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1.
Cell Mol Gastroenterol Hepatol ; 17(4): 639-656, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38199279

RESUMO

BACKGROUND & AIMS: Chronic inflammatory illnesses are debilitating and recurrent conditions associated with significant comorbidities, including an increased risk of developing cancer. Extensive tissue remodeling is a hallmark of such illnesses, and is both a consequence and a mediator of disease progression. Despite previous characterization of epithelial and stromal remodeling during inflammatory bowel disease, a complete understanding of its impact on disease progression is lacking. METHODS: A comprehensive proteomic pipeline using data-independent acquisition was applied to decellularized colon samples from the Muc2 knockout (Muc2KO) mouse model of colitis for an in-depth characterization of extracellular matrix remodeling. Unique proteomic profiles of the matrisomal landscape were extracted from prepathologic and overt colitis. Integration of proteomics and transcriptomics data sets extracted from the same murine model produced network maps describing the orchestrating role of matrisomal proteins in tissue remodeling during the progression of colitis. RESULTS: The in-depth proteomic workflow used here allowed the addition of 34 proteins to the known colon matrisomal signature. Protein signatures of prepathologic and pathologic colitic states were extracted, differentiating the 2 states by expression of small leucine-rich proteoglycans. We outlined the role of this class and other matrisomal proteins in tissue remodeling during colitis, as well as the potential for coordinated regulation of cell types by matrisomal ligands. CONCLUSIONS: Our work highlights a central role for matrisomal proteins in tissue remodeling during colitis and defines orchestrating nodes that can be exploited in the selection of therapeutic targets.


Assuntos
Colite , Proteômica , Camundongos , Animais , Matriz Extracelular/metabolismo , Colite/patologia , Doença Crônica , Progressão da Doença
2.
Proc Natl Acad Sci U S A ; 120(42): e2303774120, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37816052

RESUMO

Although robustly expressed in the disease-free (DF) breast stroma, CD36 is consistently absent from the stroma surrounding invasive breast cancers (IBCs). In this study, we primarily observed CD36 expression in adipocytes and intralobular capillaries within the DF breast. Larger vessels concentrated in interlobular regions lacked CD36 and were instead marked by the expression of CD31. When evaluated in perilesional capillaries surrounding ductal carcinoma in situ, a nonobligate IBC precursor, CD36 loss was more commonly observed in lesions associated with subsequent IBC. Peroxisome proliferator-activated receptor γ (PPARγ) governs the expression of CD36 and genes involved in differentiation, metabolism, angiogenesis, and inflammation. Coincident with CD36 loss, we observed a dramatic suppression of PPARγ and its target genes in capillary endothelial cells (ECs) and pericytes, which typically surround and support the stability of the capillary endothelium. Factors present in conditioned media from malignant cells repressed PPARγ and its target genes not only in cultured ECs and pericytes but also in adipocytes, which require PPARγ for proper differentiation. In addition, we identified a role for PPARγ in opposing the transition of pericytes toward a tumor-supportive myofibroblast phenotype. In mouse xenograft models, early intervention with rosiglitazone, a PPARγ agonist, demonstrated significant antitumor effects; however, following the development of a palpable tumor, the antitumor effects of rosiglitazone were negated by the repression of PPARγ in the mouse stroma. In summary, PPARγ activity in healthy tissues places several stromal cell types in an antitumorigenic state, directly inhibiting EC proliferation, maintaining adipocyte differentiation, and suppressing the transition of pericytes into tumor-supportive myofibroblasts.


Assuntos
Neoplasias da Mama , Animais , Feminino , Humanos , Camundongos , Adipócitos/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Rosiglitazona/farmacologia
3.
bioRxiv ; 2023 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-37333362

RESUMO

Esophageal adenocarcinoma arises from Barrett's esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients' paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barrett's esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming.

4.
Gastroenterology ; 165(2): 374-390, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37196797

RESUMO

BACKGROUND & AIMS: Elements of field cancerization, including atrophic gastritis, metaplasia, and dysplasia, promote gastric cancer development in association with chronic inflammation. However, it remains unclear how stroma changes during carcinogenesis and how the stroma contributes to progression of gastric preneoplasia. Here we investigated heterogeneity of fibroblasts, one of the most important elements in the stroma, and their roles in neoplastic transformation of metaplasia. METHODS: We used single-cell transcriptomics to evaluate the cellular heterogeneity of mucosal cells from patients with gastric cancer. Tissue sections from the same cohort and tissue microarrays were used to identify the geographical distribution of distinct fibroblast subsets. We further evaluated the role of fibroblasts from pathologic mucosa in dysplastic progression of metaplastic cells using patient-derived metaplastic gastroids and fibroblasts. RESULTS: We identified 4 subsets of fibroblasts within stromal cells defined by the differential expression of PDGFRA, FBLN2, ACTA2, or PDGFRB. Each subset was distributed distinctively throughout stomach tissues with different proportions at each pathologic stage. The PDGFRα+ subset expanded in metaplasia and cancer compared with normal, maintaining a close proximity with the epithelial compartment. Co-culture of metaplasia- or cancer-derived fibroblasts with gastroids showing the characteristics of spasmolytic polypeptide-expressing metaplasia-induced disordered growth, loss of metaplastic markers, and increases in markers of dysplasia. Culture of metaplastic gastroids with conditioned media from metaplasia- or cancer-derived fibroblasts also promoted dysplastic transition. CONCLUSIONS: These findings indicate that fibroblast associations with metaplastic epithelial cells can facilitate direct transition of metaplastic spasmolytic polypeptide-expressing metaplasia cell lineages into dysplastic lineages.


Assuntos
Mucosa Gástrica , Neoplasias Gástricas , Humanos , Mucosa Gástrica/patologia , Neoplasias Gástricas/patologia , Hiperplasia , Metaplasia/patologia , Fibroblastos/metabolismo
5.
Proteomics ; 23(7-8): e2200021, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36228107

RESUMO

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition (DIA) strategies, and stringent statistical processing to analyze "Tumor" and matched adjacent histologically normal ("Matched Normal") tissues from patients with LSCC. Overall, 1802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as "extracellular." Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the "Tumor" compared to "Matched Normal" tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of "Tumor" and "Matched Normal" tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets.


Assuntos
Carcinoma de Células Escamosas , Proteômica , Humanos , Matriz Extracelular/metabolismo , Pulmão/metabolismo , Carcinoma de Células Escamosas/patologia , Inflamação/metabolismo , Proteínas da Matriz Extracelular/metabolismo
6.
Cell Syst ; 13(8): 644-664.e8, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35863345

RESUMO

The rise and fall of estrogen and progesterone across menstrual cycles and during pregnancy regulates breast development and modifies cancer risk. How these hormones impact each cell type in the breast remains poorly understood because they act indirectly through paracrine networks. Using single-cell analysis of premenopausal breast tissue, we reveal a network of coordinated transcriptional programs representing the tissue-level response to changing hormone levels. Our computational approach, DECIPHER-seq, leverages person-to-person variability in breast composition and cell state to uncover programs that co-vary across individuals. We use differences in cell-type proportions to infer a subset of programs that arise from direct cell-cell interactions regulated by hormones. Further, we demonstrate that prior pregnancy and obesity modify hormone responsiveness through distinct mechanisms: obesity reduces the proportion of hormone-responsive cells, whereas pregnancy dampens the direct response of these cells to hormones. Together, these results provide a comprehensive map of the cycling human breast.


Assuntos
Mama , Progesterona , Mama/metabolismo , Comunicação Celular , Estrogênios/metabolismo , Feminino , Humanos , Obesidade/metabolismo , Gravidez , Progesterona/metabolismo
8.
Cell Signal ; 28(12): 1923-1932, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27705752

RESUMO

Endogenous Plastic Somatic (ePS) cells isolated from adult human tissues exhibit extensive lineage plasticity in vitro and in vivo. Here we visualize these rare ePS cells in a latent state, i.e. lacking SOX2, OCT3/4 and NANOG (SON) expression, in non-diseased breast specimens through immunohistochemical analysis of previously identified ePS-specific biomarkers (CD73+, EpCAM+ and CD90-). We also report a novel mechanism by which these latent ePS cells acquire SON expression and plasticity in vitro. Four extracellular factors are necessary for the acquisition of SON expression and lineage plasticity in ePS cells: adenosine (which is produced by the 5' ecto-nucleotidase CD73 and activates in turn the PKA-dependent IL6/STAT3 pathway through the adenosine receptor ADORA2b), IL6, FGF2 and ACTIVIN A. Blocking any pathway component renders ePS cells incapable of SON expression and lineage plasticity. Notably, hESCs do not use adenosine or IL6 nor they express CD73 or ADORA2b and inhibition of adenosine signaling does not ablate their plasticity. Therefore, the data presented here delineate novel circuitry and physiological signals for accessing SON expression in rare, undifferentiated human cells.


Assuntos
5'-Nucleotidase/metabolismo , Plasticidade Celular , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Adenosina/metabolismo , Adulto , Animais , Comunicação Autócrina , Biomarcadores/metabolismo , Mama/metabolismo , Diferenciação Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Alimentadoras/citologia , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Ligadas por GPI/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Camundongos , Receptor A2B de Adenosina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Antígenos Thy-1/metabolismo
9.
Genes Dev ; 30(9): 1002-19, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27151975

RESUMO

The tumor stroma is no longer seen solely as physical support for mutated epithelial cells but as an important modulator and even a driver of tumorigenicity. Within the tumor stromal milieu, heterogeneous populations of fibroblast-like cells, collectively termed carcinoma-associated fibroblasts (CAFs), are key players in the multicellular, stromal-dependent alterations that contribute to malignant initiation and progression. This review focuses on novel insights into the contributions of CAFs to disease progression, emergent events leading to the generation of CAFs, identification of CAF-specific biomarkers predictive of disease outcome, and recent therapeutic approaches aimed at blunting or reverting detrimental protumorigenic phenotypes associated with CAFs.


Assuntos
Carcinoma/patologia , Fibroblastos/patologia , Animais , Carcinoma/fisiopatologia , Carcinoma/terapia , Progressão da Doença , Humanos
10.
Sci Rep ; 5: 15153, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26472542

RESUMO

3D tissue culture models are utilized to study breast cancer and other pathologies because they better capture the complexity of in vivo tissue architecture compared to 2D models. However, to mimic the in vivo environment, the mechanics and geometry of the ECM must also be considered. Here, we studied the mechanical environment created in two 3D models, the overlay protocol (OP) and embedded protocol (EP). Mammary epithelial acini features were compared using OP or EP under conditions known to alter acinus organization, i.e. collagen crosslinking and/or ErbB2 receptor activation. Finite element analysis and active microrheology demonstrated that OP creates a physically asymmetric environment with non-uniform mechanical stresses in radial and circumferential directions. Further contrasting with EP, acini in OP displayed cooperation between ErbB2 signalling and matrix crosslinking. These differences in acini phenotype observed between OP and EP highlight the functional impact of physical symmetry in 3D tissue culture models.


Assuntos
Modelos Biológicos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular , Colágeno/química , Combinação de Medicamentos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Análise de Elementos Finitos , Humanos , Laminina/química , Pinças Ópticas , Fenótipo , Proteoglicanas/química , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Reologia , Transdução de Sinais , Estresse Mecânico
11.
Org Biomol Chem ; 13(45): 11078-86, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26400105

RESUMO

Cyclooxygenases (COXs) are enzymes that play a vital role in the inflammatory cascade through the generation of prostaglandins. Their over-expression has been implicated in numerous diseases. In particular, over-expression of COX-2 has been shown to be a predictive biomarker for progression of pre-malignant lesions towards invasive cancer in various tissues. This makes the early detection of COX-2 expressing lesions of high clinical relevance. Herein we describe the development of the first self-immolating trigger which targets COXs. We incorporated our trigger design into 2 activatable fluorogenic probes and demonstrated COX-specific activation in vitro. Experimental data revealed probe activation was likely caused by solvent-exposed amino acids on the surface of the COXs. Overall, the probes reported here mark the first step towards developing self-immolating imaging/therapeutic agents targeted to specific COXs.


Assuntos
Aspirina/análogos & derivados , Aspirina/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Animais , Linhagem Celular , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 2/análise , Humanos , Camundongos , Modelos Moleculares , Imagem Óptica , Ovinos , Espectrometria de Fluorescência , Suínos
12.
Nat Commun ; 6: 6351, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25690954

RESUMO

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.


Assuntos
Mama/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Mama/citologia , Ciclo Celular , Diferenciação Celular , Separação Celular , Cromatina/química , Imunoprecipitação da Cromatina , Ilhas de CpG , Epigenômica , Células Epiteliais/citologia , Éxons , Feminino , Citometria de Fluxo , Genoma Humano , Histonas/química , Humanos , Íntrons , Cariotipagem , MicroRNAs/metabolismo , Análise de Sequência de RNA , Transcrição Gênica
13.
Mol Cell ; 50(4): 552-64, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23706820

RESUMO

Cellular stress results in profound changes in RNA and protein synthesis. How cells integrate this intrinsic, p53-centered program with extracellular signals is largely unknown. We demonstrate that TGF-ß1 signaling interferes with the stress response through coordinate transcriptional and translational repression of p53 levels, which reduces p53-activated transcription, and apoptosis in precancerous cells. Mechanistically, E2F-4 binds constitutively to the TP53 gene and induces transcription. TGF-ß1-activated Smads are recruited to a composite Smad/E2F-4 element by an E2F-4/p107 complex that switches to a Smad corepressor, which represses TP53 transcription. TGF-ß1 also causes dissociation of ribosomal protein RPL26 and elongation factor eEF1A from p53 mRNA, thereby reducing p53 mRNA association with polyribosomes and p53 translation. TGF-ß1 signaling is dominant over stress-induced transcription and translation of p53 and prevents stress-imposed downregulation of Smad proteins. Thus, crosstalk between the TGF-ß and p53 pathways defines a major node of regulation in the cellular stress response, enhancing drug resistance.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Fator de Transcrição E2F4/genética , Fator de Transcrição E2F4/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/citologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/metabolismo
14.
Sci Rep ; 3: 1449, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23618955

RESUMO

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Modelos Biológicos , Metástase Neoplásica/patologia , Metástase Neoplásica/fisiopatologia , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Tamanho Celular , Sobrevivência Celular , Simulação por Computador , Humanos
15.
Proc Natl Acad Sci U S A ; 110(12): 4598-603, 2013 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-23487770

RESUMO

We identified cell surface markers associated with repression of p16(INK4a)/cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical determinant in the acquisition of a plastic state. These cell surface markers allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation is poised to transcribe plasticity markers, OCT3/4, SOX2, and NANOG, at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo, and teratoma assays demonstrated that either a directly sorted (uncultured) or a single-cell (clonogenic) cell population from primary tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal, and mesodermal. In contrast to other cells that express OCT3/4, SOX2, and NANOG, these human endogenous plastic somatic cells are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Antígenos de Diferenciação/biossíntese , Mama/citologia , Mama/metabolismo , Separação Celular/métodos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas de Homeodomínio/biossíntese , Humanos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Fatores de Transcrição SOXB1/biossíntese
16.
Nature ; 486(7403): 395-9, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22495314

RESUMO

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Evolução Molecular , Mutação/genética , Alelos , Neoplasias da Mama/diagnóstico , Células Clonais/metabolismo , Células Clonais/patologia , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Mutação Puntual/genética , Medicina de Precisão , Reprodutibilidade dos Testes , Análise de Sequência de RNA
17.
Cancer Res ; 69(2): 565-72, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19147570

RESUMO

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Neoplasias da Mama/patologia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Ciclina D1/antagonistas & inibidores , Ciclina D1/metabolismo , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Fase G1/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos
18.
Cancer Res ; 67(18): 8662-70, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17875706

RESUMO

Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.


Assuntos
Mama/efeitos dos fármacos , Mama/efeitos da radiação , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos da radiação , Fator de Crescimento Transformador beta/farmacologia , Mama/metabolismo , Mama/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Mesoderma/efeitos da radiação , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
19.
Am J Physiol Cell Physiol ; 291(5): C880-6, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16774987

RESUMO

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1(-/-); Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1(-/-) mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1(-/-) erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 +/- 3.2 in 4.1(-/-) vs. 9.8 +/- 1.3 mmol/10(13) cell x h in control mice), with an abnormal dependence on osmolality (EC(50) = 417 +/- 42 in 4.1(-/-) vs. 460 +/- 35 mosmol/kgH(2)O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered (K(0.5) = 489.7 +/- 0.7 vs. 537.0 +/- 0.56 nM in control mice), the V(max) of the H-induced Na/H exchange activity was markedly elevated in 4.1(-/-) erythrocytes (V(max) 91.47 +/- 7.2 compared with 46.52 +/- 5.4 mmol/10(13) cell x h in control mice). Na/H exchange activation by okadaic acid was absent in 4.1(-/-) erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically downregulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of 4.1(-/-) erythrocytes.


Assuntos
Proteínas Sanguíneas/deficiência , Eritrócitos/metabolismo , Transporte de Íons/fisiologia , Animais , Proteínas Sanguíneas/metabolismo , Bumetanida/farmacologia , Volume de Eritrócitos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Soluções Hipertônicas/metabolismo , Transporte de Íons/efeitos dos fármacos , Soluções Isotônicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Ácido Okadáico/farmacologia , Concentração Osmolar , Ouabaína/farmacologia , Ácidos Ftálicos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sódio/sangue , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Simportadores/metabolismo , Cotransportadores de K e Cl-
20.
J Biotechnol ; 116(2): 145-51, 2005 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-15664078

RESUMO

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.


Assuntos
Mapeamento Cromossômico/métodos , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica/métodos , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Proteínas Recombinantes/biossíntese , Animais , Diferenciação Celular/genética , Linhagem Celular , Ratos
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