RESUMO
Pancreatic ductal adenocarcinoma (PDA) has one of the worst prognoses of all cancers. Mucin 1 (MUC1), a transmembrane mucin glycoprotein, is a key modulator of several signaling pathways that affect oncogenesis, motility and metastasis. Its expression is known to be associated with poor prognosis in patients. However, the precise mechanism remains elusive. We report a novel association of MUC1 with platelet-derived growth factor-A (PDGFA). PDGFA is one of the many drivers of tumor growth, angiogenesis and metastasis in PDA. Using mouse PDA models as well as human samples, we show clear evidence that MUC1 regulates the expression and secretion of PDGFA. This, in turn, influences proliferation and invasion of pancreatic cancer cells leading to higher tumor burden in vivo. In addition, we reveal that MUC1 overexpressing cells are heavily dependent on PDGFA both for proliferation and invasion, whereas MUC1-null cells are not. Moreover, PDGFA and MUC1 are critical for translocation of ß catenin to the nucleus for oncogenesis to ensue. Finally, we elucidate the underlying mechanism by which MUC1 regulates PDGFA expression and secretion in pancreatic cancer cells. We show that MUC1 associates with Hif1-α, a known transcription factor involved in controlling PDGFA expression. Furthermore, MUC1 facilitates Hif1-α translocation to the nucleus. In summary, we have demonstrated that MUC1-induced invasion and proliferation occurs via increased exogenous production of PDGFA. Thus, impeding MUC1 regulation of PDGFA signaling may be therapeutically beneficial for patients with PDA.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucina-1/fisiologia , Neoplasias Pancreáticas/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fator de Crescimento Derivado de Plaquetas/genética , Ligação Proteica , Transporte Proteico , Carga Tumoral , beta Catenina/metabolismoRESUMO
The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.
Assuntos
Colite/fisiopatologia , Colo/metabolismo , Mucosa Intestinal/metabolismo , Mucina-1/metabolismo , Mucina-2/metabolismo , Animais , Translocação Bacteriana , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Colo/efeitos dos fármacos , Sulfato de Dextrana , Suscetibilidade a Doenças , Regulação para Baixo , Vida Livre de Germes , Mucosa Intestinal/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-1/genética , Mucina-2/genética , Peptidoglicano/farmacologia , Índice de Gravidade de Doença , Linfócitos T/patologiaRESUMO
Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic ductal adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. EMT was significantly reduced when MUC1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT-PCR and western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared with cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with ß-catenin and translocate to the nucleus, thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and ß-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer.
Assuntos
Transição Epitelial-Mesenquimal , Mucina-1/metabolismo , Neoplasias Pancreáticas/patologia , Sequência de Aminoácidos , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mucina-1/genética , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMO
Mice transgenic for MUC1 (mucin 1) and polyomavirus middle T (PyMT) develop mammary carcinomas within 15 weeks with 100% penetrance. PyMT-induced mammary tumorigenesis is closely correlated with robust telomerase expression and activity. To assess the role of telomerase activation and telomere maintenance in mammary carcinoma tumorigenesis, we generated mice expressing MUC1 and PyMT (MMT mice) but deficient in the telomerase RNA component, mTerc, on the C57BL/6 background. Successive generational intercrosses of mTerc(-/-)MMT mice produced cohorts with progressively shorter telomeres that were audited for mammary tumor formation. Relative to MMT (N=14) and G0 mTerc(+/-) female controls (G0=14), mTerc(-/-)MMT females (G1=11, G2=15, G3=15 and G4=5) showed decreased tumor volumes and increased tumor latency-MMT=95.6 days; G0 mTerc(+/-)MMT=98.6 days versus G1, G2, G3 and G4 mTerc(-/-)MMT mice with latencies of 122.6, 138.9, 140.7 and 220.9 days, respectively (controls versus G1-G4, P<0.005). The progressive impairment of lung metastasis was also observed with each successive mTerc(-/-)MMT generation. The impairment of tumorigenesis was associated with decreased proliferation of mammary epithelial and tumor cells and increased apoptosis of tumor cells. Together, these results indicate that, in the setting of viral oncoprotein mammary tumorigenesis, telomerase-dependent telomere maintenance facilitates the formation and metastatic progression of mammary tumors.
Assuntos
Neoplasias Mamárias Experimentais/genética , Oncogenes/genética , Telomerase/deficiência , Telômero/patologia , Animais , Ciclo Celular/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Mamárias Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Mucina-1/genética , Oncogenes/fisiologia , RNARESUMO
A MUC1-based vaccine was used in a preclinical model of colon cancer. The trial was conducted in a MUC1-tolerant immune competent host injected with MC38 colon cancer cells expressing MUC1. The vaccine included: MHC class I-restricted MUC1 peptides, MHC class II-restricted pan-helper-peptide, unmethylated CpG oligodeoxynucleotide, and granulocyte macrophage-colony stimulating factor. Immunization was successful in breaking MUC1 self-tolerance, and in eliciting a robust anti-tumor response. The vaccine stimulated IFN-gamma-producing CD4(+) helper and CD8(+) cytotoxic T cells against MUC1 and other undefined MC38 tumor antigens. In the prophylactic setting, immunization caused complete rejection of tumor cells, while in the therapeutic regimen, tumor burden was significantly reduced.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Modelos Animais de Doenças , Imunoterapia/métodos , Mucina-1/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Linhagem Celular Tumoral , Neoplasias do Colo/fisiopatologia , Neoplasias do Colo/prevenção & controle , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunização , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Mucina-1/administração & dosagem , Mucina-1/metabolismo , Mucinas , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologiaRESUMO
UNLABELLED: The immunohistochemical detection (IHC) of MUC1-CT employing a polyclonal antibody (CT33) in relation to CT2 monoclonal antibody (MAb) was analyzed. Western blot (WB) was used to determine the molecular mass of CT. MATERIALS AND METHODS: We studied 163 breast and 89 colorectal cancer specimens, 10 breast and 14 colorectal benign conditions, and 12 breast and 20 colorectal normal samples. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and WB. A nonparametric statistical analysis was employed; data were standardized and a Kendall-Tau correlation was applied. RESULTS: By IHC, 146/163 (90%) and 151/163 (93%) of breast cancer were positive with CT33 and CT2, respectively; a statistically significant correlation was obtained (t=0.5199). Seven out of ten (70%) benign breast specimens were positive with CT33 while all samples stained with CT2; in normal breast sample tissues, all were positive with both Abs. In colorectal cancer samples, both antibodies stained 47/89 (53%) samples; CT2 reacted in 13/14 (93%) of benign samples while CT33 showed a positive reaction in 9/14 (64%) of benign specimens. In normal samples, CT2 showed staining in 17/20 (85%) of samples and CT33 was reactive in 12/20 (60%). By WB, in breast and colorectal cancer samples, similar results were obtained with both antibodies: a main band at about 30kDa which represents the smaller subunit. CONCLUSION: CT33 polyclonal antibody has demonstrated its efficacy to detect MUC1 in breast and colorectal cancer tissues with similar reactivity to CT2. It is worthwhile to affirm that CT33 is a good indicator of MUC1 expression.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias Colorretais/metabolismo , Mucina-1/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/metabolismo , Biomarcadores Tumorais , Mama/anatomia & histologia , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Fracionamento Celular , Colo/anatomia & histologia , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Humanos , Técnicas Imunoenzimáticas , Mucina-1/imunologia , Proteínas de Transporte de Cátions Orgânicos/imunologia , Reto/anatomia & histologia , Reto/metabolismo , Reto/patologiaRESUMO
Ovarian cancer represents the fifth leading cause of cancer death among women in the United States, with >16 000 deaths expected this year. This study was carried out to investigate the potential of sodium iodide symporter (NIS)-mediated radioiodide therapy as a novel approach for ovarian cancer treatment. Radioiodide is routinely and effectively used for the treatment of benign and malignant thyroid disease as a result of native thyroidal expression of NIS, which mediates iodide uptake. In vitro gene transfer studies in ovarian cancer cells revealed a 12- and five-fold increase in iodide uptake when transduced with Ad/CMV/NIS or Ad/MUC1/NIS, respectively. Western blot/immunohistochemistry confirmed NIS protein expression. In vivo ovarian tumor xenografts were infected with the adenoviral constructs. (123)I imaging revealed a clear image of the CMV/NIS-transduced tumor, with a less intense image apparent following infection with MUC1/NIS. Therapeutic doses of (131)I following CMV/NIS infection caused a mean 53% reduction in tumor volume (P<0.0001). MUC1/NIS-transduced tumors did not regress, although at 8 weeks following therapy, tumor volume was significantly less that of control animals (166 versus 332%, respectively, P<0.05). This study represents a promising first step investigating the potential for NIS-mediated radioiodide imaging and therapy of ovarian tumors.
Assuntos
Terapia Genética/métodos , Radioisótopos do Iodo , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/terapia , Simportadores/genética , Adenoviridae/genética , Animais , Western Blotting/métodos , Linhagem Celular Tumoral , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Imuno-Histoquímica/métodos , Radioisótopos do Iodo/uso terapêutico , Camundongos , Mucina-1/genética , Transplante de Neoplasias , Regiões Promotoras Genéticas , Cintilografia , Simportadores/metabolismo , Transdução Genética/métodos , Transplante HeterólogoRESUMO
MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfoma/metabolismo , Mucina-1/metabolismo , Linfócitos T/metabolismo , Adulto , Sequência de Aminoácidos , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proliferação de Células , Humanos , Interleucina-2/metabolismo , Células Jurkat , Lectinas Tipo C , Ativação Linfocitária , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Mucina-1/genética , Fosforilação , RNA Interferente Pequeno/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Homologia de Sequência de AminoácidosRESUMO
MUC4 is highly expressed in human pancreatic tumours and pancreatic tumour cell lines, but is minimally or not expressed in normal pancreas or chronic pancreatitis. Here, we investigated the aberrant regulation of MUC4 expression in vivo using clonal human pancreatic tumour cells (CD18/HPAF) grown either orthotopically in the pancreas (OT) or ectopically in subcutaneous tissue (SC) in the nude mice. Histological examination of the OT and SC tumours showed moderately differentiated and anaplastic morphology, respectively. The OT tumour cells showed metastases to distant lymph nodes and faster tumour growth (P<0.01) compared to the SC tumours. The MUC4 transcripts in OT tumours were very high compared to the undetectable levels in SC tumours. The SC tumour cells regained their ability to express MUC4 transcripts after in vitro culture. Immunohistochemical analysis using MUC4-specific polyclonal antiserum confirmed the results obtained by Northern blot analysis. Interestingly, the OT tumours showed expression of TGFbeta2 compared to no expression in SC, suggesting a possible link between MUC4 and TGFbeta2. The MUC4 expression, morphology, and metastasis of human pancreatic tumour cells are regulated by a local host microenvironment. TGFbeta2 may serve as an interim regulator of this function.
Assuntos
Biomarcadores Tumorais/análise , Regulação Neoplásica da Expressão Gênica , Imunossupressores/farmacologia , Mucinas/biossíntese , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/fisiopatologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Northern Blotting , Coristoma , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Mucina-4 , Fator de Crescimento Transformador beta2 , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.
Assuntos
Células Dendríticas/imunologia , Mucina-1/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Antígeno CD11c/imunologia , Células Cultivadas , Citoplasma , Células Dendríticas/citologia , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Baço/citologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Fusions of autologous tumor cells with allogeneic dendritic cells (DC) represent an approach for the induction of antitumor immunity. In the present studies, we investigated the antitumor effects of vaccinating MUC1-transgenic (MUC1.Tg) mice with MC38/MUC1 carcinoma cells fused to allogeneic DC from BALB/c mice (allo-DC, H-2(d)) or syngeneic DC from C57BL/6 mice (syn-DC, H-2(b)). Both allo and syn fusion cells (FC/MUC1) expressed MHC class II, costimulatory molecules, and the MUC1 antigen. Allo-FC/MUC1 exhibited dual expression of MHC class I haplotypes (H-2(d)/H-2(b))and MUC1 antigen. By contrast, only H-2(b) and MUC1 antigen were expressed by syn-FC/MUC1. CTLs from MUC1.Tg mice immunized with allo- or syn-FC/MUC1 fusion cells lysed MC38/MUC1 targets. Moreover, immunization with allo- or syn-FC/MUC1 was effective in eliminating established MUC1-positive pulmonary metastases in MUC1.Tg mice. These results indicate that immunization of MUC1.Tg mice with syn- or allo-FC/MUC1 is effective in reversing immunologic unresponsiveness to MUC1 antigen and inducing immunity against MUC1-positive tumors. The findings in the present study have broader clinical implications for fusion cell vaccines.
Assuntos
Vacinas Anticâncer/imunologia , Fusão Celular , Células Dendríticas/imunologia , Mucina-1/imunologia , Animais , Feminino , Antígenos de Histocompatibilidade Classe I/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucina-1/análise , Mucina-1/genética , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , VacinaçãoRESUMO
Recent discoveries regarding the identification of tumor-associated antigens and antigen presentation have made successful immunotherapy strategies possible with little, if any, toxicity. Here, we describe transgenic mammary, pancreas, prostate, stomach and lung adenocarcinoma animal models that can be used to study various immunotherapeutic strategies. The challenge in developing a tumor vaccine is effective antigen presentation that elicits anti-tumor immune responses without precipitating autoimmunity. Clinical trials must be preceded by appropriate animal studies to demonstrate that the concepts can be translated into efficacious therapy for cancer. Although many xenograph or transplantable tumor models have been used, the most effective studies are in spontaneous tumor models. These models are clinically relevant, as tumors arise in an appropriate tissue background and in a host conditioned by the physiological events of neoplastic progression and tumorigenesis and in the context of a viable immune system.
Assuntos
Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Modelos Animais de Doenças , Imunoterapia/métodos , Adenocarcinoma/patologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/terapia , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/terapiaRESUMO
MUC1 is a large, heavily glycosylated mucin expressed on the apical surfaces of most simple, secretory epithelia including the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 was thought to be an epithelial-specific protein, it is now known to be expressed on a variety of hematopoietic cells as well. Mucins function in protection and lubrication of epithelial surfaces. Transmembrane mucins, which contain cytoplasmic tail domains, appear to have additional functions through their abilities to interact with many proteins involved in signal transduction and cell adhesion. The goal of this review is to highlight recent discoveries that suggest that MUC1 may be a multifunctional protein, located on the surfaces of cells as a sensor of the environment, poised to signal to the interior when things go awry.
Assuntos
Neoplasias da Mama/metabolismo , Mucina-1/fisiologia , Animais , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismoRESUMO
We have reported previously that MUC1 transgenic mice with spontaneous tumors of the pancreas (designated MET) naturally develop MHC class I-restricted, MUC1-specific CTLs as tumors progress (P. Mukherjee et al., J. Immunol., 165: 3451-3460, 2000). From these MET mice, we have isolated, expanded, and cloned naturally occurring MUC1-specific CTLs in vitro. In this report, we show that the CTL line is predominantly CD8+ T cells and expresses T-cell receptor Vbeta chains 5.1/5.2, 11, 13, and 2 and Valpha chains 2, 8.3, 3.2, and 11.1/11.2. These CTLs recognize several epitopes on the MUC1 tandem repeat with highest affinity to APGSTAPPA. The CTL clone, on the other hand, is 100% CD8+ cells and expresses a single Vbeta chain of 5.1/5.2 and Valpha2. It recognizes only the H-2Db class I-restricted epitope of MUC1, APGSTAPPA. When adoptively transferred, the CTLs were effective in eradicating MUC1-expressing injected tumor cells including mammary gland cells (C57mg) and B16 melanomas. These results suggest that MUC1-specific CTLs are capable of possibly preventing, or at least substantially delaying, MUC1-expressing tumor formation. To our knowledge, this is the first evidence that demonstrates that the naturally occurring MUC1-specific CTLs isolated from one tumor model has antitumor effects on other MUC1-expressing tumors in vivo. Therefore, our data confirm that MUC1 is an important tumor rejection antigen and can serve as a target for immunotherapy.
Assuntos
Mucina-1/química , Linfócitos T Citotóxicos/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Separação Celular , Epitopos , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Melanoma Experimental , Camundongos , Camundongos Transgênicos , Peptídeos/química , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de TempoRESUMO
MUC1 is a large (>400 kDa), heavily glycosylated transmembrane protein that is aberrantly expressed on greater than 90% of human breast carcinomas and subsequent metastases. The precise function of MUC1 overexpression in tumorigenesis is unknown, although various domains of MUC1 have been implicated in cell adhesion, cell signaling, and immunoregulation. Stimulation of the MDA-MB-468 breast cancer line as well as mouse mammary glands with epidermal growth factor results in the co-immunoprecipitation of MUC1 with a tyrosine-phosphorylated protein of approximately 180 kDa. We have generated transgenic lines overexpressing full-length (MMF), cytoplasmic tail deleted (DeltaCT), or tandem repeat deleted (DeltaTR)-human MUC1 under the control of the mouse mammary tumor virus promoter to further examine the role of MUC1 in signaling and tumorigenesis. Immunoprecipitation experiments revealed that full-length transgenic MUC1 physically associates with all four erbB receptors, and co-localizes with erbB1 in the lactating gland. Furthermore, we detected a sharp increase in ERK1/2 activation in MUC1 transgenic mammary glands compared with Muc1 null and wild-type animals. These results point to a novel function of increased MUC1 expression, potentiation of erbB signaling through the activation of mitogenic MAP kinase pathways.
Assuntos
Receptores ErbB/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucina-1/metabolismo , Animais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mucina-1/genética , Fosfoproteínas/metabolismo , Fosfotirosina , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Receptor ErbB-4RESUMO
We investigated the influence of organ-specific parameters on tolerance and immunity to human MUC1. C57Bl/6 mice (wild-type) and C57Bl/6 transgenic for MUC1 (MUC1.Tg) were challenged in the pancreas with Panc02-MUC1, a C57Bl/6-syngeneic pancreatic cancer cell line expressing human MUC1. Wild-type mice produced immune responses to MUC1 when presented on tumor cells growing in the pancreas; however, the responses to tumors in the pancreas were less effective than responses produced by tumor challenge at the s.c. site. Tumor immunity specific for MUC1 was produced in wild-type mice by two different procedures: (i) s.c. immunization of wild-type mice with a low dose of Panc02-MUC1 or (ii) adoptive transfer of spleen and lymph node cells harvested from wild-type mice previously immunized s.c. with Panc02-MUC1. This demonstrates that immune responses to MUC1 presented at the s.c. site can be detected and adoptively transferred. MUC1.Tg mice were immunologically tolerant to MUC1; however, some immunological protection against orthotopic challenge with Panc02-MUC1 was conferred by adoptive transfer of CD4+ and CD8+ T cells from wild-type mice. These results show that it is more difficult to produce immune responses to tumors growing at the pancreatic site than the s.c. site. Panc02-MUC1 cells growing in the pancreas were accessible to the immune system, and immune responses evoked by s.c. presentation of this molecule in wild-type mice were effective in rejecting tumor cells in the pancreas of both wild-type and MUC1.Tg mice. No effective anti-tumor immune responses against MUC1 were produced in MUC1.Tg mice.
Assuntos
Rejeição de Enxerto/imunologia , Mucina-1/imunologia , Pâncreas/imunologia , Neoplasias Pancreáticas/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Rejeição de Enxerto/patologia , Humanos , Imunidade Celular/genética , Imunidade Celular/imunologia , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias/imunologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Análise de Sobrevida , Células Tumorais Cultivadas/transplanteAssuntos
Bioquímica/história , Anticorpos Monoclonais/metabolismo , Glicosilação , História do Século XX , Proteínas de Homeodomínio/química , Humanos , Leite Humano/química , Leite Humano/imunologia , Mucina-1/imunologia , Neoplasias/história , Neoplasias/metabolismo , Fatores de Transcrição/químicaRESUMO
Pancreatic cancer is a highly aggressive, treatment refractory disease and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas over-express altered forms of a tumor-associated antigen, MUC1 (an epithelial mucin glycoprotein), which is a target for immunotherapy. Using a clinically relevant mouse model of pancreas cancer that demonstrates peripheral and central tolerance to human MUC1 and develops spontaneous tumors of the pancreas, we have previously reported the presence of functionally active, low affinity, MUC1-specific precursor cytotoxic T cells (pCTLs). Hypothesis for this study is that MUC1-based immunization may enhance the low level MUC1-specific immunity that may lead to an effective anti-tumor response. Data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-gamma and are cytolytic against MUC1-expressing tumor cells in vitro. However, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. We demonstrate that the CTL tolerance could be reversed at least in vitro with the use of anti-CD40 co-stimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-beta that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+ CD25+ T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy.
Assuntos
Mucina-1/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Modelos Animais de Doenças , Progressão da Doença , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mucina-1/biossíntese , Mucina-1/genética , Neoplasias Pancreáticas/metabolismo , Taxa de Sobrevida , Fator de Crescimento Transformador beta/metabolismo , Evasão Tumoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Mice transgenic for the human MUC1 carcinoma-associated antigen (MUC1.Tg) are tolerant to immunization with MUC1 antigen. Recent studies, however, have demonstrated that immunization of MUC1.Tg mice with fusions of MUC1-positive tumour and dendritic cells (FC/MUC1) reverses MUC1 unresponsiveness and results in rejection of established MUC1-positive pulmonary metastases. Here we demonstrate that lymph node cells from MUC1.Tg mice immunized with the FC/MUC1 fusion cells proliferate in response to MUC1 antigen by a mechanism dependent on the function of CD4, major histocompatibility complex (MHC) class II, B7-1, B7-2, CD28, CD40 and CD40 ligand. The findings demonstrate that stimulation of lymph node cells with MUC1 results in selection of MUC1-specific CD8+ T cells. We show that the CD8+ T cells exhibit MUC1-specific cytotoxic T lymphocyte (CTL) activity by recognition of MUC1 peptides presented in the context of MHC class I molecules Kb and Db. The MUC1-specific CD8+ T cells also exhibit antitumour activity against MUC1-positive metastases, but with no apparent reactivity against normal tissues. These results indicate that immunization of MUC1.Tg mice with FC/MUC1 reverses immunological unresponsiveness to MUC1 by presentation of MUC1 peptides in the presence of costimulatory signals and generates MHC-restricted MUC1-specific CD8+ T cells.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Mucina-1/imunologia , Animais , Divisão Celular/imunologia , Fusão Celular , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Epitopos de Linfócito T/análise , Imunização , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.