Publications by authors named "Paola Monti"

50 Publications

Review of Histological Grading Systems in Veterinary Medicine.

Vet Pathol 2021 Mar 26:300985821999831. Epub 2021 Mar 26.

Department of Veterinary Medicine (DIMEVET), University of Milano, Lodi (LO), Italy.

Tumor grading is a method to quantify the putative clinical aggressiveness of a neoplasm based on specific histological features. A good grading system should be simple, easy to use, reproducible, and accurately segregate tumors into those with low versus high risk. The aim of this review is to summarize the histological and, when available, cytological grading systems applied in veterinary pathology, providing information regarding their prognostic impact, reproducibility, usefulness, and shortcomings. Most of the grading schemes used in veterinary medicine are developed for common tumor entities. Grading systems exist for soft tissue sarcoma, osteosarcoma, multilobular tumor of bone, mast cell tumor, lymphoma, mammary carcinoma, pulmonary carcinoma, urothelial carcinoma, renal cell carcinoma, prostatic carcinoma, and central nervous system tumors. The prognostic relevance of many grading schemes has been demonstrated, but for some tumor types the usefulness of grading remains controversial. Furthermore, validation studies are available only for a minority of the grading systems. Contrasting data on the prognostic power of some grading systems, lack of detailed instructions in the materials and methods in some studies, and lack of data on reproducibility and validation studies are discussed for the relevant grading systems. Awareness of the limitations of grading is necessary for pathologists and oncologists to use these systems appropriately and to drive initiatives for their improvement.
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http://dx.doi.org/10.1177/0300985821999831DOI Listing
March 2021

A comparison of cytological quality between fine-needle aspiration and non-aspiration techniques for obtaining ultrasound-guided samples from canine and feline lymph nodes.

Vet Rec 2021 03 10;188(6):e25. Epub 2021 Feb 10.

Dick White Referrals, Diagnostic Pathology, Cambridge, UK.

Background: In small animal medicine, ultrasound-guided fine-needle lymph node sampling plays a pivotal role in the diagnostic investigation of a range of pathologies including the staging of neoplastic disease. Traditionally fine needle aspiration cytology (FNAC) has been employed to produce samples, but fine needle non-aspiration cytology (FNNAC) has been suggested to generate superior sample quality and diagnosticity.

Methods: In a randomised control trial, 104 canine and feline lymph nodes were each sampled by both techniques. The cytological samples were then submitted to pathologists who were blinded to the technique used to generate each sample. They determined if the sample was diagnostic or non-diagnostic and graded the sample in terms of the degree of cellularity, cellular preservation and haemodilution.

Results: It was found that lymph node samples obtained using the FNAC technique were more likely to be diagnostic (p = 0.043) than samples obtained using the FNNAC technique. In addition, FNAC samples had significantly higher cellularity than FNNAC counterparts (P = 0.043). No significant difference in cell preservation or haemodilution was found between samples from the FNAC and FNNAC groups.

Conclusion: In this study, FNAC was superior to non-aspiration cytology for the sampling of canine and feline lymph nodes as it generated a higher number of diagnostic samples with greater cellularity.
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http://dx.doi.org/10.1002/vetr.25DOI Listing
March 2021

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

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

Effects of PM Exposure on the Methylation of Clock Genes in a Population of Subjects with Overweight or Obesity.

Int J Environ Res Public Health 2021 Jan 27;18(3). Epub 2021 Jan 27.

EPIGET-Epidemiology, Epigenetics and Toxicology Lab., Department of Clinical Sciences and Community Health, Università degli Studi di Milano, 20122 Milan, Italy.

The expression of clock genes, regulating the synchronization of metabolic and behavioral processes with environmental light/dark cycles, is regulated by methylation and might be influenced by short-term exposure to airborne particulate matter (PM), especially in individuals that are hypersensitive to proinflammatory cues. The present study aimed to evaluate the effects of PM and PM on the methylation profile of the clock genes , , , , , , and in a population of 200 women with obesity. A significant association between PM exposure and the methylation of clock genes was found, namely, this was negative for gene and positive for the , , , and genes. PM was negatively associated with methylation of gene and positively with methylation of gene. Evidence was observed for effect modification from body mass index (BMI) regarding the gene: as PM increases, DNA methylation increases significantly for relatively low BMI values (BMI = 25), while it decreases in participants with severe obesity (BMI = 51). PM may therefore alter the epigenetic regulation of clock genes, possibly affecting circadian rhythms. Future studies are needed to clarify how alterations in clock gene methylation are predictive of disease development and how obesity can modulate the adverse health effects of PM.
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http://dx.doi.org/10.3390/ijerph18031122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908270PMC
January 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

Orbital paraganglioma in a dog.

Tierarztl Prax Ausg K Kleintiere Heimtiere 2018 Dec 18;46(6):410-415. Epub 2019 Jan 18.

A 10-year-old Rottweiler presented with right-sided moderately painful exophthalmia, blindness, absence of dazzle and pupillary light reflexes, a swollen optic nerve head and ventrolateral indentation of the globe. On magnetic resonance imaging, a 3 x 2 x 2 cm mass with a fluid filled center and contrast-enhancing periphery was noted posteriolateral of the globe. Orbital ultrasound was used for a guided fine needle aspirate of the mass. Cytology revealed moderate numbers of polygonal cells with lightly basophilic cytoplasm. Several cells showed nuclear pseudoinclusions. Histopathology following exenteration of the orbit revealed an infiltrative, extradural neoplasm surrounding the optic nerve. Cells were arranged in packets. Neoplastic cells were immunopositive for neuron specific enolase, synaptophysin and chromogranin A and immunonegative for cytokeratin. Findings were consistent with an extra-adrenal paraganglioma (neuroendocrine tumour). Although complete excision could not be confirmed on histopathology, the owners reported no apparent tumour recurrence 25 months after surgery. In conclusion a paraganglioma should be considered as a differential diagnosis of an orbital mass.
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http://dx.doi.org/10.1055/s-0038-1677406DOI Listing
December 2018

Validation of a spectrophotometric method for GGT measurement in canine urine and determination of the urine GGT-to-creatinine ratio reference interval and biological variation in 41 healthy dogs.

J Vet Diagn Invest 2019 Jan 18;31(1):33-39. Epub 2018 Nov 18.

Dick White Diagnostics, Dick White Referrals, Six Mile Bottom, Cambridgeshire, UK.

The urine gamma-glutamyl transferase (GGT)-to-creatinine ratio has been used to monitor patients at risk of acute renal injury. We validated the spectrophotometric quantification of GGT in urine in a commercial biochemistry analyzer. The assay was precise, accurate, and linear. Intra-assay precision was 3.59% in 4 samples, with GGT concentrations of 47-195 U/L. Inter-assay precision in 3 samples with activities of 11-51 U/L was 7.74%. Accuracy was 97.3%, with an absolute bias of 2.7 U/L. Urine GGT was unaffected by hematuria, hemoglobinuria, or bacteriuria. Urine GGT was stable at 20°C and 4°C for up to 3 d. Storage by freezing at -20°C resulted in a significant reduction in enzyme activity. A pH outside the range of 6.5-8 resulted in reduced GGT activity. The biological variation of urine GGT-to-creatinine ratio provided an index of individuality of 1.6, indicating that a population-based reference interval (RI) can be used. The reference change value was calculated, and an increase in consecutive measurements >43% is required to be regarded as significant. The urine GGT-to-creatinine ratio RI obtained in a population of 41 healthy dogs was 8.5-28.5 U/g.
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http://dx.doi.org/10.1177/1040638718812927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505752PMC
January 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

Extra-enteric Blastocystis infection in a duck.

J Vet Diagn Invest 2019 May 20;31(3):382-384. Epub 2018 Jul 20.

Dick White Referrals, Six Mile Bottom, Cambridgeshire, UK.

Cell structures morphologically consistent with Blastocystis were aspirated from a subcutaneous facial swelling in a 13-mo-old pet duck. On PCR analysis and sequencing, the organism was confirmed as Blastocystis sp. subtype 7. Blastocystis is a single-celled protist that is found in the intestinal tract of many species, including mammals, birds, reptiles, amphibians, and insects. A complete understanding of the lifecycle and pathogenesis of the parasite remains elusive. Blastocystis has been implicated in human and animal disease; however, its role is controversial given that it is commonly found among healthy gut microbiota. Infection with Blastocystis outside the intestinal tract has been reported only rarely in humans. Our case of subcutaneous Blastocystis infection in a duck is a novel presentation of a ubiquitous, generally asymptomatic, parasite or commensal of the intestinal tract.
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http://dx.doi.org/10.1177/1040638718791215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838701PMC
May 2019

Canine Clitoral Carcinoma: A Clinical, Cytologic, Histopathologic, Immunohistochemical, and Ultrastructural Study.

Vet Pathol 2018 07 14;55(4):501-509. Epub 2018 Feb 14.

6 Department of Small Animal Clinical Science, Institute of Veterinary Science, University of Liverpool, Liverpool, UK.

Vaginal and vulvar tumors are uncommon in dogs. Knowledge of canine primary clitoral neoplasia is restricted to a few case reports, and only carcinomas have been reported. Cytologic and histologic features reported in the literature seem to overlap with those of canine apocrine gland anal sac adenocarcinoma (AGASA). Clinical features also recall those of canine AGASA, such as locoregional metastases and hypercalcemia of malignancy (HM). In this study, 6 cases of primary canine clitoral carcinomas (CCCs), with and without HM, were investigated by means of cytology, histopathology, electron microscopy, and immunohistochemistry for neuroendocrine markers including chromogranin A (CGA), synaptophysin (SYN), neuron-specific enolase (NSE), and S-100. In all 6 tumors, cytologic findings were consistent with malignant epithelial neoplasia of apocrine gland origin. The tumors examined were classified into 3 different histological patterns representing different degrees of differentiation: tubular, solid, and rosette type. Both CGA and SYN were mildly expressed in 2 of 6 tumors, while NSE was consistently expressed in all 6 cases. None of the tumors were S-100 positive. Transmission electron microscopy revealed electron-dense cytoplasmic granules compatible with neuroendocrine granules in all 6 cases. CCCs presented clinicopathologic features resembling AGASAs with neuroendocrine characteristics, and 2 of 6 neoplasms were considered as carcinomas with neuroendocrine differentiation and were positive for 3 neuroendocrine markers. CCCs can often present with HM, and long-term outcome is likely poor. Our study concludes that CCC seems to be a rare tumor, but it might be underestimated because of the overlapping features with AGASA. Further studies should aim to define the true incidence of this disease.
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http://dx.doi.org/10.1177/0300985818759772DOI Listing
July 2018

Synovial cell sarcoma in a dog: A misnomer-Cytologic and histologic findings and review of the literature.

Vet Clin Pathol 2018 Jun 6;47(2):181-185. Epub 2018 Feb 6.

Dick White Referrals, DWR Diagnostics, Cambridgeshire, UK.

A 4-year-old Irish Setter was presented with a history of progressive left pelvic limb lameness. Orthopedic examination revealed pain on manipulation of the left stifle. Radiographs showed an osteolytic lesion in the subchondral bone of the medial tibial condyle. Fine-needle aspirates were taken, and cytology revealed numerous cohesive clusters of plump, oval to spindloid cells often with perivascular distribution and moderate cellular atypia. A diagnosis of sarcoma was made with synovial cell sarcoma (SCS) and histiocytic sarcoma being the 2 main differentials. Histopathology confirmed the diagnosis of sarcoma and provided the same differentials. All neoplastic cells were positive for vimentin, and approximately 5% of them also stained with pan-cytokeratin using immunohistochemical staining methods. Neoplastic cells did not express CD18. The combination of this immunohistochemical profile and cell morphology was consistent with an SCS. Synovial cell sarcoma is a rare and poorly understood canine tumor entity. This is the first extensive description of the cytologic features of this neoplasm. The literature was also reviewed, focusing on comparative aspects of dogs and people, with a special emphasis on the cell of origin and diagnostic tools. Controversies regarding the nomenclature of this tumor are also presented. The authors propose a new term (cytokeratin-positive joint-associated sarcoma) for addressing this neoplasm until the cell of origin of this tumor is elucidated.
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http://dx.doi.org/10.1111/vcp.12590DOI Listing
June 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

What is your diagnosis? Perianal mass in a dog.

Vet Clin Pathol 2015 Dec 15;44(4):615-6. Epub 2015 Oct 15.

DWR Diagnostics, Dick White Referrals, Newmarket, UK.

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http://dx.doi.org/10.1111/vcp.12297DOI Listing
December 2015

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

∆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

Method of comparison--making the right choice.

Authors:
Paola Monti

J Small Anim Pract 2014 Mar;55(3):125-6

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http://dx.doi.org/10.1111/jsap.12195DOI Listing
March 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