RESUMO
Mesothelin (MSLN) is a cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed in many cancers. Mesothelin has been implicated to play role/s in cell adhesion and multiple signaling pathways. Mucin-16/CA125 is an enormous cell-surface glycoprotein, also normally expressed on mesothelium and implicated in the progression and metastasis of several cancers, and directly binds mesothelin. However, the precise biological function/s of mesothelin and mucin-16/CA125 remain mysterious. We report protein engineering and recombinant production, qualitative and quantitative binding studies, and a crystal structure determination elucidating the molecular-level details governing recognition of mesothelin by mucin-16/CA125. The interface is small, consistent with the â¼micromolar binding constant and is free of glycan-mediated interactions. Sequence comparisons and modeling suggest that multiple mucin-16/CA125 modules can interact with mesothelin through comparable interactions, potentially generating a high degree of avidity at the cell surface to overcome the weak affinity, with implications for functioning and therapeutic interventions.
Assuntos
Antígeno Ca-125 , Proteínas Ligadas por GPI , Mesotelina , Modelos Moleculares , Ligação Proteica , Mesotelina/metabolismo , Humanos , Antígeno Ca-125/metabolismo , Antígeno Ca-125/química , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Cristalografia por Raios X , Sítios de Ligação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Engenharia de Proteínas , Proteínas de MembranaRESUMO
Mesothelin (MSLN) overexpression (OE) is a frequent finding in ovarian carcinomas and increases cell survival and tumor aggressiveness. Since cancer stem cells (CSCs) contribute to pathogenesis, chemoresistance and malignant behavior in ovarian cancer (OC), we hypothesized that MSLN expression could be creating a favorable environment that nurtures CSCs. In this study, we analyzed the expression of MSLN and CSC markers SOX2 and ALDH1 by immunohistochemistry (IHC) in different model systems: primary high-grade serous carcinomas (HGSCs) and OC cell lines, including cell lines that were genetically engineered for MSLN expression by either CRISPR-Cas9-mediated knockout (Δ) or lentivirus-mediated OE. Cell lines, wild type and genetically engineered, were evaluated in 2D and 3D culture conditions and xenografted in nude mice. We observed that MSLN was widely expressed in HGSC, and restricted expression was observed in OC cell lines. In contrast, SOX2 and ALDH1 expression was limited in all tissue and cell models. Most importantly, the expression of CSC markers was independent of MSLN expression, and manipulation of MSLN expression did not affect CSC markers. In conclusion, MSLN expression is not involved in driving the CSC phenotype.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Cistadenocarcinoma Seroso/patologia , Mesotelina/metabolismo , Neoplasias Ovarianas/patologia , Retinal Desidrogenase/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Estudos Retrospectivos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. METHODS: We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody-drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma. RESULTS: Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth. CONCLUSIONS: These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo.
Assuntos
Imunoconjugados/administração & dosagem , Maitansina/análogos & derivados , Mesotelina/genética , Mesotelina/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Timoma/tratamento farmacológico , Neoplasias do Timo/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Imunoconjugados/farmacologia , Maitansina/administração & dosagem , Maitansina/farmacologia , Camundongos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Timoma/genética , Timoma/metabolismo , Neoplasias do Timo/genética , Neoplasias do Timo/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Given the heterogeneity of solid tumors, single-target CAR-T cell therapy often leads to recurrence, especially in ovarian cancer (OV). Here, we constructed a Tandem-CAR targeting two antigens with secretory activity (IL-12) to improve the effects of CAR-T cell therapy. Twenty coexpressed upregulated genes were identified from the GEO database, and we found FOLR1 (folate receptor 1) and MSLN (mesothelin) were specifically and highly expressed in cancer tissues and only 11.25% of samples were negative for both antigens. We observed an increased proliferation rate for these three CAR-T cells, and Tandem CAR-T cells could efficiently lyse antigen-positive OV cells in vitro and secrete higher levels of cytokines than single-target CAR-T cells. More importantly, in vivo experiments indicated that Tandem CAR-T cells markedly decreased tumor volume, exhibited enhanced antitumor activity, and prolonged mouse survival. Furthermore, the infiltration and persistence of T cells in the Tandem-CAR group were higher than those in the MSLN-CAR and Control-T groups but comparable to those in the FOLR1-CAR group. Collectively, this study demonstrated that Tandem CAR-T cells secreting IL-12 could enhance immunotherapeutic effects by reducing tumor antigen escape and increasing T cell functionality, which could be a promising therapeutic strategy for OV and other solid tumors.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Receptor 1 de Folato/metabolismo , Mesotelina/metabolismo , Neoplasias Ovarianas/terapia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Citocinas/genética , Citocinas/metabolismo , Bases de Dados Genéticas , Feminino , Receptor 1 de Folato/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-12/metabolismo , Mesotelina/genética , Camundongos , Camundongos Nus , Transcriptoma , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLNKO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLNKO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLNWT) or MSLNKO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLNKO mice compared to MSLNWT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.
Assuntos
Células Epiteliais/metabolismo , Mesotelina/genética , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/genética , Células Estromais/metabolismo , Microambiente Tumoral/genética , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Células Epiteliais/patologia , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Mesotelina/deficiência , Mesotelina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Células Estromais/patologiaRESUMO
Chimeric antigen receptor (CAR) T cells have been successfully used for the treatment of hematological malignancies including acute and chronic lymphoblastic leukemia. However, results of CAR T cell projects in solid tumors have been less impressive to date, partly because of immunosuppressive tumor microenvironment (TME). It is widely known that high adenosine production is an important factor causing tumor-induced immunosuppression in TME, and adenosine mediates the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). Previous studies have shown that adenosine generated by cancer cells significantly inhibits T cell anti-tumor activity through binding and then activating adenosine 2A receptors (A2aRs) of T cells. Based on the previous work, in our study, we evaluated whether A2aR disruption by shRNA could enhance the anti-tumor function of anti-mesothelin (MSLN) CAR T cells both in vitro and in vivo. For this goal above, we used MSLN-positive human ovarian serous carcinoma cells (SKOV3) and human colon cancer cells (HCT116) as target cancer cells while MSLN-negative human ovarian cancer cells (ES2) as non-target cancer cells. We observed that targeting cell-intrinsic A2aR through shRNA overexpression caused significant A2aR disruption in CAR T cells and profoundly increased CAR T cell efficacy in both CAR T cell cytokine production and cytotoxicity towards MSLN-positive cancer cells in vitro. More importantly, in SKOV3 xenograft mouse models, anti-MSLN CAR-T cells significantly reduced the tumor burden compared with non-transduced T cells, and the anti-tumor activity of A2aR-disrupted anti-MSLN CAR-T cells was stronger than that of wild-type anti-MSLN CAR-T cells. Altogether, our study showed enhanced anti-tumor efficacy caused by shRNA-mediated A2aR disruption in anti-MSLN CAR T cells both in vitro and in vivo, which proved that shRNA-mediated modification of gene expression might be an excellent strategy for improving CAR T cell function in immunosuppressive tumor microenvironment (TME) and could potentially improve the outcome of treatment in clinical trials.
Assuntos
Carcinoma Epitelial do Ovário/metabolismo , Mesotelina/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HCT116 , Células HEK293 , Humanos , Tolerância Imunológica/fisiologia , Imunoterapia Adotiva/métodos , Camundongos , Microambiente Tumoral/fisiologiaRESUMO
Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.
Assuntos
Adenocarcinoma/terapia , Mesotelina/administração & dosagem , Mesotelioma/terapia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose , Proliferação de Células , Feminino , Engenharia Genética , Humanos , Masculino , Mesotelina/genética , Mesotelina/metabolismo , Mesotelioma/genética , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Objective: CA125/MUC16 is an O-glycosylated protein that is expressed on the surfaces of ovarian epithelial cells. This molecule is a widely used tumor-associated marker for diagnosis of ovarian cancer. Recently, CA125 was shown to be involved in ovarian cancer metastasis. The purpose of this study was to investigate the mechanism of CA125 during ovarian cancer metastasis. Methods: We analyzed the Oncomine and CSIOVDB databases to determine the expression levels of DKK1 in ovarian cancer. DKK1 expression levels were upregulated or downregulated and applied with CA125 to Transwell and Western blot assays to ascertain the underlying mechanism by which CA125 stimulates cell migration via the SGK3/FOXO3 pathway. Anti-mesothelin antibodies (anti-MSLN) were used to block CA125 stimulation. Then the expression levels of DKK1were tested by enzyme-linked immunosorbent assay (ELISA) to eliminate the blocking effect of anti-MSLN to CA125 stimulation. Xenograft mouse models were used to detect the effects of CA125 and anti-MSLN on ovarian cancer metastasis in vivo. Results: DKK1 levels were downregulated in ovarian tumor tissues according to the analyses of two databases and significantly correlated with FIGO stage, grade and disease-free survival in ovarian cancer patients. DKK1 levels were downregulated by CA125 stimulation in vitro. Overexpression of DKK1 reversed the ability of exogenous CA125 to mediate cell migration by activating the SGK3/FOXO3 signaling pathway. Anti-MSLN abrogated the DKK1 reduction and increased the apoptosis of ovarian cancer cells. The use of anti-MSLN in xenograft mouse models significantly reduced tumor growth and metastasis accelerated by CA125. Conclusions: These experiments revealed that the SGK3/FOXO3 pathway was activated, wherein decreased expression of DKK1 was caused by CA125, which fuels ovarian cancer cell migration. Mesothelin is a potential therapeutic target for the treatment of ovarian cancer metastasis.