RESUMEN
Ewing sarcoma (ES) is an aggressive cancer diagnosed in adolescents and young adults. The fusion oncoprotein (EWSR1::FLI1) that drives Ewing sarcoma is known to downregulate TGFBR2 expression (part of the TGFß receptor). Because TGFBR2 is downregulated, it was thought that TGFß likely plays an inconsequential role in Ewing biology. However, the expression of TGFß in the Ewing tumor immune microenvironment (TIME) and functional impact of TGFß in the TIME remains largely unknown given the historical lack of immunocompetent preclinical models. Here, we use single-cell RNAseq analysis of human Ewing tumors to show that immune cells, such as NK cells, are the largest source of TGFß production in human Ewing tumors. We develop a humanized (immunocompetent) mouse model of ES and demonstrate distinct TME signatures and metastatic potential in these models as compared to tumors developed in immunodeficient mice. Using this humanized model, we study the effect of TGFß inhibition on the Ewing TME during radiation therapy, a treatment that both enhances TGFß activation and is used to treat aggressive ES. Utilizing a trivalent ligand TGFß TRAP to inhibit TGFß, we demonstrate that in combination with radiation, TGFß inhibition both increases ES immune cell infiltration and decreases lung metastatic burden in vivo . The culmination of these data demonstrates the value of humanized models to address immunobiologic preclinical questions in Ewing sarcoma and suggests TGFß inhibition as a promising intervention during radiation therapy to promote metastatic tumor control.
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Merosin-deficient congenital muscular dystrophy (MDC1A) is an autosomal recessive disorder caused by mutations in the LAMA2 gene, resulting in a defective form of the extracellular matrix protein laminin-α2 (LAMA2). Individuals diagnosed with MDC1A exhibit progressive muscle wasting and declining neuromuscular functions. No treatments for this disorder are currently available. We previously showed that postnatal Lama1 upregulation, achieved through CRISPR activation (CRISPRa), compensates for Lama2 deficiency and prevents neuromuscular pathophysiology in a mouse model of MDC1A. In this study, we assessed the feasibility of upregulating human LAMA1 as a potential therapeutic strategy for individuals with MDC1A, regardless of their mutations. We hypothesized that CRISPRa-mediated upregulation of human LAMA1 would compensate for the lack of LAMA2 and rescue cellular abnormalities in MDC1A fibroblasts. Global transcriptomic and pathway enrichment analyses of fibroblasts collected from individuals carrying pathogenic LAMA2 mutations, compared with healthy controls, indicated higher expression of transcripts encoding proteins that contribute to wound healing, including Transforming Growth Factor-ß (TGF-ß) and Fibroblast Growth Factor (FGF). These findings were supported by wound-healing assays indicating that MDC1A fibroblasts migrated significantly more rapidly than the controls. Subsequently, we treated the MDC1A fibroblasts with SadCas9-2XVP64 and sgRNAs targeting the LAMA1 promoter. We observed robust LAMA1 expression, which was accompanied by significant decreases in cell migration and expression of FGFR2, TGF-ß2, and ACTA2, which are involved in the wound-healing mechanism in MDC1A fibroblasts. Collectively, our data suggest that CRISPRa-mediated LAMA1 upregulation may be a feasible mutation-independent therapeutic approach for MDC1A. This strategy might be adapted to address other neuromuscular diseases and inherited conditions in which strong compensatory mechanisms have been identified.
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Ewing sarcoma is a fusion oncoprotein-driven primary bone tumor. A subset of patients (~10%) with Ewing sarcoma are known to harbor germline variants in a growing number of genes involved in DNA damage repair. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. We thus questioned the impact of BARD1 loss on Ewing cell sensitivity to DNA damage and the Ewing sarcoma transcriptome. We demonstrate that PSaRC318 cells, a novel patient-derived cell line harboring a pathogenic BARD1 variant, are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that BARD1 loss enhances cell sensitivity to PARP inhibition plus radiation. Additionally, RNA-seq analysis revealed that loss of BARD1 results in the upregulation of GBP1 (guanylate-binding protein 1), a protein whose expression is associated with variable response to therapy depending on the adult carcinoma subtype examined. Here, we demonstrate that GBP1 contributes to the enhanced sensitivity of BARD1 deficient Ewing cells to DNA damage. Together, our findings demonstrate the impact of loss-of function mutations in DNA damage repair genes, such as BARD1, on Ewing sarcoma treatment response.
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Neoplasias Óseas , Tumores Neuroectodérmicos Periféricos Primitivos , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Daño del ADN/genética , Reparación del ADN/genética , Neoplasias Óseas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Unión al GTP/genética , Proteína BRCA1/genéticaRESUMEN
MALT1 is the effector protein of the CARMA/Bcl10/MALT1 (CBM) signalosome, a multiprotein complex that drives pro-inflammatory signaling pathways downstream of a diverse set of receptors. Although CBM activity is best known for its role in immune cells, emerging evidence suggests that it plays a key role in the pathogenesis of solid tumors, where it can be activated by selected G protein-coupled receptors (GPCR). Here, we demonstrated that overexpression of GPCRs implicated in breast cancer pathogenesis, specifically the receptors for Angiotensin II and thrombin (AT1R and PAR1), drove a strong epithelial-to-mesenchymal transition (EMT) program in breast cancer cells that is characteristic of claudin-low, triple-negative breast cancer (TNBC). In concert, MALT1 was activated in these cells and contributed to the dramatic EMT phenotypic changes through regulation of master EMT transcription factors including Snail and ZEB1. Importantly, blocking MALT1 signaling, through either siRNA-mediated depletion of MALT1 protein or pharmacologic inhibition of its activity, was effective at partially reversing the molecular and phenotypic indicators of EMT. Treatment of mice with mepazine, a pharmacologic MALT1 inhibitor, reduced growth of PAR1+, MDA-MB-231 xenografts and had an even more dramatic effect in reducing the burden of metastatic disease. These findings highlight MALT1 as an attractive therapeutic target for claudin-low TNBCs harboring overexpression of one or more selected GPCRs. IMPLICATIONS: This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.
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Neoplasias de la Mama Triple Negativas , Animales , Línea Celular Tumoral , Movimiento Celular , Claudinas/farmacología , Claudinas/uso terapéutico , Transición Epitelial-Mesenquimal , Humanos , Ratones , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Receptor PAR-1/uso terapéutico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative signaling node in a variety of biological processes ranging from cell growth and proliferation to migration and chemotaxis. As such, GRK2 is now implicated as playing a role in the molecular pathogenesis of a broad group of diseases including heart failure, cancer, depression, neurodegenerative disease, and others. In addition to its long-known canonical role in the phosphorylation and desensitization of G protein-coupled receptors (GPCRs), recent studies have shown that GRK2 also modulates a diverse array of other molecular processes via newly identified GRK2 kinase substrates and via a growing number of protein-protein interaction binding partners. GRK2 belongs to the 7-member GRK family. It is a multidomain protein containing a specific N-terminal region (referred to as αN), followed by a regulator of G protein signaling homology (RH) domain, an AGC (Protein kinase A, G, C serine/threonine kinase family) kinase domain, and a C-terminal pleckstrin homology (PH) domain. GPCRs mediate the activity of many regulators of the immune system such as chemokines and leukotrienes, and thus GRK proteins may play key roles in modulating the lymphocyte response to these factors. As one of the predominant GRK family members expressed in immune cells, GRK2's canonical and noncanonical actions play an especially significant role in normal immune cell function as well as in the development and progression of disorders of the immune system. This review summarizes our current state of knowledge of the roles of GRK2 in lymphocytes. We highlight the diverse functions of GRK2 and discuss how ongoing investigation of GRK2 in lymphocytes may inform the development of new therapies for diseases associated with lymphocyte dysregulation.
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Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Linfocitos/metabolismo , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
The CBM complex, which is composed of the proteins CARMA1, BCL10, and MALT1, serves multiple pivotal roles as a mediator of T-cell receptor and B-cell receptor-dependent NF-κB induction and lymphocyte activation. CARMA1, BCL10, and MALT1 are each proto-oncoproteins and dysregulation of CBM signaling, as a result of somatic gain-of-function mutation or chromosomal translocation, is a hallmark of multiple lymphoid malignancies including Activated B-cell Diffuse Large B-cell Lymphoma. Moreover, loss-of-function as well as gain-of-function germline mutations in CBM complex proteins have been associated with a range of immune dysregulation syndromes. A wealth of detailed structural information has become available over the past decade through meticulous interrogation of the interactions between CBM components. Here, we review key findings regarding the biochemical nature of these protein-protein interactions which have ultimately led the field to a sophisticated understanding of how these proteins assemble into high-order filamentous CBM complexes. To date, approaches to therapeutic inhibition of the CBM complex for the treatment of lymphoid malignancy and/or auto-immunity have focused on blocking MALT1 protease function. We also review key studies relating to the structural impact of MALT1 protease inhibitors on key protein-protein interactions.
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Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Guanilato Ciclasa/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 10 de la LLC-Linfoma de Células B/fisiología , Linfocitos B/metabolismo , Proteínas Adaptadoras de Señalización CARD/fisiología , Guanilato Ciclasa/fisiología , Humanos , Activación de Linfocitos/fisiología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/fisiología , FN-kappa B/metabolismo , Mapas de Interacción de Proteínas/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunologíaRESUMEN
The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB-dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow-derived mast cells from MALT1 knockout (MALT1-/-) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1-/- and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell-derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.
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Células Endoteliales/metabolismo , Hipersensibilidad/metabolismo , Mastocitos/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Células Endoteliales/inmunología , Femenino , Histamina/inmunología , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Activación de Linfocitos/inmunología , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Receptores de IgE/inmunología , Receptores de IgE/metabolismoRESUMEN
Antigen receptor-dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein-coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.
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Carcinogénesis/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteínas Oncogénicas/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Femenino , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Humanos , Células Jurkat , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Endogámicos NOD , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteínas Oncogénicas/genéticaRESUMEN
Protease-activated receptor 1 (PAR1), a thrombin-responsive G protein-coupled receptor (GPCR), is implicated in promoting metastasis in multiple tumor types, including both sarcomas and carcinomas, but the molecular mechanisms responsible remain largely unknown. We previously discovered that PAR1 stimulation in endothelial cells leads to activation of NF-κB, mediated by a protein complex comprised of CARMA3, Bcl10, and the MALT1 effector protein (CBM complex). Given the strong association between NF-κB and metastasis, we hypothesized that this CBM complex could play a critical role in the PAR1-driven metastatic progression of specific solid tumors. In support of our hypothesis, we demonstrate that PAR1 stimulation results in NF-κB activation in both osteosarcoma and breast cancer, which is suppressed by siRNA-mediated MALT1 knockdown, suggesting that an intact CBM complex is required for the response in both tumor cell types. We identify several metastasis-associated genes that are upregulated in a MALT1-dependent manner after PAR1 stimulation in cancer cells, including those encoding the matrix remodeling protein, MMP9, and the cytokines, IL-1ß and IL-8. Further, exogenous expression of PAR1 in MCF7 breast cancer cells confers highly invasive and metastatic behavior which can be blocked by CRISPR/Cas9-mediated MALT1 knockout. Importantly, we find that PAR1 stimulation induces MALT1 protease activity in both osteosarcoma and breast cancer cells, an activity that is mechanistically linked to NF-κB activation and potentially other responses associated with aggressive phenotype. Several small molecule MALT1 protease inhibitors have recently been described that could therefore represent promising new therapeutics for the prevention and/or treatment of PAR1-driven tumor metastasis.
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Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , FN-kappa B/metabolismo , Osteosarcoma/patología , Receptor PAR-1/metabolismo , Animales , Apoptosis , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , FN-kappa B/genética , Osteosarcoma/genética , Osteosarcoma/metabolismo , Receptor PAR-1/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastatic and relapsed Ewing sarcoma typically afflicts the adolescent population and is largely fatal. These bone tumors are most commonly driven by the fusion oncoprotein EWS-FLI1. Ewing tumors demonstrate significant intra-tumoral heterogeneity, and individual tumor cells can express highly variable and dynamic levels of EWS-FLI1. Recent studies revealed that the EWS-FLI1 oncoprotein level (high versus low expression) greatly influences the behavior of Ewing tumor cells. As compared to cells with high EWS-FLI1, Ewing cells in the EWS-FLI1 low state demonstrate an increased propensity for metastasis. In light of these observations, we sought to determine how tumor cell EWS-FLI1 level influences the anti-tumor cell immune response. Since ICAM-1, which can promote tumor cell/T-cell interaction and T-cell activation, is highly expressed on EWS-FLI1 low cells, we hypothesized that EWS-FLI1 low cells would be more susceptible to T-cell mediated tumor cell apoptosis as compared to cells with high EWS-FLI1. Unexpectedly, we found that EWS-FLI1 low cells are more resistant to T-cell mediated apoptosis than EWS-FLI1 high cells. We investigated the potential mechanisms by which EWS-FLI1 level might influence the T-cell anti-tumor response, and discovered that low EWS-FLI1 expression results in upregulation of PD-L1 and PD-L2, both important ligands for the PD-1 immune checkpoint receptor on T-cells. We demonstrated that blocking PD-1 results in a greater increase of T-cell mediated killing of EWS-FLI1 low tumor cells as compared to cells with higher EWS-FLI1 expression. Our studies suggest that Ewing cells in the EWS-FLI1 low expression state may serve as a niche of tumor immune-evasion.
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Pediatric and adult patients with recurrent/refractory Burkitt lymphoma (BL) continue to have poor outcomes, emphasizing the need for newer therapeutic agents. Bruton's tyrosine kinase (BTK) is activated following B-cell receptor stimulation and in part regulates normal B-cell development. Ibrutinib, a selective and irreversible BTK inhibitor, has been efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, and marginal zone lymphoma. In this study, we investigated the efficacy of ibrutinib alone and in selective adjuvant combinations against BL in-vitro and in a human BL xenografted immune-deficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse model. Our data demonstrated that phospho-BTK level was significantly reduced in BL cells treated with ibrutinib (p < 0.001). Moreover, we observed a significant decrease in cell proliferation as well as significant decrease in IC50 of ibrutinib in combination with dexamethasone, rituximab, obinutuzumab, carfilzomib, and doxorubicin (p < 0.001). In-vivo studies demonstrated ibrutinib treated mice had a significantly prolonged survival with median survival of mice following ibrutinib treatment (32 days) (24 days) (p < 0.02). In conclusion, our findings demonstrate the significant in-vitro and preclinical in-vivo effects of ibrutinib in BL. Based on our preclinical results in this investigation, there is an on-going clinical trial comparing overall survival in children and adolescents with relapsed/refractory BL treated with chemoimmunotherapy with or without ibrutinib (NCT02703272).
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The CARMA-Bcl10-MALT1 (CBM) signalosome is an intracellular protein complex composed of a CARMA scaffolding protein, the Bcl10 linker protein, and the MALT1 protease. This complex was first recognized because the genes encoding its components are targeted by mutation and chromosomal translocation in lymphoid malignancy. We now know that the CBM signalosome plays a critical role in normal lymphocyte function by mediating antigen receptor-dependent activation of the pro-inflammatory, pro-survival NF-κB transcription factor, and that deregulation of this signaling complex promotes B-cell lymphomagenesis. More recently, we and others have demonstrated that a CBM signalosome also operates in cells outside of the immune system, including in several solid tumors. While CARMA1 (also referred to as CARD11) is expressed primarily within lymphoid tissues, the related scaffolding protein, CARMA3 (CARD10), is more widely expressed and participates in a CARMA3-containing CBM complex in a variety of cell types. The CARMA3-containing CBM complex operates downstream of specific G protein-coupled receptors (GPCRs) and/or growth factor receptor tyrosine kinases (RTKs). Since inappropriate expression and activation of GPCRs and/or RTKs underlies the pathogenesis of several solid tumors, there is now great interest in elucidating the contribution of CARMA3-mediated cellular signaling in these malignancies. Here, we summarize the key discoveries leading to our current understanding of the role of CARMA3 in solid tumor biology and highlight the current gaps in our knowledge.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Biomarcadores , Humanos , FN-kappa B/metabolismo , Neoplasias/patología , Transducción de SeñalRESUMEN
Intestinal transplantation is a successful treatment for children with intestinal failure, but has many potential complications. PTLD, a clinically and histologically diverse malignancy, occurs frequently after intestinal transplantation and can be fatal. The management of this disease is particularly challenging. The rejection-prone intestinal allograft requires high levels of immunosuppression, a precondition for PTLD. While EBV infection clearly plays a role in disease pathogenesis, the relatively naïve immune system of children is another likely contributor. As a result, pediatric intestine recipients have a higher risk of developing PTLD than other solid organ recipients. Other risk factors for disease development such as molecular and genomic changes that precipitate malignant transformation are not fully understood, especially among children. Studies on adults have started to describe the molecular pathogenesis of PTLD, but the genomic landscape of the malignancy remains largely undefined in pediatric intestinal transplant patients. In this review, we describe what is known about PTLD in pediatric patients after intestinal transplant and highlight current knowledge gaps to better direct future investigations in the pediatric population.
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Intestinos/trasplante , Trastornos Linfoproliferativos/etiología , Complicaciones Posoperatorias , Humanos , Trastornos Linfoproliferativos/diagnóstico , Trastornos Linfoproliferativos/terapia , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/terapiaRESUMEN
The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NFκB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NFκB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell-intrinsic responses that include proliferation, migration, and invasion. In addition, CBM-dependent activation of NFκB elicited cancer cell-extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NFκB signaling in AGTR1+ breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II-dependent NFκB signaling pathways to improve the treatment of this breast cancer subset.Significance: These findings offer a mechanistic rationale to explore the repurposing of drugs that target angiotensin action to improve the treatment of AGTR1-expressing breast cancers. Cancer Res; 78(5); 1225-40. ©2017 AACR.
Asunto(s)
Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Neoplasias de la Mama/patología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , FN-kappa B/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de Angiotensina/metabolismo , Animales , Apoptosis , Proteína 10 de la LLC-Linfoma de Células B/antagonistas & inhibidores , Proteína 10 de la LLC-Linfoma de Células B/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Adaptadoras de Señalización CARD/antagonistas & inhibidores , Proteínas Adaptadoras de Señalización CARD/genética , Movimiento Celular , Proliferación Celular , Embrión de Pollo , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , FN-kappa B/genética , Neovascularización Patológica , Pronóstico , ARN Interferente Pequeño/genética , Receptor de Angiotensina Tipo 1/genética , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.
Asunto(s)
Caspasas/inmunología , Cisteína Endopeptidasas/inmunología , Células Endoteliales/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Proteínas de Neoplasias/inmunología , Trombina/farmacología , Animales , Transporte Biológico , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/inmunología , Caspasas/genética , Línea Celular , Cisteína Endopeptidasas/genética , Enzima Desubiquitinante CYLD , Células Endoteliales/citología , Células Endoteliales/inmunología , Regulación de la Expresión Génica , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Ratones , Ratones Transgénicos , Microtúbulos/ultraestructura , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/genética , FN-kappa B/inmunología , Proteínas de Neoplasias/genética , Permeabilidad/efectos de los fármacos , Cultivo Primario de Células , Receptor PAR-1/genética , Receptor PAR-1/inmunología , Transducción de Señal , Trombina/metabolismoRESUMEN
Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin's lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to generate a stable, constitutively active fragment. Thus, API2-MALT1 harnesses multiple unique pathways to achieve deregulated NF-κB activation. Emerging data from our group and others have also detailed additional gain-of-function activities of API2-MALT1 that extend beyond NF-κB activation. Specifically, API2-MALT1 recruits and subverts multiple other signaling factors, including LIM domain and actin-binding protein 1 (LIMA1) and Smac/DIABLO. Like NIK, LIMA1 represents a unique substrate for API2-MALT1 protease activity, but unlike NIK, its cleavage sets in motion a major NF-κB-independent pathway for promoting oncogenesis. In this review, we highlight the most recent results characterizing these unique and diverse gain-of-function activities of API2-MALT1 and how they contribute to lymphomagenesis.
RESUMEN
MALT1 is the only known paracaspase and is a critical mediator of B- and T-cell receptor signalling. The function of the MALT1 gene is subverted by oncogenic chimeric fusions arising from the recurrent t(11;18)(q21;q21) aberration, which is the most frequent translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. API2-MALT1-positive MALT lymphomas manifest antibiotic resistance and aggressive clinical behaviour with poor clinical outcome. However, the mechanisms underlying API2-MALT1-induced MALT lymphomagenesis are not fully understood. Here we show that API2-MALT1 induces paracaspase-mediated cleavage of the tumour suppressor protein LIMA1. LIMA1 binding by API2-MALT1 is API2 dependent and proteolytic cleavage is dependent on MALT1 paracaspase activity. Intriguingly, API2-MALT1-mediated proteolysis generates a LIM domain-only (LMO)-containing fragment with oncogenic properties in vitro and in vivo. Importantly, primary MALT lymphomas harbouring the API2-MALT1 fusion uniquely demonstrate LIMA1 cleavage fragments. Our studies reveal a novel paracaspase-mediated oncogenic gain-of-function mechanism in the pathogenesis of MALT lymphoma.
Asunto(s)
Carcinogénesis/metabolismo , Proteínas del Citoesqueleto/metabolismo , Linfoma de Células B de la Zona Marginal/fisiopatología , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Células HEK293 , Humanos , Linfoma de Células B de la Zona Marginal/metabolismo , Espectrometría de Masas , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Proteolisis , ARN Interferente Pequeño/genética , Ensayo de Tumor de Célula MadreRESUMEN
Angiotensin (Ang) II is a potent mediator of both hypertension and cardiac damage; however, the mechanisms by which this occur remain unclear. B-cell lymphoma/leukemia 10 (Bcl10) is a member of the CBM signalosome, which links Ang II and nuclear factor-κB signaling. We hypothesized that Bcl10 is pivotal in the pathogenesis of Ang II-induced cardiac damage. Ang II infusion in mice lacking Bcl10 resulted in reduced cardiac fibrosis, less cellular infiltration, and improved arrhythmogenic electric remodeling, despite a similar degree of hypertension or cardiac hypertrophy. Adoptive transfer of bone marrow (BM), whereby Bcl10 knockout or wildtype BM was transferred to their opposite genotype recipients, revealed the dual importance of Bcl10 within both cardiac and immune cells. Loss of Bcl10 in cardiac cells resulted in reduced expression of genes important for the adhesion and recruitment of immune cells. In vitro experiments demonstrated that adhesion of monocytes to Ang II-treated endothelial cells also required Bcl10. Additionally, Bcl10 deficiency in macrophages reduced their intrinsic migratory ability. To address the role of BM-derived fibroblasts in the formation of cardiac fibrosis, we explored whether Bcl10 is also important for the infiltration of BM-derived (myo)fibroblasts into the heart. The transfer of green fluorescent protein positive wildtype BM into Bcl10 knockout recipient mice revealed a reduced number of noncardiac (myo)fibroblasts compared with those wildtype recipients. Our results demonstrate the significant role of Bcl10 in multiple cell types important for the generation of Ang II-induced cardiac damage and electric remodeling and may provide a new avenue for therapeutic intervention.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Angiotensina II/efectos adversos , Remodelación Atrial/fisiología , Cardiopatías/inducido químicamente , Cardiopatías/fisiopatología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteína 10 de la LLC-Linfoma de Células B , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Fibrosis , Cardiopatías/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Miocardio/metabolismo , Miocardio/patología , FN-kappa B/metabolismoRESUMEN
Aggressive histiocytic lesions are uncommon in the pediatric population. These neoplasms occur in isolation or after therapy for other types of hematopoietic malignancy such as T-cell acute lymphoblastic leukemia. The etiology of these lesions is poorly understood, and no definitive standard of care has been established for patients with these diagnoses. Here, we report the success of thalidomide treatment for 2 subtypes of histiocytic proliferation--metastatic histiocytic sarcoma and extracutaneous juvenile xanthogranuloma--in pediatric patients. Our findings highlight the importance of considering thalidomide therapy in this unique and difficult to treat patient population.
Asunto(s)
Sarcoma Histiocítico/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Talidomida/uso terapéutico , Xantogranuloma Juvenil/tratamiento farmacológico , Adolescente , Niño , Femenino , Sarcoma Histiocítico/epidemiología , Sarcoma Histiocítico/etiología , Humanos , Michigan/epidemiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Pronóstico , Xantogranuloma Juvenil/epidemiología , Xantogranuloma Juvenil/etiologíaRESUMEN
Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. With high-fat feeding, FFAs activate NF-kB in target tissues, initiating negative crosstalk with insulin signaling. However, the mechanisms underlying FFA-dependent NF-kB activation remain unclear. Here, we demonstrate that the saturated FA, palmitate, requires Bcl10 for NF-kB activation in hepatocytes. Uptake of palmitate, metabolism to diacylglycerol, and subsequent activation of protein kinase C (PKC) appear to mechanistically link palmitate with Bcl10, known as a central component of a signaling complex that, along with CARMA3 and MALT1, activates NF-kB downstream of selected cell surface receptors. Consequently, Bcl10-deficient mice are protected from hepatic NF-kB activation and insulin resistance following brief high-fat diet, suggesting that Bcl10 plays a major role in the metabolic consequences of acute overnutrition. Surprisingly, while CARMA3 also participates in the palmitate response, MALT1 is completely dispensable, thereby revealing an apparent nonclassical role for Bcl10 in NF-kB signaling.