RESUMO
The PPARγ coactivator 1α (PGC-1α) is a transcriptional regulator of mitochondrial biogenesis and oxidative metabolism. Recent studies have highlighted a fundamental role of PGC-1α in promoting breast cancer progression and metastasis, but the physiological role of this coactivator in the development of mammary glands is still unknown. First, we show that PGC-1α is highly expressed during puberty and involution, but nearly disappeared in pregnancy and lactation. Then, taking advantage of a newly generated transgenic mouse model with a stable and specific overexpression of PGC-1α in mammary glands, we demonstrate that the re-expression of this coactivator during the lactation stage leads to a precocious regression of the mammary glands. Thus, we propose that PGC-1α action is non-essential during pregnancy and lactation, whereas it is indispensable during involution. The rapid preadipocyte-adipocyte transition, together with an increased rate of apoptosis promotes a premature mammary glands involution that cause lactation defects and pup growth retardation. Overall, we provide new insights in the comprehension of female reproductive cycles and lactation deficiency, thus opening new roads for mothers that cannot breastfeed.
Assuntos
Lactação/genética , Glândulas Mamárias Animais/metabolismo , Mitocôndrias/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Adipócitos/metabolismo , Animais , Apoptose/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Lactação/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , GravidezRESUMO
The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.
RESUMO
Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/fisiopatologia , Mesilato de Imatinib/uso terapêutico , Paraganglioma/tratamento farmacológico , Paraganglioma/fisiopatologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Mesilato de Imatinib/farmacologia , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Organogênese/efeitos dos fármacos , Organogênese/fisiologia , Paraganglioma/genética , Paraganglioma/patologia , Cultura Primária de Células , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway. METHODS: We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays. RESULTS: We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8. CONCLUSIONS: In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.
Assuntos
Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Proteína Supressora de Tumor p53/genética , Regiões 3' não Traduzidas , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Farnesoid X receptor (FXR) is the master regulator of bile acid (BA) homeostasis because it controls BA synthesis, influx, efflux, and detoxification in the gut/liver axis. Deregulation of BA homeostasis has been linked to hepatocellular carcinoma (HCC), and spontaneous hepatocarcinogenesis has been observed in FXR-null mice. This dreaded liver neoplasm has been associated with both FXR gene deletion and BA-mediated metabolic abnormalities after inactivation of FXR transcriptional activity. In the present study, we addressed the hypothesis that intestinal selective FXR reactivation would be sufficient to restore the fibroblast growth factor 15 (FGF15)/cholesterol-7alpha-hydroxylase (Cyp7a1) enterohepatic axis and eventually provide protection against HCC. To this end, we generated FXR-null mice with re-expression of constitutively active FXR in enterocytes (FXR(-/-)iVP16FXR) and corresponding control mice (FXR(-/-)iVP16). In FXR-null mice, intestinal selective FXR reactivation normalized BA enterohepatic circulation along with up-regulation of intestinal FXR transcriptome and reduction of hepatic BA synthesis. At 16 months of age, intestinal FXR reactivation protected FXR-null mice from spontaneous HCC development that occurred in otherwise FXR-null mice. Activation of intestinal FXR conferred hepatoprotection by restoring hepatic homeostasis, limiting cellular proliferation through reduced cyclinD1 expression, decreasing hepatic inflammation and fibrosis (decreased signal transducer and activator of transcription 3 activation and curtailed collagen deposition). CONCLUSION: Intestinal FXR is sufficient to restore BA homeostasis through the FGF15 axis and prevent progression of liver damage to HCC even in the absence of hepatic FXR. Intestinal-selective FXR modulators could stand as potential therapeutic intervention to prevent this devastating hepatic malignancy, even if carrying a somatic FXR mutation.
Assuntos
Ácidos e Sais Biliares/metabolismo , Carcinoma Hepatocelular/etiologia , Mucosa Intestinal/metabolismo , Neoplasias Hepáticas/etiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Envelhecimento/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação para Baixo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Genes cdc , Homeostase , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
The mucosa of the small intestine is renewed completely every 3-5 d throughout the entire lifetime by small populations of adult stem cells that are believed to reside in the bottom of the crypts and to migrate and differentiate into all the different populations of intestinal cells. When the cells reach the apex of the villi and are fully differentiated, they undergo cell death and are shed into the lumen. Reactive oxygen species (ROS) production is proportional to the electron transfer activity of the mitochondrial respiration chain. ROS homeostasis is maintained to control cell death and is finely tuned by an inducible antioxidant program. Here we show that peroxisome proliferator-activated receptor-γ coactivator-1ß (PGC-1ß) is highly expressed in the intestinal epithelium and possesses dual activity, stimulating mitochondrial biogenesis and oxygen consumption while inducing antioxidant enzymes. To study the role of PGC-1ß gain and loss of function in the gut, we generated both intestinal-specific PGC-1ß transgenic and PGC-1ß knockout mice. Mice overexpressing PGC-1ß present a peculiar intestinal morphology with very long villi resulting from increased enterocyte lifespan and also demonstrate greater tumor susceptibility, with increased tumor number and size when exposed to carcinogens. PGC-1ß knockout mice are protected from carcinogenesis. We show that PGC-1ß triggers mitochondrial respiration while protecting enterocytes from ROS-driven macromolecule damage and consequent apoptosis in both normal and dysplastic mucosa. Therefore, PGC-1ß in the gut acts as an adaptive self-point regulator, capable of providing a balance between enhanced mitochondrial activity and protection from increased ROS production.
Assuntos
Neoplasias do Colo/patologia , Enterócitos/citologia , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Fatores de Transcrição/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose , Carcinogênese , Transporte de Elétrons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genéticaRESUMO
Farnesoid X receptor (FXR, NR1H4) is a bile acid-activated transcription factor that belongs to the nuclear receptor superfamily. It is highly expressed in the enterohepatic system, where it senses bile acid levels to consequently reduce their synthesis while inducing their detoxification. Bile acids are intestinal tumor promoters and their concentrations have to be tightly regulated. Indeed, reduced expression of FXR in the intestine increases colorectal cancer susceptibility in mice, whereas its activation can promote apoptosis in genetically modified cells. Notably, despite the broad knowledge of the FXR enterohepatic transcriptional activity, the molecular mechanisms regulating FXR expression in the intestine are still unknown. Herein, by combining both gain and loss of function approaches and FXR promoter activity studies, we identified caudal-related homeobox 2 (CDX2) transcription factor as a positive regulator of FXR expression in the enterocytes. Our results provide a putative novel tool for modulating FXR expression against bile acid-related colorectal cancer progression.
Assuntos
Polipose Adenomatosa do Colo/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Transcrição Gênica , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Sítios de Ligação , Fator de Transcrição CDX2 , Linhagem Celular Tumoral , Proteínas de Homeodomínio/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND AND AIMS: Augmenter of Liver Regeneration is a protein encoded by the Growth Factor Erv1-Like gene. Its biological properties are crucial for cell survival since knock-out mice for Growth Factor Erv1-Like gene do not survive. In this study, we injected hepatotropic adenoviral particles harboring oligonucleotide sequences against Growth Factor Erv1-Like gene into 70% partially hepatectomized rats and studied the effect of gene silencing on the progression liver regeneration. METHODS: Partially hepatectomized rats were divided into three groups of animals and, before surgery, received either phosphate buffer saline, or adenoviral particles alone or adenoviral particles harboring the oligonucleotide silencing sequence. In each group, rats were sacrificed at 12, 24 and 48 h after surgery. Liver tissues were collected to analyze the expression of Augmenter of Liver Regeneration, Bax, Bcl-2 and activated Caspase-9 and -3, as well as hepatocyte proliferation and apoptosis, polyamines levels and histological and ultrastructural features. RESULTS: Growth Factor Erv1-Like gene silencing reduced the compensatory hepatocellular proliferation triggered by surgery through (i) the reduction of polyamines synthesis, hepatocyte proliferation and anti-apoptotic gene expression and (ii) the increase of pro-apoptotic gene expression and caspase activation. CONCLUSIONS: For the first time, using a technique of gene silencing in vivo, our results demonstrate that Growth Factor Erv1-Like gene knock-down, i.e., the lack of Augmenter of Liver Regeneration, modifies the expression of genes involved in cell apoptosis and inhibits early phase of DNA synthesis. As a consequence, a promotion of cell death and a reduction of cell proliferation occurs.
Assuntos
Apoptose/genética , Hepatopatias/genética , Regeneração Hepática/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/biossíntese , Proteínas/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Inativação Gênica , Hepatectomia , Humanos , Hepatopatias/terapia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , RatosRESUMO
Premenopausal breast cancer (BC) is one of the most common cancers of women in rural Africa and part of the disease load may be related to hereditary predisposition, including mutations in the BRCA1 gene. However, the BRCA1 mutations associated with BC in Africa are scarcely characterized. We report here 33 BRCA1 point mutations, among which 2 novel missense variants, found in 59 Central Sudanese premenopausal BC patients. The high fractions of mutations with intercontinental and uniquely African distribution (17/33, 51.5 % and 14/33, 42.4 %, respectively) are in agreement with the high genetic diversity expected in an African population. Overall 24/33 variants (72.7 %) resulted neutral; 8/33 of unknown significance (24.3 %, including the 2 novel missense mutations); 1 (3.0 %) overtly deleterious. Notably, in silico studies predict that the novel C-terminal missense variant c.5090G>A (p.Cys1697Tyr) affects phosphopeptide recognition by the BRCA1 BRCT1 domain and may have a pathogenic impact. Genetic variation and frequency of unique or rare mutations of uncertain clinical relevance pose significant challenges to BRCA1 testing in Sudan, as it might happen in other low-resource rural African contexts.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Predisposição Genética para Doença/genética , Mutação Puntual , Pré-Menopausa , Adulto , Sequência de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/genética , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Estrutura Quaternária de Proteína , Sudão , Adulto JovemRESUMO
Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The "NOTCH signaling pathway" was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.
Assuntos
Epigênese Genética/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Paraganglioma/genética , Paraganglioma/patologia , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Western Blotting , Caspases/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Análise Mutacional de DNA , Imunofluorescência , Humanos , Imuno-Histoquímica , Lentivirus/genética , Análise em Microsséries , Microscopia Imunoeletrônica , Nervos Periféricos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Succinato Desidrogenase/genética , TransfecçãoRESUMO
Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.
Assuntos
Diferenciação Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Adulto , Idoso , Células CACO-2 , Polaridade Celular , Colo/citologia , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Mucosa Intestinal/citologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Transporte ProteicoRESUMO
BACKGROUND: SEL1L gene product is implicated in the endoplasmic reticulum (ER)-associated protein degradation and Unfolded Protein Response pathways. This gene and associated miRNAs have been indicated as predictive and prognostic markers of pancreatic cancer. AIM: Explore the role of SEL1L in colorectal cancer (CRC) progression. METHODS: SEL1L expression was analysed immunohistochemically in 153 adenomas and 71 CRCs from African American and North Italian patients. The distribution of stained cells was determined by computing median and inter quartile range. The receiver operating characteristics plot was used as discriminate power of SEL1L expression, CRC diagnosis and the effects on patient survival. RESULTS: SEL1L was low in normal mucosa and confined to few scattered cells at the base crypt of the villi and in the foveolar glandular compartment. The highest levels were in Paneth cells within the lysosomes. The enterocytic progenitor cells and mature enterocytes showed less cytoplasmic staining. In CRCs, SEL1L expression significantly correlated with the progression from adenoma to carcinoma (P = 0.0001) being stronger in well-to-moderately differentiated cancers. No correlation was found with other clinicopathological characteristics or ethnicity. CONCLUSIONS: SEL1L expression is a potential CRC tissue biomarker since its expression is significantly higher in adenoma cells with respect to normal mucosa. The levels of expression decrease sensibly in undifferentiated CRC cancers. Interestingly, Paneth cells contain high levels of SEL1L protein that could indicate pre-neoplastic mucosa undergoing neoplastic transformation. Since SEL1L's major function lies within ER stress and active ERAD response, it may identify CRCs with differentiated secretory phenotype and acute cellular stress.
Assuntos
Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica , Neoplasias Colorretais/diagnóstico , Proteínas/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/diagnóstico , Adenoma/metabolismo , Adenoma/patologia , Western Blotting , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Taxa de Sobrevida , Análise Serial de Tecidos , Resposta a Proteínas não DobradasRESUMO
Cholesterol homeostasis is critical for cellular proliferation. Liver X receptor (LXR) alpha and beta are the nuclear receptors responsible for regulation of cholesterol metabolism. In physiological conditions, high intracellular cholesterol levels cause increased synthesis of oxysterols, which activate LXR, thus triggering a transcriptional response for cholesterol secretion and catabolism. Here we employed a mouse model of partial hepatectomy (PH) to dissect the molecular pathways connecting cholesterol homeostasis, cellular proliferation, and LXR. First, we show that hepatic cholesterol content increases after PH, whereas the entire LXR transcriptome is down-regulated. Although LXR messenger RNA (mRNA) levels are unmodified, LXR target genes are significantly down-regulated on day 1 after PH and restored to control levels on day 7, when the liver reaches normal size. The inactivation of LXR following PH is related to the reduced oxysterol availability by way of decreased synthesis, and increased sulfation and secretion. On the contrary, cholesterol synthesis is up-regulated, and extracellular matrix remodeling is enhanced. Second, we show that reactivation of LXR by way of a synthetic ligand determines a negative modulation of hepatocyte proliferation. This effect is sustained by the reactivation of hepatic cholesterol catabolic and secretory pathways, coupled with a significant reduction of cholesterol biosynthesis. Our data unveil a previously unrecognized and apparently paradoxical scenario of LXR modulation. During liver regeneration LXR activity is abated in spite of increasing intracellular cholesterol levels. Turning off LXR-transcriptional pathways is crucial to guaranteeing the requisite intracellular cholesterol levels of regenerating hepatocytes. In line with this hypothesis, pharmacological LXR reactivation during PH significantly reduces liver regeneration capacity.
Assuntos
Colesterol/metabolismo , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Receptores Nucleares Órfãos/fisiologia , Animais , Proliferação de Células , Matriz Extracelular/metabolismo , Hepatectomia , Regeneração Hepática , Receptores X do Fígado , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Receptores Nucleares Órfãos/antagonistas & inibidores , Receptores Nucleares Órfãos/genética , Triglicerídeos/metabolismoRESUMO
Juvenile breast cancer is rare and poorly known. We studied a series of five breast cancer patients diagnosed within 25 years of age that included two adolescents, 12- and 15-years-old, and 3 young women, 21-, 21-, and 25-years-old, respectively. All cases were scanned for germline mutations along the entire BRCA1/2 coding sequences and TP53 exons 4-10, using protein truncation test, denaturing high performance liquid chromatography and direct sequencing. Paraffin-embedded primary tumors (available for 4/5 cases), and a distant metastasis (from the 15-years-old) were characterized for histological and molecular tumor subtype, human papilloma virus (HPV) types 16/18 E6 sequences and tumor-associated mutations in TP53 exons 5-8. A BRCA2 germline mutation (p.Ile2490Thr), previously reported in breast cancer and, as compound heterozygote, in Fanconi anemia, was identified in the 21-year-old patient diagnosed after pregnancy, negative for cancer family history. The tumor was not available for study. Only germline polymorphisms in BRCA1/2 and/or TP53 were detected in the other cases. The tumors of the 15- and 12-years-old were, respectively, classified as glycogen-rich carcinoma with triple negative subtype and as secretory carcinoma with basal subtype. The tumors of the 25-year-old and of the other 21-year-old were, respectively, diagnosed as infiltrating ductal carcinoma with luminal A subtype and as lobular carcinoma with luminal B subtype. No somatic TP53 mutations were found, but tumor-associated HPV 16 E6 sequences were retrieved from the 12- and 25-year-old, while both HPV 16 and HPV 18 E6 sequences were found in the tumor of the 15-year-old and in its associated metastasis. Blood from the 15- and 25-year-old, diagnosed with high-stage disease, resulted positive for HPV 16 E6. All the HPV-positive cases were homozygous for arginine at TP53 codon 72, a genotype associated with HPV-related cancer risk, and the tumors showed p16(INK4A) immunostaining, a marker of HPV-associated cancers. Notably menarche at 11 years was reported for the two adolescents, while the 25-year-old was diagnosed after pregnancy and breast-feeding. Our data suggest that high-risk HPV infection is involved in a subset of histopathologically heterogeneous juvenile breast carcinomas associated with menarche or pregnancy and breast-feeding. Furthermore we implicate BRCA2 in a juvenile breast carcinoma diagnosed at 21 years of age, 4 years after an early full-term pregnancy, in absence of cancer family history.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/virologia , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Carcinoma Lobular/virologia , Criança , Primers do DNA/química , Primers do DNA/genética , DNA Viral/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos , Mutação em Linhagem Germinativa/genética , Humanos , Técnicas Imunoenzimáticas , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/secundário , Neoplasias Ovarianas/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Reação em Cadeia da Polimerase , Gravidez , Fatores de Risco , Adulto JovemRESUMO
A series of 2-heteroarylthioalkanoic acids were synthesized through systematic structural modifications of clofibric acid and evaluated for human peroxisome proliferator-activated receptor alpha (PPARalpha) transactivation activity, with the aim of obtaining new hypolipidemic compounds. Some thiophene and benzothiazole derivatives showing a good activation of the receptor alpha were screened for activity against the PPARgamma isoform. The gene induction of selected compounds was also investigated in the human hepatoma cell line.
Assuntos
Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , PPAR alfa/agonistas , Enxofre/química , Linhagem Celular Tumoral , Ácido Clofíbrico/síntese química , Ácido Clofíbrico/química , Humanos , Oxigênio/química , PPAR alfa/genética , Estereoisomerismo , Ativação TranscricionalRESUMO
Intraluminal phospholipids affect micellar solubilization and absorption of cholesterol. We here study cholesterol transport from taurocholate-phospholipid-cholesterol micelles to CaCo2 cells, and associated effects on ABC-A1 mediated cholesterol efflux. Micellar incorporation of egg-yolk-phosphatidylcholine markedly increased apical retention of the sterol with decreased expression of ABC-A1, an effect that is prevented by synthetic liver X receptor (LXR) or retinoid X receptor (RXR) agonists. On the other hand, incorporation of lyso-phosphatidylcholine (LysoPC) increased ABC-A1-HDL-dependent basolateral cholesterol efflux, an effect that is abated when LXR is silenced. Thus, the modulation of cholesterol metabolism via intraluminal phospholipids is related to the activity of the oxysterol nuclear receptor LXR.
Assuntos
Colesterol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metabolismo dos Lipídeos , Micelas , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Biológico , Células CACO-2 , Proteínas de Ligação a DNA/genética , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Germline and somatic genomic rearrangement play a relevant role in the pathogenesis of genetic disorders, and their identification is a fundamental task in molecular diagnosis. However, screening for structural genomic abnormalities is often not included in routine mutational analyses and consequently the proportion of rearrangements playing a pathogenic role in several genetic disorders is likely to be underestimated. A wide range of molecular techniques for the detection of large genomic rearrangements has been developed: some methods have the power to screen the whole genome, others are designed to analyze one or few loci that are known to be involved in a specific disease; some may detect balanced rearrangements, while others only unbalanced rearrangements; some are suitable for detection of germline abnormalities, yet others also detect somatic abnormalities. This review provides a brief summary of principles, applications and limitations of the methods available for the screening of genomic rearrangements, focusing on multiplex PCR-based protocols that are currently employed in routine detection of extended germline genomic deletions or duplications. Future developments based on microarray platforms and high-throughput sequencing are also discussed.
Assuntos
Rearranjo Gênico , Genoma Humano , Reação em Cadeia da Polimerase/métodos , Células Germinativas , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The etiology of breast cancer in Africa is scarcely investigated. Breast cancer was responsible for 456/2,233 cancer patients (20.4%) ascertained between 1999 and 2004 at Gezira University, Central Sudan. Male breast cancer accounted for 16/456 patients (3.5%), 275/440 female patients (62.5%) were premenopausal and 150/440 cases (34%) occurred in women with > or =5 childbirths. We characterized for germline BRCA1/2 mutations a one-year series of patients (34 females, 1 male) selected by diagnosis within age 40 years or male gender. Overall 33/35 patients were found to carry 60 BRCA1/2 variants, of which 17 (28%) were novel, 22 (37%) reported in populations from various geographic areas and 21 (35%) reported worldwide. Detected variants included 5 truncating mutations, one of which (in BRCA2) was in the male patient. The 55 non-truncating variants included 3 unclassified variants predicted to affect protein product and not co-occurring with a truncating mutation in the same gene. Patients were from different tribes but AMOVA showed that most BRCA1/2 variation was within individuals (86.41%) and patients clustered independently of tribe in a phylogenetic tree. Cluster analysis based on age at cancer diagnosis and reproductive variables split female patients in two clusters that, by factor analysis, were explained by low versus high scores of the total period occupied by pregnancies and lactation. The cluster with low scores comprised all 4 patients with truncating mutations and 3 of the 4 carriers of unclassified variants predicted to affect protein product. Our findings suggest that in Central Sudan BRCA1/2 represent an important etiological factor of breast cancer in males and young women less exposed to pregnancy and lactation. Factors other than BRCA1/2 may contribute to breast cancer in young highly multiparous women who breast-fed for prolonged periods.