MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function

p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21 waf1 transcription. p21 waf1 protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance

Long non-coding MIR205HG depletes Hsa-miR-590-3p leading to unrestrained proliferation in head and neck squamous cell carcinoma

Over 70% of head & neck squamous cell carcinoma (HNSCC) patients carry TP53 oncogenic mutations. Here we studied the role of specific tumor-derived mutant p53 proteins in the aberrant transcription of long non-coding (lnc) MIR205HG gene in head and neck cancer cells. Methods: To understand the role of lncMIR205HG, that we showed to be transcriptionally regulated by mutant p53 in HNSCC, we have employed siRNA and shRNA in CAL27 and FaDu HNSCC cell lines to suppress p53 gene expression in ChIP assays and RT-qPCR. We validated our findings in a cohort of 522 HNSCC patients from The Cancer Genome Atlas Data Portal (TCGA). We further evaluated our results in 63 HNSCC tumor samples collected at our institute, 32 of which were characterized by mutated TP53 (missense mutations) while 31 were characterized by wild-type TP53. Results: Maturation of pre-MIR205HG transcript produces two non-coding RNAs, lncMIR205HG and hsa-miR-205-5p. Down-regulation of lncMIR205HG expression significantly reduced cell proliferation, cell migration and clonogenic activity of head and neck cancer cells. Expression of MIR205HG was significantly increased in HNSCC with mutated TP53 when compared with matched non-tumoral tissues. Furthermore, MIR205HG expression levels were significantly higher in tumoral samples with mutant p53 than in tumoral tissues expressing wild-type p53. Mechanistically, MIR205HG depletes endogenous miR-590-3p leading to increased cyclin B, cdk1, and YAP protein expression. Conclusions: Taken together, these findings identify a transcriptional and post-transcriptional molecular network that includes mutant p53 protein, lncMIR205HG, YAP, and other proliferation-related genes, which are enriched in HNSCC patients with poor prognosis

p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data

<p>Abstract</p> <p>Background</p> <p>The p53 gene family consists of the three genes p53, p63 and p73, which have polyhedral non-overlapping functions in pivotal cellular processes such as DNA synthesis and repair, growth arrest, apoptosis, genome stability, angiogenesis, development and differentiation. These genes encode sequence-specific nuclear transcription factors that recognise the same responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumour progression or developmental disease. The discovery of several protein isoforms with antagonistic roles, which are produced by the expression of different promoters and alternative splicing, widened the complexity of the scenario of the transcriptional network of the p53 family members. Therefore, the identification of the genes transactivated by p53 family members is crucial to understand the specific role for each gene in cell cycle regulation. We have combined a genome-wide computational search of p53 family REs and microarray analysis to identify new direct target genes. The huge amount of biological data produced has generated a critical need for bioinformatic tools able to manage and integrate such data and facilitate their retrieval and analysis.</p> <p>Description</p> <p>We have developed the p53FamTaG database (p53 FAMily TArget Genes), a modular relational database, which contains p53 family direct target genes selected in the human genome searching for the presence of the REs and the expression profile of these target genes obtained by microarray experiments. p53FamTaG database also contains annotations of publicly available databases and links to other experimental data.</p> <p>The genome-wide computational search of the REs was performed using PatSearch, a pattern-matching program implemented in the DNAfan tool. These data were integrated with the microarray results we produced from the overexpression of different isoforms of p53, p63 and p73 stably transfected in isogenic cell lines, allowing the comparative study of the transcriptional activity of all the proteins in the same cellular background.</p> <p>p53FamTaG database is available free at <url>http://www2.ba.itb.cnr.it/p53FamTaG/</url></p> <p>Conclusion</p> <p>p53FamTaG represents a unique integrated resource of human direct p53 family target genes that is extensively annotated and provides the users with an efficient query/retrieval system which displays the results of our microarray experiments and allows the export of RE sequences. The database was developed for supporting and integrating high-throughput <it>in silico</it> and experimental analyses and represents an important reference source of knowledge for research groups involved in the field of oncogenesis, apoptosis and cell cycle regulation.</p

miR-10b*, a master inhibitor of the cell cycle, is down-regulated in human breast tumours

Deregulated proliferation is a hallmark of cancer cells. Here, we show that microRNA-10b* is a master regulator of breast cancer cell proliferation and is downregulated in tumoural samples versus matched peritumoural counterparts. Two canonical CpG islands (5kb) upstream from the precursor sequence are hypermethylated in the analysed breast cancer tissues. Ectopic delivery of synthetic microRNA-10b* in breast cancer cell lines or into xenograft mouse breast tumours inhibits cell proliferation and impairs tumour growth in vivo, respectively. We identified and validated in vitro and in vivo three novel target mRNAs of miR-10b* (BUB1, PLK1 and CCNA2), which play a remarkable role in cell cycle regulation and whose high expression in breast cancer patients is associated with reduced disease-free survival, relapse-free survival and metastasis-free survival when compared to patients with low expression. This also suggests that restoration of microRNA-10b* expression might have therapeutic promise

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

Crosstalk between c-Jun and TAp73α/β contributes to the apoptosis–survival balance

The p53-family member p73 plays a role in various cellular signaling pathways during development and growth control and it can have tumor suppressor properties. Several isoforms of p73 exist with considerable differences in their function. Whereas the functions of the N-terminal isoforms (TA and ΔNp73) and their opposing pro- and antiapoptotic roles have become evident, the functional differences of the distinct C-terminal splice forms of TAp73 have remained unclear. Here, we characterized the global genomic binding sites for TAp73α and TAp73β by chromatin immunoprecipitation sequencing as well as the transcriptional responses by performing RNA sequencing. We identified a specific p73 consensus binding motif and found a strong enrichment of AP1 motifs in close proximity to binding sites for TAp73α. These AP1 motif-containing target genes are selectively upregulated by TAp73α, while their mRNA expression is repressed upon TAp73β induction. We show that their expression is dependent on endogenous c-Jun and that recruitment of c-Jun to the respective AP1 sites was impaired upon TAp73β expression, in part due to downregulation of c-Jun. Several of these AP1-site containing TAp73α-induced genes impinge on apoptosis induction, suggesting an underlying molecular mechanism for the observed functional differences between TAp73α and TAp73β

ZEB1 Links p63 and p73 in a Novel Neuronal Survival Pathway Rapidly Induced in Response to Cortical Ischemia

Background: Acute hypoxic/ischemic insults to the forebrain, often resulting in significant cellular loss of the cortical parenchyma, are a major cause of debilitating injury in the industrialized world. A clearer understanding of the pro-death/ pro-survival signaling pathways and their downstream targets is critical to the development of therapeutic interventions to mitigate permanent neurological damage. Methodology/Principal Findings: We demonstrate here that the transcriptional repressor ZEB1, thought to be involved in regulating the timing and spatial boundaries of basic-Helix-Loop-Helix transactivator-mediated neurogenic determination/ differentiation programs, functions to link a pro-survival transcriptional cascade rapidly induced in cortical neurons in response to experimentally induced ischemia. Employing histological, tissue culture, and molecular biological read-outs, we show that this novel pro-survival response, initiated through the rapid induction of p63, is mediated ultimately by the transcriptional repression of a pro-apoptotic isoform of p73 by ZEB1. We show further that this phylogenetically conserved pathway is induced as well in the human cortex subjected to episodes of clinically relevant stroke. Conclusions/Significance: The data presented here provide the first evidence that ZEB1 induction is part of a protective response by neurons to ischemia. The stroke-induced increase in ZEB1 mRNA and protein levels in cortical neurons is both developmentally and phylogenetically conserved and may therefore be part of a fundamental cellular response to thi

Mutant p53 as a guardian of the cancer cell

Forty years of research have established that the p53 tumor suppressor provides a major barrier to neoplastic transformation and tumor progression by its unique ability to act as an extremely sensitive collector of stress inputs, and to coordinate a complex framework of diverse effector pathways and processes that protect cellular homeostasis and genome stability. Missense mutations in the TP53 gene are extremely widespread in human cancers and give rise to mutant p53 proteins that lose tumor suppressive activities, and some of which exert trans-dominant repression over the wild-type counterpart. Cancer cells acquire selective advantages by retaining mutant forms of the protein, which radically subvert the nature of the p53 pathway by promoting invasion, metastasis and chemoresistance. In this review, we consider available evidence suggesting that mutant p53 proteins can favor cancer cell survival and tumor progression by acting as homeostatic factors that sense and protect cancer cells from transformation-related stress stimuli, including DNA lesions, oxidative and proteotoxic stress, metabolic inbalance, interaction with the tumor microenvironment, and the immune system. These activities of mutant p53 may explain cancer cell addiction to this particular oncogene, and their study may disclose tumor vulnerabilities and synthetic lethalities that could be exploited for hitting tumors bearing missense TP53 mutations