review is focused on recent findings leaving no doubt that N-terminally truncated p73 proteins are operative during oncogenesis, thus underscoring its significance as a marker for disease severity in patients and as target for cancer therapy.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

Effects of inducible overexpression of DNp73alpha 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 DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha 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 DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha 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.     

Hypoxia-induced DNp73 stabilization regulates Vegf-A expression and tumor angiogenesis similar to TAp73

P73, the homolog of p53, exists in 2 major forms: either as a pro-apoptotic TAp73 or an amino-terminally truncated DNp73, the latter lacking the first transactivation domain. While TAp73s tumor suppressive functions have been established, DNp73 is an anti-apoptotic protein conferring chemoresistance and is associated with poor survival. However, both forms are variably overexpressed in many human cancers. In this context, we have recently demonstrated that TAp73 is stabilized by hypoxia, a tumor-relevant condition that is associated with cell survival, via HIF-1α-mediated suppression of Siah1 E3 ligase that degrades TAp73. Consequently, hypoxic signals lead to TAp73-mediated activation of several angiogenic genes and blood vessel formation, thereby supporting tumorigenesis. We show here that, similar to TAp73, DNp73 is stabilized by hypoxia in a HIF-1α-dependent manner, which otherwise is degraded by Siah1. Moreover, DNp73 is capable of inducing the expression of Vegf-A, the prototypic angiogenic gene, and loss of DNp73 expression results in reduction in tumor vasculature and size. These data therefore indicate a common mode of regulation for both p73 forms by hypoxia, resulting in the promotion of angiogenesis and tumor growth, highlighting common functionality of these antagonistic proteins under specific physiological contexts.     

Regulation of HSF1-responsive gene expression by N-terminal truncated form of p73alpha

DNp73 is a transactivation domain (TAD)-truncated form of p73. The ability of DNp73alpha to regulate gene expression was examined using reporter assays with luciferase gene constructs. Among various promoter-regulated reporter genes tested, heat shock factor (HSF)-responsive gene expression was selectively activated by DNp73alpha, but not by other p73-isoforms with TAD and DNp73beta. Deletion of TAD endowed p73alpha with the ability to activate HSF-responsive gene expression, but deletion of N-terminal proline-rich domain (PRD) rendered both DNp73alpha and the TAD-deleted p73alpha inactive. Considering the inability of DNp73beta, which is the C-terminus-truncated form of DNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DNp73alpha to be able to activate the HSF-dependent gene expression. In addition to the reporter gene expression, both DNp73alpha and TAD-deleted p73alpha activated the expression of an endogenous gene, hsp70, corresponding with an increase in the active form of HSF1. Taken together, these results demonstrate that TAD-truncated p73alpha can activate HSF-dependent gene expression via induction of active HSF1.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

Change of conformation of the DNA-binding domain of p53 is the only key element for binding of and interference with p73

Xenopus p53 has biological and biochemical properties similar to those of human p53, except for optimal temperature. The frog protein is fully active at 30 degrees C and inactive at 37 degrees C, leading to a temperature-sensitive behavior similar to that of the human mutant p53Ala(143) and the murine mutant p53Val(135). Using hybrid proteins between human and Xenopus expressed from artificial p53 minigenes, we have been able to demonstrate that change of conformation of the DNA-binding domain is the major determinant of this heat sensitivity. It has been reported that some human tumor-derived p53 mutants can engage in a physical association with p73, thus inhibiting its transactivating properties. The mechanism of this association remains to be elucidated. The nature of the mutant p53 that can engage in this association also remains controversial. Using the unique opportunity of the temperature sensitivity of Xenopus p53, we demonstrate that binding of and interference with p73 require a change of conformation in the p53 protein. This interaction occurs through the DNA-binding domain of p53 only when it is in a denatured state. These results reinforce the notion that mutant p53 with a conformational change can act as a down-regulator of the p73 pathway in human cancer and could confer a selective advantage to the tumor.     

p73 is required for endothelial cell differentiation,migration and the formation of vascular networks regulating VEGF and TGFβ signaling

Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.Vascular system formation is one of the earliest events during organogenesis.¹ The original vascular plexus is established by vasculogenesis, through differentiation and assembly of mesodermal precursors.² The angiogenesis process allows the formation of new blood vessels from the existing vasculature and is perturbed in many diseases, including cancer.³ Although efforts have been made to identify factors that control vascular development, the understanding of the molecular networks remains incomplete.The formation of new capillaries and the remodeling of preexisting blood vessels is linked by signal transduction pathways.⁴ The members of the p53 family (p53, p73 and p63) coordinate cell proliferation, migration and differentiation, and could act as regulators of vascular development. TP73 function in angiogenesis is quite controversial,^{5, 6, 7} and it has never been addressed using developmental models.TP73 has a dual nature that resides in the existence of TA and DNp73 variants. TAp73 is capable of transactivating p53 targets^{8, 9, 10} whereas DNp73 can act as p53 and TAp73 repressor.^{11, 12, 13} TP73 final outcome will depend upon the differential expression of the TA/DNp73 isoforms in each cellular context, as they can execute synergic, as well as antagonist, functions.TP73 role during development is emphasized by the p73-knockout mice (Trp73−/−, p73KO from now on) multiple growth defects.¹⁴ These mice, which lack all p73 isoforms, exhibit gastrointestinal and cranial hemorrhages,¹⁴ suggestive of vascular fragility. Furthermore, TAp73 directly regulates GATA-1,⁸ which is essential for endothelial and hematopoietic differentiation.^{15, 16} This compounded information led us to hypothesize that p73 could be implicated in the regulation of vasculogenesis/angiogenesis.Regulation of these processes involves a broad range of signaling molecules essential for vascular growth and stability,¹⁷ such as vascular endothelial growth factor (VEGF)¹⁸ and transforming growth factor-β (TGF-β).¹⁹ TGF-β operates as a rheostat that controls endothelial cell (EC) differentiation, having an inhibitory effect on EC migration and proliferation by the TGF-β/TGFRI (ALK5)/Smad2/3 pathway, while the TβRII–ALK5/ALK1 complex activates Smad1/5/8, ID1 expression and a pro-angiogenic state.^{20, 21, 22}Regulation of the TGF-β and VEGF pathways by p53 family members has been documented.^{23, 24} However, p73''s function in these pathways during development remains largely unexplored. In this work, we have used mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSCs) as models that recapitulate early vascular morphogenesis.^{25, 26, 27} ESC and iPSC form multi-cellular aggregates (embryoid bodies, EBs) that, under appropriate conditions, generate functional EC.²⁸ mESC and iPSC differentiation capacity into ECs has been fully addressed.^{29, 30} We have also performed retinal vascularization analysis to assess vascular processes in vivo.^{31, 32}We demonstrate that p73 deficiency perturbs density and stability of mouse retinal development by affecting VEGF and TGF-β signaling. Furthermore, p73 is necessary for the assembly of vascular structures under physiological conditions in mESC and iPSC. Moreover, DNp73 positively affects angiogenesis through regulation of the TGF-β pathway in human umbilical vein cells (HUVEC) and DNp73-overexpression results in enhanced angiogenic potential of B16-F10 melanoma cells.     

Full-length p73alpha represses drug-induced apoptosis in small cell lung carcinoma cells

The p73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the NH2 terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the COOH terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaNp73 isoforms exert a dominant-negative effect on p73 by blocking their transactivation activity and hence the ability to induce apoptosis. Considerable efforts are made to identify the functional diversity of the COOH-terminal p73 variants. In this study, we found that p73alpha inhibited drug-induced apoptosis in small cell lung carcinoma cells, whereas p73beta promoted it. p73alpha prevented Bax activation, mitochondrial dysfunction, and caspase activation. In addition, p73alpha was also able to reduce apoptosis induced by the BH3-only protein PUMA (p53 up-regulated modulator of apoptosis). Furthermore, we discovered that p73alpha is able to inhibit the pro-apoptotic effect of p73beta, demonstrating the existence of equilibrium between these two p73 isoforms. In conclusion, the reported overexpression of p73alpha in certain tumor types, and our findings that p73alpha exerts anti-apoptotic functions, indicate a potential oncogenic activity for p73.     

p73 induces apoptosis by different mechanisms

p73, like its homologue, the tumor suppressor p53, is able to induce apoptosis in several cell types. This property is important for the involvement of p73 in cancer development and therapy. However, in contrast with p53, the TAp73 gene has two distinct promoters coding for two protein isoforms with opposite effects: while the transactivation proficient TAp73 shows pro-apoptotic effects, the amino-terminal-deleted DeltaNp73 has an anti-apoptotic function. Indeed, the relative expression of these two proteins is related to the prognosis of several cancers. Here we discuss recent developments in the control of p73-induced apoptosis. First, TAp73 induces ER stress via the direct transactivation of Scotin. Second, TAp73 induces the mitochondrial pathway by directly transactivating both Bax and the BH3 only protein PUMA promoters. While the first transactivation is weak, and not sufficient to trigger apoptosis (at least in the in vitro cellular models so far evaluated), the induction of PUMA is strong and lethal. Third, the promoter of the death receptor CD95 contains a p53 responsive element and preliminary experiments suggest that TAp73 also activates the death receptor pathway. In addition, TAp73 is able to transactivate its own second promoter, thus inducing the expression of the anti-apoptotic DeltaNp73 isoform. Therefore, the balance between TAp73 and DeltaNp73 finely regulates cellular sensitivity to death.     

The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins

The p97/VCP ATPase complex facilitates the extraction and degradation of ubiquitinated proteins from larger structures. We therefore studied if p97 participates to the rapid degradation of myofibrillar proteins during muscle atrophy. Electroporation of a dominant negative p97 (DNp97), but not the WT, into mouse muscle reduced fibre atrophy caused by denervation and food deprivation. DNp97 (acting as a substrate-trap) became associated with specific myofibrillar proteins and its cofactors, Ufd1 and p47, and caused accumulation of ubiquitinated components of thin and thick filaments, which suggests a role for p97 in extracting ubiquitinated proteins from myofibrils. DNp97 expression in myotubes reduced overall proteolysis by proteasomes and lysosomes and blocked the accelerated proteolysis induced by FoxO3, which is essential for atrophy. Expression of p97, Ufd1 and p47 increases following denervation, at times when myofibrils are rapidly degraded. Surprisingly, p97 inhibition, though toxic to most cells, caused rapid growth of myotubes (without enhancing protein synthesis) and hypertrophy of adult muscles. Thus, p97 restrains post-natal muscle growth, and during atrophy, is essential for the accelerated degradation of most muscle proteins.     

In good times and bad: p73 in cancer

It is widely accepted in cancer biology that p53 has a tumor suppressive activity, which is lost during tumorigenesis most frequently by mutations in the p53 gene. The discovery of p73 as the first homologue of p53 raised immediate expectations about p53-like tumor suppressor activities. Although tumors show changes in the expression level of p73 and differences in the expression pattern of p73 isoforms, mutations are not commonly observed making it difficult to infer p73's role in tumorigenesis. An experimental model of human cell transformation closely mimics the expression changes observed in cancer patients and provides novel insights into the regulation and function of p73 in the various steps on the road to malignant transformation. p53-like isoforms of p73 (TAp73) are upregulated early during the transformation process in response to RB pathway alterations and block progression to the fully transformed state. Antagonists of TAp73 such as the dominant-negative p73 protein ΔNp73 overcome this block and pave the way to full transformation. Here we review these findings in the context of patient data and recent advances on molecular aspects of p73 function and discuss the implications for p73 as a target for cancer therapy.