RESUMEN
BACKGROUND: IL-17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation. METHODS: Flow-cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL-17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses. RESULTS: We identified Th17 cells as the main population of IL-17A- and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL-17A and its receptor subunit IL-17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL-17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro-inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL-17A and TNF synergistically induced the Th17-promoting cytokines IL-6 and IL-1ß as well as the Th17-attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor-promoting role of Th17 cells in OC. CONCLUSIONS: Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL-17A/TNF-mediated induction of mesothelial-mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.
Asunto(s)
Neoplasias Ováricas , Células Th17 , Humanos , Femenino , Interleucina-17/metabolismo , Citocinas/metabolismo , Neoplasias Ováricas/metabolismo , Inflamación/metabolismo , Microambiente TumoralRESUMEN
A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated T cells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the T cell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.
RESUMEN
Introduction: We here thought to dissect the inflammatory signature in lesions of three skin disorders, which show a common adaptive immune response against autoantigens of the skin but are characterized by diverging clinical phenotypes. Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are type-2-dependent, IgG autoantibody-driven blistering disorders of mucous membranes and skin, which target desmoglein (Dsg)3 and bullous pemphigoid (BP)180, respectively. In contrast, lichen planus (LP) is a common chronic inflammatory disease of the skin and mucous membranes with a pronounced dermal T cell infiltrate. We previously identified peripheral type 1 and 17 T cell responses against Dsg3 and BP180 in a cohort of LP patients strongly suggesting that the underlying inflammatory T cell signature may drive the evolving phenotype. Methods: Paraffin-embedded skin biopsies from well-characterized patients with LP (n=31), BP (n=19), PV (n=9), and pemphigus foliaceus (PF) (n=2) were analysed. Areas with the most prominent inflammatory infiltrate were excised with punch biopsies and tissue microarrays (TMA) containing multiple biopsies were created. Using multicolor immunofluorescence, the inflammatory infiltrate was stained with antibodies against multiple cellular markers, i. e. CD3ϵ, CD4, CD15, TCR-δ, the cytokine IL-17A, and the transcription factors, T-bet and GATA-3. Results: In LP, there was a higher number of CD4+ T cells expressing T-bet compared to GATA-3. In contrast, CD4+ T cells in PV and BP skin lesions more frequently expressed GATA-3 than T-bet. IL-17A+ cells and IL-17A+ T cells were found to a similar extent in all the three disorders. IL-17A+ granulocytes were more predominant in BP than in LP or PV. Of note, the majority of IL-17A+ cells in LP were neither T cells nor granulocytes. Discussion: Our findings in inflammatory skin infiltrates clearly show a predominant type 1 signature in LP in contrast to a preponderance of type 2 T cells in PV and BP. In contrast to LP, granulocytes and to a much lesser extent CD3+ T cells were a cellular source of IL-17A in BP and PV. These data strongly suggest that different inflammatory cell signatures drive evolving clinically diverse phenotypes of LP, PV and BP despite common target antigens of the skin.
Asunto(s)
Liquen Plano , Penfigoide Ampolloso , Pénfigo , Humanos , Interleucina-17 , Autoanticuerpos , Linfocitos T CD4-Positivos/patologíaRESUMEN
Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.
Asunto(s)
Manosa , Manosiltransferasas , Animales , Glicosilación , Mamíferos/metabolismo , Manosa/metabolismo , Manosiltransferasas/genética , Manosiltransferasas/metabolismoRESUMEN
Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.
Asunto(s)
Moléculas de Adhesión Celular , Semaforinas , Anticuerpos de Dominio Único , Animales , Ratones , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Transducción de Señal , Moléculas de Adhesión Celular/metabolismoRESUMEN
Metastatic prostate cancer is essentially incurable and is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We show here that prostate epithelial cell-specific expression of a mutant form of Plexin-B1 (P1597L) which was identified in metastatic deposits in patients with prostate cancer, significantly increases metastasis, in particular metastasis to distant sites, in two transgenic mouse models of prostate cancer (PbCre+Ptenfl /flKrasG12V and PbCre+Ptenfl /flp53fl/ fl ). In contrast, prostate epithelial cell-specific expression of wild-type (WT) Plexin-B1 in PbCre+Ptenfl /flKrasG12V mice significantly decreases metastasis, showing that a single clinically relevant Pro1597Leu amino-acid change converts Plexin-B1 from a metastasis-suppressor to a metastasis-promoter. Furthermore, PLXNB1P1597L significantly increased invasion of tumor cells into the prostate stroma, while PLXNB1WT reduced invasion, suggesting that Plexin-B1 has a role in the initial stages of metastasis. Deletion of RhoA/C or PDZRhoGEF in Ptenfl /flKrasG12VPLXNB1P1597L mice suppressed metastasis, implicating the Rho/ROCK pathway in this phenotypic switch. Germline deletion of Plexin-B1, to model anti-Plexin-B1 therapy, significantly decreased invasion and metastasis in both models. Our results demonstrate that Plexin-B1 plays a complex yet significant role in metastasis in mouse models of prostate cancer and is a potential therapeutic target to block the lethal spread of the disease. Significance: Few therapeutic targets have been identified specifically for preventing locally invasive/oligometastatic prostate cancer from becoming more widely disseminated. Our findings suggest Plexin-B1 signaling, particularly from the clinically relevant P1597L mutant, is such a target.
Asunto(s)
Antineoplásicos , Neoplasias de la Próstata , Humanos , Masculino , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Neoplasias de la Próstata/genética , Ratones TransgénicosRESUMEN
Peptic ulcer disease is a frequent clinical problem with potentially serious complications such as bleeding or perforation. A decisive factor in the pathogenesis of peptic ulcers is gastric acid, the secretion of which is controlled by the hormone gastrin released from gastric G cells. However, the molecular mechanisms regulating gastrin plasma concentrations are poorly understood. Here, we identified a semaphorin-plexin signaling pathway that operates in gastric G cells to inhibit gastrin expression on a transcriptional level, thereby limiting food-stimulated gastrin release and gastric acid secretion. Using a systematic siRNA screening approach combined with biochemical, cell biology, and in vivo mouse experiments, we found that the RasGAP protein Rasal1 is a central mediator of plexin signal transduction, which suppresses gastrin expression through inactivation of the small GTPase R-Ras. Moreover, we show that Rasal1 is pathophysiologically relevant for the pathogenesis of peptic ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs), a main risk factor of peptic ulcers in humans. Last, we show that application of recombinant semaphorin 4D alleviates peptic ulcer disease in mice in vivo, demonstrating that this signaling pathway can be harnessed pharmacologically. This study unravels a mode of G cell regulation that is functionally important in gastric homeostasis and disease.
Asunto(s)
Úlcera Péptica , Semaforinas , Animales , Moléculas de Adhesión Celular , Proteínas Activadoras de GTPasa , Gastrinas/efectos adversos , Gastrinas/metabolismo , Humanos , Ratones , Proteínas del Tejido Nervioso , Úlcera Péptica/inducido químicamente , Transducción de SeñalRESUMEN
Osteoporosis and multiple sclerosis are highly prevalent diseases with limited treatment options. In light of these unmet medical needs, novel therapeutic approaches are urgently sought. Previously, the activation of the transmembrane receptor Plexin-B1 by its ligand semaphorin 4D (Sema4D) has been shown to suppress bone formation and promote neuroinflammation in mice. However, it is unclear whether inhibition of this receptor-ligand interaction by an anti-Plexin-B1 antibody could represent a viable strategy against diseases related to these processes. Here, we raised and systematically characterized a monoclonal antibody directed against the extracellular domain of human Plexin-B1, which specifically blocks the binding of Sema4D to Plexin-B1. In vitro, we show that this antibody inhibits the suppressive effects of Sema4D on human osteoblast differentiation and mineralization. To test the therapeutic potential of the antibody in vivo, we generated a humanized mouse line, which expresses transgenic human Plexin-B1 instead of endogenous murine Plexin-B1. Employing these mice, we demonstrate that the anti-Plexin-B1 antibody exhibits beneficial effects in mouse models of postmenopausal osteoporosis and multiple sclerosis in vivo. In summary, our data identify an anti-Plexin-B1 antibody as a potential therapeutic agent for the treatment of osteoporosis and multiple sclerosis.
Asunto(s)
Anticuerpos Monoclonales , Antígenos CD , Esclerosis Múltiple , Proteínas del Tejido Nervioso , Osteoporosis Posmenopáusica , Receptores de Superficie Celular , Semaforinas , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ligandos , Ratones , Esclerosis Múltiple/terapia , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Osteoporosis Posmenopáusica/terapia , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Semaforinas/antagonistas & inhibidores , Semaforinas/metabolismoRESUMEN
Epithelial monolayer formation depends on the architecture and composition of the microtubule cytoskeleton. Microtubules control bidirectional trafficking and determine the positioning of structural cellular proteins. We studied the role of tubulin tyrosination in epithelial cell shape and motility. Tubulin tyrosine ligase (TTL), the enzyme that adds tyrosine to the carboxy terminus of detyrosinated α-tubulin, was depleted or overexpressed in 2D epithelial monolayers as well as in 3D intestinal organoids. We demonstrate qualitatively and quantitatively that in the absence of TTL the cells comprise high levels of detyrosinated tubulin, change their shape into an initial flat morphology and retardedly acquire a differentiated columnar epithelial cell shape. Enhanced adhesion and accelerated migration patterns of TTL-knockout cells combined with reverse effects in TTL-overexpressing cells indicate that the loss of TTL affects the organization of cell adhesion foci. Precipitation of detyrosinated tubulin with focal adhesion scaffold components coincides with increased quantities and persistence of focal adhesion plaques. Our results indicate that the equilibrium between microtubules enriched in detyrosinated or tyrosinated tubulin modulates epithelial tissue formation, cell morphology, and adhesion.
RESUMEN
The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , División Celular/fisiología , Células Epidérmicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Células Madre/metabolismo , Animales , Carcinoma Basocelular/patología , Proteínas Portadoras/metabolismo , Adhesión Celular , Proliferación Celular , Desarrollo Embrionario/fisiología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Uniones Intercelulares , Queratinocitos , Ratones , Mitosis , Morfogénesis , OrganogénesisRESUMEN
Aberrant fear is a cornerstone of several psychiatric disorders. Consequently, there is large interest in elucidation of signaling mechanisms that link extracellular cues to changes in neuronal function and structure in brain pathways that are important in the generation and maintenance of fear memory and its behavioral expression. Members of the Plexin-B family of receptors for class 4 semaphorins play important roles in developmental plasticity of neurons, and their expression persists in some areas of the adult nervous system. Here, we aimed to elucidate the role of Semaphorin 4C (Sema4C) and its cognate receptor, Plexin-B2, in the expression of contextual and cued fear memory, setting a mechanistic focus on structural plasticity and exploration of contributing signaling pathways. We observed that Plexin-B2 and Sema4C are expressed in forebrain areas related to fear memory, such as the anterior cingulate cortex, amygdala and the hippocampus, and their expression is regulated by aversive stimuli that induce fear memory. By generating forebrain-specific Plexin-B2 knockout mice and analyzing fear-related behaviors, we demonstrate that Sema4C-PlexinB2 signaling plays a crucial functional role in the recent and remote recall of fear memory. Detailed neuronal morphological analyses revealed that Sema4C-PlexinB2 signaling largely mediates fear-induced structural plasticity by enhancing dendritic ramifications and modulating synaptic density in the adult hippocampus. Analyses on signaling-related mutant mice showed that these functions are mediated by PlexinB2-dependent RhoA activation. These results deliver important insights into the mechanistic understanding of maladaptive plasticity in fear circuits and have implications for novel therapeutic strategies against fear-related disorders.
Asunto(s)
Miedo , Memoria , Proteínas del Tejido Nervioso , Semaforinas , Animales , Moléculas de Adhesión Celular , Ratones , Proteínas del Tejido Nervioso/genética , Neuronas , Semaforinas/genéticaRESUMEN
A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of proteins that are upregulated in the plasma of OC patients. Using binders targeting 368 different plasma proteins, we compared 20 plasma samples from HGSC patients (OC-plasma) with 20 plasma samples from individuals with non-malignant gynecologic disorders (N-plasma). We identified 176 proteins with significantly higher levels in OC-plasma compared to N-plasma by PEA (p < 0.05 by U-test; Benjamini-Hochberg corrected), which are mainly implicated in immune regulation and metastasis-associated processes, such as matrix remodeling, adhesion, migration and proliferation. A number of these proteins have not been reported in previous studies, such as BCAM, CDH6, DDR1, N2DL-2 (ULBP2), SPINT2, and WISP-1 (CCN4). Of these SPINT2, a protease inhibitor mainly derived from tumor cells within the HGSC microenvironment, showed the highest significance (p < 2 × 10-7) similar to the previously described IL-6 and PVRL4 (NECTIN4) proteins. Results were validated by means of the aptamer-based 1.3 k SOMAscan proteomic platform, which revealed a high inter-platform correlation with a median Spearman ρ of 0.62. Likewise, ELISA confirmed the PEA data for 10 out of 12 proteins analyzed, including SPINT2. These findings suggest that in contrast to other entities SPINT2 does not act as a tumor suppressor in HGSC. This is supported by data from the PRECOG and KM-Plotter meta-analysis databases, which point to a tumor-type-specific inverse association of SPINT2 gene expression with survival. Our data also demonstrate that both the PEA and SOMAscan affinity proteomics platforms bear considerable potential for the unbiased discovery of novel disease-associated biomarkers.
RESUMEN
The peritoneal fluid (ascites), replete with abundant tumor-promoting factors and extracellular vesicles (EVs) reflecting the tumor secretome, plays an essential role in ovarian high-grade serous carcinoma (HGSC) metastasis and immune suppression. A comprehensive picture of mediators impacting HGSC progression is, however, not available. Methods: Proteins in ascites from HGSC patients were quantified by the aptamer-based SOMAscan affinity proteomic platform. SOMAscan data were analyzed by bioinformatic methods to reveal clinically relevant links and functional connections, and were validated using the antibody-based proximity extension assay (PEA) Olink platform. Mass spectrometry was used to identify proteins in extracellular microvesicles released by HGSC cells. Results: Consistent with the clinical features of HGSC, 779 proteins in ascites identified by SOMAscan clustered into groups associated either with metastasis and a short relapse-free survival (RFS), or with immune regulation and a favorable RFS. In total, 346 proteins were linked to OC recurrence in either direction. Reanalysis of 214 of these proteins by PEA revealed an excellent median Spearman inter-platform correlation of ρ=0.82 for the 46 positively RFS-associated proteins in both datasets. Intriguingly, many proteins strongly associated with clinical outcome were constituents of extracellular vesicles. These include proteins either linked to a poor RFS, such as HSPA1A, BCAM and DKK1, or associated with a favorable outcome, such as the protein kinase LCK. Finally, based on these data we defined two protein signatures that clearly classify short-term and long-term relapse-free survivors. Conclusion: The ascites secretome points to metastasis-promoting events and an anti-tumor response as the major determinants of the clinical outcome of HGSC. Relevant proteins include both bone fide secreted and vesicle-encapsulated polypeptides, many of which have previously not been linked to HGSC recurrence. Besides a deeper understanding of the HGSC microenvironment our data provide novel potential tools for HGSC patient stratification. Furthermore, the first large-scale inter-platform validation of SOMAscan and PEA will be invaluable for other studies using these affinity proteomics platforms.
Asunto(s)
Cistadenocarcinoma Seroso/patología , Neoplasias Ováricas/patología , Proteómica/métodos , Microambiente Tumoral , Ascitis/metabolismo , Ascitis/patología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Proteínas Sanguíneas/análisis , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/mortalidad , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , RecurrenciaRESUMEN
The peritoneal fluid of ovarian carcinoma patients promotes cancer cell invasion and metastatic spread with lysophosphatidic acid (LPA) as a potentially crucial mediator. However, the origin of LPA in ascites and the clinical relevance of individual LPA species have not been addressed. Here, we show that the levels of multiple acyl-LPA species are strongly elevated in ascites versus plasma and are associated with short relapse-free survival. Data derived from transcriptome and secretome analyses of primary ascite-derived cells indicate that (a) the major route of LPA synthesis is the consecutive action of a secretory phospholipase A2 (PLA2 ) and autotaxin, (b) that the components of this pathway are coordinately upregulated in ascites, and (c) that CD163+CD206+ tumor-associated macrophages play an essential role as main producers of PLA2 G7 and autotaxin. The latter conclusion is consistent with mass spectrometry-based metabolomic analyses of conditioned medium from ascites cells, which showed that tumor-associated macrophages, but not tumor cells, are able to produce 20:4 acyl-LPA in lipid-free medium. Furthermore, our transcriptomic data revealed that LPA receptor (LPAR) genes are expressed in a clearly cell type-selective manner: While tumor cells express predominantly LPAR1-3, macrophages and T cells also express LPAR5 and LPAR6 at high levels, pointing to cell type-selective LPA signaling pathways. RNA profiling identified cytokines linked to cell motility and migration as the most conspicuous class of LPA-induced genes in macrophages, suggesting that LPA exerts protumorigenic properties at least in part via the tumor secretome.
Asunto(s)
Lisofosfolípidos/biosíntesis , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Transducción de Señal , Microambiente Tumoral , Ascitis/metabolismo , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Metaboloma , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Ováricas/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Resultado del Tratamiento , Microambiente Tumoral/genética , Regulación hacia Arriba/genéticaRESUMEN
The accumulation of intratumoral CD8+ T cells is associated with the survival of high grade serous ovarian carcinoma patients, but it is unclear which CD8+ T cell subsets contribute to this effect and how they are affected by the peritoneal tumor microenvironment. Here, we provide evidence for a functional link between long relapse-free survival, accumulation of CD8+ effector memory T (TEM) cells in peritoneal effusion (ascites), and the level of the CD8+ TEM attracting chemokine CXCL9, produced by macrophages as a major source. We also propose a novel mechanism by which the tumor microenvironment could contribute to T cell dysfunction and shorter survival, i.e., diminished expression levels of essential signaling proteins, including STAT5B, PLCγ1 and NFATc2. CD8+ TEM cells in ascites, CXCL9 levels and the expression of crucial signal transduction proteins may therefore be important biomarkers to gauge the efficiency of immune therapies and potentially represent therapeutic targets.
RESUMEN
Ovarian cancer is characterized by early transcoelomic metastatic spread via the peritoneal fluid, where tumor cell spheroids (TU), tumor-associated T cells (TAT), and macrophages (TAM) create a unique microenvironment promoting cancer progression, chemoresistance, and immunosuppression. However, the underlying signaling mechanisms remain largely obscure. To chart these signaling networks, we performed comprehensive proteomic and transcriptomic analyses of TU, TAT, and TAM from ascites of ovarian cancer patients. We identify multiple intercellular signaling pathways driven by protein or lipid mediators that are associated with clinical outcome. Beyond cytokines, chemokines and growth factors, these include proteins of the extracellular matrix, immune checkpoint regulators, complement factors, and a prominent network of axon guidance molecules of the ephrin, semaphorin, and slit families. Intriguingly, both TU and TAM from patients with a predicted short survival selectively produce mediators supporting prometastatic events, including matrix remodeling, stemness, invasion, angiogenesis, and immunosuppression, whereas TAM associated with a longer survival express cytokines linked to effector T-cell chemoattraction and activation. In summary, our study uncovers previously unrecognized signaling networks in the ovarian cancer microenvironment that are of potential clinical relevance.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Microambiente Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Proteómica , Transducción de SeñalRESUMEN
Semaphorins and their transmembrane receptors, Plexins, are key regulators of axon guidance and development of neuronal connectivity. B-type Plexins respond to Class IV semaphorins and mediate a variety of developmental functions. Here we report that the expression of Plexin-B2 and its high-affinity ligand, Sema4C, persists in peripheral sensory neurons in adult life and is markedly increased in states of persistent pain in mice. Genetic deletion of Sema4C as well as adult-onset loss of Plexin-B2 leads to impairment of the development and duration of inflammatory hypersensitivity. Remarkably, unlike the neurodevelopmental functions of Plexin-B2 that solely rely on Ras signaling, we obtained genetic and pharmacological evidence for a requirement of RhoA-ROCK-dependent mechanisms as well as TRPA1 sensitization in pronociceptive functions of Sema4C-Plexin-B2 signaling in adult life. These results suggest important roles for Plexin-B2 signaling in sensory function that may be of therapeutic relevance in pathological pain.Semaphorins and their receptors are involved in neurodevelopment, but their functions in the adult nervous system are not fully understood. This study finds that semaphorin 4C and its receptor Plexin B are expressed in sensory neurons and are pronociceptive in a mouse model of inflammatory pain.
Asunto(s)
Inflamación , Proteínas del Tejido Nervioso/genética , Nocicepción , Dolor/genética , Semaforinas/genética , Células Receptoras Sensoriales/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Proteínas del Tejido Nervioso/metabolismo , Dolor/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Transducción de Señal , Canal Catiónico TRPA1/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
The reciprocal interplay of cancer cells and host cells is an indispensable prerequisite for tumor growth and progression. Cells of both the innate and adaptive immune system, in particular tumor-associated macrophages (TAMs) and T cells, as well as cancer-associated fibroblasts enter into a malicious liaison with tumor cells to create a tumor-promoting and immunosuppressive tumor microenvironment (TME). Ovarian cancer, the most lethal of all gynecological malignancies, is characterized by a unique TME that enables specific and efficient metastatic routes, impairs immune surveillance, and mediates therapy resistance. A characteristic feature of the ovarian cancer TME is the role of resident host cells, in particular activated mesothelial cells, which line the peritoneal cavity in huge numbers, as well as adipocytes of the omentum, the preferred site of metastatic lesions. Another crucial factor is the peritoneal fluid, which enables the transcoelomic spread of tumor cells to other pelvic and peritoneal organs, and occurs at more advanced stages as a malignancy-associated effusion. This ascites is rich in tumor-promoting soluble factors, extracellular vesicles and detached cancer cells as well as large numbers of T cells, TAMs, and other host cells, which cooperate with resident host cells to support tumor progression and immune evasion. In this review, we summarize and discuss our current knowledge of the cellular and molecular interactions that govern this interplay with a focus on signaling networks formed by cytokines, lipids, and extracellular vesicles; the pathophysiologial roles of TAMs and T cells; the mechanism of transcoelomic metastasis; and the cell type selective processing of signals from the TME.
RESUMEN
Plexins comprise a family of transmembrane receptors for semaphorins. Plexins of the B- and D-subfamily interact with the receptor tyrosine kinase ErbB-2, and this interaction has been shown to be functionally relevant for various biological processes including tumor metastasis and bone formation. Binding of semaphorins to B- and D-subfamily plexins results in the activation of ErbB-2, which in turn phosphorylates these plexins. This phosphorylation triggers the activation of the small GTPases RhoA and RhoC downstream of B-subfamily plexins. Here we describe a methodology that allows the analysis of ErbB-2-mediated plexin phosphorylation and signaling.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptor ErbB-2/metabolismo , Tirosina/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Humanos , Células MCF-7 , Fosforilación , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor ßPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.