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1.
Mol Cell ; 82(13): 2472-2489.e8, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35537449

RESUMO

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Proteínas F-Box , Histona-Lisina N-Metiltransferase , Histona Desmetilases com o Domínio Jumonji , Proteínas do Grupo Polycomb , Proteína SMARCB1 , Cromatina/genética , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Células Tumorais Cultivadas/metabolismo
2.
Int J Gynecol Pathol ; 40(4): 324-332, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32897971

RESUMO

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.


Assuntos
Aborto Espontâneo/metabolismo , Acetilglucosamina/metabolismo , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Queratina-8/metabolismo , Vimentina/metabolismo , Aborto Espontâneo/imunologia , Acetilglucosamina/imunologia , Vilosidades Coriônicas/imunologia , Vilosidades Coriônicas/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Gravidez , Primeiro Trimestre da Gravidez/imunologia , Primeiro Trimestre da Gravidez/metabolismo , Trofoblastos/imunologia , Trofoblastos/metabolismo , Regulação para Cima
3.
Lab Invest ; 94(4): 409-21, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24535260

RESUMO

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.


Assuntos
Carcinoma Ductal Pancreático/imunologia , Macrófagos/fisiologia , Pâncreas/patologia , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/fisiologia , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Metaplasia , Camundongos , Neoplasias Pancreáticas/patologia , Receptor Cross-Talk
4.
Nat Commun ; 15(1): 7303, 2024 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-39181868

RESUMO

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.


Assuntos
Micrognatismo , Regiões Promotoras Genéticas , Proteínas Repressoras , Tumor Rabdoide , Proteína SMARCB1 , Fatores de Transcrição , Animais , Humanos , Masculino , Camundongos , Anormalidades Múltiplas , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Sistemas CRISPR-Cas , Face/anormalidades , Deformidades Congênitas do Pé/genética , Deformidades Congênitas do Pé/metabolismo , Deformidades Congênitas da Mão , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Micrognatismo/genética , Micrognatismo/metabolismo , Mutação , Pescoço/anormalidades , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/metabolismo , Proteína SMARCB1/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica
5.
Dev Dyn ; 241(3): 583-94, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22275141

RESUMO

BACKGROUND: The assembly of distinct proteins into tight junctions results in the formation of a continuous barrier that regulates the paracellular flux of water, ions, and small molecules across epithelia. The claudin protein family encompasses numerous major structural components of tight junctions. These proteins specify the permeability characteristics of tight junctions and consequently, some of the physiological properties of epithelia. Furthermore, defective claudin expression has been found to correlate with some diseases, tumor progression, and defective morphogenesis. Investigating the pattern of claudin expression during embryogenesis or in certain pathological conditions is necessary to begin disclosing the role of these proteins in health and disease. RESULTS: This study analyzed the expression of several claudins during mouse pancreas organogenesis and in pancreatic intraepithelial neoplasias of mouse and human origin. CONCLUSIONS: Our results underscored a distinctive, dynamic distribution of certain claudins in both the developing pancreas and the pancreatic epithelium undergoing neoplastic transformation.


Assuntos
Transformação Celular Neoplásica , Claudinas/metabolismo , Morfogênese , Pâncreas/embriologia , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Animais , Claudinas/genética , Epitélio/embriologia , Epitélio/metabolismo , Epitélio/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Pâncreas/metabolismo , Ductos Pancreáticos/embriologia , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Células Tumorais Cultivadas
6.
Cancers (Basel) ; 15(14)2023 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-37509392

RESUMO

Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy.

7.
Ultrastruct Pathol ; 36(6): 387-99, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23181508

RESUMO

Integrins mediate cell adhesion to the extracellular matrix. Integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site and has been associated with high malignant potential in breast cancer cells, signaling the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including RGD peptides, have been used as potential anti-cancer agents. In the present work, the effect of the linear RGD hexapeptide GRGDSP was studied, for the first time, on breast tumor explants, as well as on well-spread human breast cancer cells from primary cultures, using the explant technique, to clarify the role of this peptide in the suppression of breast cancer cell migration. The results showed that incubation of breast tumor explants with RGD peptide at the beginning of culture development inhibited completely the migration of cancer cells out of the tissue fragment as revealed by electron microscopy. RGD incubation of well-spread breast cancer cells from primary culture resulted in rounding and shrinkage of the cells accompanied by altered distribution of integrin alphavbeta3 and concomitant F-actin cytoskeletal disorganization, as revealed by immunofluorescence. Electron immunocytochemistry showed aggregation of integrin alphavbeta3 at the cell periphery and its detection in noncoated vesicles. However, Western immunoblotting showed no change in beta3 subunit expression, despite the altered distribution of the integrin alphavbeta3. In light of the above, it appears that the RGD peptide plays an important role in the modulation of cell motility and in the perturbation of cell attachment affecting the malignant potential of breast cancer cells in primary cultures.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Integrina alfaVbeta3/antagonistas & inibidores , Oligopeptídeos/farmacologia , Actinas/metabolismo , Antineoplásicos/metabolismo , Western Blotting , Neoplasias da Mama/ultraestrutura , Carcinoma Ductal de Mama/ultraestrutura , Forma Celular/efeitos dos fármacos , Feminino , Humanos , Integrina alfaVbeta3/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Oligopeptídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Técnicas de Cultura de Tecidos , Células Tumorais Cultivadas
8.
Clin Cancer Res ; 26(18): 4995-5006, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32631955

RESUMO

PURPOSE: Rhabdoid tumors are devastating pediatric cancers in need of improved therapies. We sought to identify small molecules that exhibit in vitro and in vivo efficacy against preclinical models of rhabdoid tumor. EXPERIMENTAL DESIGN: We screened eight rhabdoid tumor cell lines with 481 small molecules and compared their sensitivity with that of 879 other cancer cell lines. Genome-scale CRISPR-Cas9 inactivation screens in rhabdoid tumors were analyzed to confirm target vulnerabilities. Gene expression and CRISPR-Cas9 data were queried across cell lines and primary rhabdoid tumors to discover biomarkers of small-molecule sensitivity. Molecular correlates were validated by manipulating gene expression. Subcutaneous rhabdoid tumor xenografts were treated with the most effective drug to confirm in vitro results. RESULTS: Small-molecule screening identified the protein-translation inhibitor homoharringtonine (HHT), an FDA-approved treatment for chronic myelogenous leukemia (CML), as the sole drug to which all rhabdoid tumor cell lines were selectively sensitive. Validation studies confirmed the sensitivity of rhabdoid tumor to HHT was comparable with that of CML cell lines. Low expression of the antiapoptotic gene BCL2L1, which encodes Bcl-XL, was the strongest predictor of HHT sensitivity, and HHT treatment consistently depleted Mcl-1, the synthetic-lethal antiapoptotic partner of Bcl-XL. Rhabdoid tumor cell lines and primary-tumor samples expressed low BCL2L1, and overexpression of BCL2L1 induced resistance to HHT in rhabdoid tumor cells. Furthermore, HHT treatment inhibited rhabdoid tumor cell line and patient-derived xenograft growth in vivo. CONCLUSIONS: Rhabdoid tumor cell lines and xenografts are highly sensitive to HHT, at least partially due to their low expression of BCL2L1. HHT may have therapeutic potential against rhabdoid tumors.


Assuntos
Mepesuccinato de Omacetaxina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Tumor Rabdoide/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Mepesuccinato de Omacetaxina/uso terapêutico , Humanos , Camundongos , Tumor Rabdoide/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genética
9.
Cancer Cell Int ; 9: 19, 2009 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-19664271

RESUMO

BACKGROUND: NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies. RESULTS: The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures. CONCLUSION: The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

10.
Cancer Cell ; 35(1): 95-110.e8, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30595504

RESUMO

Biallelic inactivation of SMARCB1, encoding a member of the SWI/SNF chromatin remodeling complex, is the hallmark genetic aberration of atypical teratoid rhabdoid tumors (ATRT). Here, we report how loss of SMARCB1 affects the epigenome in these tumors. Using chromatin immunoprecipitation sequencing (ChIP-seq) on primary tumors for a series of active and repressive histone marks, we identified the chromatin states differentially represented in ATRTs compared with other brain tumors and non-neoplastic brain. Re-expression of SMARCB1 in ATRT cell lines enabled confirmation of our genome-wide findings for the chromatin states. Additional generation of ChIP-seq data for SWI/SNF and Polycomb group proteins and the transcriptional repressor protein REST determined differential dependencies of SWI/SNF and Polycomb complexes in regulation of diverse gene sets in ATRTs.


Assuntos
Cromatina/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Tumor Rabdoide/metabolismo , Proteína SMARCB1/metabolismo , Teratoma/metabolismo , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Proteína SMARCB1/química , Análise de Sequência de DNA , Análise de Sobrevida
11.
Cancer Cell Int ; 7: 16, 2007 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-17910753

RESUMO

BACKGROUND: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. RESULTS: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. CONCLUSION: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

12.
Sci Rep ; 7(1): 11144, 2017 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-28894253

RESUMO

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Animais , Biomarcadores Tumorais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Instabilidade Genômica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neoplasias Pancreáticas/mortalidade , Tolerância a Radiação/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
13.
Neoplasia ; 18(3): 172-84, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26992918

RESUMO

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.


Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Homeodomínio/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Supressoras de Tumor/genética , Células Acinares/patologia , Animais , Transformação Celular Neoplásica/patologia , Ceruletídeo/administração & dosagem , Heterozigoto , Proteínas de Homeodomínio/biossíntese , Humanos , Inflamação/genética , Inflamação/patologia , Metaplasia/genética , Metaplasia/patologia , Camundongos , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Proteínas Supressoras de Tumor/biossíntese
14.
Diabetes ; 65(3): 687-98, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26631740

RESUMO

Transcription factor expression fluctuates during ß-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature ß-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with ß-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature ß-cells has no functional consequences; in contrast, Prox1 overexpression in immature ß-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal ß-cells. Also, we uncovered substantial deficiency in ß-cells that overexpress Prox1 of the key regulator of ß-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-ßH1 ß-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal ß-cells.


Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Proteínas de Homeodomínio/genética , Hiperglicemia/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Animais Recém-Nascidos , Linhagem Celular , Imunoprecipitação da Cromatina , Simulação por Computador , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Teste de Tolerância a Glucose , Humanos , Células Secretoras de Insulina/citologia , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real
15.
Gene Expr Patterns ; 14(1): 19-29, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24140890

RESUMO

The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis.


Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Apoptose , Sequência de Bases , Caspase 3/metabolismo , Embrião de Mamíferos/citologia , Evolução Molecular , Etiquetas de Sequências Expressas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Tecido Nervoso/embriologia , Proteínas Nucleares/química , Fosfoproteínas/química , Gravidez , Ratos , Ratos Wistar , Proteína X Associada a bcl-2/metabolismo
16.
Biochem Biophys Res Commun ; 323(3): 796-801, 2004 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-15381070

RESUMO

The NUCKS gene is located on human chromosome 1q32.1 and consists of seven exons and six introns. The gene lacks a TATA box but contains two Inr elements, two GC boxes, and one consensus-binding site for E2F-1. NUCKS is expressed in all human adult and foetal tissues investigated, and has all the features of being a housekeeping gene. Both data searches and Western immunoblotting experiments show that a homologous protein is present in fish, amphibians, and birds but not in insects and yeast, suggesting that NUCKS is a vertebrate specific gene. In all the species investigated, the protein contains several consensus phosphorylation sites for cyclin-dependent kinases and CK-2, and we have shown that the fish protein (like mammalian NUCKS) indeed is a substrate for CDK1 and CK-2 in vitro. The NUCKS protein is also conserved with respect to a DNA-binding domain previously characterised in mammals, and two putative bipartite nuclear localisation signals.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 1/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Anfíbios , Animais , Aves , Peixes , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
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