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AIMS: Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB-prone animal model. MATERIALS AND METHODS: We generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)-dependent MB-prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1-depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA-Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs. RESULTS: We found that monoallelic Nbn deletion increases SmoA1-dependent MB incidence. In addition to a defective DDR, Nbn+/- GCPs show increased clonogenicity compared to Nbn+/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full NbnKO impairs MB development both in SmoA1 mice and in an SHH-driven tumour allograft. CONCLUSIONS: Our study indicates that Nbn is haploinsufficient for SHH-MB development whereas full NbnKO is epistatic on SHH-driven MB development, thus revealing a gene dosage-dependent effect of Nbn inactivation on SHH-MB development.
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Proteínas de Ciclo Celular , Neoplasias Cerebelares , Proteínas de Ligação a DNA , Meduloblastoma , Animais , Proteínas de Ciclo Celular/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/genética , Dosagem de Genes , Genes Essenciais , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos TransgênicosRESUMO
Hereditary breast and ovarian cancer are mainly linked to mutations in BRCA1 and BRCA2 genes which confer a similar cumulative risk of developing breast cancer. Importantly, while BRCA2 mutation carriers generally have a lower cumulative risk for ovarian cancer, mutations clustered in the central portion of BRCA2 are associated with a higher proportion of ovarian compared with breast cancer cases. The boundaries of this ovarian cancer cluster region (OCCR) have been tentatively defined within a 3.3 kb region of BRCA2 exon 11, and herein, we reassessed these boundaries using our series of Italian breast/ovarian cancer families. We used direct sequencing to investigate BRCA mutations in 367 breast/ovarian cancer families. We also studied the association between the location of the mutations and the ovarian cancer phenotype in our cohort of BRCA2-mutated families. We observed the novel c.7309_7309delA frameshift mutation and the c.7007G>A deleterious mutation in BRCA2 exons 14 and 13, respectively, in five independent Italian families characterized by a high proportion of ovarian cancer cases. Of note, a significantly higher proportion of ovarian versus breast cancer cases was associated not only with mutations in the previously defined OCCR (OR = 5.91; p = 0.004), but also with the exon 13-14 region (OR = 7.37; p = 0.001) in our BRCA2-mutated families. Our data provide initial evidence for a novel putative OCCR in BRCA2 exons 13-14.
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Proteína BRCA2/genética , Neoplasias da Mama Masculina/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama Masculina/epidemiologia , Éxons , Feminino , Mutação em Linhagem Germinativa , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/patologia , LinhagemRESUMO
The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.
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Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Humanos , Camundongos , Proteínas de Ciclo Celular , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Proteômica , Fatores de Transcrição/genética , Transferases , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Reactivation of the HMGA1 protoncogene is very frequent in human cancer, but still very little is known on the molecular mechanisms leading to this event. Prompted by the finding of putative E2F binding sites in the human HMGA1 promoter and by the frequent deregulation of the RB/E2F1 pathway in human carcinogenesis, we investigated whether E2F1 might contribute to the regulation of HMGA1 gene expression. Here we report that E2F1 induces HMGA1 by interacting with a 193 bp region of the HMGA1 promoter containing an E2F binding site surrounded by three putative Sp1 binding sites. Both gain and loss of function experiments indicate that Sp1 functionally interacts with E2F1 to promote HMGA1 expression. However, while Sp1 constitutively binds HMGA1 promoter, it is the balance between different E2F family members that tunes the levels of HMGA1 expression between quiescence and proliferation. Finally, we found increased HMGA1 expression in pituitary and thyroid tumors developed in Rb(+/-) mice, supporting the hypothesis that E2F1 is a novel important regulator of HMGA1 expression and that deregulation of the RB/E2F1 path might significantly contribute to HMGA1 deregulation in cancer.
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Fator de Transcrição E2F1/metabolismo , Proteína HMGA1a/genética , Neoplasias Hipofisárias/metabolismo , Fator de Transcrição Sp1/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Imunoprecipitação da Cromatina , Fator de Transcrição E2F1/genética , Proteína HMGA1a/metabolismo , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Neoplasias Hipofisárias/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteína do Retinoblastoma/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/genética , Neoplasias da Glândula Tireoide/genética , Ativação TranscricionalRESUMO
Hypomorphic mutations in MRN complex genes are frequently found in cancer, supporting their role as oncosuppressors. However, unlike canonical oncosuppressors, MRN proteins are often overexpressed in tumor tissues, where they actively work to counteract DSBs induced by both oncogene-dependent RS and radio-chemotherapy. Moreover, at the same time, MRN genes are also essential genes, since the constitutive KO of each component leads to embryonic lethality. Therefore, even though it is paradoxical, MRN genes may work as oncosuppressive, oncopromoting, and essential genes. In this review, we discussed how alterations in the MRN complex impact the physiopathology of cancer, in light of our recent discoveries on the gene-dosage-dependent effect of NBS1 in Medulloblastoma. These updates aim to understand whether MRN complex can be realistically used as a prognostic/predictive marker and/or as a therapeutic target for the treatment of cancer patients in the future.
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The Hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development and adult tissue homeostasis. On the other end, dysregulated Hh signaling triggers a prolonged mitogenic response that may prompt abnormal cell proliferation, favoring tumorigenesis. Indeed, about 30% of medulloblastomas (MBs), the most common malignant childhood cerebellar tumors, exhibit improper activation of the Hh signaling. The oncosuppressor KCASH2 has been described as a suppressor of the Hh signaling pathway, and low KCASH2 expression was observed in Hh-dependent MB tumor. Therefore, the study of the modulation of KCASH2 expression may provide fundamental information for the development of new therapeutic approaches, aimed to restore physiological KCASH2 levels and Hh inhibition. To this end, we have analyzed the TATA-less KCASH2 proximal promoter and identified key transcriptional regulators of this gene: Sp1, a TF frequently overexpressed in tumors, and the tumor suppressor p53. Here, we show that in WT cells, Sp1 binds KCASH2 promoter on several putative binding sites, leading to increase in KCASH2 expression. On the other hand, p53 is involved in negative regulation of KCASH2. In this context, the balance between p53 and Sp1 expression, and the interplay between these two proteins determine whether Sp1 acts as an activator or a repressor of KCASH2 transcription. Indeed, in p53-/- MEF and p53 mutated tumor cells, we hypothesize that Sp1 drives promoter methylation through increased expression of the DNA methyltransferase 1 (DNMT1) and reduces KCASH2 transcription, which can be reversed by Sp1 inhibition or use of demethylating agents. We suggest therefore that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells where p53 functionality is lost. This work may open new venues for novel therapeutic multidrug approaches in the treatment of Hh-dependent tumors carrying p53 deficiency.
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MYCN drives aggressive behavior and refractoriness to chemotherapy, in several tumors. Since MYCN inactivation in clinical settings is not achievable, alternative vulnerabilities of MYCN-driven tumors need to be explored to identify more effective and less toxic therapies. We previously demonstrated that PARP inhibitors enhance MYCN-induced replication stress and promote mitotic catastrophe, counteracted by CHK1. Here, we showed that PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of SHH-dependent medulloblastoma, their combination being more effective in MYCN amplified and MYCN overexpressing cells compared to MYCN non-amplified cells. Although the MYCN amplified IMR-32 cell line carrying the p.Val2716Ala ATM mutation showed the highest sensitivity to the drug combination, this was not related to ATM status, as indicated by CRISPR/Cas9-based correction of the mutation. Suboptimal doses of the CHK1 inhibitor MK-8776 plus the PARP inhibitor olaparib led to a MYCN-dependent accumulation of DNA damage and cell death in vitro and significantly reduced the growth of four in vivo models of MYCN-driven tumors, without major toxicities. Our data highlight the combination of PARP and CHK1 inhibitors as a new potential chemo-free strategy to treat MYCN-driven tumors, which might be promptly translated into clinical trials.
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Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Sinergismo Farmacológico , Feminino , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Mutação , Neuroblastoma/genética , Neuroblastoma/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: BRCA1/2 VUSs represent an important clinical issue in risk assessment for the breast/ovarian cancer families (HBOC) families. Among them, some occurring within the intron-exon boundary may lead to aberrant splicing process by altering or creating de novo splicing regulatory elements or unmasking cryptic splice site. Defining the impact of these potential splice variants at functional level is important to establish their pathogenic role. METHODS: Genomic DNA was extracted from peripheral blood sample of a young woman affected with breast cancer belonging to a HBOC family and the entire coding regions of the BRCA1 and BRCA2 genes were amplified using the Ion AmpliSeq BRCA1 and BRCA2 Panel. The BRCA2 c.682-2delA variant has been characterized by RT-PCR analysis performed on mRNA extracted from blood and lymphoblastoid cell line. RESULTS: We demonstrated that a novel BRCA2 c.682-2delA variant at the highly conserved splice consensus site in intron 8 disrupts the canonical splice acceptor site generating a truncated protein as predicted by several bioinformatics tools. Segregations analysis in the family and LOH performed on proband breast cancer tissue further confirmed its classification as pathogenic variant. CONCLUSION: Combining different methodologies, we characterized this new BRCA2 variant and provided findings of clinical utility for its classification as pathogenic variant.
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Proteína BRCA2/genética , Deleção de Genes , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Sítios de Splice de RNA , Adulto , Sequência Conservada , Síndrome Hereditária de Câncer de Mama e Ovário/patologia , Humanos , Perda de HeterozigosidadeRESUMO
Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB.
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Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APCMin/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lisina/metabolismo , Camundongos Nus , Fases de Leitura Aberta/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/química , Peptídeos/metabolismo , Poliaminas/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/química , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
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.
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The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.
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Antineoplásicos/uso terapêutico , Proteínas Correpressoras/efeitos dos fármacos , Proteínas Hedgehog/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Neoplasias/tratamento farmacológico , Fenformin/uso terapêutico , Animais , Antineoplásicos/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Camundongos , Fenformin/farmacologiaRESUMO
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.
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BACKGROUND: Genetic testing for BRCA1/2 germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients. METHODS: We first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel BRCA1 LGRs was obtained via long-range PCR and direct sequencing of the DNA products. RESULTS: In our cohort, the newly defined DQ-based algorithm detected 3/3 BRCA1 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6-99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6-99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel BRCA1 deletions, involving exons 16-17, 21-22 and 20, have been characterized. CONCLUSIONS: Our study defined a DQ-based algorithm to identify BRCA1 LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.
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The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDAapproved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HHGLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HHregulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogenactivated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogenactivated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HHregulated transcription factor. Phosphorylation occurred at multiple residues in the Cterminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 1433. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.
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Proteínas Hedgehog/genética , MAP Quinase Quinase Quinase 1/genética , Meduloblastoma/genética , Proteína GLI1 em Dedos de Zinco/genética , Anilidas/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Piridinas/administração & dosagem , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Growth and patterning of the cerebellum is compromised if granule cell precursors do not properly expand and migrate. During embryonic and postnatal cerebellar development, the Hedgehog pathway tightly regulates granule cell progenitors to coordinate appropriate foliation and lobule formation. Indeed, granule cells impairment or defects in the Hedgehog signaling are associated with developmental, neurodegenerative and neoplastic disorders. So far, scant and inefficient cellular models have been available to study granule cell progenitors, in vitro. Here, we validated a new culture method to grow postnatal granule cell progenitors as hedgehog-dependent neurospheres with prolonged self-renewal and ability to differentiate into granule cells, under appropriate conditions. Taking advantage of this cellular model, we provide evidence that Ptch1-KO, but not the SMO-M2 mutation, supports constitutive and cell-autonomous activity of the hedgehog pathway.
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Diferenciação Celular , Cerebelo/metabolismo , Proteínas Hedgehog , Células-Tronco Neurais/metabolismo , Transdução de Sinais , Receptor Smoothened , Animais , Cerebelo/citologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMO
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.
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Cutaneous squamous cell carcinoma (cSCC) is the second most common type of non-melanoma skin cancer. Platinum-based regimens have been an integral part of palliative care for patients with locally advanced or metastatic disease. There is no evidence of efficacy for later lines of chemotherapy and no targeted therapy has been introduced as 'standard of care'. Here we report on the case of an elderly cSCC patient, resistant to conventional therapy, however successfully treated with anti-epidermal growth factor receptor (EGFR) agent (Cetuximab) in addition to a daily dose of Curcumin phospholipid. The patient responded to treatment and experienced no recurrence for 11 months with only minor skin-related toxicity. To our knowledge, this is the first report of clinical evidence that an anti EGFR targeted therapy with a daily oral dose of Curcumin phospholipid is well tolerated and results in a highly effective disease control in a heavily pretreated cSCC patient.
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The introduction of multigene panel testing for hereditary breast/ovarian cancer screening has greatly improved efficiency, speed, and costs. However, its clinical utility is still debated, mostly due to the lack of conclusive evidences on the impact of newly discovered genetic variants on cancer risk and lack of evidence-based guidelines for the clinical management of their carriers. In this pilot study, we aimed to test whether a systematic and multiparametric characterization of newly discovered mutations could enhance the clinical utility of multigene panel sequencing. Out of a pool of 367 breast/ovarian cancer families Sanger-sequenced for BRCA1 and BRCA2 gene mutations, we selected a cohort of 20 BRCA1/2-negative families to be subjected to the BROCA-Cancer Risk Panel massive parallel sequencing. As a strategy for the systematic characterization of newly discovered genetic variants, we collected blood and cancer tissue samples and established lymphoblastoid cell lines from all available individuals in these families, to perform segregation analysis, loss-of-heterozygosity and further molecular studies. We identified loss-of-function mutations in 6 out 20 high-risk families, 5 of which occurred on BRCA1, CHEK2 and ATM and are esteemed to be risk-relevant. In contrast, a novel RAD50 truncating mutation is most likely unrelated to breast cancer. Our data suggest that integrating multigene panel testing with a pre-organized, multiparametric characterization of newly discovered genetic variants improves the identification of risk-relevant alleles impacting on the clinical management of their carriers.
Assuntos
Predisposição Genética para Doença , Testes Genéticos/métodos , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Hidrolases Anidrido Ácido , Adulto , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Quinase do Ponto de Checagem 2/genética , Estudos de Coortes , Análise Mutacional de DNA/métodos , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação com Perda de Função , Pessoa de Meia-Idade , Projetos PilotoRESUMO
MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors.