ΔN Isoforms Research Articles

The dual role of p63 in cancer.

The p53 family is made up of three transcription factors: p53, p63, and p73. These proteins are well-known regulators of cell function and play a crucial role in controlling various processes related to cancer progression, including cell division, proliferation, genomic stability, cell cycle arrest, senescence, and apoptosis. In response to extra- or intracellular stress or oncogenic stimulation, all members of the p53 family are mutated in structure or altered in expression levels to affect the signaling network, coordinating many other pivotal cellular processes. P63 exists as two main isoforms (TAp63 and ΔNp63) that have been contrastingly discovered; the TA and ΔN isoforms exhibit distinguished properties by promoting or inhibiting cancer progression. As such, p63 isoforms comprise a fully mysterious and challenging regulatory pathway. Recent studies have revealed the intricate role of p63 in regulating the DNA damage response (DDR) and its impact on diverse cellular processes. In this review, we will highlight the significance of how p63 isoforms respond to DNA damage and cancer stem cells, as well as the dual role of TAp63 and ΔNp63 in cancer.

Computational Modeling of TP63-TP53 Interaction and Rational Design of Inhibitors: Implications for Therapeutics.

Tumor protein p63 (TP63) is a member of the TP53 protein family that are important for development and in tumor suppression. Unlike TP53, TP63 is rarely mutated in cancer, but instead different TP63 isoforms regulate its activity. TA isoforms (TAp63) act as tumor suppressors, whereas ΔN isoforms are strong drivers of squamous or squamous-like cancers. Many of these tumors become addicted to ΔN isoforms and removal of ΔN isoforms result in cancer cell death. Furthermore, some TP53 conformational mutants (TP53CM) gain the ability to interact with TAp63 isoforms and inhibit their antitumorigenic function, while indirectly promoting tumorigenic function of ΔN isoforms, but the exact mechanism of TP63-TP53CM interaction is unclear. The changes in the balance of TP63 isoform activity are crucial to understanding the transition between normal and tumor cells. Here, we modeled TP63-TP53CM complex using computational approaches. We then used our models to design peptides to disrupt the TP63-TP53CM interaction and restore antitumorigenic TAp63 function. In addition, we studied ΔN isoform oligomerization and designed peptides to inhibit its oligomerization and reduce their tumorigenic activity. We show that some of our peptides promoted cell death in a TP63 highly expressed cancer cell line, but not in a TP63 lowly expressed cancer cell line. Furthermore, we performed kinetic-binding assays to validate binding of our peptides to their targets. Our computational and experimental analyses present a detailed model for the TP63-TP53CM interaction and provide a framework for potential therapeutic peptides for the elimination of TP53CM cancer cells.

Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via β-Catenin/TCF/LEF Functions.

SummaryThe Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as β-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a β-catenin-dependent manner. Finally, β-catenin (ΔC) isoforms can rescue β-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.

Tmub1 Suppresses Hepatocellular Carcinoma by Promoting the Ubiquitination of ΔNp63 Isoforms

Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) inhibits hepatocyte proliferation during liver regeneration, but its role in hepatocellular carcinoma (HCC) has yet to be revealed. In this study, we show that the levels of Tmub1 were significantly lower in HCC tissues and cells than they were in adjacent tissues and normal hepatic cells, and the low levels of Tmub1 indicated a poor prognosis in HCC patients. Xenograft growth assay revealed that Tmub1 represses HCC growth in vivo. In addition, Tmub1 formed a protein complex with apoptosis-associated protein tumor protein 63 (p63), especially with the ΔN isoforms (ΔNp63α, β, and γ). Further loss- and gain-of-function analyses indicated that Tmub1 promotes apoptosis of Hep3B and MHCC-LM3 cells. Tmub1 decreased the protein expression of ΔNp63, and the pro-apoptotic effect of Tmub1 can be reversed by ΔNp63 isoforms (α, β, and γ). Additionally, we report that Tmub1 promotes the ubiquitination and degradation of ΔNp63 proteins. Finally, we confirmed in HCC tissues that Tmub1 is negatively correlated with ΔNp63 and positively correlated with the level of apoptosis. Taken together, Tmub1 suppresses HCC by enhancing the ubiquitination and degradation of ΔNp63 isoforms to induce HCC cell apoptosis. These findings provide a potential strategy for the management of HCC.

P63 modulates the expression of the WDFY2 gene which is implicated in cancer regulation and limb development.

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.

Expression of p53-family members in odontogenic cysts

Expression of p53-family members in odontogenic cysts

Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, induces apoptosis in TP53-deficient cancer cells

BackgroundThe p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated ΔN isoform that inhibits TAp73 and p53 and thus, acts as an oncogene.ResultsBy employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch. Finally, PpIX showed potent antitumor effect and inhibited the growth of xenograft human tumors in mice.ConclusionOur findings may in future contribute to the successful repurposing of PpIX into clinical practice.

Abstract 4440: Differential regulation of p63 isoforms by alternative polyadenylation

Abstract p63, a transcription factor belonging to the p53 family, plays vital roles in epidermal development and tumorigenesis. p63 expresses from two different promoters generating two classes of proteins, TAp63 and ΔNp63 that differ in their N-terminus. Moreover, alternative splicing of the TA and ΔN isoforms in the C-terminus creates additional isoforms (α-ϵ); thus generating five TA and five ΔN p63 isoforms. The factors that give rise to this repertoire of p63 isoforms are not very well understood. Alternative polyadenylation (APA) can generate multiple isoforms of a gene with alternative 3' ends based on the polyadenylation site selection during cleavage and polyadenylation of mRNA. APA can give rise to isoforms that differ in the coding region (CDS APA) or that have varying lengths of 3' UTR (3' UTR APA). Recently, PABPN1 protein that binds the 3' poly(A) tail of mRNAs has emerged as a key regulator of APA. Here, we show that PABPN1 differentially regulates p63α and p63γ isoforms by alternative polyadenylation. Specifically, knockdown of PABPN1 increases the levels of p63γ mRNA as well as protein by regulating CDS APA of p63 gene. Moreover, knockdown of PABPN1 also generates p63α mRNAs with varying 3' UTR lengths. Although the mRNA levels of p63α remain unchanged upon PABPN1 KD, p63α protein is significantly decreased, suggesting that PABPN1 also regulates p63α protein translation. Interestingly, we also show that PABPN1 is a p63α target and that p63 transcriptionally regulates PABPN1 by binding to its promoter forming a negative feedback loop. Taken together, our study identifies PABPN1 as a novel regulator of various p63 isoforms by APA and that p63 in turn regulates PABPN1 transcriptionally. Citation Format: Shakur Mohibi, Jin Zhang, Xinbin Chen. Differential regulation of p63 isoforms by alternative polyadenylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4440.

A double dealing tale of p63: an oncogene or a tumor suppressor.

As a member of tumor suppressor p53 family, p63, a gene encoding versatile protein variant, has been documented to correlate with cancer formation and progression, though it is rarely mutated in cancer patients. However, it has long been controversial on whether p63 is an oncogene or a tumor suppressor. Here, we comprehensively reviewed reports on roles of p63 in development, tumorigenesis and tumor progression. According to data from molecular cell biology, genetic models and clinic research, we conclude that p63 may act as either an oncogene or a tumor suppressor gene in different scenarios: TA isoforms of p63 gene are generally tumor-suppressive through repressing cell proliferation, survival and metastasis; ΔN isoforms, however, may initiate tumorigenesis via promoting cell proliferation and survival, but inhibit tumor metastasis and progression; effects of p63 on tumor formation and progression depend on the context of the whole p53 family, and either amplification or loss of p63 gene locus can break the balance to cause tumorigenesis.

STXBP4 Drives Tumor Growth and Is Associated with Poor Prognosis through PDGF Receptor Signaling in Lung Squamous Cell Carcinoma.

Purpose: Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be an SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis.Experimental Design: We carried out a clinicopathologic analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft-agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells.Results: Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman rank correlation ρ = 0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P < 0.001), disease stage (P = 0.030), and pleural involvement (P = 0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death, and survival in cancer. Platelet-derived growth factor receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft-agar and xenograft assays.Conclusions: STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC. Clin Cancer Res; 23(13); 3442-52. ©2017 AACR.

TAp63γ and ΔNp63γ are regulated by RBM38 via mRNA stability and have an opposing function in growth suppression

The p63 gene is expressed as TAp63 from the P1 promoter and as ΔNp63 from the P2 promoter. Through alternative splicing, five TA and five ΔN isoforms (α-ε) are expressed. Isoforms α-β and δ share an identical 3’ untranslated region (3’UTR) whereas isoform γ has a unique 3’UTR. Recently, we found that RBM38 RNA-binding protein is a target of p63 and RBM38 in turn regulates p63α/β expression via mRNA stability. However, it is uncertain whether p63γ has a unique biological activity and whether p63γ is regulated by RBM38. Here, we found that the levels of ΔNp63γ transcript and protein are induced upon overexpression of RBM38 but decreased by RBM38 knockdown. Conversely, we found that the levels of ΔNp63β transcript and protein are decreased by ectopic expression of RBM38 but increased by RBM38 knockdown, consistent with our previous report. Interestingly, RBM38 increases the half-life of p63γ mRNA by binding to a GU-rich element in p63γ 3’UTR. In contrast, our previous studies showed that RBM38 decreases the half-life of p63α/β mRNAs by binding to AU-/U-rich elements in their 3’UTR. We also found that knockout of p63γ in ME180 and HaCaT cells, in which ΔNp63 isoforms are predominant, inhibits cell proliferation and migration, suggesting that ΔNp63γ has a pro-growth activity. In contrast, we found that knockout of TAp63γ in MIA PaCa-2 cells, in which TAp63 isoforms are predominant, promotes cell proliferation, migration, and inhibits cellular senescence. Taken together, we conclude that ΔNp63γ has an oncogenic potential whereas TAp63γ is a tumor suppressor.

Role of p63 and p73 isoforms on the cell death in patients with hepatocellular carcinoma submitted to orthotopic liver transplantation.

Background & AimsPatients with hepatocellular carcinoma (HCC) submitted to orthotopic liver transplantation (OLT) have a variable 5-year survival rate limited mostly by tumor recurrence. The etiology, age, sex, alcohol, Child-Pugh, and the immunesuppressor have been associated with tumour recurrence. The expression of ΔNp73 is related to the reduced survival of patients with HCC. The study evaluated the expression of p63 and p73 isoforms and cell death receptors, and their relation to tumour recurrence and survival. The results were in vitro validated in HCC cell lines.MethodsHCC sections from patients submitted to OLT were used. The in vitro study was done in differentiated hepatitis B virus (HBV)-expressing Hep3B and control HepG2 cells. The expression of cell death receptors and cFLIPS/L, caspase-8 and -3 activities, and cell proliferation were determined in control and p63 and p73 overexpressing HCC cells.ResultsThe reduced tumor expression of cell death receptors and TAp63 and TAp73, and increased ΔNp63 and ΔNp73 expression were associated with tumor recurrence and reduced survival. The in vitro study demonstrated that HBV-expressing Hep3B vs HepG2 cells showed reduced expression of p63 and p73, cell death receptors and caspase activation, and increased cFLIPL/cFLIPS ratio. The overexpression of TAp63 and TAp73 exerted a more potent pro-apoptotic and anti-proliferative effects in Hep3B than HepG2-transfected cells which was related to cFLIPL upregulation.ConclusionsThe reduction of TAp63 and TAp73 isoforms, rather than alteration of ΔN isoform expression, exerted a significant functional repercussion on cell death and proliferation in HBV-expressing HepB cells.

Stem and progenitor cells of the mammalian olfactory epithelium: Taking poietic license.

The capacity of the olfactory epithelium (OE) for lifelong neurogenesis and regeneration depends on the persistence of neurocompetent stem cells, which self-renew as well as generating all of the cell types found within the nasal epithelium. This Review focuses on the types of stem and progenitor cells in the epithelium and their regulation. Both horizontal basal cells (HBCs) and some among the population of globose basal cells (GBCs) are stem cells, but the two types plays vastly different roles. The GBC population includes the basal cells that proliferate in the uninjured OE and is heterogeneous with respect to transcription factor expression. From upstream in the hierarchy to downstream, GBCs encompass 1) Sox2+ /Pax6+ stem-like cells that are totipotent and self-renew over the long term, 2) Ascl1+ transit-amplifying progenitors with a limited capacity for expansive proliferation, and 3) Neurog1+ /NeuroD1+ immediate precursor cells that make neurons directly. In contrast, the normally quiescent HBCs are activated to multipotency and proliferate when sustentacular cells are killed, but not when only OSNs die, indicating that HBCs are reserve stem cells that respond to severe epithelial injury. The master regulator of HBC activation is the ΔN isoform of the transcription factor p63; eliminating ΔNp63 unleashes HBC multipotency. Notch signaling, via Jagged1 ligand on Sus cells and Notch1 and Notch2 receptors on HBCs, is likely to play a major role in setting the level of p63 expression. Thus, ΔNp63 becomes a potential therapeutic target for reversing the neurogenic exhaustion characteristic of the aged OE. J. Comp. Neurol. 525:1034-1054, 2017. © 2016 Wiley Periodicals, Inc.

Differentiating Small Cell Carcinoma From Squamous Cell Carcinoma in Cytologic Specimens

Distinguishing small cell carcinoma (SCC) from poorly differentiated squamous cell carcinoma (SCCA) can be challenging on fine needle aspiration (FNA). p63 immunocytochemistry is frequently used to support the presence of squamous differentiation. However, issues relating to specificity have arisen, and the common pan-p63 clone (4A4) has been challenged by antibodies specific for the ΔN isoform of p63 (p40), which show superior specificity for SCCA. A total of 50 SCC and 25 SCCA FNA cell blocks were stained with antibodies for p40, p63, K903, and CK5/6. For each marker, both extent and intensity of immunoreactivity were recorded, blinded to diagnosis, and H-scores (extent×intensity) were calculated. A total of 4%, 34%, 4%, and 2% of SCCs were positive for p40, p63, K903, and CK5/6, respectively. The rate of p63 positivity was significantly higher than for the other markers. Median H-scores in p63-positive SCCs (40) and SCCAs (270) were significantly different (P<0.0001), though, and 94% of p63-positive SCCs showed an H-score <150. SCCs frequently express p63 (34%). In the SCCA versus SCC differential diagnosis, p40 and K903 are each marginally more sensitive (92%) than p63 (88%), whereas CK5/6 boasts the greatest specificity (98%). p63's poor specificity (66%) can be improved to 94%, if an H-score ≥150 is used as the cutoff for a positive result.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

BackgroundThe transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field.ResultsHere we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt’s and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions.ConclusionsIn this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1793-9) contains supplementary material, which is available to authorized users.

Elevated H3K18 acetylation in airway epithelial cells of asthmatic subjects.

BackgroundEpigenetic adjustments of the chromatin architecture through histone modifications are reactive to the environment and can establish chromatin states which are permissive or repressive to gene expression. Epigenetic regulation of gene expression is cell specific and therefore, it is important to understand its contribution to individual cellular responses in tissues like the airway epithelium which forms the mucosal barrier to the inhaled environment within the lung. The airway epithelium of asthmatics is abnormal with dysregulation of genes such as epidermal growth factor receptor (EGFR), the ΔN isoform of the transcription factor p63 (ΔNp63), and signal transducer and activator of transcription 6 (STAT6), integral to differentiation, proliferation, and inflammation. It is important to establish in diseases like asthma how histone modifications affect tissue responses such as proliferation and differentiation.ObjectivesTo characterize the global histone acetylation and methylation status in the epithelium of asthmatic compared to healthy subjects and to identify the impact of these variations on genes involved in epithelial functions.MethodsWhole lungs were obtained from healthy and asthmatic subjects (n = 6) from which airway epithelial cells (AECs) were isolated and airway sections were taken for analysis of histone lysine acetylation and methylation by immunohistochemistry. AECs were subjected to chromatin immunoprecipitation (ChIP) using anti-H3K18ac and anti-H3K4me2 antibodies followed by RT-PCR targeting ΔNp63, EGFR, and STAT6. AECs were also treated with TSA and changes in ΔNp63, EGFR, and STAT6 expression were determined.ResultsWe identified an increase in the acetylation of lysine 18 on histone 3 (H3K18ac) and trimethylation of lysine 9 on histone 3 (H3K9me3) in the airway epithelium of asthmatic compared to healthy subjects. We found increased association of H3K18ac around the transcription start site of ΔNp63, EGFR, and STAT6 in AECs of asthmatics. However, we were unable to modify the expression of these genes with the use of the HDAC inhibitor TSA in healthy subjects.DiscussionThe airway epithelium from asthmatic subjects displays increased acetylation of H3K18 and association of this mark around the transcription start site of ΔNp63, EGFR, and STAT6. These findings suggest a complex interaction between histone modifications and gene regulation in asthma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0254-y) contains supplementary material, which is available to authorized users.

Abstract SY18-03: Therapeutically targeting the p53 pathway using miRNA-based approaches in mouse models of cancer

Abstract The tumor suppressor gene, p53, is commonly mutated or lost in human cancer, and p53 reactivation has been shown to suppress tumors in-vivo. This strategy has proven difficult to implement therapeutically. One alternative strategy to overcome p53 loss is to manipulate the p53 family members, p63 and p73, which interact with p53 extensively to suppress tumorigenesis. Both p63 and p73 are composed of multiple isoforms. The isoforms can be placed into two groups: the TA isoforms, which structurally resemble p53 and act as tumor suppressors, and the ΔN isoforms, which bind to p53, TAp63, and TAp73 and inhibit their function, thus acting as oncogenes. Many current therapeutic strategies to target p53 focus on p53 solely, ignoring the existence of the other p53 family members. Over the last decade, we have learned that this family of genes acts together to suppress tumorigenesis and metastasis; therefore, a clear understanding of the cross talk between the p53 family as a whole is needed to effectively treat cancers with alterations in the p53 family of genes. Recently, my laboratory has shown that in vivo inactivation of ΔNp63 or ΔNp73 in the p53-deficient mouse model of thymic lymphoma leads to massive tumor regression and increased survival through upregulation of TAp63 and TAp73 and subsequent transactivation of a large network of genes and miRNAs identified through RNA sequencing (Venkatanarayan et al., Nature, 2014 Nov 17, doi: 10.1038/nature13910). Importantly, we have previously shown that Dicer and DGCR8 are transcriptional targets of p63 (Su et al., Nature, 2010 Oct 21;467(7318):986-90 and Chakravarti et al., Proc Natl Acad Sci U S A, 2014 Feb 4;111(5):E572-81. doi: 10.1073/pnas.1319743111). To identify inhibitors of ΔNp63 or ΔNp73 as a means to identify compounds that may be used to target the p53 pathway therapeutically, we screened a drug library of more than 850 FDA approved compounds. We found several small molecules that were able to reduce ΔNp63 protein stability and a specific set of miRNAs essential for the effectiveness of the drugs. In vivo analyses of these compounds as well as of these miRNA inhibitors have shown their efficacy in counteracting tumor formation and progression in genetically engineered mouse models deficient for p53 and in patient derived xenograft (PDX) mouse models with tumors that have p53 mutations. In summary, we have now identified multiple modalities to therapeutically target the p53 pathway through manipulation of p63 and p73. Citation Format: Elsa R. Flores. Therapeutically targeting the p53 pathway using miRNA-based approaches in mouse models of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY18-03. doi:10.1158/1538-7445.AM2015-SY18-03

IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo.

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the β cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.

Abstract 3402: ΔN-p63α and TA-p63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites

Abstract TP63 is a member of the p53 gene family that encodes for up to ten different TA- and ΔN- isoforms through differential promoter usage and alternative C-terminal splicing. The TA isoforms (TA-p63α, β, γ, δ and ε) contain the N-terminal transactivation domain (TA1), whereas the ΔN isoforms (ΔN-p63α, β, γ, δ and ε) are transcribed from an internal promoter (P2) and lack the TA1 domain. A second C-terminal transactivation domain (TA2) present in all p63α and β isoforms has been reported. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance (germline TP63 mutations are causative for a subset of human ectodermal dysplasia syndromes -EDs-), TA- and ΔN-p63 isoforms play an important role in tumorigenesis. More recently, p63 was shown to modulate apoptosis in the female and male germ line in response to DNA damage. All isoforms share a large, immunoglobulin-like folded DNA binding domain that is responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to the p53 RE. Since the ΔN-p63 isoforms lack the N-terminal transactivation domain, it was originally proposed that these proteins might act primarily as oncogenes through dominant-negative mechanisms. However, different studies indicate that ΔN-p63 protein itself can be transcriptionally active. For example, ΔN-p63α may directly contribute to tumorigenesis by up-regulating the chaperone protein Hsp70, which displays proliferative and anti-apoptotic functions or by repressing pro-apoptotic genes. Transcriptional activation of specific genes by ΔN-p63α, namely VDR and Id-3, has been also associated with an anti-tumorigenic role, i.e. a decrease in cell invasion. Using a defined functional assay in yeast where p63 isoform and RE sequence are the only variables (more than 80 different REs were tested), as well as mammalian-transcription assays (gene reporter assays, qPCR measurements, western blotting), we demonstrated that human TA- and ΔN-p63α proteins exhibited differences in transactivation specificity. In fact 21 REs were identified that exhibited higher or selective responsiveness to ΔN-p63α. These differences were not observed with the related p73 and p53 proteins isoforms and were dependent on specific features of the RE sequences. Based on gene annotations, we propose that cis-element sequence features might have been selected along with evolutionarily conserved, intrinsic differences in cooperative DNA binding of p63 proteins, to establish tighter control of the apoptotic processes. Citation Format: Yari Ciribilli, Alessandra Bisio, Paola Monti, Giorgia Foggetti, Ivan Raimondi, Paola Campomenosi, Paola Menichini, Gilberto Fronza, Alberto Inga. ΔN-p63α and TA-p63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3402. doi:10.1158/1538-7445.AM2014-3402

A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins.

p28 is an anionic, amphipathic, cell-penetrating peptide derived from the cupredoxin azurin that binds to the DNA-binding domain (DBD) of the tumor suppressor protein, p53, and induces a post-translational increase in the level of wild type and mutated p53 in a wide variety of human cancer cells. As p63 and p73, additional members of the p53 superfamily of proteins, also appear to be involved in the cellular response to cancer therapy and are reportedly required for p53-induced apoptosis, we asked whether p28 also binds to p63 and p73. Atomic force spectroscopy demonstrates that p28 forms a stable, high-affinity complex with full-length p63, the DBD of p63, and full-length p73. Exposure to p28 decreased the level of TAp63α and ΔNp63α, the truncated form of p63, in p53 wild type and mutated human breast cancer cells, respectively. p28 increased the level of TAp73α, but not ΔNp73α, in the same breast cancer cell lines. In contrast, p28 increased the level of the TA and ΔN isoforms of p63 in p53 wild type, but not in p53 mutated melanoma cells, while decreasing TA p73α in p53 wild type and mutated human melanoma cells. All changes were mirrored by an associated change in the expression of the HECT E3 ligases Itch/AIP4, AIP5, and the RING E3 ligase Pirh2, but not in the receptor for activated C kinase or the RING E3 ligases Mdm2 and Cop1. Collectively, the data suggest that molecules such as p28 bind with high affinity to the DBD of p63 and p73 and alter their expression independent of the Mdm2 and Cop1 pathways.