Il ripristino della via di segnale del TGF-b si associa ad una riduzione del potenziale metastatico delle cellule di tiroide trasformate dall’oncogene K-ras

Il Trasforming Growth Factor-beta1 (TGF-b1) è una citochina multifunzionale in grado di controllare, attraverso il legame ai suoi recettori, numerosi processi cellulari come la proliferazione, la differenziazione, la plasticità, lo sviluppo, l’embriogenesi, l’adesività, la motilità e l’apoptosi. Abbiamo precedentemente dimostrato che le cellule di tiroide di ratto trasformate dall’oncogene K-ras, K10, sono resistenti all’azione inibitoria della crescita del TGF-b1, visto che mostrano una diminuita espressione del recettore di tipo II (TbRII) e che i cloni ottenuti transfettando il TbRII, revertono parzialmente il loro fenotipo maligno riducendo in maniera statisticamente significativa la crescita ancoraggio-dipendente e indipendente e la loro tumorigenicità (numero di metastasi artificiali e spontanee) rispetto alle cellule parentali altamente maligne, quando trapiantate in topi nudi atimici. Lo scopo di questo lavoro è di chiarire gli eventi molecolari coinvolti in questa modulazione del potenziale tumorigenico delle cellule di tiroide di ratto trasformate dall’oncogene K-ras over-esprimenti il TbRII. Il nostro lavoro dimostra che il TbRII iperespresso nelle cellule di tiroide di ratto trasformate da K-ras è un recettore funzionale, indispensabile per il recupero della responsività al fattore di crescita ed essenziale per ripristinare in queste cellule un comportamento simile a quello delle cellule di controllo. Il TbRII è responsabile di una forte riduzione dell’adesione e della motilità delle cellule di tiroide trasformate, K10, altamente maligne. Tale modulazione del comportamento adesivo e migratorio non appare determinata da modificazioni dei livelli di espressione dell’integrina alfa5 beta1 e della sua subunità alfa5 ma, probabilmente, dell’avidità/affinità dell’integrina presa in esame per il suo specifico substrato. In queste stesse cellule, l’iperespressione del recettore, inoltre, sembra essere associata ad una riduzione dell’espressione della proteina RhoA che, visto il coinvolgimento di RhoA nei processi migratori e nel mantenimento della polarità cellulare, spiegherebbe ulteriormente la ridotta capacità migratoria dei cloni rispetto alla linea parentale e la loro diversa morfologia cellulare, e ad un parziale recupero dell’espressione della E-Caderina a livello della membrana plasmatica. Molto intrigante appare, inoltre, il risultato che i livelli di espressione della MMP2, una metalloproteasi coinvolta fortemente nel processo metastatico, la cui attività nei tumori è spesso accentuata, sono molto ridotti nelle cellule iperesprimenti il TbRII rispetto ai corrispondenti controlli e molto più simili a quelli della linea normale FRTL-5. Questi risultati, considerati nel loro complesso, suggeriscono che il ripristino in queste cellule di un recettore per il TGF-b funzionale, possa essere utile a limitare la disseminazione e la propagazione del tumore

The role of peroxiredoxins in cancer

Peroxiredoxins (PRDXs) are a ubiquitously expressed family of small (22-27 kDa) non-seleno peroxidases that catalyze the peroxide reduction of H2O2, organic hydroperoxides and peroxynitrite. They are highly involved in the control of various physiological functions, including cell growth, differentiation, apoptosis, embryonic development, lipid metabolism, the immune response, as well as cellular homeostasis. Although the protective role of PRDXs in cardiovascular and neurological diseases is well established, their role in cancer remains controversial. Increasing evidence suggests the involvement of PRDXs in carcinogenesis and in the development of drug resistance. Numerous types of cancer cells, in fact, are characterized by an increase in reactive oxygen species (ROS) production, and often exhibit an altered redox environment compared with normal cells. The present review focuses on the complex association between oxidant balance and cancer, and it provides a brief account of the involvement of PRDXs in tumorigenesis and in the development of chemoresistance

Il ripristino della via di segnale del TGF-b si associa ad una riduzione del potenziale metastatico delle cellule di tiroide trasformate dall’oncogene K-ras

Il Trasforming Growth Factor-beta1 (TGF-b1) è una citochina multifunzionale in grado di controllare, attraverso il legame ai suoi recettori, numerosi processi cellulari come la proliferazione, la differenziazione, la plasticità, lo sviluppo, l’embriogenesi, l’adesività, la motilità e l’apoptosi. Abbiamo precedentemente dimostrato che le cellule di tiroide di ratto trasformate dall’oncogene K-ras, K10, sono resistenti all’azione inibitoria della crescita del TGF-b1, visto che mostrano una diminuita espressione del recettore di tipo II (TbRII) e che i cloni ottenuti transfettando il TbRII, revertono parzialmente il loro fenotipo maligno riducendo in maniera statisticamente significativa la crescita ancoraggio-dipendente e indipendente e la loro tumorigenicità (numero di metastasi artificiali e spontanee) rispetto alle cellule parentali altamente maligne, quando trapiantate in topi nudi atimici. Lo scopo di questo lavoro è di chiarire gli eventi molecolari coinvolti in questa modulazione del potenziale tumorigenico delle cellule di tiroide di ratto trasformate dall’oncogene K-ras over-esprimenti il TbRII. Il nostro lavoro dimostra che il TbRII iperespresso nelle cellule di tiroide di ratto trasformate da K-ras è un recettore funzionale, indispensabile per il recupero della responsività al fattore di crescita ed essenziale per ripristinare in queste cellule un comportamento simile a quello delle cellule di controllo. Il TbRII è responsabile di una forte riduzione dell’adesione e della motilità delle cellule di tiroide trasformate, K10, altamente maligne. Tale modulazione del comportamento adesivo e migratorio non appare determinata da modificazioni dei livelli di espressione dell’integrina alfa5 beta1 e della sua subunità alfa5 ma, probabilmente, dell’avidità/affinità dell’integrina presa in esame per il suo specifico substrato. In queste stesse cellule, l’iperespressione del recettore, inoltre, sembra essere associata ad una riduzione dell’espressione della proteina RhoA che, visto il coinvolgimento di RhoA nei processi migratori e nel mantenimento della polarità cellulare, spiegherebbe ulteriormente la ridotta capacità migratoria dei cloni rispetto alla linea parentale e la loro diversa morfologia cellulare, e ad un parziale recupero dell’espressione della E-Caderina a livello della membrana plasmatica. Molto intrigante appare, inoltre, il risultato che i livelli di espressione della MMP2, una metalloproteasi coinvolta fortemente nel processo metastatico, la cui attività nei tumori è spesso accentuata, sono molto ridotti nelle cellule iperesprimenti il TbRII rispetto ai corrispondenti controlli e molto più simili a quelli della linea normale FRTL-5. Questi risultati, considerati nel loro complesso, suggeriscono che il ripristino in queste cellule di un recettore per il TGF-b funzionale, possa essere utile a limitare la disseminazione e la propagazione del tumore

EGF and TGF-β1 Effects on Thyroid Function

Normal epithelial thyroid cells in culture are inhibited by TGF-β1. Instead, transformed thyroid cell lines are frequently resistant to its growth inhibitory effect. Loss of TGF-β responsiveness could be due to a reduced expression of TGF-β receptors, as shown in transformed rat thyroid cell lines and in human thyroid tumors, or to alterations of other genes controlling TGF-β signal transduction pathway. However, in thyroid neoplasia, a complex pattern of alterations occurring during transformation and progression has been identified. Functionally, TGF-β1 acts as a tumor suppressor in the early stage of transformation or as a tumor promoter in advanced cancer. This peculiar pleiotropic behaviour of TGF-β may result from cross-talk with signalling pathways mediated by other growth factors, among which EGF-like ligands play an important role. This paper reports evidences on TGF-β1 and EGF systems in thyroid tumors and on the cross-talk between these growth factors in thyroid cancer

PO-076 Molecular analysis of BRCA-negative breast and/or ovarian cancer families by multigene panel testing

Introduction About 5%–10% of the hereditary breast and/or ovarian cancer (BC/BOC) is associated with an autosomal dominant genetic susceptibility due to highly penetrant mutations of the BRCA1/2 genes. In particular, BRCA1/2 gene mutations are found in 25%–30% of the BC families subjected to genetic testing. These numbers suggest the possible involvement of other genes in BC/BOC genetic predisposition and a fraction of these cases remains to be assigned to specific genetic factors. Here we report on the application of the NGS multigene panel to a group of BRCA1/2 mutation negative BC/BOC cases, in order to identify germline mutations that could further explain BC/BOC genetic susceptibility. Material and methods We selected a group of 27 BRCA1/2 negative BC and BOC families on the basis of a clear dominant inheritance pattern and/or a moderate/high BRCAPro score. We performed a genomic screening by a comprehensive multi-gene custom panel of 29 cancer-related genes, using Ion Torrent platform (Thermo Fisher Scientific). Results and discussions In three cases (11%) we found mutations described as pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/) in ATM, MUTYH and PALB2 genes. In the series analysed, the most frequently altered genes were APC and ATM (15%) but were also identified mutations in MSH6 and TP53 (11%), MUTYH and RAD51B (7%), MRE11, EPCAM, BRIP1, CHEK2, PALB2, BARD1, STK11 and RAD50 (4%). In particular, we found six genomic variants of uncertain significance (VUS) in MSH6, ATM, BRIP1, RAD50 and APC genes; nine genomic variants of conflicting interpretations of pathogenicity in MUTYH, MRE11, TP53, APC, MSH6, CHEK2, EPCAM and ATM genes and eight genomic variants not reported in ClinVar in APC, RAD51B, STK11, TP53, ATM and BARD1 genes predicted deleterious by in silico analysis. Their biological significance and involvement in the development of the pathology is still unknown today. Only six patients were negative for the presence of mutations in the 29 genes analysed. Conclusion Preliminary results of this study suggest that NGS could offer a great contribution to identify the genetic component of susceptibility to BC/BOC and could potentially be used with implications for clinical management and counselling of patients and their families. Moreover, our results suggest that multigene testing approach may benefit appropriately selected patients, especially those with increased risk of BC/BOC development

Molecular profiling of male breast cancer by multigene panel testing: Implications for precision oncology

Introduction: Compared with breast cancer (BC) in women, BC in men is a rare disease with genetic and molecular peculiarities. Therapeutic approaches for male BC (MBC) are currently extrapolated from the clinical management of female BC, although the disease does not exactly overlap in males and females. Data on specific molecular biomarkers in MBC are lacking, cutting out male patients from more appropriate therapeutic strategies. Growing evidence indicates that Next Generation Sequencing (NGS) multigene panel testing can be used for the detection of predictive molecular biomarkers, including Tumor Mutational Burden (TMB) and Microsatellite Instability (MSI). Methods: In this study, NGS multigene gene panel sequencing, targeting 1.94 Mb of the genome at 523 cancer-relevant genes (TruSight Oncology 500, Illumina), was used to identify and characterize somatic variants, Copy Number Variations (CNVs), TMB and MSI, in 15 Formalin-Fixed Paraffin-Embedded (FFPE) male breast cancer samples. Results and discussion: A total of 40 pathogenic variants were detected in 24 genes. All MBC cases harbored at least one pathogenic variant. PIK3CA was the most frequently mutated gene, with six (40.0%) MBCs harboring targetable PIK3CA alterations. CNVs analysis showed copy number gains in 22 genes. No copy number losses were found. Specifically, 13 (86.7%) MBCs showed gene copy number gains. MYC was the most frequently amplified gene with eight (53.3%) MBCs showing a median fold-changes value of 1.9 (range 1.8-3.8). A median TMB value of 4.3 (range 0.8-12.3) mut/Mb was observed, with two (13%) MBCs showing high-TMB. The median percentage of MSI was 2.4% (range 0-17.6%), with two (13%) MBCs showing high-MSI. Overall, these results indicate that NGS multigene panel sequencing can provide a comprehensive molecular tumor profiling in MBC. The identification of targetable molecular alterations in more than 70% of MBCs suggests that the NGS approach may allow for the selection of MBC patients eligible for precision/targeted therapy

A simplified genomic profiling approach predicts outcome in metastatic colorectal cancer

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on TP53 and/or RAS genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of TP53/RAS, the expected response is much worse compared to patients with exclusive TP53/RAS mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy

Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer

Background Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. Methods We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). Results By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. Discussion Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches

Clinical Multigene Panel Sequencing Identifies Distinct Mutational Association Patterns in Metastatic Colorectal Cancer

Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases