PRIMA-1 induces autophagy in cancer cells carrying mutant or wild type p53

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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

Emerging role of Proline dehydrogenase in 2D and 3D growth of lung cancer cells resistant to tyrosine kinase inhibitors

Lung cancer is the leading cause of cancer death. Lung adenocarcinoma (LUAD), the prevalent subtype, is characterized by recurrent molecular alterations, such as EGFR mutations. These mutations make tumors susceptible to therapy with Tyrosine-Kinase Inhibitors (TKIs). However, resistance mechanisms occur and lead to cancer progression. Identification of prognostic and predictive biomarkers would be beneficial. Proline dehydrogenase (PRODH) is a mitochondrial flavoenzyme that catalyzes proline oxidation, electrons derived from the reaction can be used for ATP or ROS production. Thus, PRODH plays a critical role in cancer, affecting cellular processes such as survival, senescence, and apoptosis. We found higher PRODH expression in clinical LUAD samples, but not in LUAD cell lines. Interestingly, we observed increased PRODH expression in EGFR-mutant lung cell lines with in vitro acquired resistance to TKIs, compared to the TKI-sensitive parental lines. We hypothesize that PRODH may influence metastatic and survival capabilities during the development of resistance in lung cancer cells. The HCC827 cell line, harboring an EGFR exon 19 deletion, and its derivative, HCC827-GR5, with acquired resistance to Gefitinib, a second-generation TKI, were grown in both 2D and 3D to examine the role of PRODH in the TKI resistance. PRODH was either silenced or inhibited to evaluate its influence on cell survival, viability, and apoptosis. PRODH transcript and protein were increased in EGFR-mutant LUAD cell lines with in vitro acquired resistance to TKIs. Moreover, we observed that PRODH inhibition strongly impaired the survival and proliferation ability of HCC827-GR5 cells. In TKI-resistant cells, PRODH inhibition determined an increased expression of cell cycle arrest (p21 mRNA) and apoptotic (PUMA mRNA, cleaved caspase 8) markers, together with an increase in Annexin V positive cells, suggesting that PRODH inhibition may induce a combination of growth arrest and cell death. Furthermore, PRODH inhibition affected the 3D growth and viability of TKI-resistant, EGFR-mutated, lung cancer cells. Our results suggest that PRODH may be involved in the onset of TKI resistance. Specifically, PRODH inhibition or silencing impaired the growth of EGFR mutant, TKI-resistant cells in both 2D and 3D, while did not affect their TKI-sensitive parental cells. PRODH may potentially serve as a biomarker for predicting TKI-resistant LUAD therapy

A dual role of Proline Dehydrogenase in Lung Cancer

Lung cancer is still a global health problem. Present therapeutic approaches, among which Tyrosine Kinase Inhibitors (TKIs), have improved outcomes of patients with lung adenocarcinoma (LUAD). However, resistance mechanisms occur. Proline dehydrogenase (PRODH) could be a novel target and/or biomarker of therapy response in EGFR mutant LUADs. In mitochondria, PRODH oxidizes proline to pyrroline 5-carboxylate. The electrons produced during the reaction can be used to generate ATP or ROS species, thus affecting several cellular processes, such as survival, senescence, and apoptosis, playing a critical role in cancer. Using different cellular models, we have shown a negative correlation between EGFR and PRODH expression, which appears to be mediated by STAT3. We hypothesized that STAT3 decreases PRODH expression, by recognizing specific response elements (REs) in the PRODH gene (Aim 1).Concurrently, we observed an increase in PRODH expression in EGFR mutant LUAD cell lines with in vitro acquired resistance to EGFR TKIs, compared to their parental cell lines, and asked if PRODH could play a role in acquired resistance (Aim 2). Luciferase assays were carried out in different cell lines using constructs carrying PRODH intronic regions containing putative STAT3 binding sites identified bioinformatically. To investigate PRODH role in acquired resistance to TKIs, we carried out PRODH silencing or inhibition in HCC827 cells, which carry EGFR exon 19 p.E746-A750del, and in its derivative HCC827-GR5 (2nd gen. TKI, gefitinib-resistant) cells, grown in 2D or 3D, and evaluated viability, and apoptosis. Results 1. We observed luciferase activity upon STAT3 inhibition in DU145 cells compared to those treated with vehicle. Luciferase activity instead decreased when we ectopically expressed STAT3 in NCI-H1299 cells. Our data suggest that STAT3 may directly bind its specific REs in the PRODH gene to repress its expression and favor cell growth. Results 2. We observed that PRODH inhibition strongly impaired 2D and 3D growth of EGFR mutant, resistant LUAD cells but instead favored the growth of the parental cell line, in line with previous data. Interestingly, in TKI-resistant cells, PRODH inhibition determined an increased expression of cell cycle arrest (p21) and apoptotic (PUMA, caspase 8) markers. From the data shown here, PRODH may exert different roles in early- and late-stage lung cancer and upon acquired drug resistance, based on the activated signaling pathways

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