TP63 mutations are frequent in cutaneous melanoma, support UV etiology, but their role in melanomagenesis is unclear

In contrast to TP53, cancer development is rarely associated with mutations in the TP63 and TP73 genes. Recently, next generation sequencing analysis revealed that TP63 mutations are frequent, specifically in cutaneous melanomas. Cutaneous melanoma represents 4% of skin cancers but it is responsible for 80% of skin cancer related deaths. In the present study, we first determined whether all three members of the P53 family of transcription factors were found mutated in cutaneous melanomas by retrieving all TP53, TP63 and TP73 mutations from cBioPortal (http://www.cbioportal.org/). TP53 and TP63 were frequently mutated [15.0% (91/605) and 14.7% (89/605), respectively], while TP73 [1.5% (9/605)] was more rarely mutated (p<0.0001). A UV-mutation fingerprint was recognized for TP63 and TP73 genes. Then, we tried to evaluate the potential role of TP63 mutations as drivers or passengers in the tumorigenic process. In the former case, the amino acid substitutions should cause significant functional consequences on the main biochemical activity of the P63 protein, namely transactivation. The predicted effects of specific amino acid substitutions by two bioinformatics tools were rather different. Using a yeast-based functional assay, the observed hotspot mutant R379CP63 protein exhibited a substantial residual activity compared to the wild-type (>70%). This result does not support a major role of the mutant P63 protein in melanomagenesis while it is still consistent with the TP63 gene being a recorder of UV exposure. The TP63 mutation spectrum from cutaneous melanomas, when compared with that observed at the germinal level in patients affected by P63-associated diseases [ectodermal dysplasia syndromes, (EDs)], revealed significant differences. The TP63 mutations were more frequent at CpGs sites (p<0.0001) in EDs and at PyPy sites (p<0.0001) in cutaneous melanomas. The two spectra differed significantly (p<0.0001). We conclude that TP63 mutations are frequent in cutaneous melanoma, support UV etiology, but their role in melanomagenesis is unclear

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\u2019 encoding proteins with different N-terminal regions ( 06N 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

Tumor suppressor pathways shape EGFR-driven lung tumor progression and response to treatment.

In vivo modeling combined with CRISPR/Cas9-mediated somatic genome editing has contributed to elucidating the functional importance of specific genetic alterations in human tumors. Our recent work uncovered tumor suppressor pathways that affect EGFR-driven lung tumor growth and sensitivity to tyrosine kinase inhibitors and reflect the mutational landscape and treatment outcomes in the human disease

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

9noneTP63 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.Ciribilli, Yari; Bisio, Alessandra; Monti, Paola; Foggetti, Giorgia; Raimondi, Ivan; Campomenosi, Paola; Menichini, Paola; Fronza, Gilberto; Inga, AlbertoCiribilli, Yari; Bisio, Alessandra; Monti, Paola; Foggetti, Giorgia; Raimondi, Ivan; Campomenosi, Paola; Menichini, Paola; Fronza, Gilberto; Inga, Albert

EEC- and ADULT-AssociatedTP63Mutations Exhibit Functional Heterogeneity Toward P63 Responsive Sequences

TP63 germ-line mutations are responsible for a group of human ectodermaldysplasia syndromes, underlining the key role of P63 in the development ofectoderm-derived tissues. Here, we report the identification of two TP63 alleles,G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identifiedG134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized inyeast, studied in a mammalian cell line and modeled based on the crystalstructure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showedboth complete loss of transactivation function and ability to interfere overwild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63\u3b1-specific REs derived from genesclosely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinctfunctional effect of each mutant. The present results highlight the importance ofintegrating different functional endpoints that take in account the features ofP63 proteins' target sequences to examine the impact of TP63 mutations and theassociated clinical variability

DataSheet1_Mutant p53K120R expression enables a partial capacity to modulate metabolism.pdf

The TP53 tumor suppressor gene is one of the most studied gene in virtue of its ability to prevent cancer development by regulating apoptosis, cell cycle arrest, DNA repair, autophagy and senescence. Furthermore, the modulation of metabolism by P53 is fundamental for tumor suppressor activity. Studies in mouse models showed that mice carrying TP53 mutations affecting the acetylation in the DNA binding domain still retain the ability to transactivate genes involved in metabolism. Noteworthy, mice expressing the triple 3KR or the single K117R mutant do not show early on-set tumor development in contrast to TP53−/− mice. Interestingly, the mouse K117R mutation corresponds to the human tumor-derived K120R modification, which abrogates P53-dependent activation of apoptosis without affecting growth arrest. In this study, we investigated the property of the human P53 K120R mutant in the regulation of metabolism by analyzing the transcriptional specificity in yeast- and mammalian-based reporter assays, the metabolic phenotype associated to its expression in colon cancer HCT116TP53−/− cells and the induction of P53 targets and proteins involved in the antioxidant response. These properties were analyzed in comparison to wild type P53 protein, the human triple mutant corresponding to mouse 3KR and the cancer hot-spot R273H mutant. We confirm the selective functionality of P53 K120R mutant, which shows a transcriptional activity on cell cycle arrest but not on apoptotic targets. Interestingly, this mutant shows a partial transactivation activity on p53 response element belonging to the metabolic target TIGAR. Moreover, we observe a significant uncoupling between oxygen consumption and ATP production associated with higher lipid peroxidation level in all P53 mutants carrying cells with respect to wild type P53 expressing cells. Noteworthy, in the absence of a pro-oxidative challenge, cells expressing K120R mutant retain a partial capacity to modulate glucose metabolism, limiting lipid peroxidation with respect to the other P53 mutants carrying cells. Lastly, especially in presence of human 3KR mutant, a high expression of proteins involved in the antioxidant response is found. However, this response does not avoid the increased lipid peroxidation, confirming that only wild type P53 is able to completely counteract the oxidative stress and relative damages.</p

Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation

Abstract Drug resistance is the major obstacle in successfully treating high-risk neuroblastoma. The aim of this study was to investigate the basis of etoposide-resistance in neuroblastoma. To this end, a MYCN-amplified neuroblastoma cell line (HTLA-230) was treated with increasing etoposide concentrations and an etoposide-resistant cell line (HTLA-ER) was obtained. HTLA-ER cells, following etoposide exposure, evaded apoptosis by altering Bax/Bcl2 ratio. While both cell populations shared a homozygous TP53 mutation encoding a partially-functioning protein, a mono-allelic deletion of 13q14.3 locus, where the P53 inducible miRNAs 15a/16-1 are located, and the consequent miRNA down-regulation were detected only in HTLA-ER cells. This event correlated with BMI-1 oncoprotein up-regulation which caused a decrease in p16 tumor suppressor content and a metabolic adaptation of HTLA-ER cells. These results, taken collectively, highlight the role of miRNAs 15a/16-1 as markers of chemoresistance

Genetic Determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo.

In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2-the strongest drivers of growth in a KRAS-driven model-reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment. SIGNIFICANCE: By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease.This article is highlighted in the In This Issue feature, p. 1601