Recently, we established that a C>T single nucleotide polymorphism (SNP) in the promoter of the VEGF receptor FLT1 gene generates a (1/2) site p53 response element (RE-T) that results in p53 responsiveness of the promoter. The transcriptional control required an estrogen receptor (ER) (1/2) site response element (ERE1) 225 nt upstream to the RE-T.

Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity.

Cutaneous melanoma is the most aggressive form of skin cancer. Despite the significant advances in the management of melanoma in recent decades, it still represents a challenge for clinicians. The TP53 gene, the guardian of the genome, which is altered in more than 50% of human cancers, is rarely mutated in melanoma. More recently, researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of the p53-dependent responses. We analyzed the expression of p53 and p73 isoforms both at the RNA and protein level in a panel of melanoma-derived cell lines with different TP53 and BRAF status, in normal conditions or upon treatment with common anti-cancer DNA damaging agents or targeted therapy. Using lentiviral vectors, we also generated stable clones of H1299 p53 null cells over-expressing the less characterized isoforms Δ160p53α, Δ160p53β, and Δ160p53γ. Further, we obtained two melanoma-derived cell lines resistant to BRAF inhibitor vemurafenib. We observed that melanoma cell lines expressed a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable one. We demonstrated for the first time that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and that Δ160p53γ can localize in perinuclear foci; moreover, all Δ160p53 isoforms can stimulate proliferation and in vitro migration. Lastly, vemurafenib-resistant melanoma cells showed an altered expression of p53 and p73 isoforms, namely an increased expression of potentially pro-oncogenic Δ40p53β and a decrease in tumor-suppressive TAp73β. We therefore propose that p53 family isoforms can play a role in melanoma cells' aggressiveness.

TP63 is a member of the TP53 gene family that encodes for up to ten different TA and ∆N isoforms through alternative promoter usage and alternative splicing. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance, P63, through differential expression of its isoforms, plays important roles in tumorigenesis. All P63 isoforms share an immunoglobulin-like folded DNA binding domain responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to that of the canonical p53 RE. Using a defined assay in yeast, where P63 isoforms and RE sequences are the only variables, and gene expression assays in human cell lines, we demonstrated that human TA- and ∆N-P63α proteins exhibited differences in transactivation specificity not observed with the corresponding P73 or P53 protein isoforms. These differences 1) were dependent on specific features of the RE sequence, 2) could be related to intrinsic differences in their oligomeric state and cooperative DNA binding, and 3) appeared to be conserved in evolution. Sicen genotoxic stress can change relative ratio of TA- and ∆N-P63α protein levels, the different transactivation specificity of each P63 isoform could potentially influence cellular responses to specific stresses.

The tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair.

Fluctuations in mRNA levels only partially contribute to determine variations in mRNA availability for translation, producing the well-known poor correlation between transcriptome and proteome data. Recent advances in microscopy now enable researchers to obtain high resolution images of ribosomes on transcripts, providing precious snapshots of translation in vivo. Here we propose RiboAbacus, a mathematical model that for the first time incorporates imaging data in a predictive model of transcript-specific ribosome densities and translational efficiencies. RiboAbacus uses a mechanistic model of ribosome dynamics, enabling the quantification of the relative importance of different features (such as codon usage and the 5' ramp effect) in determining the accuracy of predictions. The model has been optimized in the human Hek-293 cell line to fit thousands of images of human polysomes obtained by atomic force microscopy, from which we could get a reference distribution of the number of ribosomes per mRNA with unmatched resolution. After validation, we applied RiboAbacus to three case studies of known transcriptome-proteome datasets for estimating the translational efficiencies, resulting in an increased correlation with corresponding proteomes. RiboAbacus is an intuitive tool that allows an immediate estimation of crucial translation properties for entire transcriptomes, based on easily obtainable transcript expression levels.

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.

c-Myc's role in pulmonary cancer metabolism is uncertain. We therefore investigated c-Myc activity in papillary lung adenocarcinomas (PLAC). Genomics revealed 90 significantly regulated genes (> 3-fold) coding for cell growth, DNA metabolism, RNA processing and ribosomal biogenesis and bioinformatics defined c-Myc binding sites (TFBS) at > 95% of up-regulated genes. EMSA assays at 33 novel TFBS evidenced DNA binding activity and ChIP-seq data retrieved from public repositories confirmed these to be c-Myc bound. Dual-luciferase gene reporter assays developed for RNA-Terminal-Phosphate-Cyclase-Like-1(RCL1), Ribosomal-Protein-SA(RPSA), Nucleophosmin/Nucleoplasmin-3(NPM3) and Hexokinase-1(HK1) confirmed c-Myc functional relevance and ChIP assays with HEK293T cells over-expressing ectopic c-Myc demonstrated enriched c-Myc occupancy at predicted TFBS for RCL1, NPM3, HK1 and RPSA. Note, c-Myc recruitment on chromatin was comparable to the positive controls CCND2 and CDK4. Computational analyses defined master regulators (MR), i.e. heterogeneous nuclear ribonucleoprotein A1, nucleolin, the apurinic/apyrimidinic endonuclease 1, triosephosphate-isomerase 1, folate transporter (SLC19A1) and nucleophosmin to influence activity of up to 90% of PLAC-regulated genes. Their expression was induced by 3-, 3-, 6-, 3-, 11- and 7-fold, respectively. STRING analysis confirmed protein-protein-interactions of regulated genes and Western immunoblotting of fatty acid synthase, serine hydroxyl-methyltransferase 1, arginine 1 and hexokinase 2 showed tumor specific induction. Published knock down studies confirmed these proteins to induce apoptosis by disrupting neoplastic lipogenesis, by endorsing uracil accumulation and by suppressing arginine metabolism and glucose-derived ribonucleotide biosynthesis. Finally, translational research demonstrated high expression of MR and of 47 PLAC up-regulated genes to be associated with poor survival in lung adenocarcinoma patients (HR 3.2 p < 0.001) thus, providing a rationale for molecular targeted therapies in PLACs.

We present a new model of ESR1 network regulation based on analysis of Doxorubicin, Estradiol, and TNFα combination treatment in MCF-7. We used Doxorubicin as a therapeutic agent, TNFα as marker and mediator of an inflammatory microenvironment and 17β-Estradiol (E2) as an agonist of Estrogen Receptors, known predisposing factor for hormone-driven breast cancer, whose pharmacological inhibition reduces the risk of breast cancer recurrence. Based on the results of transcriptomics analysis, we found 71 differentially expressed genes that are specific for the combination treatment with Doxorubicin + Estradiol + TNFα in comparison with single or double treatments. The responsiveness to the triple treatment was examined for seven genes by qPCR, of which six were validated, and then extended to four additional cell lines differing for p53 and/or ER status. The results of differential regulation enrichment analysis highlight the role of the ESR1 network that included 36 of 71 specific differentially expressed genes. We propose that the combined activation of p53 and NF-kB transcription factors significantly influences ligand-dependent, ER-driven transcriptional responses, also of the ESR1 gene itself. These results provide a model of coordinated interaction of TFs to explain the Doxorubicin, E2 and TNFα induced repression mechanisms.

Many recent studies using ChIP-seq approaches cross-referenced to trascriptome data and also to potentially unbiased in vitro DNA binding selection experiments are detailing with increasing precision the p53-directed gene regulatory network that, nevertheless, is still expanding. However, most experiments have been conducted in established cell lines subjected to specific p53-inducing stimuli, both factors potentially biasing the results.

Androgen Receptor (AR) and Estrogen Receptors (ERs) are key nuclear receptors that can cooperate in orchestrating gene expression programs in multiple tissues and diseases, targeting binding elements in promoters and distant enhancers. We report the unbiased identification of enhancer elements bound by AR and ER-α whose activity can be allele-specific depending on the status of nearby Single Nucleotide Polymorphisms (SNP). ENCODE data were computationally mined to nominate genomic loci with: (i) chromatin signature of enhancer activity from activation histone marks, (ii) binding evidence by AR and ER-α, (iii) presence of a SNP. Forty-one loci were identified and two, on 1q21.3 and 13q34, selected for characterization by gene reporter, Chromatin immunoprecipitation (ChIP) and RT-qPCR assays in breast (MCF7) and prostate (PC-3) cancer-derived cell lines. We observed allele-specific enhancer activity, responsiveness to ligand-bound AR, and potentially influence on the transcription of closely located genes (RAB20, ING1, ARHGEF7, ADAM15). The 1q21.3 variant, rs2242193, showed impact on AR binding in MCF7 cells that are heterozygous for the SNP. Our unbiased genome-wide search proved to be an efficient methodology to discover new functional polymorphic regulatory regions (PRR) potentially acting as risk modifiers in hormone-driven cancers and overall nominated SNPs in PRR across 136 transcription factors.

The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.

Breast cancer treatment often includes Doxorubicin as adjuvant as well as neoadjuvant chemotherapy. Despite its cytotoxicity, cells can develop drug resistance to Doxorubicin. Uncovering pathways and mechanisms involved in drug resistance is an urgent and critical aim for breast cancer research oriented to improve treatment efficacy. Here we show that Doxorubicin and other chemotherapeutic drugs induce the expression of ETV7, a transcriptional repressor member of ETS family of transcription factors. The ETV7 expression led to DNAJC15 down-regulation, a co-chaperone protein whose low expression was previously associated with drug resistance in breast and ovarian cancer. There was a corresponding reduction in Doxorubicin sensitivity of MCF7 and MDA-MB-231 breast cancer cells. We identified the binding site for ETV7 within DNAJC15 promoter and we also found that DNA methylation may be a factor in ETV7-mediated DNAJC15 transcriptional repression. These findings of an inverse correlation between ETV7 and DNAJC15 expression in MCF7 cells in terms of Doxorubicin resistance, correlated well with treatment responses of breast cancer patients with recurrent disease, based on our analyses of reported genome-wide expression arrays. Moreover, we demonstrated that ETV7-mediated Doxorubicin-resistance involves increased Doxorubicin efflux via nuclear pumps, which could be rescued in part by DNAJC15 up-regulation. With this study, we propose a novel role for ETV7 in breast cancer, and we identify DNAJC15 as a new target gene responsible for ETV7-mediated Doxorubicin-resistance. A better understanding of the opposing impacts of Doxorubicin could improve the design of combinatorial adjuvant regimens with the aim of avoiding resistance and relapse.

Unlike other tumours, TP53 is rarely mutated in melanoma; however, it fails to function as a tumour suppressor. We assume that its functions might be altered through interactions with several families of proteins, including p53/p73, NME and GLI. To elucidate the potential interplay among these families we analysed the expression profiles of aforementioned genes and proteins in a panel of melanoma cell lines, metastatic melanoma specimens and healthy corresponding tissue. Using qPCR a higher level of NME1 gene expression and lower levels of Δ40p53β, ΔNp73, GLI1, GLI2 and PTCH1 were observed in tumour samples compared to healthy tissue. Protein expression of Δ133p53α, Δ160p53α and ΔNp73α isoforms, NME1 and NME2, and N'ΔGLI1, GLI1FL, GLI2ΔN isoforms was elevated in tumour tissue, whereas ∆Np73β was downregulated. The results in melanoma cell lines, in general, support these findings. In addition, we correlated expression profiles with clinical features and outcome. Higher Δ133p53β and p53α mRNA and both GLI1 mRNA and GLI3R protein expression had a negative impact on the overall survival. Shorter overall survival was also connected with lower p53β and NME1 gene expression levels. In conclusion, all examined genes may have implications in melanoma development and functional inactivity of TP53.

The tumor suppressor p53 was previously shown to markedly up-regulate the expression of the PRODH gene, encoding the proline dehydrogenase (PRODH) enzyme, which catalyzes the first step in proline degradation. Also PRODH2, which degrades 4-hydroxy-L-proline, a product of protein (e.g. collagen) catabolism, was recently described as a p53 target. Here, we confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin. We established that over-expression of TAp73β or TAp63β is sufficient to induce PRODH expression in p53-null cells and that PRODH expression parallels the modulation of endogenous p73 by genotoxic drugs in several cell lines. The p53, p63, and p73-dependent transcriptional activation was linked to specific intronic response elements (REs), among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay. p53 occupancy measurements were validated in HCT116 and MCF7 human cell lines. Conversely, PRODH2 was not responsive to p63 nor p73 and, at best, could be considered a weak p53 target. In fact, minimal levels of PRODH2 transcript induction by genotoxic stress was observed exclusively in one of four p53 wild-type cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed very weak responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH, but not PRODH2, expression is under the control of p53 family members, specifically p53 and p73. This supports a deeper link between proteins of the p53-family and metabolic pathways, as PRODH modulates the balance of proline and glutamate levels and those of their derivative alpha-keto-glutarate (α-KG) under normal and pathological (tumor) conditions.

[This corrects the article DOI: 10.18632/oncotarget.21981.].

TP63 is a member of the TP53 gene family, sharing a common gene structure that produces two groups of mRNAs' encoding proteins with different N-terminal regions (ΔN and TA isoforms); both transcripts are also subjected to alternative splicing mechanisms at C-terminus, generating a variety of isoforms. p63 is a master regulator of epidermal development and homoeostasis as well as an important player in tumorigenesis and cancer progression with both oncogenic and tumour suppressive roles. A number of studies have aimed at the identification of p63 target genes, allowing the dissection of the molecular pathways orchestrated by the different isoforms. In the present study we investigated in more detail the p63 responsiveness of the WDFY2 (WD repeat and FYVE domain containing 2) gene, encoding for an endosomal protein identified as a binding partner of the PI-3K/AKT signalling pathway. We showed that overexpression of different p63 isoforms was able to induce WDFY2 expression in TP53-null cells. The p63-dependent transcriptional activation was associated with specific response elements (REs) that have been identified by a bioinformatics tool and validated by yeast- and mammal-based assays. Interestingly, to confirm that WDFY2 belongs to the p63 network of cancer regulation, we analysed the impact of WDFY2 alterations, by showing its frequent deletion in different types of tumours and suggesting its expression level as a prognostic biomarker. Lastly, we identified a chromosomal translocation involving the WDFY2 locus in a patient affected by a rare congenital limb anomaly, indicating WDFY2 as a possible susceptibility gene placed downstream p63 in the network of limb development.

Cancer stem cells (CSCs) represent a population of cells within the tumor able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy and radiotherapy. In this work, we show a new role for ETV7, a transcriptional repressor member of the ETS family, in promoting breast cancer stem-like cells plasticity and resistance to chemo- and radiotherapy in breast cancer (BC) cells. We observed that MCF7 and T47D BC-derived cells stably over-expressing ETV7 showed reduced sensitivity to the chemotherapeutic drug 5-fluorouracil and to radiotherapy, accompanied by an adaptive proliferative behavior observed in different culture conditions. We further noticed that alteration of ETV7 expression could significantly affect the population of breast CSCs, measured by CD44+/CD24low cell population and mammosphere formation efficiency. By transcriptome profiling, we identified a signature of Interferon-responsive genes significantly repressed in cells over-expressing ETV7, which could be responsible for the increase in the breast CSCs population, as this could be partially reverted by the treatment with IFN-β. Lastly, we show that the expression of the IFN-responsive genes repressed by ETV7 could have prognostic value in breast cancer, as low expression of these genes was associated with a worse prognosis. Therefore, we propose a novel role for ETV7 in breast cancer stem cells' plasticity and associated resistance to conventional chemotherapy and radiotherapy, which involves the repression of a group of IFN-responsive genes, potentially reversible upon IFN-β treatment. We, therefore, suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens, possibly involving the therapeutic use of IFN-β, with the aim of avoiding resistance development and relapse in breast cancer.

The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective.