Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer.

p63 inhibits metastasis. Here, we show that p63 (both TAp63 and ΔNp63 isoforms) regulates expression of miR-205 in prostate cancer (PCa) cells, and miR-205 is essential for the inhibitory effects of p63 on markers of epithelial-mesenchymal transition (EMT), such as ZEB1 and vimentin. Correspondingly, the inhibitory effect of p63 on EMT markers and cell migration is reverted by anti-miR-205. p53 mutants inhibit expression of both p63 and miR-205, and the cell migration, in a cell line expressing endogenous mutated p53, can be abrogated by pre-miR-205 or silencing of mutated p53. In accordance with this in vitro data, ΔNp63 or miR-205 significantly inhibits the incidence of lung metastasis in vivo in a mouse tail vein model. Similarly, one or both components of the p63/miR-205 axis were absent in metastases or colonized lymph nodes in a set of 218 human prostate cancer samples. This was confirmed in an independent clinical data set of 281 patients. Loss of this axis was associated with higher Gleason scores, an increased likelihood of metastatic and infiltration events, and worse prognosis. These data suggest that p63/miR-205 may be a useful clinical predictor of metastatic behavior in prostate cancer.

Neuronal differentiation by TAp73 is mediated by microRNA-34a regulation of synaptic protein targets.

The p53-family member TAp73 is a transcription factor that plays a key role in many biological processes. Here, we show that p73 drives the expression of microRNA (miR)-34a, but not miR-34b and -c, by acting on specific binding sites on the miR-34a promoter. Expression of miR-34a is modulated in parallel with that of TAp73 during in vitro differentiation of neuroblastoma cells and cortical neurons. Retinoid-driven neuroblastoma differentiation is inhibited by knockdown of either p73 or miR-34a. Transcript expression of miR-34a is significantly reduced in vivo both in the cortex and hippocampus of p73(-/-) mice; miR-34a and TAp73 expression also increase during postnatal development of the brain and cerebellum when synaptogenesis occurs. Accordingly, overexpression or silencing of miR-34a inversely modulates expression of synaptic targets, including synaptotagmin-1 and syntaxin-1A. Notably, the axis TAp73/miR-34a/synaptotagmin-1 is conserved in brains from Alzheimer's patients. These data reinforce a role for TAp73 in neuronal development.

Pharmacoproteomic study of the natural product Ebenfuran III in DU-145 prostate cancer cells: the quantitative and temporal interrogation of chemically induced cell death at the protein level.

A naturally occurring benzofuran derivative, Ebenfuran III (Eb III), was investigated for its antiproliferative effects using the DU-145 prostate cell line. Eb III was isolated from Onobrychis ebenoides of the Leguminosae family, a plant endemic in Central and Southern Greece. We have previously reported that Eb III exerts significant cytotoxic effects on certain cancer cell lines. This effect is thought to occur via the isoprenyl moiety at the C-5 position of the molecule. The study aim was to gain a deeper understanding of the pharmacological effect of Eb III on DU-145 cell death at the translational level using a relative quantitative and temporal proteomics approach. Proteins extracted from the cell pellets were subjected to solution phase trypsin proteolysis followed by iTRAQ-labeling. The labeled tryptic peptide extracts were then fractionated using strong cation exchange chromatography and the fractions were analyzed by nanoflow reverse phase ultraperformance liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry analysis using a hybrid QqTOF platform. Using this approach, we compared the expression levels of 1360 proteins analyzed at ≤ 1% global protein false discovery rate (FDR), commonly present in untreated (control, vehicle only) and Eb III-treated cells at the different exposure time points. Through the iterative use of Ingenuity Pathway Analysis with hierarchical clustering of protein expression patterns, followed by bibliographic research, the temporal regulation of the Calpain-1, ERK2, PAR-4, RAB-7, and Bap31 proteins were identified as potential nodes of multipathway convergence to Eb III induced DU-145 cell death. These proteins were further verified with Western blot analysis. This gel-free, quantitative 2DLC-MS/MS proteomics method effectively captured novel modulated proteins in the DU-145 cell line as a response to Eb III treatment. This approach also provided greater insight to the multifocal and combinatorial signaling pathways implicated in Eb III-induced cell death.

Differential control of TAp73 and DeltaNp73 protein stability by the ring finger ubiquitin ligase PIR2.

p73 is a p53-related transcription factor with fundamental roles in development and tumor suppression. Transcription from two different promoters on the p73 gene results in generation of transcriptionally active TAp73 isoforms and dominant negative DeltaNp73 isoforms with opposing pro- and anti-apoptotic functions. Therefore, the relative ratio of each isoform is an important determinant of the cell fate. Proteasomal degradation of p73 is mediated by polyubiquitination-dependent and -independent processes both of which appear, thus far, to lack selectivity for the TAp73 and DeltaNp73 isoforms. Here, we describe the characterization of another transcriptional target of TAp73; a ring finger domain ubiquitin ligase p73 Induced RING 2 protein (PIR2). Although PIR2 was initially identified a p53-induced gene (p53RFP), low abundance of PIR2 transcript in mouse embryonic fibroblasts of TAp73 KO mice compared with WT mice and comparison of PIR2 mRNA and protein levels following TAp73 or p53 overexpression substantiate TAp73 isoforms as strong inducers of PIR2. Although PIR2 expression was induced by DNA damage, its expression did not alter apoptotic response or cell cycle profile per se. However, coexpression of PIR2 with TAp73 or DeltaNp73 resulted in an increase of the TA/DeltaNp73 ratio, due to preferential degradation of DeltaNp73. Finally, PIR2 was able to relieve the inhibitory effect of DeltaNp73 on TAp73 induced apoptosis following DNA damage. These results suggest that PIR2, by being induced by TAp73 and degrading DeltaNp73, differentially regulates TAp73/DeltaNp73 stability, and, hence, it may offer a therapeutic approach to enhance the chemosensitivity of tumor cells.

Induction of TAp63 by histone deacetylase inhibitors.

TAp63 belongs to the p53-tumour suppressor family and is capable of transactivating a set of target genes to induce cell cycle arrest and apoptosis. We showed that treatment of cancer cells with chemo-therapeutic drugs or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) results in induction of TAp63 expression, which is in turn related with chemosensitivity. Indeed, induction of TAp63 by TSA affects sensitivity to chemo-therapeutic drugs via the cleavage of the trans-inhibitory domain of TAp63 by active caspases, resulting in generation of a transcriptionally hyper-active TAp63 fragment. Therefore therapeutic approaches that enhance TAp63 expression may offer an improvement in the management of chemoresistant tumours. In this study we tested the abilities of different HDAC inhibitors to induce TAp63 expression. We discovered that two HDAC inhibitors belonging to the hydroxamate group, namely TSA and LBH589, are the most efficient inducers of TAp63 expression. Finally, we found that induction of TAp63 expression in HCT116 cells depends on p53, as p53-negative HCT116 cells failed to induce significant TAp63 expression following treatment with different HDAC inhibitors.

p73 and p63 regulate the expression of fibroblast growth factor receptor 3.

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.

Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis.

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.

Human papillomavirus E7 induces p63 expression to modulate DNA damage response.

Cervical cancer is the third most common malignancy diagnosed in women worldwide. The major aetiological factor underlying the malignant transformation of cervical cells is the persistent infection with high-risk human papillomaviruses (HR-HPV), with more than 99% of cases expressing viral sequences. Here, we report a previously unknown mechanism driven by high-risk human papillomavirus E7 protein to modulate response to DNA damage in cervical cancer cells. Our data shows that HR-HPV E7 oncoprotein induces the transcription of the p53-family member p63, which modulates DNA damage response pathways, to facilitate repair of DNA damage. Based on our findings, we proposed a model, where HR-HPV could interfere with the sensitivity of transformed cells to radiation therapy by modulating DNA damage repair efficiency. Importantly, we have shown for the first time a critical role for p63 in response to DNA damage in cervical cancer cells.

Pir2/Rnf144b is a potential endometrial cancer biomarker that promotes cell proliferation.

Endometrial cancer is one of the most common gynaecological cancers in developed countries. Its incidence has increased 20% over the last decade and the death rate has increased >100% over the past two decades. Current models for prediction of prognosis and treatment response are suboptimal, and as such biomarkers to support clinical decision-making and contribute to individualised treatment are needed. In this study, we show that the E3-ubiquitin ligase PIR2/RNF144B is a potential targetable biomarker in endometrial cancer. At transcript level, it is expressed both in normal endometrium and tumour samples, but at protein level, it is expressed in tumours only. By using endometrial cancer cell lines, we demonstrated that PIR2/RNF144B is stabilised via phosphorylation downstream of GSK3ß and this is necessary for the proliferation of endometrial cancer cells, in the absence of oestrogenic growth stimuli. Here, inactivation of GSK3ß activity is associated with loss of PIR2/RNF144B protein and consequent inhibition of cell proliferation. Our results, therefore, substantiate PIR2/RNF144B as a novel candidate for targeted therapy in endometrial cancer.

Generation of DeltaTAp73 proteins by translation from a putative internal ribosome entry site.

p73 belongs to a family of transcription factors, including p53 and p63, that mediate response to DNA damage and cellular stress by inducing DNA repair, cell cycle arrest, and apoptosis. TP73 gene contains two promotors and several splice variants resulting in up to 24 possible permutations of p73 proteins which underlies the complexity of the family and its regulatory mechanisms. p73 variants lacking the N-terminal, denoted as DeltaTAp73, are not transcriptionally competent and they act in a dominant negative fashion over TAp73. DeltaTAp73 isoforms can be generated by alternative promotor usage, giving rise to DeltaNp73, or alternative splicing of exons 2, 3 or 2, and 3 together. Such transcript isoforms potentially produce oncogenic proteins and they were shown to be present in primary tumors and tumor-derived cell lines. We investigated the possibility of additional mechanisms by which p73 protein could be regulated and discovered a putative internal ribosome entry site (IRES) in exon 2. Translation initiation of TAp73 mRNA results in a DeltaNp73-like peptide, thus demonstrating an additional mechanism whereby a DeltaTA p73 protein is produced from a transcript originally generated from the P1 promotor of the p73 gene.

Urocortin suppresses endometrial cancer cell migration via CRFR2 and its system components are differentially modulated by estrogen.

Urocortin (UCN1) peptide shares structural and functional homology with corticotropin-releasing factor (CRF). UCN1 is significantly reduced in endometrial adenocarcinoma compared to healthy controls. However, there are no data which evaluate the effects of UCN1 in the endometrium, or how it is modulated. We used proliferation and transwell assays to determine the effect of UCN1 on the proliferation and migration of Ishikawa and HEC1A cells. We also determined the expression levels of UCN1 and its receptors produced by estrogen receptor agonists, and the effect of UCN1 on estrogen receptor expression, using quantitative polymerase chain reaction. UCN1 suppressed migration of endometrial cancer cells in vitro. This effect appears to be specific to CRF receptor 2 (CRFR2), as selective antagonism of CRFR2 but not CRFR1 completely eliminated suppression of migration. Activation of ERA reduced UCN1 expression, but only had a small effect on the expression of CRFR1. However, expression of CRFR2 was more notably reduced at both the mRNA and protein levels by activation of ERB. UCN1 in turn reduced both ERA and ERB expression, as assessed by real-time quantitative PCR. We demonstrate that UCN1 significantly suppresses the migration of endometrial cancer cells but has no effect on their proliferation. Thus, loss of UCN1 in endometrial cancer may promote invasion and metastatic spread. There is a complex relationship between the UCN1 system and estrogen receptors, which may provide insights into endometrial carcinogenesis, a disease known to be driven by estrogen excess.

p63 and p73, members of the p53 gene family, transactivate PKCdelta.

The p53 family comprises three genes that encode for p53, p63 and p73. These genes have a significant degree of sequence homology, especially in the central sequence-specific DNA-binding domain. The high homology among the three DNA-binding domains indicates that these transcription factors have identical residues interacting with DNA, and thus potentially can recognize the same transcriptional targets. In this study, we demonstrate that PKCdelta is induced by p63 and p73 in Saos2 cells. The putative human PKCdelta promoter harbours three p53-like binding sites (RE I, RE II, RE III). In order to confirm the transactivation of PKCdelta by p53 family members, we performed transcription assays using the entire or selected regions of the promoter upstream of a luciferase reporter gene. The results obtained demonstrated that, at least in vitro, the p53 family members tested (TAp63alpha, TAp73alpha, DeltaNp63alpha, but not DeltaNp73alpha) were able to drive transcription from the PKCdelta promoter.

Regulation of myocardial interleukin-6 expression by p53 and STAT1.

Cardiovascular diseases are a major cause of morbidity and mortality worldwide. The interferon inducible transcriptional activator signal transducer and activator of transcription-1 (STAT1) and p53 are two critical transcriptional factors that have pivotal roles in cardiac biology and pathology. Here we describe a novel interplay between these two key players that critically regulate the levels of the pleiotropic interleukin 6 (IL6) in the heart. We provide in vivo evidence to demonstrate that, in cardiac tissues, STAT1 is a positive regulator of IL6 expression and it competes with the suppressive effect of p53 to sustain basal IL6 levels. Induction of IL6 expression in response to interferon gamma (IFNγ), a well-characterized activator of STAT1, parallels that of STAT1 phosphorylation and induction of STAT1 target genes, such as the interferon regulatory factor-1 (IRF-1), major histocompatibility complex class II transactivator (C2ta), and ß2-microglobulin (B2m). Furthermore, hearts from STAT1 knockout mice fail to induce IL6 expression in response to IFNγ. More importantly, we showed that this regulatory system is not functional in mouse embryonic fibroblasts, suggesting that activation of IL6 expression by STAT1 may be tissue specific. IL6 is a major effector of inflammation and cardiac hypertrophy, two major processes involved in heart failure, and therefore, understanding the molecular mechanisms regulating IL6 expression will enable better therapies and treatments for cardiovascular disease patients.

Relative expression of TAp73 and ΔNp73 isoforms.

The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative ΔNp73 isoform. Interestingly, we detected higher levels of ΔNp73 in some mouse tissues, suggesting that ΔNp73 may have a physiological role in these tissues.

MicroRNA Control of Invasion and Metastasis Pathways.

Despite recent advances, cancer remains a leading cause of death worldwide. In developed countries, the incidence of colorectal and breast cancer has been stable, but no improvement in prognosis has been observed if the patient presents with metastases at diagnosis. This fact highlights the importance of therapeutic approaches targeting cellular invasion and metastasis programs as the next step in cancer treatment. During carcinoma progression a process called epithelial-mesenchymal transition (EMT) results in enhanced invasion and motility which is directly linked with loss of epithelial polarity and epithelial junctions, migration permissive cytoskeleton alterations, and the acquisition of mesenchymal properties. The recent discovery of microRNAs (miRNAs) controlling key cellular pathways has opened a new era in understanding how EMT pathways are modulated. In this review, we classify EMT regulating proteins according to their cellular localization (membrane, cytoplasmic, and nuclear), and summarize the current knowledge on how they are controlled by miRNAs and propose potential miRNAs for the transcripts that may control their expression.

p73: a multifunctional protein in neurobiology.

p73, a transcription factor of the p53 family, plays a key role in many biological processes including neuronal development. Indeed, mice deficient for both TAp73 and ΔNp73 isoforms display neuronal pathologies, including hydrocephalus and hippocampal dysgenesis, with defects in the CA1-CA3 pyramidal cell layers and the dentate gyrus. TAp73 expression increases in parallel with neuronal differentiation and its ectopic expression induces neurite outgrowth and expression of neuronal markers in neuroblastoma cell lines and neural stem cells, suggesting that it has a pro-differentiation role. In contrast, ΔNp73 shows a survival function in mature cortical neurons as selective ΔNp73 null mice have reduced cortical thickness. Recent evidence has also suggested that p73 isoforms are deregulated in neurodegenerative pathologies such as Alzheimer's disease, with abnormal tau phosphorylation. Thus, in addition to its increasingly accepted contribution to tumorigenesis, the p73 subfamily also plays a role in neuronal development and neurodegeneration.

p73 in Cancer.

p73 is a tumor suppressor belonging to the p53 family of transcription factors. Distinct isoforms are transcribed from the p73 locus. The use of 2 promoters at the N-terminus allows the expression of an isoform containing (TAp73) or not containing (ΔNp73) a complete N-terminal transactivation domain, with the latter isoform capable of a dominant negative effect over the former. In addition, both N-terminal variants are alternatively spliced at the C-terminus. TAp73 is a bona fide tumor suppressor, being able to induce cell death and cell cycle arrest; conversely, ΔNp73 shows oncogenic properties, inhibiting TAp73 and p53 functions. Here, we discuss the latest findings linking p73 to cancer. The generation of isoform specific null mice has helped in dissecting the contribution of TA versus ΔNp73 isoforms to tumorigenesis. The activity of both isoforms is regulated transcriptionally and by posttranslational modification. p73 dysfunction, particularly of TAp73, has been associated with mitotic abnormalities, which may lead to polyploidy and aneuploidy and thus contribute to tumorigenesis. Although p73 is only rarely mutated in cancer, the tumor suppressor actions of TAp73 are inhibited by mutant p53, a finding that has important implications for cancer therapy. Finally, we discuss the expression and role of p73 isoforms in human cancer, with a particular emphasis on the neuroblastoma cancer model. Broadly, the data support the hypothesis that the ratio between TAp73 and ΔNp73 is crucial for tumor progression and therapeutic response.

p53 is cleaved by caspases generating fragments localizing to mitochondria.

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.

p63 is upstream of IKK alpha in epidermal development.

The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (DeltaNp63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKKalpha, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKKalpha and Ets-1, respectively. We also found that DeltaNp63 upregulates IKKalpha indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKKalpha in epithelial development.