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Epithelial-mesenchymal transition (EMT) is an embryonic program that is reactivated in cancer and regulates the invasion and metastasis of tumor cells. Zinc finger E-box binding homeobox 2 (ZEB2) induces EMT by upregulating matrix metalloproteinases (MMP), yet MMP genes lack ZEB2 binding motif in their promoters. Recently, expression of MMPs was associated to the activation of ETS1 transcription factor; however, a link between ZEB2 and ETS proto-oncogene 1, transcription factor (ETS1) remains to be elucidated. Hence, we investigated the transcriptional regulation of ETS1 by ZEB2 after our initial observation that ZEB2 and ETS1 are coexpressed in hepatocellular carcinoma cells (HCCs). Chromatin immunoprecipitation and luciferase reporter assays clearly showed that ZEB2 binds to E-box sequences on the promoter of ETS1. Elevated expression of ETS1 was found in DLD-ZEB2 and A431-ZEB2 inducible systems, and knockdown of ZEB2 caused an explicit downregulation of ETS1 in shZEB2-SNU398 and shZEB2-SK-HEP-1 cells. Repression of ETS1 expression in ZEB2-induced conditions substantially impaired the migration and invasive capacities of DLD1 cells. Mechanistically, knockdown of ETS1 in ZEB2-expressing cells resulted in the downregulation of established ZEB2 targets TWIST and MMP9. Correlation analyses in HCC lines, cancer complementary DNA arrays, and The Cancer Genome Atlas RNA-sequencing data set revealed that ZEB2 and ETS1 are coexpressed, and their expressions in human tumors show a highly significant positive correlation. Our results demonstrated that ZEB2 acts as an upstream regulator of ETS1 and, in turn, ETS1 maintains ZEB2-induced EMT. These findings add another level of complexity to the understanding of ZEB2 in the invasion and metastasis of cancer cells, and put ZEB2/ETS1 axis as a novel therapeutic target in human malignancies.

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions. For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets. Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.

Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms—association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS–ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS–ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing. This work was supported by grants from the National Health and Medical Research Council of Australia & SFI Deposited by bulk import

p53, p63 and p73 make a family of transcription factors that play a vital role in development and cancer. All p53 family members have more than one promoter producing Transactivating (TA) and Dominant Negative (DeltaN) isoforms and their mRNAs are subjected to extensive splicing at 3' end to produce multiple protein products. p53 is usually inactivated by point mutations during tumorigenesis, whereas the expression levels and p63 and p73 are modulated to give tumor cells a selective advantage. In this study, aiming to find novel targets of the p53 family members, we identified FGFR3 as a gene transcriptionally controlled by p63 and p73. FGFR3 has been implicated in development and tumor biology as activating mutations of this gene was described in skeletal disorders, non-invasive skin conditions and superficial bladder cancers. We found that TAp73, TAp63 and DeltaNp63 was capable of inducing FGFR3. siRNA mediated downregulation of DeltaNp63 decreased endogenous FGFR3 protein levels. Our findings of this new link between p53 family proteins and FGFR3 may help understanding the transition of superficial bladder cancers to an invasive phenotype.

Zinc finger transcription factors of the Snail/Slug and ZEB-1/SIP1 families control epithelial-mesenchymal transitions in development in cancer. Here, we studied SIP1-regulated mesenchymal conversion of epidermoid A431 cells. We found that concomitant with inducing invasive phenotype, SIP1 inhibited expression of cyclin D1 and induced hypophosphorylation of the Rb tumor suppressor protein. Repression of cyclin D1 was caused by direct binding of SIP1 to three sequence elements in the cyclin D1 gene promoter. By expressing exogenous cyclin D1 in A431/SIP1 cells and using RNA interference, we demonstrated that the repression of cyclin D1 gene by SIP1 was necessary and sufficient for Rb hypophosphorylation and accumulation of cells in G1 phase. A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression. However, in another epithelial-mesenchymal transition model, gradual mesenchymal conversion of A431 cells induced by a dominant negative mutant of E-cadherin produced no effect on the cell cycle. We suggest that impaired G1/S phase progression is a general feature of cells that have undergone EMT induced by transcription factors of the Snail/Slug and ZEB-1/SIP1 families.

p73, unlike p53, is expressed as a number of isomeric forms. Alternative splicing at the 3' end of p73 transcript, together with the usage of a second promoter downstream of exon 3, can generate up to 24 p73 isoforms. Variants lacking the TA domain (DeltaN isoforms) are induced by TAp73 and by p53, and inhibit their transcriptional activity. However, understanding the complex biology of p73 has been handicapped by the lack of high affinity specific antibodies for the different isoforms. Here, we report the characterization, by Western blotting and immunoprecipitation, of three new polyclonal antisera recognizing all p73 isoforms, only DeltaN isoforms or only p73alpha, and which have advantages of affinity and specificity over previously available antibodies.

Epithelial–mesenchymal transition (EMT) is an embryonic trans-differentiation programme that is implicated in organ formation by facilitating the formation of highly motile cells with stem cell capabilities.1 Most tissues of cells derived from ectoderm or mesoderm retain certain elements of EMT and mesenchymal properties are their differentiation markers. On the other hand, cells of organs derived from endoderm only experience EMT and its reversal (mesenchymal-epithelial transition) at certain phases of development and become differentiated secretory epithelial cells. As de-differentiation is a common feature of cancer progression, acquisition of mesenchymal markers accompanied with the loss of epithelial features can be observed in most carcinomas and considered as the reason of cancer spread (metastasis). For the reasons that (1) more than 85% of all cancers are of epithelial origin and (2) epithelial (differentiated) and mesenchymal (undifferentiated) carcinoma cells are morphologically very different, EMT has been mostly studied in carcinoma setting. However, there is growing evidence suggesting that cancers of organs derived from mesoderm (e.g., hematopoietic malignancies and sarcomas) or ectoderm (e.g., glioblastomas and melanomas) also activate EMT programs to acquire an undifferentiated state and become resistant to conventional therapies.2 A good example of this is a recent article by Sanchez-Tillo et al. that investigated the role of EMT-inducing transcription factor ZEB1 in mantle cell lymphoma (MCL) progression.3 MCL is a B-cell malignancy and considered as the rarest form of non-Hodgkin lymphoma.4 Despite this statistical data, it is responsible for a significant portion of B-cell malignancy related mortality because it is an aggressive cancer with a continuous relapse pattern.5 In addition, most MCL patients show poor response to chemotherapy. The translocation of cyclin D1 to immunoglobulin heavy chain enhancer locus t(11;14)(q13;q32) is the genetic hallmark of MCLs; however Wnt, p53, Notch pathway aberrations and pro-survival gene overexpression (Mcl1 and Bcl2 overexpression) are also commonly observed. Sanchez-Tillo et al. investigated the expression of EMT-inducing transcription factor ZEB1 in a cohort of MCL patient samples.3 They found ZEB1 overexpression in half of the samples with a strong correlation with nuclear β-catenin presence (Wnt signalling active). In addition, the clinico-pathological analysis of patient data revealed ZEB1 positivity as a marker for shorter overall survival. This led them to investigate the mechanisms of ZEB1 upregulation and functional contribution of ZEB1 expression to the pathology of MCL. ZEB1 promoter has been previously reported (by the same group) to contain two TCF–LEF binding sites allowing β-catenin–TCF4 binding in colorectal cancer setting.6 They validated the presence of endogenous β-catenin on the respective DNA motifs of ZEB1 promoter by chromatin immunoprecipitation and also showed that ZEB1 mRNA levels are changing upon stimulation with Wnt-3a or knockdown of endogenous β-catenin using two different MCL cell lines. Following this observation, they also investigated if modulation of β-catenin signalling by chemical inhibitors such as salinomycin can alter ZEB1 expression. Salinomycin treatment, indeed, was able to decrease endogenous ZEB1 expression. These results mark the potential to control acquired ZEB1 expression by targeting Wnt pathway and inhibit its functional consequences in MCL. The cellular consequences of ZEB1 overexpression in MCL have not been investigated, however, carcinomas expressing ZEB1 were shown to have canonical hallmarks of EMT such as increased invasiveness and metastasis, resistance to chemotherapy and decreased proliferation.7 Sanchez-Tillo et al. systematically tested all these aspects in MCL by knocking down ZEB1 or β-catenin using MCL cell lines. They found that ZEB1 downregulation decreases baseline cell viability and proliferation. The gene expression of ZEB1-expressing cells indicated activation of survival and repression of cell death pathways as validated by increased levels of myc, cyclin D1, CENPF, Ki67, Bcl2, MCL1 and decreased p53, bax and PMAIP1 mRNAs. In addition, they also investigated whether ZEB1 expression confers resistance to chemotherapy. In line with their previous data regarding changes in baseline cell viability, ZEB1 downregulation increased sensitivity to commonly used chemotherapeutic drugs used in MCL treatment, such as Doxorubicin, Cytarabine and Gemcitabine but not Vincristine. The effect of ZEB1 knockdown on chemosensitization was also confirmed by pre-treatment of MCL cell lines with Salinomycin. EMT-induced chemoresistance has been described in different carcinoma models; however, the cellular mechanism of this phenomenon still remains to be clarified.8 The reasons for chemoresistance can be at least 3-fold. Cancer cells can alter drug influx/efflux to favour less chemotherapeutic agents in the cell (e.g., activate multidrug resistance proteins), they can activate efficient detoxification (e.g., by glutathione S-transferase) or enable efficient DNA repair to escape DNA damage-induced apoptosis. In this article, Sanchez-Tillo et al. investigated the regulation of drug influx/efflux pathways by ZEB1. Using two different MCL cell lines and validating by IHC in primary MCL samples, they showed that critical drug transporters are regulated by ZEB1 to favour survival of Wnt signalling active cancer cells. The authors also extended their in vitro studies to animal models. Subcutaneous injection of Granta-519 cells expressing or silenced ZEB1 showed significantly different responses; ZEB1 silenced cells showing delays in tumour growth and enhanced sensitivity to Doxorubicin treatment. Finally, they explored whether salinomycin can synergize with Doxorubicin for an enhanced response to chemotherapy. Using established MCL cell lines and primary cells from MCL patients, they showed that sublethal doses of Salinomycin and Doxorubicin can synergize and effectively kill MCL cells. These results support their in vivo data and provide evidence that β-catenin-activated ZEB1 expression contributes to tumour progression and chemoresistance (Figure 1). Figure 1 Role of EMT-inducing transcription factor ZEB1 in MCL. In carcinoma cells, activation of distinct signalling pathways such as TGFβ, Wnt and receptor tyrosine kinase (RTK) converges to ZEB1 for a fully de-differentiated and mesenchymal (EMT) phenotype. ... What is next? A couple of things remained to be done and questions answered to make use of the full potential of these exciting findings. First of all, Sanchez-Tillo et al. did not show the clinico-pathological variables of their patient cohort. In recent years, anti-B cell-based immunotherapeutic regimens, such as the anti-CD20 antibody rituximab, gained attention as an alternative in the treatment of B-cell malignancies. The provision of detailed patient data may allow stratification of MCL cases and potentially mark subgroups that can or cannot be treated with chemo and/or immunotherapies. In addition, similar correlative studies have to be performed using independent patient cohorts to externally validate the clinical importance of ZEB1 in MCL and allow clinical translation. Another point to be clarified is the functional link between ZEB1-induced alterations in drug influx/efflux and the actual presence of drugs in MCL cells. Genotoxic agents such as Doxorubicin can be imported through cell membrane by passive diffusion and effectively retained in the cell as DNA-bound form so they cannot be exported by drug transporters. This question can be easily answered due to the fluorescence nature of Doxorubicin using normal and transformed B cells, in the presence and absence of ZEB1. Finally, activation of EMT pathways in carcinoma results in generation of malignant cells with stem cell properties. A critical question that remains to be answered is whether ZEB1-expressing MCL cells are de-differentiated enough to be considered as B-cell precursors. As a follow-up of this work, it will be fascinating to explore if the tumour-promoting features of ZEB1 in MCL cells are also conserved in other malignancies, particularly in other mesoderm-derived tissues such as sarcomas or cancers such as hepatoblastomas that β-catenin activation is an early event in tumourigenesis.

As Eugene Garfield said, while it is easy to recognize a good paper, it could be more difficult to recognize a bad paper. In fact, the results could be weak, but the conclusion could still be right, even though not fully supported by the data shown. Preliminary reports could also fall in this category. After all, it was not a Cell but a BBRC paper – only a little BBRC of three impact factor – describing a novel experimental model in which to study ATP-dependent proteolysis.1 In a lysate from rabbit reticulocytes, where the proteolytic activity was not due to lysosomes (pH optimum of 7.8), they separated two fractions in a DEAE cellulose column, each one individually inactive, but after recombination of the two fractions ATP-dependent proteolysis was reconstituted. Immediately, Hershko and his young assistant, Ciechanover, went for a sabbatical to the Fox Chase Cancer Center in Philadelphia to work with Irwin Rose, and the three began unveiling the E1-E2-E3 ligase components of the Ubiquitin-Proteasome System (UPS),2 see for example some recent reviews.3, 4 For this work, Ciechanover, Hershko, and Rose were granted the 2004 Nobel Prize. Five clinical trials based on UPS inhibition were in progress in the same year:5 (i) Velcade-TM (dipeptide boronic acid, Bortezomid, PS-341, Millenium Pharmaceuticals Inc.) phase III, FDA approved for relapsed and refractory multiple myeloma and other solid cancers; it stabilizes cell-cycle and proapaoptotic proteins, inhibits antiapoptotic proteins, and affects tumor microenvironment; (ii) MLN519 (lactacystin derivative, PS-519, Millenium Pharmaceuticals Inc.) phase I for acute stroke and myocardial infections; it is a potent anti-inflammatory and neuroprotective compound; (iii) Epoxomicin-Eponemycin (Streptomyces epoxyketones) preclinical; it has cytotoxic effects in various tumor cells; (iv) NLVS (trileucine vinyl-sulfone) preclinical; it is an irreversible inhibitor of trypsin- and chemotrypsin-like proteasome activities; (v) Ritonavir (Peptidomimetic protease inhibitor; Abbott) phase II for AIDS and tumor patients; it is an HIV protease inhibitor, also inhibits chemotrypsin-like activity of proteasome. While the timeline of this story is outlined by Ciechanover himself,6 the present book describes the most advanced ongoing clinical trials on UPS inhibition. It is of particular interest that this book review comes out in the same issue containing manuscripts from scientists that have originated in this field.7, 8, 9, 10

Cataloged from PDF version of article. Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development. © 2010 Elsevier Inc.

Epithelial mesenchymal transitions (EMT), the generation of motile mesenchymal cells from epithelial sheets, are differentiation programs which take place at several critical steps of embryonic development and in metastatic cancer. Recent data have shown that the transcription factors which are master regulators of EMT also regulate cell cycle progression, apoptosis and senescence. In light of these new observations, the role of these factors in human cancer may be broader than previously anticipated. Here we review recent literature on non-EMT functions of EMT-controlling transcription factors. We will mainly focus on transcription factors belonging to the ZEB family, but some important results obtained by investigators studying other key EMT regulators, Snail and Twist are also discussed.

G-protein coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This super-family of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In the current study we demonstrate that a classical Gq/11-coupled GPCR, the M3-muscarinic receptor, was able to regulate apoptosis via receptors that are endogenously expressed in the human neuroblastoma cell line SH-SY5Y and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M3-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, sub-cellular trafficking and modification of p53 in a manner that is in part dependent on the cell type.

p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The alpha isoforms of TAp63 and DeltaNp63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63alpha can directly inhibit its activity through an intramolecular interaction; similarly, DeltaNp63alpha can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63alpha isoforms is cleaved by activated caspases. Cleavage of DeltaNp63alpha relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63alpha results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63.

Although signal transducer and activator of transcription 1 (STAT1) mediated regulation of p53 transcription and apoptosis has been previously reported, modulation of other members of the p53 family of transcription factors remains poorly understood. In this study, we found that STAT1 and TA-p73 can interact directly and that p73-mediated Bax promoter activity was observed to be reduced by STAT1 expression in a p53-independent manner for which STAT1 Tyrosine-701 and Serine-727 are key residues. This study presents the first report physically linking STAT1 and TA-p73 signalling and highlights the modulation of the Bax promoter in the context of IFN-γ stimulation.

The epidermis is a multi-layered stratified epithelium continuously renewed by differentiating keratinocytes that develops by the action of p63, a member of the p53 family. The TP63 contains two promoters, resulting in the expression of different proteins, containing (TAp63) or not (DeltaNp63) an amino-terminal transactivation domain, which contribution in skin formation is not fully understood. We found that p63 binds and transactivate GATA-3 promoter, which in turn transactivate IKKalpha, two pivotal regulators of epithelial development. Indeed, GATA-3 is a regulator of cell lineage in skin and hair follicles formation. To further study the relationship between GATA-3 and p63 isoforms here we investigated their expression during keratinocyte differentiation, in human epidermis and hair follicle.

The p53 tumor suppressor protein exerts most of its anti-tumorigenic activity by transcriptionally activating several pro-apoptotic genes. Accumulating evidence also suggests a transcription-independent function of p53 during apoptosis. It has recently been shown that, when activated, a fraction of p53 translocates to mitochondria, causing cytochrome c release. We now demonstrate a caspase-dependent cleavage of p53 resulting in the generation of four fragments, two of which lack a nuclear localization signal and consequently localize to cytosol. Moreover, these two fragments translocate to mitochondria and induce mitochondrial membrane depolarization in the absence of transcriptional activity. This novel feature of p53 supports the model whereby cytosolic p53 exerts major functions in apoptosis and also suggests the presence of a positive feedback loop in which activated caspases cleave p53 to augment mitochondrial membrane depolarization.

The function of p73, a transcription factor belonging to the p53 family, is finely regulated by its steady-state protein stability. p73 protein degradation/stabilization can be regulated by mechanisms in part dependent on the ubiquitin proteasome system (UPS): (i) Itch/NEDD4-like UPS degradation, (ii) NEDD8 UPS degradation, and (iii) NQO1 20S proteasome-dependent (but ubiquitin-independent) breakdown. Here, we show that, in vitro, Calpain I can cleave p73 at two distinct sites: the first proline-rich region and within the oligomerization domain. Consequently, different p73 isoforms can be degraded by calpains, i.e., both N-terminal isoforms (TAp73 and DeltaNp73) as well as the C-terminal isoforms (alpha, beta, gamma, delta). Moreover, overexpression of the specific endogenous calpain inhibitor, calpastatin, in cultured cells increased the steady-state p73 level. This suggests that calpains may play a physiological role in the regulation of p73 protein stability.

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 TP73 gene, first identified in 1997, encodes the p73 protein, a p53 tumour suppressor homologue. The lack of mutations in the TP73 gene in cancers and the developmentally abnormal phenotype of the TP73 knockout mouse suggest a different function for TP73 gene derived proteins. An alternative promoter in the third intron of the TP73 gene, which produces a transcription deficient and dominant negative protein (DeltaNp73), produces increased complexity in the function of p73 proteins. Functional studies of transcriptionally active (TAp73; regulated by the first promoter) and DeltaNp73 (regulated by the second promoter) show that the TP73 gene encodes both a candidate tumour suppressor (TAp73) and an oncogene (DeltaNp73), with pro-apoptotic and anti-apoptotic properties, respectively. This "two in one" gene architecture leads us to make an in silico search for other probable promoter regions and transcription start sites in different introns of the TP73 gene. To identify such regulatory regions, we have analysed the genomic structure of human and mouse TP73 genes. We have found introns 1 and 4 to be extremely large and relatively conserved in size in mouse and human, in addition to intron 3, which includes the DeltaNp73 promoter. We have further characterized these introns by transcription factor binding motifs, transcription initiation sites, open reading frames, and splicing using six programs that can successfully identify the already characterized promoters. Our results suggest the presence of a candidate 2 kbp genomic DNA in the first intron of human TP73 gene, harbouring 2 putative promoter regions, together with a similar region within intron 1 of the mouse gene.