Publications by authors named "Gilberto Fronza"

56 Publications

Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay.

Genes (Basel) 2021 Feb 15;12(2). Epub 2021 Feb 15.

Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy.

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.
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http://dx.doi.org/10.3390/genes12020277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919268PMC
February 2021

Validation of the Alternative International Prognostic Score-E (AIPS-E): Analysis of Binet stage A chronic lymphocytic leukemia patients enrolled into the O-CLL1-GISL protocol.

Eur J Haematol 2021 Mar 4. Epub 2021 Mar 4.

Hematology Unit AO of Cosenza, Cosenza, Italy.

Objectives: To validate the predictive value on time to first treatment (TTFT) of AIPS-E and IPS-E evaluated in an independent cohort of newly diagnosed and non-referred Binet stage A CLL patients enrolled in the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540).

Methods: A cohort of 292 newly diagnosed Binet A CLL cases has been enrolled in the study. Patients from several Italian Institutions were prospectively enrolled within 12 months of diagnosis into the O-CLL1-GISL protocol.

Results: The majority of patients were male (62%); median age was 60.4 years, 102 cases (34.9%) showed unmutated IGHV genes, 8 cases (2.8) the presence of del(11q)/del(17p), 142 cases (48.6%) the presence of palpable lymph nodes and 146 cases (50%) and ALC > 15 × 10 /l. After a median follow-up of 7.2 years, 130 patients underwent treatment. According to the AIPS-E, 96 patients were classified as low-risk, 128 as intermediate-risk, and 68 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.71 (P < .0001) for predicting TTFT. According to IPS-E, 77 patients were classified as low-risk, 135 as intermediate-risk, and 80 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.705 (P < .0001) for predicting TTFT.

Conclusions: Our data confirm an accurate prognostic utility of both AIPS-E and IPS-E at the individual patient level. These data may be useful for a precise stratification of early-stage patients.
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http://dx.doi.org/10.1111/ejh.13614DOI Listing
March 2021

Potential Role of miRNAs in the Acquisition of Chemoresistance in Neuroblastoma.

J Pers Med 2021 Feb 7;11(2). Epub 2021 Feb 7.

Department of Experimental Medicine, University of Genova, 16100 Genova, Italy.

Neuroblastoma (NB) accounts for about 8-10% of pediatric cancers, and the main causes of death are the presence of metastases and the acquisition of chemoresistance. Metastatic NB is characterized by amplification that correlates with changes in the expression of miRNAs, which are small non-coding RNA sequences, playing a crucial role in NB development and chemoresistance. In the present study, miRNA expression was analyzed in two human -amplified NB cell lines, one sensitive (HTLA-230) and one resistant to Etoposide (ER-HTLA), by microarray and RT-qPCR techniques. These analyses showed that miRNA-15a, -16-1, -19b, -218, and -338 were down-regulated in ER-HTLA cells. In order to validate the presence of this down-regulation in vivo, the expression of these miRNAs was analyzed in primary tumors, metastases, and bone marrow of therapy responder and non-responder pediatric patients. Principal component analysis data showed that the expression of miRNA-19b, -218, and -338 influenced metastases, and that the expression levels of all miRNAs analyzed were higher in therapy responders in respect to non-responders. Collectively, these findings suggest that these miRNAs might be involved in the regulation of the drug response, and could be employed for therapeutic purposes.
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http://dx.doi.org/10.3390/jpm11020107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916079PMC
February 2021

Antitumor Effects of PRIMA-1 and PRIMA-1 (APR246) in Hematological Malignancies: Still a Mutant P53-Dependent Affair?

Cells 2021 Jan 7;10(1). Epub 2021 Jan 7.

Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy.

Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repair, cell migration, autophagy, and cell metabolism, the tumor suppressor gene is a key player for cellular homeostasis. gene is mutated in more than 50% of human cancers, although its overall dysfunction may be even more frequent. mutations are detected in a lower percentage of hematological malignancies compared to solid tumors, but their frequency generally increases with disease progression, generating adverse effects such as resistance to chemotherapy. Due to the crucial role of P53 in therapy response, several molecules have been developed to re-establish the wild-type P53 function to mutant P53. PRIMA-1 and its methylated form PRIMA-1 (also named APR246) are capable of restoring the wild-type conformation to mutant P53 and inducing apoptosis in cancer cells; however, they also possess mutant P53-independent properties. This review presents the activities of PRIMA-1 and PRIMA-1/APR246 and describes their potential use in hematological malignancies.
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http://dx.doi.org/10.3390/cells10010098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827888PMC
January 2021

Comparison of ibrutinib and idelalisib plus rituximab in real-life relapsed/resistant chronic lymphocytic leukemia cases.

Eur J Haematol 2021 Apr 28;106(4):493-499. Epub 2021 Jan 28.

Biothecnology Research Unit, AO of Cosenza, Cosenza, Italy.

Objectives: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only.

Methods: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study.

Results: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders.

Conclusions: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.
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http://dx.doi.org/10.1111/ejh.13573DOI Listing
April 2021

MicroRNA-Mutant P53 Crosstalk in Chemoresistance: A Hint to Monitor Therapy Outcome.

Microrna 2020 ;9(5):322-335

Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, Largo R. Benzi 10, Genoa, Italy.

The chemoresistance of cancer cells is a multifactorial mechanism in which de-regulated apoptotic pathways, the oxidative response and cancer cell migration play a crucial role. A key player in the control of such pathways is the tumor suppressor gene TP53, also defined as the "guardian of the genome", encoding the P53 tetrameric transcription factor. P53, following cell injuries, can activate the transcription of several target genes crucial for the induction of apoptosis, cell cycle arrest, modulation of senescence, DNA repair, autophagy and metabolism. Importantly, TP53 gene is mutated in nearly 50% of human cancers, implying an altered expression of target genes in cancer cells. The presence of TP53 mutations can also affect the expression of several small noncoding RNAs (microRNAs or miRNAs) involved in the same regulation of the apoptotic signaling, cell cycle regulation and cell migration. In mutant P53 expressing tumors, some miRNAs resulted in being down-regulated, while others appeared to be up-regulated as demonstrated by in vitro and in vivo studies. Thus, the expression level of specific P53 responsive miRNAs could be used as a marker of cancer progression and therapy performance. In the present review, we will summarize the role of P53-related miRNAs and their clinical relevance in monitoring therapy outcome and progression of cancers with mutant P53.
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http://dx.doi.org/10.2174/2211536609666201209151659DOI Listing
January 2020

Heterogeneity of Mutations and P53 Protein Residual Function in Cancer: Does It Matter?

Front Oncol 2020 28;10:593383. Epub 2020 Oct 28.

Mutagenesis and Cancer Prevention Unit, Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS) Ospedale Policlinico San Martino, Genoa, Italy.

The human locus, located on the short arm of chromosome 17, encodes a tumour suppressor protein which functions as a tetrameric transcription factor capable of regulating the expression of a plethora of target genes involved in cell cycle arrest, apoptosis, DNA repair, autophagy, and metabolism regulation. is the most commonly mutated gene in human cancer cells and germ-line mutations are responsible for the cancer-prone Li-Fraumeni syndrome. When mutated, the gene generally presents missense mutations, which can be distributed throughout the coding sequence, although they are found most frequently in the central DNA binding domain of the protein. mutations represent an important prognostic and predictive marker in cancer. The presence of a mutation does not necessarily imply a complete P53 inactivation; in fact, mutant P53 proteins are classified based on the effects on P53 protein function. Different models have been used to explore these never-ending facets of mutations, generating abundant experimental data on their functional impact. Here, we briefly review the studies analysing the consequences of mutations on P53 protein function and their possible implications for clinical outcome. The focus shall be on Chronic Lymphocytic Leukemia (CLL), which also has generated considerable discussion on the role of mutations for therapy decisions.
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http://dx.doi.org/10.3389/fonc.2020.593383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655923PMC
October 2020

Time to first treatment and P53 dysfunction in chronic lymphocytic leukaemia: results of the O-CLL1 study in early stage patients.

Sci Rep 2020 10 28;10(1):18427. Epub 2020 Oct 28.

Biotechnology Research Unit, Aprigliano, A.O./ASP of Cosenza, 87100, Cosenza, Italy.

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.
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http://dx.doi.org/10.1038/s41598-020-75364-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595214PMC
October 2020

TP53 dysfunction in chronic lymphocytic leukemia: clinical relevance in the era of B-cell receptors and BCL-2 inhibitors.

Expert Opin Investig Drugs 2020 Aug 27;29(8):869-880. Epub 2020 Jun 27.

Department of Experimental Medicine, University of Genoa , Genoa, Italy.

Introduction: Patients with dysfunction, assessed by del(17p) or mutations, respond poorly to chemo-immunotherapy and fare better with the new therapies (BCR and BCL-2 inhibitors); however, it is unclear whether their response is similar to that of patients without anomalies or whether there is currently an adequate determination of dysfunction.

Area Covered: A literature search was undertaken on clinical trials and real-world experience data on patients with dysfunction treated with different protocols. Moreover, data on the biological function and on the tests currently employed for its assessment were reviewed.

Expert Opinion: Although dysfunction has less negative influence on the new biological therapies, patients with these alterations, particularly those with biallelic inactivation of , have a worst outcome with these therapies than those without alterations. At present, a determination of , particularly with next generation sequencing (NGS) methodologies, may be sufficient for the identifications of the patients unsuitable for chemo-immunotherapy, although integration with del(17p) would be advisable. For the future, more extensive determinations of the status, including functional assays, may become part of the current armamentarium for a better patient stratification and treatment with newer protocols.
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http://dx.doi.org/10.1080/13543784.2020.1783239DOI Listing
August 2020

NEAT1 Long Isoform Is Highly Expressed in Chronic Lymphocytic Leukemia Irrespectively of Cytogenetic Groups or Clinical Outcome.

Noncoding RNA 2020 Mar 13;6(1). Epub 2020 Mar 13.

Department of Oncology and Hemato-oncology, University of Milan, 20122 Milan, Italy.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lymphocytic leukemia (CLL) are still open questions. Herein, we investigated the significance of the lncRNA NEAT1 in CLL. We examined NEAT1 expression in 310 newly diagnosed Binet A patients, in normal CD19+ B-cells, and other types of B-cell malignancies. Although global NEAT1 expression level was not statistically different in CLL cells compared to normal B cells, the median ratio of NEAT1_2 long isoform and global NEAT1 expression in CLL samples was significantly higher than in other groups. NEAT1_2 was more expressed in patients carrying mutated genes. Concerning cytogenetic aberrations, NEAT1_2 expression in CLL with trisomy 12 was lower with respect to patients without alterations. Although global NEAT1 expression appeared not to be associated with clinical outcome, patients with the lowest NEAT1_2 expression displayed the shortest time to first treatment; however, a multivariate regression analysis showed that the NEAT1_2 risk model was not independent from other known prognostic factors, particularly the IGHV mutational status. Overall, our data prompt future studies to investigate whether the increased amount of the long NEAT1_2 isoform detected in CLL cells may have a specific role in the pathology of the disease.
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http://dx.doi.org/10.3390/ncrna6010011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7151605PMC
March 2020

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

Biosci Rep 2019 12;39(12)

Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, Largo R. Benzi, 10, Genoa 16132, Italy.

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.
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http://dx.doi.org/10.1042/BSR20192114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914664PMC
December 2019

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations.

Biochim Biophys Acta Gen Subj 2020 01 16;1864(1):129440. Epub 2019 Sep 16.

LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal. Electronic address:

Background: Half of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.

Methods And Results: By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.

Conclusions: SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.

General Significance: This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.
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http://dx.doi.org/10.1016/j.bbagen.2019.129440DOI Listing
January 2020

Yeast As a Chassis for Developing Functional Assays to Study Human P53.

J Vis Exp 2019 08 4(150). Epub 2019 Aug 4.

Department CIBIO, University of Trento;

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.
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http://dx.doi.org/10.3791/59071DOI Listing
August 2019

Autophagy induced by SAHA affects mutant P53 degradation and cancer cell survival.

Biosci Rep 2019 02 19;39(2). Epub 2019 Feb 19.

Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, Genoa 16132, Italy

Missense mutations in the gene produce mutant p53 (mutp53) proteins which may acquire oncogenic properties favoring chemoresistance, cell migration, and metastasis. The exploitation of cellular pathways that promote mutp53 degradation may reduce cell proliferation and invasion as well as increase the sensitivity to anticancer drugs, with a strong impact on current cancer therapies. In the last years, several molecules have been characterized for their ability to induce the degradation of mutp53 through the activation of autophagy. Here, we investigated the correlation between autophagy and mutp53 degradation induced by suberoylanilide hydroxamic acid (SAHA), an FDA-approved histone deacetylase inhibitor. In the human cancer lines MDA-MB-231 (mutp53-R280K) and DLD1 (mutp53-S241F), SAHA induced a significant mutp53 degradation. However, such degradation correlated with autophagy induction only in MDA-MB-231 cells, being counteracted by autophagy inhibition, which also increased SAHA-induced cell death. Conversely, in DLD1 cells SAHA triggered a low level of autophagy despite promoting a strong decrease in mutp53 level, and autophagy inhibition did not change either mutp53 levels or sensitivity to this drug. We conclude that autophagy can be a relevant pathway for mutp53 degradation induced by SAHA, but its contribution to mutp53 destabilization and the consequences on cell death are likely context-dependent.
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http://dx.doi.org/10.1042/BSR20181345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379511PMC
February 2019

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

Sci Rep 2018 09 13;8(1):13762. Epub 2018 Sep 13.

Department of Experimental Medicine, General Pathology Section, University of Genova, Genova, Italy.

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.
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http://dx.doi.org/10.1038/s41598-018-32195-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137223PMC
September 2018

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

Oncol Rep 2017 Oct 14;38(4):1985-1994. Epub 2017 Aug 14.

UOC Mutagenesis, Ospedale Policlinico San Martino, I-16132 Genova, Italy.

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.
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http://dx.doi.org/10.3892/or.2017.5903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652947PMC
October 2017

Gambogic acid counteracts mutant p53 stability by inducing autophagy.

Biochim Biophys Acta Mol Cell Res 2017 Feb 27;1864(2):382-392. Epub 2016 Nov 27.

U.O.C. Mutagenesi, IRCCS AOU San Martino-IST, 16132 Genova, Italy. Electronic address:

Mutant p53 (mutp53) proteins are frequently present at higher levels than the wild-type (wt) protein in tumors, and some of them can acquire oncogenic properties. Consistently, knockdown of mutp53 protein in human cancer cell lines leads to reduced cell proliferation and invasion as well as to an increased sensitivity to some anticancer drugs. Therefore, the exploitation of cellular pathways and/or molecules that promote mutp53 degradation may have a therapeutic interest. Recently, autophagy is emerging as an important pathway involved in the stability of mutp53. In this paper, we explored the autophagic potential of gambogic acid (GA), a molecule that stimulates the degradation of mutp53 and increases the sensitivity of cancer cells to chemotherapeutic agents. We demonstrated that GA may induce mutp53 degradation through autophagy in cancer cells expressing the p53-R280K (MDA-MB-231) and the p53-S241F (DLD1) proteins. The inhibition of autophagy with bafilomycin A1 or chloroquine counteracted mutp53 degradation by GA. However, the autophagy induction and mutp53 degradation affected cell survival and proliferation only at low GA concentrations. At higher GA concentrations, when cells undergo massive apoptosis, autophagy is no longer detectable by immuno-fluorescence analysis. We concluded that autophagy is a relevant pathway for mutp53 degradation in cancer cells but it contributes only partially to GA-induced cell death, in a time and dose-dependent manner.
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http://dx.doi.org/10.1016/j.bbamcr.2016.11.023DOI Listing
February 2017

Human transcription factors in yeast: the fruitful examples of P53 and NF-кB.

FEMS Yeast Res 2016 11 27;16(7). Epub 2016 Sep 27.

Centre for Integrative Biology, CIBIO, University of Trento, via Sommarive 9, 38123, Trento, Italy.

The observation that human transcription factors (TFs) can function when expressed in yeast cells has stimulated the development of various functional assays to investigate (i) the role of binding site sequences (herein referred to as response elements, REs) in transactivation specificity, (ii) the impact of polymorphic nucleotide variants on transactivation potential, (iii) the functional consequences of mutations in TFs and (iv) the impact of cofactors or small molecules. These approaches have found applications in basic as well as applied research, including the identification and the characterisation of mutant TF alleles from clinical samples. The ease of genome editing of yeast cells and the availability of regulated systems for ectopic protein expression enabled the development of quantitative reporter systems, integrated at a chosen chromosomal locus in isogenic yeast strains that differ only at the level of a specific RE targeted by a TF or for the expression of distinct TF alleles. In many cases, these assays were proven predictive of results in higher eukaryotes. The potential to work in small volume formats and the availability of yeast strains with modified chemical uptake have enhanced the scalability of these approaches. Next to well-established one-, two-, three-hybrid assays, the functional assays with non-chimeric human TFs enrich the palette of opportunities for functional characterisation. We review ∼25 years of research on human sequence-specific TFs expressed in yeast, with an emphasis on the P53 and NF-кB family of proteins, highlighting outcomes, advantages, challenges and limitations of these heterologous assays.
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http://dx.doi.org/10.1093/femsyr/fow083DOI Listing
November 2016

The CDKN2A/p16(INK) (4a) 5'UTR sequence and translational regulation: impact of novel variants predisposing to melanoma.

Pigment Cell Melanoma Res 2016 Mar 17;29(2):210-21. Epub 2015 Dec 17.

Department of Internal Medicine and Medical Specialties, DiMI, University of Genoa, Genoa, Italy.

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5'UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.-27del23, c.-93-91delAGG) were classified as causal mutations (score ≥3), along with the c.-21C>T, c.-34G>T, and c.-56G>T, which had already been studied by a subset of assays. The novel c.-42T>A as well as the previously described c.-67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.-14C>T, c.-20A>G, c.-25C>T+c.-180G>A, c.-30G>A, c.-40C>T, c.-45G>A, c.-59C>G, c.-87T>A, c.-252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5'UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16(INK) (4a) translation.
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http://dx.doi.org/10.1111/pcmr.12444DOI Listing
March 2016

Studying p53 family proteins in yeast: induction of autophagic cell death and modulation by interactors and small molecules.

Exp Cell Res 2015 Jan 28;330(1):164-77. Epub 2014 Sep 28.

REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto, Portugal. Electronic address:

In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions.
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http://dx.doi.org/10.1016/j.yexcr.2014.09.028DOI Listing
January 2015

Structural studies on mechanisms to activate mutant p53.

Subcell Biochem 2014 ;85:119-32

Instituto de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City, D.F., Mexico,

The design of a broad-spectrum cancer drug would provide enormous clinical benefits to treat cancer patients. Most of cancerous cells have a mutation in the p53 gene that results in an inactive mutant p53 protein. For this reason, p53 is a prime target for the development of a broad-spectrum cancer drug. To provide the atomic information to rationally design a drug to recover p53 activity is the main goal of the structural studies on mutant p53. We review three mechanisms that influence p53 activity and provide information about how reactivation of mutant p53 can be achieved: stabilization of the active conformation of the DNA-binding domain of the protein, suppression of missense mutations in the DNA-binding domain by a second-site mutation, and increased transactivation.
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http://dx.doi.org/10.1007/978-94-017-9211-0_7DOI Listing
December 2014

∆N-P63α and TA-P63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites.

Oncotarget 2014 Apr;5(8):2116-30

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4039150PMC
http://dx.doi.org/10.18632/oncotarget.1845DOI Listing
April 2014

TP53 mutants in the tower of babel of cancer progression.

Hum Mutat 2014 Jun 14;35(6):689-701. Epub 2014 Mar 14.

Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.

Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive, interfering, contact, structural, unfolded, misfolded, dimeric, monomeric, non-cooperative, unstable, supertrans, superstable, intragenic suppressor. TP53 mutants are many, more than 2,000 in fact, and they can be very diverse. Sporadic; germline; gain-of-function (GoF); oncogenic; rebel-angel; yin and yang; prion-like; metastasis-inducer; mediator of chemo-resistance; modifier of stemness. TP53 mutants can impact important cancer clinical variables, in multiple, often subtle ways, as revealed by cell-based assays as well as animal models. Here, we review studies investigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recent findings on how TP53 mutants can exhibit GoF properties. We also review reports on TP53 mutants' impact on cancer cell transcriptomes and studies with Li-Fraumeni patients trying to classify and predict phenotypes in relation to experimentally determined transcription fingerprints. Finally, we provide an example of the complexity of correlating TP53 mutant functionality to clinical variables in sporadic cancer patients. Conflicting results and limitations of experimental approaches notwithstanding, the study of TP53 mutants has provided a rich body of knowledge, mostly available in the public domain and accessible through databases, which is beginning to impact cancer intervention strategies.
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http://dx.doi.org/10.1002/humu.22514DOI Listing
June 2014

Comparison of the biological effects of MMS and Me-lex, a minor groove methylating agent: clarifying the role of N3-methyladenine.

Mutat Res 2014 Jan 7;759:45-51. Epub 2013 Nov 7.

Molecular Mutagenesis and DNA Repair Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy. Electronic address:

N3-methyladenine (3-mA), generated by the reaction of methylating agents with DNA, is considered a highly toxic but weakly mutagenic lesion. However, due to its intrinsic instability, some of the biological effects of the adduct can result from the formation of the corresponding depurination product [an apurinic (AP)-site]. Previously, we exploited Me-lex, i.e. {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2 carboxamido}propane, a minor groove equilibrium binder with selectivity for A/T rich sequences that efficiently reacts with DNA to afford 3-mA as the dominant product, to probe the biology of this lesion. Using human p53 cDNA as a target in a yeast system, a weak increase in mutagenicity was observed in the absence of Mag1 (3-methyladenine-DNA glycosylase 1, mag1), the enzyme devoted to remove 3-mA from DNA. Moreover, a significant increase in mutagenicity occurred in the absence of the enzymes involved in the repair of AP-sites (AP endonucleases 1 and 2, apn1apn2). Since methyl methanesulfonate (MMS) has been extensively used to explore the biological effects of 3-mA, even though it produces 3-mA in low relative yield, we compared the toxicity and mutagenicity induced by MMS and Me-lex in yeast. A mutagenesis reporter plasmid was damaged in vitro by MMS and then transformed into wild-type and Translesion Synthesis (TLS) Polζ (REV3) and Polη (RAD30) deficient strains. Furthermore, a mag1rad30 double mutant strain was constructed and transformed with the DNA plasmid damaged in vitro by Me-lex. The results confirm the important role of Polζ in the mutagenic bypass of MMS and Me-lex induced lesions, with Polη contributing only towards the bypass of Me-lex induced lesions, mainly in an error-free way. Previous and present results point towards the involvement of AP-sites, derived from the depurination of 3-mA, in the observed toxicity and mutagenicity.
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http://dx.doi.org/10.1016/j.mrfmmm.2013.10.004DOI Listing
January 2014

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code.

Nucleic Acids Res 2013 Oct 26;41(18):8637-53. Epub 2013 Jul 26.

Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, TN, 38060 Italy, Molecular Mutagenesis and DNA Repair Unit, IRCSS Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, 92093, USA and Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIEHS, NIH, RTP, NC, 27709, USA.

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.
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http://dx.doi.org/10.1093/nar/gkt657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794606PMC
October 2013

P53 family members modulate the expression of PRODH, but not PRODH2, via intronic p53 response elements.

PLoS One 2013 8;8(7):e69152. Epub 2013 Jul 8.

Department of Biotechnology and Life Sciences, DBSV, University of Insubria, Varese, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069152PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3704516PMC
February 2014

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

Biochim Biophys Acta 2013 Aug 29;1833(8):1904-13. Epub 2013 Mar 29.

IRCCS Azienda Ospedaliera Universitaria San Martino-IST, Genova, Italy.

PRIMA-1 is a chemical compound identified as a growth suppressor of tumor cells expressing mutant p53. We previously found that in the MDA-MB-231 cell line expressing high level of the mutant p53-R280K protein, PRIMA-1 induced p53 ubiquitination and degradation associated to cell death. In this study, we investigated the ability of PRIMA-1 to induce autophagy in cancer cells. In MDA-MB-231 and HCT116 cells, expressing mutant or wild type p53, respectively, autophagy occurred following exposure to PRIMA-1, as shown by acridine orange staining, anti-LC3 immunofluorescence and immunoblots, as well as by electron microscopy. Autophagy was triggered also in the derivative cell lines knocked-down for p53, although to a different extent than in the parental cells expressing mutant or wild type p53. In particular, while wild type p53 limited PRIMA-1 induced autophagy, mutant p53 conversely promoted autophagy, thus sustaining cell viability following PRIMA-1 treatment. Therefore, the autophagic potential of PRIMA-1, besides being cell context dependent, could be modulated in a different way by the presence of wild type or mutant p53. Furthermore, since both cell lines lacking p53 were more sensitive to the cytotoxic effect of PRIMA-1 than the parental ones, our findings suggest that a deregulated autophagy may favor cell death induced by this drug.
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http://dx.doi.org/10.1016/j.bbamcr.2013.03.020DOI Listing
August 2013