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2.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201893

RESUMO

Lynch syndrome is a hereditary cancer-predisposing syndrome caused by germline defects in DNA mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, and PMS2. Carriers of pathogenic mutations in these genes have an increased lifetime risk of developing colorectal cancer (CRC) and other malignancies. Despite intensive surveillance, Lynch patients typically develop CRC after 10 years of follow-up, regardless of the screening interval. Recently, three different molecular models of colorectal carcinogenesis were identified in Lynch patients based on when MMR deficiency is acquired. In the first pathway, adenoma formation occurs in an MMR-proficient background, and carcinogenesis is characterized by APC and/or KRAS mutation and IGF2, NEUROG1, CDK2A, and/or CRABP1 hypermethylation. In the second pathway, deficiency in the MMR pathway is an early event arising in macroscopically normal gut surface before adenoma formation. In the third pathway, which is associated with mutations in CTNNB1 and/or TP53, the adenoma step is skipped, with fast and invasive tumor growth occurring in an MMR-deficient context. Here, we describe the association between molecular and histological features in these three routes of colorectal carcinogenesis in Lynch patients. The findings summarized in this review may guide the use of individualized surveillance guidelines based on a patient's carcinogenesis subtype.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Carcinogênese/genética , Neoplasias Colorretais Hereditárias sem Polipose/etiologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Modelos Biológicos , Fenótipo , Fatores de Risco
3.
Cell Death Dis ; 12(4): 316, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767160

RESUMO

The prognosis of locally advanced colorectal cancer (CRC) is currently unsatisfactory. This is mainly due to drug resistance, recurrence, and subsequent metastatic dissemination, which are sustained by the cancer stem cell (CSC) population. The main driver of the CSC gene expression program is Wnt signaling, and previous reports indicate that Wnt3a can activate p38 MAPK. Besides, p38 was shown to feed into the canonical Wnt/ß-catenin pathway. Here we show that patient-derived locally advanced CRC stem cells (CRC-SCs) are characterized by increased expression of p38α and are "addicted" to its kinase activity. Of note, we found that stage III CRC patients with high p38α levels display reduced disease-free and progression-free survival. Extensive molecular analysis in patient-derived CRC-SC tumorspheres and APCMin/+ mice intestinal organoids revealed that p38α acts as a ß-catenin chromatin-associated kinase required for the regulation of a signaling platform involved in tumor proliferation, metastatic dissemination, and chemoresistance in these CRC model systems. In particular, the p38α kinase inhibitor ralimetinib, which has already entered clinical trials, promoted sensitization of patient-derived CRC-SCs to chemotherapeutic agents commonly used for CRC treatment and showed a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib. Taken together, these results suggest that p38α may be targeted in CSCs to devise new personalized CRC treatment strategies.


Assuntos
Cromatina/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides/metabolismo , Processamento de Proteína Pós-Traducional/genética , beta Catenina/metabolismo , Neoplasias Colorretais/genética , Humanos , Prognóstico
4.
Genes (Basel) ; 12(3)2021 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33670833

RESUMO

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Éxons , Mutação em Linhagem Germinativa , Linhagem , Família , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade
5.
Chembiochem ; 22(9): 1597-1608, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400854

RESUMO

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (KD =42 and 84 µM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3-HSP90 binding was confirmed (KD =13 µM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.

6.
Clin Genet ; 99(3): 425-429, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33236357

RESUMO

mTOR dysregulation has been described in pathological conditions, such as cardiovascular and overgrowth disorders. Here we report on the first case of a patient with a complex congenital heart disease and an interstitial duplication in the short arm of chromosome 1, encompassing part of the mTOR gene. Our results suggest that an intragenic mTOR microduplication might play a role in the pathogenesis of non-syndromic congenital heart defects (CHDs) due to an upregulation of mTOR/Rictor and consequently an increased phosphorylation of PI3K/AKT and MEK/ERK signaling pathways in patient-derived amniocytes. This is the first report which shows a causative role of intragenic mTOR microduplication in the etiology of an isolated complex CHD.

7.
iScience ; 23(10): 101604, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33205017

RESUMO

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

8.
J Med Genet ; 57(5): 356-360, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31591141

RESUMO

Germline mutations of the APC gene, which encodes a multidomain protein of 2843 amino acid residues, cause familial adenomatous polyposis (FAP). Three FAP clinical variants are correlated with the location of APC mutations: (1) classic FAP with profuse polyposis (>1000 adenomas), associated with mutations from codon 1250 to 1424; (2) attenuated FAP (<100 adenomas), associated with mutations at APC extremities (before codon 157 and after codon 1595); (3) classic FAP with intermediate colonic polyposis (100-1000 adenomas), associated with mutations located in the remaining part of APC In an effort to decipher the clinical phenotype associated with APC C-terminal germline truncating mutations in patients with FAP, after screening APC mutations in one family whose members (n=4) developed gastric polyposis, colon oligo-polyposis and desmoid tumours, we performed a literature meta-analysis of clinically characterised patients (n=97) harbouring truncating mutations in APC C-terminus. The APC distal mutations identified in this study cluster with a phenotype characterised by colon oligo-polyposis, diffuse gastric polyposis and desmoid tumours. In conclusion, we describe a novel FAP clinical variant, which we propose to refer to as Gastric Polyposis and Desmoid FAP, that may require tailored management.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Predisposição Genética para Doença , Neoplasias Gástricas/genética , Polipose Adenomatosa do Colo/epidemiologia , Polipose Adenomatosa do Colo/patologia , Adulto , Feminino , Fibromatose Agressiva/patologia , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/patologia
9.
Cells ; 8(9)2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546924

RESUMO

Cellular stress response is a universal mechanism that ensures the survival or negative selection of cells in challenging conditions. The transcription factor Forkhead box protein O3 (FOXO3a) is a core regulator of cellular homeostasis, stress response, and longevity since it can modulate a variety of stress responses upon nutrient shortage, oxidative stress, hypoxia, heat shock, and DNA damage. FOXO3a activity is regulated by post-translational modifications that drive its shuttling between different cellular compartments, thereby determining its inactivation (cytoplasm) or activation (nucleus and mitochondria). Depending on the stress stimulus and subcellular context, activated FOXO3a can induce specific sets of nuclear genes, including cell cycle inhibitors, pro-apoptotic genes, reactive oxygen species (ROS) scavengers, autophagy effectors, gluconeogenic enzymes, and others. On the other hand, upon glucose restriction, 5'-AMP-activated protein kinase (AMPK) and mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -dependent FOXO3a mitochondrial translocation allows the transcription of oxidative phosphorylation (OXPHOS) genes, restoring cellular ATP levels, while in cancer cells, mitochondrial FOXO3a mediates survival upon genotoxic stress induced by chemotherapy. Interestingly, these target genes and their related pathways are diverse and sometimes antagonistic, suggesting that FOXO3a is an adaptable player in the dynamic homeostasis of normal and stressed cells. In this review, we describe the multiple roles of FOXO3a in cellular stress response, with a focus on both its nuclear and mitochondrial functions.


Assuntos
Núcleo Celular/química , Proteína Forkhead Box O3/metabolismo , Mitocôndrias/metabolismo , Estresse Fisiológico , Animais , Núcleo Celular/metabolismo , Humanos
10.
Cancers (Basel) ; 11(3)2019 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909600

RESUMO

Colorectal cancer (CRC) poses a formidable challenge in terms of molecular heterogeneity, as it involves a variety of cancer-related pathways and molecular changes unique to an individual's tumor. On the other hand, recent advances in DNA sequencing technologies provide an unprecedented capacity to comprehensively identify the genetic alterations resulting in tumorigenesis, raising the hope that new therapeutic approaches based on molecularly targeted drugs may prevent the occurrence of chemoresistance. Regulation of the transcription factor FOXO3a in response to extracellular cues plays a fundamental role in cellular homeostasis, being part of the molecular machinery that drives cells towards survival or death. Indeed, FOXO3a is controlled by a range of external stimuli, which not only influence its transcriptional activity, but also affect its subcellular localization. These regulation mechanisms are mediated by cancer-related signaling pathways that eventually drive changes in FOXO3a post-translational modifications (e.g., phosphorylation). Recent results showed that FOXO3a is imported into the mitochondria in tumor cells and tissues subjected to metabolic stress and cancer therapeutics, where it induces expression of the mitochondrial genome to support mitochondrial metabolism and cell survival. The current review discusses the potential clinical relevance of multidrug therapies that drive cancer cell fate by regulating critical pathways converging on FOXO3a.

11.
Aging (Albany NY) ; 10(12): 3957-3985, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538212

RESUMO

An altered metabolism is involved in the development of clear cell - renal cell carcinoma (ccRCC), and in this tumor many altered genes play a fundamental role in controlling cell metabolic activities. We delineated a large-scale metabolomic profile of human ccRCC, and integrated it with transcriptomic data to connect the variations in cancer metabolism with gene expression changes. Moreover, to better analyze the specific contribution of metabolic gene alterations potentially associated with tumorigenesis and tumor progression, we evaluated the transcription profile of primary renal tumor cells. Untargeted metabolomic analysis revealed a signature of an increased glucose uptake and utilization in ccRCC. In addition, metabolites related to pentose phosphate pathway were also altered in the tumor samples in association with changes in Krebs cycle intermediates and related metabolites. We identified NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) as the most highly expressed gene in renal cancer cells and evaluated its role in sustaining angiogenesis, chemoresistance, and mitochondrial dysfunction. Finally, we showed that silencing of NDUFA4L2 affects cell viability, increases mitochondrial mass, and induces ROS generation in hypoxia.


Assuntos
Carcinoma de Células Renais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Complexo I de Transporte de Elétrons/metabolismo , Neoplasias Renais/metabolismo , Mitocôndrias/metabolismo , Neovascularização Patológica/metabolismo , Animais , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Embrião de Galinha , Membrana Corioalantoide , Biologia Computacional , DNA Mitocondrial , Mineração de Dados , Complexo I de Transporte de Elétrons/genética , Glucose/metabolismo , Humanos , Neoplasias Renais/genética , Metabolômica , Neovascularização Patológica/genética , Espécies Reativas de Oxigênio , Transcriptoma
12.
Nucleic Acids Res ; 46(11): 5587-5600, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29733381

RESUMO

The HSF and FOXO families of transcription factors play evolutionarily conserved roles in stress resistance and lifespan. In humans, the rs2802292 G-allele at FOXO3 locus has been associated with longevity in all human populations tested; moreover, its copy number correlated with reduced frequency of age-related diseases in centenarians. At the molecular level, the intronic rs2802292 G-allele correlated with increased expression of FOXO3, suggesting that FOXO3 intron 2 may represent a regulatory region. Here we show that the 90-bp sequence around the intronic single nucleotide polymorphism rs2802292 has enhancer functions, and that the rs2802292 G-allele creates a novel HSE binding site for HSF1, which induces FOXO3 expression in response to diverse stress stimuli. At the molecular level, HSF1 mediates the occurrence of a promoter-enhancer interaction at FOXO3 locus involving the 5'UTR and the rs2802292 region. These data were confirmed in various cellular models including human HAP1 isogenic cell lines (G/T). Our functional studies highlighted the importance of the HSF1-FOXO3-SOD2/CAT/GADD45A cascade in cellular stress response and survival by promoting ROS detoxification, redox balance and DNA repair. Our findings suggest the existence of an HSF1-FOXO3 axis in human cells that could be involved in stress response pathways functionally regulating lifespan and disease susceptibility.


Assuntos
Elementos Facilitadores Genéticos , Proteína Forkhead Box O3/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Polimorfismo de Nucleotídeo Único , Estresse Fisiológico/genética , Ativação Transcricional , Regiões 5' não Traduzidas , Alelos , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Proteína Forkhead Box O3/biossíntese , Humanos , Íntrons , Longevidade/genética , Regiões Promotoras Genéticas
13.
Genome Res ; 28(6): 910-920, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29776991

RESUMO

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.


Assuntos
Inversão Cromossômica/genética , Genoma Humano/genética , Primatas/genética , Inversão de Sequência/genética , Animais , Evolução Molecular , Humanos , Anotação de Sequência Molecular , Pan troglodytes/genética
14.
Cell Death Dis ; 9(2): 231, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29445193

RESUMO

While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress and cancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. Using FoxO3A-/- cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity.


Assuntos
Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , Proteína Forkhead Box O3/genética , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular , Cisplatino/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fluoruracila/farmacologia , Proteína Forkhead Box O3/metabolismo , Edição de Genes , Genoma Mitocondrial , Células HEK293 , Humanos , Irinotecano/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Células NIH 3T3 , Fosforilação , Transdução de Sinais , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
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