RESUMO
OBJECTIVE: The objectives of this study were to characterize the well-defined endometrial cancer (EC) type I (endometrioid [EEC] G1-G2) versus the prototype of EC type II (serous [ESC]) and to evaluate the expression of specific biomarkers differentially expressed between 2 well-defined types, in those EC subtypes (such as EEC G3) disputed between types I and II. METHODS: Data from 25 patients (10 EEC G1-G2, 8 EEC G3, 5 ESC, and 2 clear cell) submitted to the surgical treatment were collected. Two-dimensional electrophoresis and mass spectrometry (MS) analysis were performed on 5 EEC G1-G2 and 5 healthy endometrial samples of the same patients. Differentially expressed proteins, such as DJ-1, were validated by Western blot. In patients with EEC G1-G2, serum levels of DJ-1, an overexpressed oncoprotein related to EC pathogenesis and progression, were evaluated and then compared with levels identified in patients with ESC and healthy controls. The DJ-1 immunohistochemical (IHC) staining was performed on neoplastic and healthy endometrium collected from the same patients. The 8 stored samples of EEC G3 were submitted to DJ-1 IHC assays. RESULTS: The 2-dimensional electrophoresis analysis identified 1040 protein spots differentially expressed in EEC G1-G2 compared with healthy endometrium. Forty-two spots were subjected to liquid chromatography-MS/MS analysis. Thirty-three up-regulated (like an annexin 2 [ANXA2] shorter isoform, CAPG [macrophage-capping protein], DJ-1/PARK7) and 9 down-regulated (like calreticulin and ubiquitin carboxyl-terminal hydrolase isozyme L1) proteins were identified and validated by Western blot. A significant increase in serum DJ-1 levels of EEC G1-G2 versus the healthy controls and in ESC versus EEC patients was observed. DJ-1 IHC score was significantly higher in ESC versus those EEC G1-G2. In 3 cases of EEC G3, the DJ-1 expression was similar to the ESC subtype. CONCLUSIONS: The identification of proteins, such as DJ-1, differentially expressed, between well-defined EC types I and II allows to make a subtype-specific presurgical diagnosis and help surgeon to safely preoperatively choose a proper surgical treatment.
Assuntos
Adenocarcinoma de Células Claras/diagnóstico , Biomarcadores Tumorais/metabolismo , Cistadenocarcinoma Seroso/diagnóstico , Neoplasias do Endométrio/classificação , Neoplasias do Endométrio/diagnóstico , Endométrio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Adenocarcinoma de Células Claras/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Estudos de Casos e Controles , Cistadenocarcinoma Seroso/metabolismo , Neoplasias do Endométrio/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Proteína Desglicase DJ-1RESUMO
We investigated the evolution of SARS-CoV-2 spread in Calabria, Southern Italy, in 2022. A total of 272 RNA isolates from nasopharyngeal swabs of individuals infected with SARS-CoV-2 were sequenced by whole genome sequencing (N = 172) and/or Sanger sequencing (N = 100). Analysis of diffusion of Omicron variants in Calabria revealed the prevalence of 10 different sub-lineages (recombinant BA.1/BA.2, BA.1, BA.1.1, BA.2, BA.2.9, BA.2.10, BA.2.12.1, BA.4, BA.5, BE.1). We observed that Omicron spread in Calabria presented a similar trend as in Italy, with some notable exceptions: BA.1 disappeared in April in Calabria but not in the rest of Italy; recombinant BA.1/BA.2 showed higher frequency in Calabria (13%) than in the rest of Italy (0.02%); BA.2.9, BA.4 and BA.5 emerged in Calabria later than in other Italian regions. In addition, Calabria Omicron presented 16 non-canonical mutations in the S protein and 151 non-canonical mutations in non-structural proteins. Most non-canonical mutations in the S protein occurred mainly in BA.5 whereas non-canonical mutations in non-structural or accessory proteins (ORF1ab, ORF3a, ORF8 and N) were identified in BA.2 and BA.5 sub-lineages. In conclusion, the data reported here underscore the importance of monitoring the entire SARS-CoV-2 genome.
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COVID-19 , Humanos , COVID-19/epidemiologia , Evolução Molecular , Genoma Viral , SARS-CoV-2/genética , Itália/epidemiologiaRESUMO
Ménière's disease (MD) is a disorder of the inner ear characterized by an insidious onset and aspecific symptoms, such as dizziness, vertigo, tinnitus, and hearing loss, that may become very debilitating. The presence of endolymphatic hydrops is a common feature in MD patients, but the pathophysiology is still largely unknown. In this study, we have used a proteomics-driven approach to identify potential biomarkers of MD. To this end, plasma was obtained from whole blood of 16 individuals previously diagnosed as suffering from MD and compared to plasma from healthy donors. A depletion of the highly abundant proteins (i.e., albumin, IgG, transferrin, etc.) was performed in order to enhance the chance of detection of the less represented ones, therefore reducing the noise-background. Two-dimensional gel electrophoresis, followed by in-gel tryptic digestion of the selected spots and LC-MS/MS analysis, allowed us to identify a set of proteins whose expression appears to be differentially modulated in patients versus controls. In particular: complement factor H and B, fibrinogen alpha and gamma chains, beta-actin and pigment epithelium derived factor are over expressed; on the other hand, the levels of beta-2 glycoprotein-1, vitamin D binding protein and apolipoprotein-1 are significantly decreased in the plasma of MD-affected individuals. Even though preliminary and not necessarily linked directly to the molecular pathogenesis of the disease, our original findings suggest that a molecular signature, represented by the plasma protein profile previously described, might represent a potentially powerful, innovative and not invasive tool for early diagnosis and clinical management of MD patients. J. Cell. Physiol. 227: 308-312, 2012. © 2011 Wiley Periodicals, Inc.
Assuntos
Doença de Meniere/sangue , Proteômica , Biomarcadores/análise , Biomarcadores/sangue , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Espectrometria de Massas , Doença de Meniere/genética , Doença de Meniere/fisiopatologia , Pessoa de Meia-IdadeRESUMO
Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the ß-myosin heavy chain (ß-MHC) gene. In this study, the expression of the mutant ß-myosin protein isoform, isolated from slow-twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin-activated ATPase activity. Isometric tension, measured in single slow-twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of ß-myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant ß-myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant ß-myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein.
Assuntos
Cardiomiopatia Hipertrófica Familiar/genética , Músculo Esquelético/metabolismo , Mutação , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/genética , Miosinas Ventriculares/genética , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adolescente , Adulto , Cardiomiopatia Hipertrófica Familiar/metabolismo , Cardiomiopatia Hipertrófica Familiar/patologia , Feminino , Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Miosinas Ventriculares/biossíntese , Miosinas Ventriculares/metabolismo , Adulto JovemRESUMO
In this study, we report on the results of SARS-CoV-2 surveillance performed in an area of Southern Italy for 12 months (from March 2021 to February 2022). To this study, we have sequenced RNA from 609 isolates. We have identified circulating VOCs by Sanger sequencing of the S gene and defined their genotypes by whole-genome NGS sequencing of 157 representative isolates. Our results indicated that B.1 and Alpha were the only circulating lineages in Calabria in March 2021; while Alpha remained the most common variant between April 2021 and May 2021 (90 and 73%, respectively), we observed a concomitant decrease in B.1 cases and appearance of Gamma cases (6 and 21%, respectively); C.36.3 and Delta appeared in June 2021 (6 and 3%, respectively); Delta became dominant in July 2021 while Alpha continued to reduce (46 and 48%, respectively). In August 2021, Delta became the only circulating variant until the end of December 2021. As of January 2022, Omicron emerged and took over Delta (72 and 28%, respectively). No patient carrying Beta, Iota, Mu, or Eta variants was identified in this survey. Among the genomes identified in this study, some were distributed all over Europe (B1_S477N, Alpha_L5F, Delta_T95, Delta_G181V, and Delta_A222V), some were distributed in the majority of Italian regions (B1_S477N, B1_Q675H, Delta_T95I and Delta_A222V), and some were present mainly in Calabria (B1_S477N_T29I, B1_S477N_T29I_E484Q, Alpha_A67S, Alpha_A701S, and Alpha_T724I). Prediction analysis of the effects of mutations on the immune response (i.e., binding to class I MHC and/or recognition of T cells) indicated that T29I in B.1 variant; A701S in Alpha variant; and T19R in Delta variant were predicted to impair binding to class I MHC whereas the mutations A67S identified in Alpha; E484K identified in Gamma; and E156G and ΔF157/R158 identified in Delta were predicted to impair recognition by T cells. In conclusion, we report on the results of SARS-CoV-2 surveillance in Regione Calabria in the period between March 2021 and February 2022, identified variants that were enriched mainly in Calabria, and predicted the effects of identified mutations on host immune response.
RESUMO
Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma.
Assuntos
Apoferritinas/genética , Inativação Gênica , Proteoma/análise , Animais , Apoferritinas/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Melanoma Experimental , Metaboloma , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteômica/métodos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
The dynamic range of plasma protein abundance, ranging from milligrams to picograms per milliliter, makes characterization of this proteome nearly impossible with current analytical methods. Plasma preprocessing by high-abundance protein depletion may concomitantly remove important diagnostic information. This article describes an original chromatographic procedure to isolate proteins bound to human serum albumin (HSA). Using HSA as an "affinity agent", we significantly improved the detection and identification of HSA ligands by two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). Some of the characterized species were not previously reported in published blood databases. Albumin-binding proteins may be classified as belonging to several putative functional categories and span a wide variety of predicted physiological functions.
Assuntos
Albumina Sérica/isolamento & purificação , Cromatografia Líquida/métodos , Humanos , Espectrometria de Massas/métodos , Proteoma/química , Albumina Sérica/químicaRESUMO
Ferritin, the principal intracellular iron-storage protein localized in the cytoplasm, nucleus, and mitochondria, plays a major role in iron metabolism. The encoding ferritin genes are members of a multigene family that includes some pseudogenes. Even though pseudogenes have been initially considered as relics of ancient genes or junk DNA devoid of function, their role in controlling gene expression in normal and transformed cells has recently been re-evaluated. Numerous studies have revealed that some pseudogenes compete with their parental gene for binding to the microRNAs (miRNAs), while others generate small interference RNAs (siRNAs) to decrease functional gene expression, and still others encode functional mutated proteins. Consequently, pseudogenes can be considered as actual master regulators of numerous biological processes. Here, we provide a detailed classification and description of the structural features of the ferritin pseudogenes known to date and review the recent evidence on their mutual interrelation within the complex regulatory network of the ferritin gene family.
Assuntos
Ferritinas/genética , Neoplasias/genética , Oxirredutases/genética , Pseudogenes/genética , Animais , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Humanos , MicroRNAs/genética , RNA Interferente Pequeno/genéticaRESUMO
The cell-microenvironment communication is essential for homing of hematopoietic stem cells in stromal niches. Recent evidences support the involvement of epithelial-to-mesenchymal (EMT) process in hematopoietic stem cell homeostasis as well as in leukemia cells invasiveness and migration capability. Here, we demonstrate that the alteration of iron homeostasis and the consequent increase of redox metabolism, mediated by the stable knock down of ferritin heavy chain (FtH), enhances the expression of CXCR4 in K562 erythroleukemia cells, thus promoting CXCL12-mediated motility. Indeed, addition of the CXCR4 receptor antagonist AMD3100 reverts this effect. Upon FtH knock down K562 cells also acquire an "EMT-like" phenotype, characterized by the increase of Snail, Slug and Vimentin with the parallel loss of E-cadherin. By using fibronectin as substrate, the cell adhesion assay further shows a reduction of cell adhesion capability in FtH-silenced K562 cells. Accordingly, confocal microscopy shows that adherent K562 control cells display a variety of protrusions while FtH-silenced K562 cells remain roundish. These phenomena are largely due to the reactive oxygen species (ROS)-mediated up-regulation of HIF-1α/CXCR4 axis which, in turn, promotes the activation of NF-κB and the enhancement of EMT features. These data are confirmed by treatments with either N-acetylcysteine (NAC) or AMD3100 or NF-κB inhibitor IκB-alpha which revert the FtH-silenced K562 invasive phenotype. Overall, our findings demonstrate the existence of a direct relationship among iron metabolism, redox homeostasis and EMT in the hematological malignancies. The effects of FtH dysregulation on CXCR4/CXCL12-mediated K562 cell motility extend the meaning of iron homeostasis in the leukemia cell microenvironment.
RESUMO
PURPOSE: The aim of this study was to explore the gene expression pattern produced by the cancer-associated BRCA1 5083del19 founder mutation by using a microarray analysis. Such a mutation, identified in a subset of familial breast cancer patients, involves a deletion at the 3' end of the BRCA1 messenger leading, in the mature protein, to the ablation of the BRCT tandem domain. EXPERIMENTAL DESIGN: We generated HeLa cells stably expressing both exogenous wild-type (HeLa/(wt)BRCA1), used as a control, and 5083del19 BRCA1 (HeLa/(5083del19)BRCA1) alleles; gene chips were then used to investigate any changes in the transcription profile induced by the 5083del19 BRCA1 mutant compared with controls. RESULTS: Among the genes showing perturbation of their expression, periostin was found to be up-regulated in HeLa/(5083del19)BRCA1 cells to an extent of 72-fold versus HeLa/(pcDNA3.1/empty) and 76-fold versus HeLa/(wt)BRCA1 cells. This finding was validated both in vitro in breast cancer cell lines harboring mutations of BRCA1 and in vivo by immunohistochemistry of breast cancer specimens bearing the 5083del19 BRCA1 mutation as well as by Western blot analysis of sera obtained from patients and healthy carriers of the same mutation. CONCLUSIONS: Our results suggest that periostin overexpression, whose product is released from cells in the extracellular fluids, might be a potential marker for early cancer detection in a specific subset of hereditary breast carcinomas triggered by cancer-associated BRCA1 mutations that affect the BRCT tandem domain.
Assuntos
Neoplasias da Mama/genética , Moléculas de Adesão Celular/genética , Regulação Neoplásica da Expressão Gênica , Genes BRCA1 , Mutação , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Efeito Fundador , Perfilação da Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
The ability of pathogens to sequester iron from their host cells and proteins affects their virulence. Moreover, iron is required for various innate host defense mechanisms as well as for acquired immune responses. Therefore, intracellular iron concentration may influence the interplay between pathogens and immune system. Here, we investigated whether changes in iron concentrations and intracellular ferritin heavy chain (FTH) abundance may modulate the expression of Major Histocompatibility Complex molecules (MHC), and susceptibility to Natural Killer (NK) cell cytotoxicity. FTH downregulation, either by shRNA transfection or iron chelation, led to MHC surface reduction in primary cancer cells and macrophages. On the contrary, mouse embryonic fibroblasts (MEFs) from NCOA4 null mice accumulated FTH for ferritinophagy impairment and displayed MHC class I cell surface overexpression. Low iron concentration, but not FTH, interfered with IFN-γ receptor signaling, preventing the increase of MHC-class I molecules on the membrane by obstructing STAT1 phosphorylation and nuclear translocation. Finally, iron depletion and FTH downregulation increased the target susceptibility of both primary cancer cells and macrophages to NK cell recognition. In conclusion, the reduction of iron and FTH may influence the expression of MHC class I molecules leading to NK cells activation.
Assuntos
Apoferritinas/metabolismo , Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ferro/metabolismo , Células Matadoras Naturais/imunologia , Animais , Apoferritinas/genética , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/genética , Desferroxamina/farmacologia , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/farmacologia , Células K562 , Células Matadoras Naturais/metabolismo , Células MCF-7 , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Interferência de RNA , Sideróforos/farmacologiaRESUMO
The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53-NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis. These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation.
Assuntos
Apoferritinas/genética , Fator de Ligação a CCAAT/metabolismo , Regulação para Baixo/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Imunoprecipitação da Cromatina , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Mitochondria are the organelles deputed to energy production, but they are also involved in carcinogenesis, cancer progression, and metastasis, playing a role in altered energy metabolism in cancer cells. Mitochondrial metabolism is connected with several mitochondrial pathways such as ROS signaling, Ca2+ homeostasis, mitophagy, and mitochondrial biogenesis. These pathways are merged in an interactive super-network that seems to play a crucial role in cancer. Germline mutations of the BRCA1 gene account for 5-10% of breast cancers and confer a risk of developing the disease 10- to 20-fold much higher than in non-carriers. By considering metabolic networks that could reconcile both genetic and non-genetic causal mechanisms in BRCA1 driven tumorigenesis, we herein based our study on the hypothesis that BRCA1 haploinsufficiency might drive metabolic rewiring in breast epithelial cells, acting as a push toward malignant transformation. Using 2D-DIGE we analyzed and compared the mitochondrial proteomic profile of sporadic breast cancer cell line (MCF7) and BRCA1 mutated breast cancer cell line (HCC1937). Image analysis was carried out with Decider Software, and proteins differentially expressed were identified by LC-MS/MS on a quadrupole-orbitrap mass spectrometer Q-Exactive. Ingenuity pathways analysis software was used to analyze the fifty-three mitochondrial proteins whose expression resulted significantly altered in response to BRCA1 mutation status. Mitochondrial Dysfunction and oxidative phosphorylation, and energy production and nucleic acid metabolism were, respectively, the canonical pathway and the molecular function mainly affected. Western blotting analysis was done to validate the expression and the peculiar mitochondrial compartmentalization of specific proteins such us HSP60 and HIF-1α. Particularly intriguing is the correlation between BRCA1 mutation status and HIF-1α localization into the mitochondria in a BRCA1 dependent manner. Data obtained led us to hypothesize an interesting connection between BRCA1 and mitochondria pathways, capable to trigger metabolic changes, which, in turn, sustain the high energetic and anabolic requirements of the malignant phenotype.
RESUMO
BRCA1 is a nuclear phosphoprotein that plays a key role in many cell functions, including DNA repair, control of transcription, recombination and cell cycle homeostasis. Inherited missense mutations in the BRCA1 gene may predispose to breast and ovarian cancer, but the molecular mechanisms underlying BRCA1-induced tumorigenesis are still to be elucidated. Functional studies performed so far have contributed to the characterization of several single-nucleotide variants, mostly located at the BRCT domain, but very little is known about modifications in the protein pattern occurring in cells carrying these mutations. To shed more light in the molecular events triggered by missense mutations affecting breast cancer susceptibility genes, we have analyzed the whole cell proteome of stably transfected HeLa cell lines bearing three distinct single aminoacid changes in the BRCA1 protein (Ser1841Asn, Met1775Arg and Trp1837Arg) by means of liquid chromatography coupled to tandem-mass spectrometry. The results show that the Met1775Arg and the Trp1837Arg do not produce significant changes in the proteomic pattern compared to cells transfected with the wild-type BRCA1 cDNA. On the other hand, a different profile is detected in the BRCA1 Ser1841Asn-bearing cell line. In this particular subset, our attention has been focused on two proteins--the tumor protein D52 (TD52) and the folate receptor alpha (FOL1)--whose expression has been already reported to be upregulated in breast cancer, as well as in other tumors. Our findings indicate that Ser1841Asn BRCA1 mutation is able to activate specific protein pathways that are not triggered by other single aminoacid changes and pinpoint to the role TD52 and FOL1 as potential markers in breast cancer patients carrying this particular BRCA1 gene alteration.
Assuntos
Proteína BRCA1/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/metabolismo , Proteínas de Transporte/biossíntese , Mutação de Sentido Incorreto , Proteínas de Neoplasias/biossíntese , Receptores de Superfície Celular/biossíntese , Substituição de Aminoácidos , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Feminino , Receptores de Folato com Âncoras de GPI , Regulação Neoplásica da Expressão Gênica/genética , Predisposição Genética para Doença , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteômica , Receptores de Superfície Celular/genéticaRESUMO
The BRCA1 tumor suppressor gene encodes a phosphoprotein involved in many cellular key functions including DNA repair, transcription regulation, cell-cycle control and apoptosis. Most of these functions are strictly related to the ability of BRCA1 to interact with the other partners of a multimeric complex called BASC. Among these components, an important role is played by the human homolog of the bacterial MutL, MLH1. In this study, we have identified the BRCA1 binding domains to MLH1 and demonstrated that three distinct mutations in one of these interaction domains can produce, in vitro, a microsatellite instability phenotype, one of the hallmarks of an imbalance in the mismatch DNA repair machinery. These data support a model in which a structural modification in a critical domain of the BRCA1 gene product secondary to single amino acid mutations, may be able, per se, to impair the DNA damage response pathway, inducing genomic instability and eventually leading to breast carcinogenesis.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Proteínas de Transporte/metabolismo , Genes BRCA1 , Instabilidade Genômica , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Sítios de Ligação , Reparo do DNA/fisiologia , Células HeLa , Humanos , Repetições de Microssatélites , Modelos Genéticos , Proteína 1 Homóloga a MutL , Fenótipo , Estrutura Terciária de Proteína , TransfecçãoRESUMO
The H ferritin promoter spans approximately 150 bp, upstream of the transcription start and is composed by two cis-elements in position -132 (A box) and -62 (B-box), respectively. The A box is recognized by the transcription factor Sp1, and the B-box by a protein complex called Bbf, which includes the CAAT binding factor NF-Y. In this study we performed a functional analysis of an H ferritin promoter allele carrying a G to T substitution adjacent to the Bbf binding site, in position -69. In vitro studies with reporter constructs revealed a significantly reduced transcriptional activity of this allele compared to that of the w.t. promoter that was mirrored by a decrease in Bbf binding. In vivo, this variant genotype is accompanied by a reduced amount of the H mRNA in peripheral blood lymphocytes.
Assuntos
Ferritinas/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Alelos , Sequência de Bases , Sítios de Ligação/genética , Fator de Ligação a CCAAT/metabolismo , DNA/genética , DNA/metabolismo , Expressão Gênica , Células HeLa , Humanos , Técnicas In Vitro , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/metabolismo , Transcrição GênicaRESUMO
Women with BRCA1 gene mutations have an increased risk for breast and ovarian cancer (BOC). Classification of missense variants as neutral or disease causing is still a challenge and has major implications for genetic counseling. BRCA1 is organized in an N-terminal ring-finger domain and two BRCT (breast cancer C-terminus) domains, involved in protein-protein interaction. The integrity of the C-terminal, BRCT repeat region is also critical for BRCA1 tumor suppressor function. Several molecular partners of BRCA1 have so far been identified; among them, the tumor suppressor protein p53 seems to play a major role. This study was aimed at evaluating the impact of two missense mutations, namely the W1837R and the S1841N, previously identified in BOC patients and located in the BRCT domain of the BRCA1 gene, on the binding capacity of this protein to p53. Co-immunoprecipitation assays of E. coli-expressed wild-type and mutated BRCTs challenged with a HeLa cell extract revealed, for the S1841N variant a significant reduction in the binding activity to p53, while the W1837R mutant showed an inverse effect. Furthermore, a clonogenic soft agar growth assay performed on HeLa cells stably transfected with either wild-type or mutant BRCA1 showed a marked decrease of the growth in wild-type BRCA1-overexpressing cells and in BRCA1S1841N-transfected cells, while no significant changes were detected in the BRCA1W1837R-transfected cells. These results demonstrate that: i) distinct single nucleotide changes in the BRCT domain of BRCA1 affect binding of this protein to the tumor suppressor p53, and ii) the two missense mutations here described are likely to play a role in breast tumorigenesis. We suggest that in vitro/in vivo experiments testing the effects of unclassified BRCA1 gene variants should therefore be taken in to consideration and that increased surveillance should be adopted in individuals bearing these two BRCA1 missense alterations.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53/metabolismo , Ágar/química , Alelos , Neoplasias da Mama/metabolismo , Proliferação de Células , Escherichia coli/metabolismo , Feminino , Células HeLa , Humanos , Técnicas In Vitro , Modelos Genéticos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Germline mutations within the mismatch repair (MMR) genes are generally found in colorectal cancer (CRC) patients with a positive family history for the presence of the neoplasia. Clinical standard criteria have been established to define hereditary-non-polyposis-colorectal cancer (HNPCC)-prone families. Interestingly, the number of MMR gene mutations found in kindreds not fulfilling these criteria is still increasing. In this work we report the identification of a novel germline mutation of the hMSH2 gene, in two CRC-bearing subjects. The two probands belong to a large kindred from South Italy with no history suggestive for cancer aggregation. On the other hand, the early-onset of the neoplasia as well as the presence of a high number of tumor infiltrating lymphocytes (TILs) in the histological specimens of both patients, prompted us to perform a comprehensive genetic analysis. This analysis included the evaluation of the microsatellite instability (MSI) status with five markers according to the National Cancer Institute recommendations, followed by direct sequencing of the hMLH1 and hMSH2 genes. Both probands were found to carry a germline missense (277 C>T) mutation leading to the change (L93F) of an amino acid residue in a highly conserved domain of the MSH2 protein. This mutation is accompanied by the loss of expression of the hMSH2 gene in the tumor tissue. Our findings suggest that in the presence of the above-mentioned criteria it may be useful to perform a molecular analysis of the MMR genes, even if the pedigree does not show marked aggregation of cancers.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas/genética , Adulto , Pareamento Incorreto de Bases , Estudos de Casos e Controles , Análise Mutacional de DNA , Reparo do DNA , Enzimas Reparadoras do DNA , Éxons , Feminino , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Itália , Masculino , Repetições de Microssatélites , Proteína 2 Homóloga a MutS , LinhagemRESUMO
Germ-line mutations in the breast cancer susceptibility BRCA1 gene account for approximately half of hereditary breast cancer cases and most of breast/ovarian cancer cases. We speculated whether breast hereditary cancers might be differentially sensitive to antitumor agents such as the mitotic spindle poisons Vinca alcaloid vinorelbine (VNR) and the taxoid docetaxel (DOC), which are commonly used in the treatment of breast cancer. We investigated the sensitivity of the BRCA1-mutated HCC1937 (derived from a BRCA1 related hereditary tumor) and BRCA1 competent MCF-7 and MDA-MB468 sporadic breast cancer cell lines to these drugs. We found that HCC1937 cells were significantly more sensitive to VNR as compared to MCF-7 or MDA-MB468 cells. Instead, BRCA1-mutated breast cancer cells exposed to DOC showed similar sensitivity as compared to BRCA1-competent MCF-7 or were less sensitive than MDA-MB468. In order to assess the role of BRCA1 in this specific pattern of chemosensitivity, we transfected the BRCA1-mutated HCC1937 cells with a full-length BRCA1 cDNA and the stable clone (HCC1937/WTBRCA1) was exposed to both drugs. Full-length BRCA1 transfection led to a significant induction of resistance to VNR, whereas only a weak but not significant increase of sensitivity to DOC was detected. Moreover, VNR induced apoptotic cell death and cytoskeletal rearrangements in HCC1937 cells. We further investigated whether a defective targeting of mitotic spindle by the mutated BRCA1 gene product might be involved in the differential sensitivity to VNR. We demonstrated that mutated BRCA1 was indeed capable of co-localizing with alpha-tubulin in the mitotic spindle, suggesting therefore that different mechanisms should account for these effects. In conclusion, our data suggest that BRCA1-mutated tumors might be differentially sensitive to anti-microtubule agents, supporting the rationale for clinical trials to improve the outcome of hereditary breast cancer patients by tailored treatments.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/genética , Genes BRCA1 , Taxoides/farmacologia , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Apoptose , Neoplasias da Mama/patologia , Citoesqueleto/patologia , Análise Mutacional de DNA , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Feminino , Mutação em Linhagem Germinativa , Humanos , Microtúbulos/patologia , Fuso Acromático/patologia , Células Tumorais Cultivadas , VinorelbinaRESUMO
BACKGROUND: Breast cancer (BC) is a leading cause of death among women. Among the major risk factors, an important role is played by familial history of BC. Germ-line mutations in BRCA1/2 genes account for most of the hereditary breast and/or ovarian cancers. Gene expression profiling studies have disclosed specific molecular signatures for BRCA1/2-related breast tumors as compared to sporadic cases, which might help diagnosis and clinical follow-up. Even though, a clear hallmark of BRCA1/2-positive BC is still lacking. Many diseases are correlated with quantitative changes of proteins in body fluids. Plasma potentially carries important information whose knowledge could help to improve early disease detection, prognosis, and response to therapeutic treatments. The aim of this study was to develop a comprehensive approach finalized to improve the recovery of specific biomarkers from plasma samples of subjects affected by hereditary BC. METHODS: To perform this analysis, we used samples from patients belonging to highly homogeneous population previously reported. Depletion of high abundant plasma proteins, 2D gel analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis were used into an integrated approach to investigate tumor-specific changes in the plasma proteome of BC patients and healthy family members sharing the same BRCA1 gene founder mutation (5083del19), previously reported by our group, with the aim to identify specific signatures. RESULTS: The comparative analysis of the experimental results led to the identification of gelsolin as the most promising biomarker. CONCLUSIONS: Further analyses, performed using a panel of breast cancer cell lines, allowed us to further elucidate the signaling network that might modulate the expression of gelsolin in breast cancer.