RESUMO
BACKGROUND: The aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell-matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis. METHODS: We used Blg-Cre; Brca1F/F; Trp53F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures. RESULTS: We show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers. CONCLUSIONS: Our data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1F/F; Trp53F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.
Assuntos
Proteína BRCA1 , Neoplasias da Mama , Integrina alfa6 , Proteína Supressora de Tumor p53 , Animais , Integrina alfa6/metabolismo , Integrina alfa6/genética , Feminino , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Camundongos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proliferação de Células , Células-Tronco/metabolismo , Deleção de Genes , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismoRESUMO
Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.
Assuntos
Integrinas/fisiologia , Lactação , Glândulas Mamárias Animais/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Citoesqueleto/fisiologia , Progressão da Doença , Feminino , Deleção de Genes , Hormônios/fisiologia , Integrina alfa3/fisiologia , Integrina alfa6/fisiologia , Integrina beta1/fisiologia , Integrina beta4/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Mutantes , Mutação , Células-Tronco Neoplásicas/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Ovário/fisiologia , Fenótipo , Gravidez , Prenhez , Prognóstico , Ligação Proteica , Multimerização ProteicaRESUMO
BACKGROUND: ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease. METHODS: Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, ß1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels. RESULTS: We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a ß1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression. CONCLUSIONS: We have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.
Assuntos
Proteínas ADAM/biossíntese , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , Invasividade Neoplásica/genética , Neoplasias de Mama Triplo Negativas/genética , Proteínas ADAM/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Camundongos , MicroRNAs/genética , RNA Interferente Pequeno , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure or functional role of these sugar moieties. Here, we report that in estrogen receptor (ER)α-negative, but not -positive, breast cancer cells ADAM8 contains N-glycosylation, which is required for its correct processing and activation. Consistently ADAM8 dimers were detected on the surface of ERα-negative breast cancer cells but not on ERα-positive ones. Site-directed mutagenesis confirmed four N-glycosylazhytion sites (Asn-67, Asn-91, Asn-436, and Asn-612) in human ADAM8. The Asn-67 and Asn-91 prodomain sites contained high mannose, whereas complex type N-glycosylation was observed on Asn-436 and Asn-612 in the active and remnant forms. The Asn-91 and Asn-612 sites were essential for its correct processing and cell surface localization, in particular its exit from the Golgi and endoplasmic reticulum, respectively. The N436Q mutation led to decreased ADAM8 stability due to enhanced lysosomal degradation. In contrast, mutation of the Asn-67 site had only modest effects on enzyme stability and processing. Thus, N-glycosylation is essential for processing, localization, stability, and activity of ADAM8.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/enzimologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteólise , Proteínas ADAM/genética , Substituição de Aminoácidos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ativação Enzimática , Estabilidade Enzimática/genética , Receptor alfa de Estrogênio , Feminino , Glicosilação , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Transporte Proteico/genéticaRESUMO
RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX-PP) released from the secreted precursor protein (Pro-LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her-2/neu-driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX-PP on MIA PaCa-2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa-2 cells in culture, LOX-PP attenuated the ERK and AKT activities and decreased the levels of the NF-κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX-PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF-κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX-PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX-PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX-PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Proteína-Lisina 6-Oxidase/farmacologia , Animais , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Precursores Enzimáticos , Feminino , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Survivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock-down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3-only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult-to-treat cancer.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Interleucina-6/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Taxa de Sobrevida , Survivina , Evasão Tumoral/imunologiaRESUMO
Integrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.
RESUMO
PURPOSE: Intrinsic activation of nuclear factor kappaB (NF-kappaB) characterizes various hematologic malignancies. In this study, we specifically address the role of NF-kappaB blockade in mediated antimyeloma activity using the IkappaB kinase-2 pharmacologic inhibitor, AS602868. EXPERIMENTAL DESIGN: Human myeloma cell lines (n = 16) and primary myeloma cells (n = 10) were tested for their sensitivity to AS602868 in terms of proliferation and apoptosis. Both in vitro and in vivo experiments were conducted. Functional mechanisms regarding the apoptotic pathways triggered by AS602868 were studied. The potential proapoptotic synergy between AS602868 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was also evaluated. RESULTS: Our results show that AS602868 efficiently targeted the canonical NF-kappaB pathway in myeloma cells and potently inhibited their growth in inducing apoptosis through Bax and caspase-3 activation. AS602868 also induced apoptosis in primary myeloma cells even in the presence of bone marrow mononuclear cells. Moreover, the IkappaB kinase-2 inhibitor targeted the paracrine effect on the bone marrow environment. Indeed, it decreased the intrinsic and myeloma-induced secretion of interleukin-6 from bone marrow stromal cells. In addition, AS602868 inhibited myeloma cell growth in the MM.1S xenograft myeloma model. Of particular interest, AS602868 strongly increased myeloma sensitivity to TRAIL in blocking TRAIL-induced NF-kappaB activation and in decreasing the expression of antiapoptotic proteins such as cFLIP and cIAP-1/2. CONCLUSIONS: Taken together, our data point out the interest to inhibit the canonical NF-kappaB pathway in myeloma and clearly encourage clinical evaluation of novel therapies based on targeting NF-kappaB, especially in combination with TRAIL.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/patologia , NF-kappa B/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Interleucina-6/metabolismo , Camundongos , Modelos Biológicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Transplante de Neoplasias , Pirimidinas/farmacologia , Receptores Imunológicos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Discovered 10 years ago, survivin has a dual role in the smooth progress of mitosis and in apoptosis resistance. Survivin plays an important physiological role in development, but is absent in differentiated adult tissues. In contrast, aberrant survivin expression is found in most human cancers because of the activation of various signalling pathways. A complex survivin network appears to intersect multiple pathways in cell biology, related to several molecular partners and fine subcellular localizations. Based on its pro-oncogenic properties, basic and translational studies have shown a growing interest in survivin that has led to consider survivin as a prognostic marker and a promising target for anti-tumoral therapies.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/fisiologia , Animais , Animais Geneticamente Modificados , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/uso terapêutico , Biomarcadores Tumorais , Vacinas Anticâncer/uso terapêutico , Ciclo Celular/fisiologia , Ensaios Clínicos Fase I como Assunto , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Desenvolvimento Embrionário/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Proteínas Inibidoras de Apoptose , Modelos Biológicos , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Frações Subcelulares/metabolismo , Survivina , Produtos do Gene tat do Vírus da Imunodeficiência Humana/uso terapêuticoRESUMO
Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC.
RESUMO
HGF/Met signaling has recently been associated with basal-type breast cancers, which are thought to originate from progenitor cells residing in the luminal compartment of the mammary epithelium. We found that ICAM-1 efficiently marks mammary luminal progenitors comprising hormone receptor-positive and receptor-negative cells, presumably ductal and alveolar progenitors. Both cell populations strongly express Met, while HGF is produced by stromal and basal myoepithelial cells. We show that persistent HGF treatment stimulates the clonogenic activity of ICAM1-positive luminal progenitors, controlling their survival and proliferation, and leads to the expression of basal cell characteristics, including stem cell potential. This is accompanied by the induction of Snai1 and Snai2, two major transcription factors triggering epithelial-mesenchymal transition, the repression of the luminal-regulatory genes Elf5 and Hey1, and claudin down-regulation. Our data strongly indicate that paracrine Met signaling can control the function of luminal progenitors and modulate their fate during mammary development and tumorigenesis.
Assuntos
Transição Epitelial-Mesenquimal , Fator de Crescimento de Hepatócito/metabolismo , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Células-Tronco/fisiologia , Animais , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Camundongos , Células-Tronco/efeitos dos fármacosRESUMO
The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via ß1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Membrana/metabolismo , Proteínas ADAM/química , Animais , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/irrigação sanguínea , Adesão Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Integrina beta1/metabolismo , Proteínas de Membrana/química , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Neovascularização Patológica/patologia , Fenótipo , Prognóstico , Estrutura Terciária de Proteína , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
Induction of epithelial-to-mesenchymal transition (EMT) by TGF-ß1 requires Ras signaling. We recently identified the transcriptional repressor Blimp-1 (PRDM1) as a downstream effector of the NF-κB, RelB/Bcl-2/Ras-driven pathway that promotes breast cancer cell migration. As the RelB/Blimp-1 pathway similarly required Ras signaling activation, we tested whether Blimp-1 plays a role in TGF-ß1-mediated EMT. Here, TGF-ß1 treatment of untransformed NMuMG mammary epithelial and MDA-MB-231 breast cancer cells was shown to induce Blimp-1 expression, which promoted an EMT signature and cell migration. TGFB1 and BLIMP1 RNA levels were correlated in patient breast tumors. BLIMP1 gene transcription was activated by TGF-ß1 via a c-Raf (RAF1) to AP-1 pathway. Blimp-1 induced expression of the EMT master regulator Snail (SNAI1) via repressing BMP-5, which inhibited Snail expression upon TGF-ß1 treatment. Interestingly, a similar cascade was observed during postnatal mouse mammary gland development. RelB expression was detected early in pregnancy followed progressively by Blimp-1 and then Snail; whereas, BMP-5 levels were high in nulliparous and regressing glands. Finally, lower BMP5 RNA levels were detected in patient breast tumors versus normal tissues, and correlated with cancer recurrence. Thus, the Ras effector Blimp-1 plays an essential role in TGF-ß1-induced EMT via repression of BMP-5 in breast cancer.
Assuntos
Proteína Morfogenética Óssea 5/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Proteína Morfogenética Óssea 5/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Immunoblotting , Células MCF-7 , Camundongos , Peptídeos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Gravidez , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively. CONCLUSIONS/SIGNIFICANCE: In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity.
Assuntos
Apoptose , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Prolina/análogos & derivados , Prolina/farmacologia , Receptor Notch2/genética , Transdução de Sinais/efeitos dos fármacos , Survivina , Tiocarbamatos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.