RESUMO
Desmoplasia is a common feature of aggressive cancers, driven by a complex interplay of protein production and degradation. Basigin is a type 1 integral membrane receptor secreted in exosomes or released by ectodomain shedding from the cell surface. Given that soluble basigin is increased in the circulation of patients with a poor cancer prognosis, we explored the putative role of the ADAM12-generated basigin ectodomain in cancer progression. We show that recombinant basigin ectodomain binds ß1 integrin and stimulates gelatin degradation and the migration of cancer cells in a matrix metalloproteinase (MMP)- and ß1-integrin-dependent manner. Subsequent in vitro and in vivo experiments demonstrated the altered expression of extracellular matrix proteins, including fibronectin and collagen type 5. Thus, we found increased deposits of collagen type 5 in the stroma of nude mice tumors of the human tumor cell line MCF7 expressing ADAM12-mimicking the desmoplastic response seen in human cancer. Our findings indicate a feedback loop between ADAM12 expression, basigin shedding, TGFß signaling, and extracellular matrix (ECM) remodeling, which could be a mechanism by which ADAM12-generated basigin ectodomain contributes to the regulation of desmoplasia, a key feature in human cancer progression.
Assuntos
Proteína ADAM12 , Basigina , Proteínas da Matriz Extracelular , Animais , Feminino , Humanos , Camundongos , Proteína ADAM12/metabolismo , Proteína ADAM12/genética , Basigina/metabolismo , Basigina/genética , Linhagem Celular Tumoral , Movimento Celular , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Células MCF-7 , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genética , Ligação Proteica , Domínios Proteicos , Integrina beta1/metabolismoRESUMO
A large number of glucagon-like-peptide-1 (GLP-1)- and peptide-YY (PYY)-producing L cells are located in the colon, but little is known about their contribution to whole body metabolism. Since bile acids (BAs) increase GLP-1 and PYY release, and since BAs spill over from the ileum to the colon, we decided to investigate the ability of BAs to stimulate colonic GLP-1 and PYY secretion. Using isolated perfused rat/mouse colon as well as stimulation of the rat colon in vivo, we demonstrate that BAs significantly enhance secretion of GLP-1 and PYY from the colon with average increases of 3.5- and 2.9-fold, respectively. Furthermore, we find that responses depend on BA absorption followed by basolateral activation of the BA-receptor Takeda-G protein-coupled-receptor 5. Surprisingly, the apical sodium-dependent BA transporter, which serves to absorb conjugated BAs, was not required for colonic conjugated BA absorption or conjugated BA-induced peptide secretion. In conclusion, we demonstrate that BAs represent a major physiological stimulus for colonic L-cell secretion. NEW & NOTEWORTHY By the use of isolated perfused rodent colon preparations we show that bile acids are potent and direct promoters of colonic glucagon-like-peptide 1 and peptide-YY secretion. The study provides convincing evidence that basolateral Takeda-G protein-coupled-receptor 5 activation is mediating the effects of bile acids in the colon and thus add to the existing literature described for L cells in the ileum.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/metabolismo , Colo/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glicoproteínas de Membrana/metabolismo , Peptídeo YY/metabolismo , Animais , Íleo/metabolismo , Absorção Intestinal/fisiologia , Células L , Camundongos , RatosRESUMO
The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.
Assuntos
Proteína ADAM12/metabolismo , Basigina/metabolismo , Proteína ADAM12/química , Proteína ADAM12/genética , Sequência de Aminoácidos , Basigina/química , Basigina/genética , Sistemas CRISPR-Cas , Linhagem Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Mutação , Especificidade por SubstratoRESUMO
Matrix metalloproteinases (MMPs), in particular MMP-2, MMP-9 and MMP-14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (a disintegrin and metalloproteinases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here, we show that there is a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell surface αVß3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14-mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVß3 integrin and MMP-14 at the tumor cell surface regulates the function of MMP-14. This interaction might point to a novel concept for the development of MMP-14-targeting drugs in treating cancer.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Gelatina/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/imunologia , Proteína ADAM12 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Xenoenxertos , Humanos , Integrina alfaVbeta3/metabolismo , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos NODRESUMO
ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.
Assuntos
Proteínas ADAM/metabolismo , Clatrina/metabolismo , Endocitose , Proteína Adaptadora GRB2/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/química , Proteína ADAM12 , Neoplasias da Mama/química , Neoplasias da Mama/enzimologia , Carcinoma/química , Endossomos/metabolismo , Feminino , Proteína Adaptadora GRB2/análise , Células HEK293 , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/química , Domínios Proteicos Ricos em Prolina , Domínios e Motivos de Interação entre ProteínasRESUMO
ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12 in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12 in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.
Assuntos
Proteínas ADAM/biossíntese , Proteínas ADAM/química , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/química , Células Endoteliais da Veia Umbilical Humana/química , Proteínas de Membrana/química , Proteínas ADAM/deficiência , Proteína ADAM12 , Animais , Neoplasias da Mama/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/patologiaRESUMO
Radiotherapy is one of the most common cancer treatments, yet, some patients require high doses to respond. Therefore, the development of new strategies leans toward personalizing therapy to avoid unnecessary burden on cancer patients. This approach prevents the administration of ineffective treatments or uses combination strategies to increase the sensitivity of cancer cells. ADAM12 has been shown to be upregulated in many cancers and correlate with poor survival and chemoresistance, thus making it a potential candidate responsible for radioresistance. Here, we show that ADAM12 expression is upregulated in response to irradiation in both mouse and human cancer cells in vitro, as well as in tumor tissues from rectal cancer patients. Interestingly, the expression of ADAM12 following radiotherapy correlates with the initial disease stage and predicts the response of rectal cancer patients to the treatment. While we found no cell-autonomous effects of ADAM12 on the response of colon cancer cells to irradiation in vitro, depletion of ADAM12 expression markedly reduced the tumor growth of irradiated cancer cells when subcutaneously transplanted in syngeneic mice. Interestingly, loss of cancer cell-derived ADAM12 expression increased the number of CD31+FAP- cells in murine tumors. Moreover, conditioned medium from ADAM12-/- colon cancer cells led to increased tube formation when added to endothelial cell cultures. Thus, it is tempting to speculate that altered tumor vascularity may be implicated in the observed effect of ADAM12 on response to radiotherapy in rectal cancer. We conclude that ADAM12 represents a promising prognostic factor for stratification of rectal cancer patients receiving radiotherapy and suggest that targeting ADAM12 in combination with radiotherapy could potentially improve the treatment response.
Assuntos
Neoplasias do Colo , Neoplasias Retais , Animais , Humanos , Camundongos , Proteína ADAM12/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/radioterapia , Regulação Neoplásica da Expressão Gênica , Prognóstico , Neoplasias Retais/genética , Neoplasias Retais/radioterapiaRESUMO
Invadopodia are dynamic actin structures at the cell surface that degrade extracellular matrix and act as sites of signal transduction. The biogenesis of invadopodia, including the mechanisms regulating their formation, composition, and turnover is not entirely understood. Here, we demonstrate that antibody ligation of ADAM12, a transmembrane disintegrin and metalloprotease, resulted in the rapid accumulation of invadopodia with extracellular matrix-degrading capacity in epithelial cells expressing the αvß3 integrin and active c-Src kinase. The induction of invadopodia clusters required an intact c-Src interaction site in the ADAM12 cytoplasmic domain, but was independent of the catalytic activity of ADAM12. Caveolin-1 and transmembrane protease MMP14/MT1-MMP were both present in the ADAM12-induced clusters of invadopodia, and cholesterol depletion prevented their formation, suggesting that lipid-raft microdomains are involved in the process. Importantly, our data demonstrate that ADAM12-mediated ectodomain shedding of epidermal growth factor receptor ligands can occur within these invadopodia. Such localized growth factor signalling offers an interesting novel biological concept highly relevant to the properties of carcinoma cells, which often show upregulated ADAM12 and ß3 integrin expression, together with high levels of c-Src kinase activity.
Assuntos
Proteínas ADAM/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM12 , Actinas/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular , Membrana Celular/genética , Matriz Extracelular/genética , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Rim/citologia , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/genética , Ligação Proteica/genética , Receptores de Vitronectina/metabolismo , Transdução de Sinais/genética , Transfecção , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Glucagon is a major regulator of metabolism and drugs targeting the glucagon receptor (GCGR) are being developed. Insight into tissue and cell-specific expression of the GCGR is important to understand the biology of glucagon and to differentiate between direct and indirect actions of glucagon. However, it has been challenging to localize the GCGR in tissue due to low expression levels and lack of specific methods. Immunohistochemistry has frequently been used for GCGR localization, but antibodies targeting G-protein-coupled-receptors may be inaccurate. We evaluated all currently commercially available GCGR antibodies. The antibody, ab75240 (Antibody no. 11) was found to perform best among the twelve antibodies tested and using this antibody we found expression of the GCGR in the kidney, liver, preadipocytes, pancreas, and heart. Three antibody-independent approaches all confirmed the presence of the GCGR within the pancreas, liver and the kidneys. GCGR expression should be evaluated by both antibody and antibody-independent approaches.
Assuntos
Glucagon , Receptores de Glucagon , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Expressão Gênica , Anticorpos/metabolismo , Fígado/metabolismoRESUMO
ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.
Assuntos
Proteínas ADAM/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ADAM/genética , Proteína ADAM12 , Sítios de Ligação/fisiologia , Ligação Competitiva , Proteína Tirosina Quinase CSK , Linhagem Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Adesões Focais/metabolismo , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Proteínas de Membrana/genética , Modelos Biológicos , Mutação/fisiologia , Fragmentos de Peptídeos/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Transporte Proteico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Vitronectina/metabolismo , Domínios de Homologia de src/fisiologia , Quinases da Família srcRESUMO
Studies on isolated pancreatic islets suggest that neuromedin U (NMU), a brain and gastrointestinal peptide, acts as a decretin hormone, inhibiting glucose-stimulated insulin secretion. We investigated whether this effect could be reproduced in vivo and in isolated perfused rat pancreas. Unlike the incretin hormone, glucagon-like peptide 1 (GLP-1), intravenous NMU administration had no effects on blood glucose and plasma insulin and glucagon in vivo. Moreover, NMU neither changed insulin, glucagon, or somatostatin secretion from isolated perfused rat pancreas, nor affected GLP-1-stimulated insulin and somatostatin secretion. For NMU to act as a decretin hormone, its secretion should increase following glucose ingestion; however, glucose did not affect NMU secretion from isolated perfused rat small intestine, which contained extractable NMU. Furthermore, the two NMU receptors were not detected in endocrine rat or human pancreas. We conclude that NMU does not act as a decretin hormone in rats.
Assuntos
Glucagon/metabolismo , Insulina/metabolismo , Intestino Delgado/metabolismo , Ilhotas Pancreáticas/metabolismo , Neuropeptídeos , Pâncreas/metabolismo , Somatostatina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Masculino , Neuropeptídeos/farmacologia , Neuropeptídeos/fisiologia , Ratos , Ratos Wistar , Receptores de Neurotransmissores/metabolismoRESUMO
ADAM12 belongs to the large family of ADAMs (a disintegrin and metalloproteases) and possesses extracellular metalloprotease and cell-binding functions, as well as intracellular signaling capacities. Interest in ADAM12 has increased recently because its expression is related to tumor progression and it is a potential biomarker for breast cancer. It is therefore important to understand ADAM12's functions. Many cellular roles for ADAM12 have been suggested. It is an active metalloprotease, and has been implicated in insulin-like growth factor (IGF) receptor signaling, through cleavage of IGF-binding proteins, and in epidermal growth factor receptor (EGFR) pathways, via ectodomain shedding of membrane-tethered EGFR ligands. These proteolytic events may regulate diverse cellular responses, such as altered cell differentiation, proliferation, migration, and invasion. ADAM12 may also regulate cell-cell and cell-extracellular matrix contacts through interactions with cell surface receptors - integrins and syndecans - potentially influencing the actin cytoskeleton. Moreover, ADAM12 interacts with several cytoplasmic signaling and adaptor molecules through its intracellular domain, thereby directly transmitting signals to or from the cell interior. These ADAM12-mediated cellular effects appear to be critical events in both biological and pathological processes. This review presents current knowledge on ADAM12 functions gained from in vitro and in vivo observations, describes ADAM12's role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.
Assuntos
Proteínas ADAM/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM12 , Animais , Cardiomegalia/enzimologia , Cardiomegalia/metabolismo , Regulação Enzimológica da Expressão Gênica , Saúde , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Doenças Musculoesqueléticas/enzimologia , Doenças Musculoesqueléticas/metabolismo , Neoplasias/enzimologia , Neoplasias/metabolismo , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/metabolismoRESUMO
OBJECTIVE: Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. METHODS: The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. RESULTS: Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or ß-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. CONCLUSION: BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.
Assuntos
Ácidos e Sais Biliares/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Mucosa Intestinal/metabolismo , Pâncreas/metabolismo , Peptídeo YY/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
PURPOSE: We have previously found ADAM12, a disintegrin and metalloprotease, to be an interesting biomarker for breast cancer. The purpose of this study was to determine the gene and protein expression profiles of ADAM12 in different grades and stages of bladder cancer. EXPERIMENTAL DESIGN: ADAM12 gene expression was evaluated in tumors from 96 patients with bladder cancer using a customized Affymetrix GeneChip. Gene expression in bladder cancer was validated using reverse transcription-PCR, quantitative PCR, and in situ hybridization. Protein expression was evaluated by immunohistochemical staining on tissue arrays of bladder cancers. The presence and relative amount of ADAM12 in the urine of cancer patients were determined by Western blotting and densitometric measurements, respectively. RESULTS: ADAM12 mRNA expression was significantly up-regulated in bladder cancer, as determined by microarray analysis, and the level of ADAM12 mRNA correlated with disease stage. Reverse transcription-PCR, quantitative PCR, and in situ hybridization validated the gene expression results. Using immunohistochemistry, we found ADAM12 protein expression correlated with tumor stage and grade. Finally, ADAM12 could be detected in the urine by Western blotting; ADAM12 was present in higher levels in the urine from patients with bladder cancer compared with urine from healthy individuals. Significantly, following removal of tumor by surgery, in most bladder cancer cases examined, the level of ADAM12 in the urine decreased and, upon recurrence of tumor, increased. CONCLUSIONS: ADAM12 is a promising biomarker of bladder cancer.
Assuntos
Proteínas ADAM/metabolismo , Carcinoma de Células de Transição/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Membrana/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Proteínas ADAM/urina , Proteína ADAM10 , Proteína ADAM12 , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adenocarcinoma/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células Escamosas/urina , Carcinoma de Células de Transição/patologia , Carcinoma de Células de Transição/cirurgia , Carcinoma de Células de Transição/urina , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Proteínas de Membrana/urina , Camundongos , Pessoa de Meia-Idade , Mucosa/metabolismo , Recidiva Local de Neoplasia/urina , Estadiamento de Neoplasias , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/urinaRESUMO
As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/patologia , Mama/citologia , Proteínas de Membrana/fisiologia , Metaloendopeptidases/fisiologia , Células 3T3-L1 , Proteínas ADAM , Proteína ADAM12 , Animais , Células CHO , Processos de Crescimento Celular/fisiologia , Cricetinae , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Células Estromais/citologiaRESUMO
PACS-2 is a multifunctional sorting protein that mediates cell homeostasis. We recently identified PACS-2 in a functional genome-wide siRNA screen for novel regulators of the metalloproteinase ADAM17, the main sheddase for ligands of the ErbB receptor family. Of note, we showed that Pacs2-/- mice have significantly reduced EGFR activity and proliferative index in the intestinal epithelium. As EGFR signaling is highly mitogenic for intestinal epithelial stem cells, and plays essential roles in intestinal epithelial regeneration and tumor development, we have now examined the role of PACS-2 in these processes. Specifically, we analyzed the role of Pacs2-deficiency in a DSS-induced colitis model as well as in the genetic ApcMin colon cancer model. We now report that loss of PACS-2 delays tissue regeneration after colonic injury with little effect on key inflammatory parameters. We did however not observe any apparent effects on tumor formation driven by excessive proliferative signaling downstream from APC-deficiency. Our findings reveal that the role of PACS-2 in regulating ADAM17-mediated shedding is not an obligate requirement for the epithelium to respond to the strong inflammatory or tumorigenic inducers in the models assessed here.
RESUMO
Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in ß cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.
Assuntos
Glicemia/análise , Insulina/análise , Falência Renal Crônica/patologia , Proglucagon/sangue , Animais , Células COS , Estudos de Casos e Controles , Células Cultivadas , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Gluconeogênese/efeitos dos fármacos , Humanos , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Falência Renal Crônica/sangue , Falência Renal Crônica/metabolismo , Masculino , Camundongos , Fosforilase Quinase/genética , Fosforilase Quinase/metabolismo , Proglucagon/farmacologia , Ratos , Ratos Wistar , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismoRESUMO
UNLABELLED: ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell-extracellular matrix interactions in the growth plate. INTRODUCTION: The disintegrin and metalloprotease ADAM12 is expressed in both osteoblasts and osteoclasts, suggesting a regulatory role of ADAM12 in bone. However, thus far, no in vivo function of ADAM12 in the skeleton has been reported. MATERIALS AND METHODS: Transgenic mice expressing the secreted form of human ADAM12, ADAM12-S, or a truncated metalloprotease-deficient form of ADAM12-S in the circulation were used to study the effects of ADAM12 on the skeleton. In addition, murine chondrocyte cultures were used to study the effect of ADAM12-S on cell-extracellular matrix interactions. RESULTS: ADAM12-S transgenic mice exhibit increased longitudinal bone growth. The increased bone length is progressive and age dependent, with a maximum increase of 17% seen in the femur from 6-month-old transgenic mice. The effect is gene dose dependent, being more pronounced in mice expressing higher levels of the transgene than in a lower-expressing line. Histological analysis revealed no alterations in the growth plate organization, but mean growth plate width was increased. Both the cellular incorporation of bromodeoxyuridine and the width of the collagen type X-positive hypertrophic zone were increased in the growth plate of ADAM12-S transgenic mice. Importantly, mice expressing a truncated form of ADAM12-S that lacked the pro- and metalloprotease domains showed no alterations in bone length, suggesting that protease activity is required for the ADAM12-S effect. In vitro studies showed that ADAM12-S inhibits chondrocyte adhesion to fibronectin and collagen type II. CONCLUSIONS: ADAM12-S stimulates bone growth in mice by modulating chondrocyte proliferation and maturation through mechanisms probably involving both metalloprotease and adhesion activities.
Assuntos
Proteínas ADAM/metabolismo , Desenvolvimento Ósseo , Osso e Ossos/anatomia & histologia , Diferenciação Celular , Condrócitos/citologia , Proteínas de Membrana/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAM12 , Animais , Desenvolvimento Ósseo/genética , Osso e Ossos/química , Osso e Ossos/metabolismo , Adesão Celular , Proliferação de Células , Condrócitos/fisiologia , Humanos , Integrinas/análise , Integrinas/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Deleção de SequênciaRESUMO
Systems-wide profiling of breast cancer has almost always entailed RNA and DNA analysis by microarray and sequencing techniques. Marked developments in proteomic technologies now enable very deep profiling of clinical samples, with high identification and quantification accuracy. We analysed 40 oestrogen receptor positive (luminal), Her2 positive and triple negative breast tumours and reached a quantitative depth of >10,000 proteins. These proteomic profiles identified functional differences between breast cancer subtypes, related to energy metabolism, cell growth, mRNA translation and cell-cell communication. Furthermore, we derived a signature of 19 proteins, which differ between the breast cancer subtypes, through support vector machine (SVM)-based classification and feature selection. Remarkably, only three proteins of the signature were associated with gene copy number variations and eleven were also reflected on the mRNA level. These breast cancer features revealed by our work provide novel insights that may ultimately translate to development of subtype-specific therapeutics.
Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteômica/métodos , Feminino , Humanos , TranscriptomaRESUMO
Incretin-based therapies are widely used for type 2 diabetes and now also for obesity, but they are associated with elevated plasma levels of pancreatic enzymes and perhaps a modestly increased risk of acute pancreatitis. However, little is known about the effects of the incretin hormone glucagon-like peptide 1 (GLP-1) on the exocrine pancreas. Here, we identify GLP-1 receptors on pancreatic acini and analyze the impact of receptor activation in humans, rodents, isolated acini, and cell lines from the exocrine pancreas. GLP-1 did not directly stimulate amylase or lipase release. However, we saw that GLP-1 induces phosphorylation of the epidermal growth factor receptor and activation of Foxo1, resulting in cell growth with concomitant enzyme release. Our work uncovers GLP-1-induced signaling pathways in the exocrine pancreas and suggests that increases in amylase and lipase levels in subjects treated with GLP-1 receptor agonists reflect adaptive growth rather than early-stage pancreatitis.