NGFR regulates stromal cell activation in germinal centers.

Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr-/- mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr-/- mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr-/- mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion.

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.

Familial Mediterranean fever in the pediatric population

Familial Mediterranean fever (FMF) is the most frequent autoinflammatory disorder characterized by short, repeated, and self-limiting crises of fever and serositis. The disease was described as autosomal recessive hereditary transmission secondary to variants of the MEFV (MEditerranean FeVer) gene, even though a variable proportion of patients only present a heterozygous variant. FMF is very common in certain ethnic groups (Turkish, Armenian, Arab, and Jewish), even though it has been described throughout the Mediterranean and elsewhere in the world. The clinical manifestations are variable, with secondary amyloidosis being the most serious complication of the disorder. Treatment and prophylaxis are mainly based on the administration of colchicine, which prevents the crises and avoids complications in most cases. This study reviews the course of seven pediatric patients diagnosed with FMF during the period 2010–2018 at a district hospital. Most of the patients were of Caucasian origin, with onset at an early age in the form of fever as the main symptom, and some patients moreover presented less frequent manifestations (pericardial effusion, sensorineural hearing loss). Two cases presented plasmatic amyloid A protein elevation that subsided with the treatment. All the patients initially received colchicine, and one of them required prescription of anakinra, which was replaced by canakinumab due to a serious adverse reaction. There were no cases of consanguinity, and all the patients were of Mediterranean origin. The subjects showed a favorable course over the years, which was attributed to the early diagnosis and treatment provided(AU)

Familial Mediterranean fever in the pediatric population.

Familial Mediterranean fever (FMF) is the most frequent autoinflammatory disorder characterized by short, repeated, and self-limiting crises of fever and serositis. The disease was described as autosomal recessive hereditary transmission secondary to variants of the MEFV (MEditerranean FeVer) gene, even though a variable proportion of patients only present a heterozygous variant. FMF is very common in certain ethnic groups (Turkish, Armenian, Arab, and Jewish), even though it has been described throughout the Mediterranean and elsewhere in the world. The clinical manifestations are variable, with secondary amyloidosis being the most serious complication of the disorder. Treatment and prophylaxis are mainly based on the administration of colchicine, which prevents the crises and avoids complications in most cases. This study reviews the course of seven pediatric patients diagnosed with FMF during the period 2010-2018 at a district hospital. Most of the patients were of Caucasian origin, with onset at an early age in the form of fever as the main symptom, and some patients moreover presented less frequent manifestations (pericardial effusion, sensorineural hearing loss). Two cases presented plasmatic amyloid A protein elevation that subsided with the treatment. All the patients initially received colchicine, and one of them required prescription of anakinra, which was replaced by canakinumab due to a serious adverse reaction. There were no cases of consanguinity, and all the patients were of Mediterranean origin. The subjects showed a favorable course over the years, which was attributed to the early diagnosis and treatment provided.

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

Could Extracellular Vesicles Contribute to Generation or Awakening of "Sleepy" Metastatic Niches?

Pre-metastatic niches provide favorable conditions for tumor cells to disseminate, home to and grow in otherwise unfamiliar and distal microenvironments. Tumor-derived extracellular vesicles are now recognized as carriers of key messengers secreted by primary tumors, signals that induce the formation of pre-metastatic niches. Recent evidence suggests that tumor cells can disseminate from the very earliest stages of primary tumor development. However, once they reach distal sites, tumor cells can persist in a dormant state for long periods of time until their growth is reactivated and they produce metastatic lesions. In this new scenario, the question arises as to whether extracellular vesicles could influence the formation of these metastatic niches with dormant tumor cells? (here defined as "sleepy niches"). If so, what are the molecular mechanisms involved? In this perspective-review article, we discuss the possible influence of extracellular vesicles in early metastatic dissemination and whether they might play a role in tumor cell dormancy. In addition, we comment whether extracellular vesicle-mediated signals may be involved in tumor cell awakening, considering the possibility that extracellular vesicles might serve as biomarkers to detect early metastasis and/or minimal residual disease (MRD) monitoring.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF V600E mutation.

Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse.

G protein-coupled receptor kinases (GRKs) in tumorigenesis and cancer progression: GPCR regulators and signaling hubs.

Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus putting forward these kinases as potentially relevant modulators of the carcinoma-fibroblast-endothelial-immune cell network fostering tumor development and dissemination. A better understanding of the alterations in different GRK isoforms taking place during cancer development and metastasis in specific tumors and cell types and of its impact in signaling pathways would help to design novel therapeutic strategies.

The influence of tumour-derived extracellular vesicles on local and distal metastatic dissemination.

Extracellular vesicles (EVs) are key mediators of intercellular communication that have been ignored for decades. Tumour cells benefit from the secretion of vesicles as they can influence the behaviour of neighbouring tumour cells within the tumour microenvironment. Several studies have shown that extracellular vesicles play an active role in pre-metastatic niche formation and importantly, they are involved in the metastatic organotropism of different tumour types. Tumour-derived EVs carry and transfer molecules to recipient cells, modifying their behaviour through a process defined as "EV-driven education". EVs favour metastasis to sentinel lymph nodes and distal organs by reinforcing angiogenesis, inflammation and lymphangiogenesis. Hence, in this review we will summarize the main mechanisms by which tumour-derived EVs regulate lymph node and distal organ metastasis. Moreover, since some cancers metastasize through the lymphatic system, we will discuss recent discoveries about the presence and function of tumour EVs in the lymph. Finally, we will address the potential value of tumour EVs as prognostic biomarkers in liquid biopsies, specially blood and lymphatic fluid, and the use of these tools as early detectors of metastases.

G-Protein-Coupled Receptor Kinase 2 as a Potential Modulator of the Hallmarks of Cancer.

Malignant features-such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis-can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions-such as cell proliferation, survival, or motility-and is involved in metabolic homeostasis, inflammation, or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to modulate carcinogenesis in a cell-type specific way.

G Protein-coupled Receptor Kinase 2 (GRK2) Promotes Breast Tumorigenesis Through a HDAC6-Pin1 Axis.

In addition to oncogenic drivers, signaling nodes can critically modulate cancer-related cellular networks to strength tumor hallmarks. We identify G-protein-coupled receptor kinase 2 (GRK2) as a relevant player in breast cancer. GRK2 is up-regulated in breast cancer cell lines, in spontaneous tumors in mice, and in a proportion of invasive ductal carcinoma patients. Increased GRK2 functionality promotes the phosphorylation and activation of the Histone Deacetylase 6 (HDAC6) leading to de-acetylation of the Prolyl Isomerase Pin1, a central modulator of tumor progression, thereby enhancing its stability and functional interaction with key mitotic regulators. Interestingly, a correlation between GRK2 expression and Pin1 levels and de-acetylation status is detected in breast cancer patients. Activation of the HDAC6-Pin1 axis underlies the positive effects of GRK2 on promoting growth factor signaling, cellular proliferation and anchorage-independent growth in both luminal and basal breast cancer cells. Enhanced GRK2 levels promote tumor growth in mice, whereas GRK2 down-modulation sensitizes cells to therapeutic drugs and abrogates tumor formation. Our data suggest that GRK2 acts as an important onco-modulator by strengthening the functionality of key players in breast tumorigenesis such as HDAC6 and Pin1.

Pre-Metastatic Niche Formation Has Taken Its TOLL.

Pre-Metastatic Niches (PMNs) result from communications between primary tumors and the microenvironment of future distant metastasis via tumor-derived factors. In this issue of Cancer Cell, Liu et al. show that TLR3 activation in lung epithelial cells by tumor exosomal RNAs triggers neutrophil recruitment, which contributes to PMN formation and metastasis.

Role of G protein-coupled receptor kinases in cell migration.

G protein-coupled receptor kinases (GRKs) are emerging as important integrative nodes in cell migration processes. Recent evidence links GRKs (particularly the GRK2 isoform) to the complex modulation of diverse aspects of cell motility. In addition to its well-established role in the desensitization of G protein-coupled receptors involved in chemotaxis, GRK2 can play an effector role in the organization of actin and microtubule networks and in adhesion dynamics, by means of novel substrates and transient interacting partners, such as the GIT1 scaffold or the cytoplasmic α-tubulin deacetylase histone deacetylase 6 (HDAC6). The overall effect of altering GRK levels or activity on chemotaxis would depend on how such different roles are integrated in a given cell type and physiological context, and may have relevant implications in inflammatory diseases or cancer progression.

Role of G protein-coupled receptor kinase 2 in tumoral angiogenesis.

Downregulation of G protein-coupled receptor kinase 2 (GRK2) in endothelial cells has recently been identified as a relevant event in the tumoral angiogenic switch. Based on the effects of altering GRK2 dosage in cell and animal models, this kinase appears to act as a hub in key signaling pathways involved in vascular stabilization and remodeling. Accordingly, decreased GRK2 expression in endothelial cells accelerates tumor growth in mice by impairing the pericytes ensheathing the vessels, thereby promoting hypoxia and macrophage infiltration. These results raise new questions regarding the mechanisms by which transformed cells trigger the decrease in GRK2 observed in human breast cancer vessels and how GRK2 modulates the interactions between different cell types that occur in the tumor microenvironment.

Developmental and tumoral vascularization is regulated by G protein-coupled receptor kinase 2.

Tumor vessel dysfunction is a pivotal event in cancer progression. Using an in vivo neovascularization model, we identified G protein-coupled receptor kinase 2 (GRK2) as a key angiogenesis regulator. An impaired angiogenic response involving immature vessels was observed in mice hemizygous for Grk2 or in animals with endothelium-specific Grk2 silencing. ECs isolated from these animals displayed intrinsic alterations in migration, TGF-ß signaling, and formation of tubular networks. Remarkably, an altered pattern of vessel growth and maturation was detected in postnatal retinas from endothelium-specific Grk2 knockout animals. Mouse embryos with systemic or endothelium-selective Grk2 ablation had marked vascular malformations involving impaired recruitment of mural cells. Moreover, decreased endothelial Grk2 dosage accelerated tumor growth in mice, along with reduced pericyte vessel coverage and enhanced macrophage infiltration, and this transformed environment promoted decreased GRK2 in ECs and human breast cancer vessels. Our study suggests that GRK2 downregulation is a relevant event in the tumoral angiogenic switch.

Roles of GRK2 in cell signaling beyond GPCR desensitization: GRK2-HDAC6 interaction modulates cell spreading and motility.

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous, essential protein kinase that is emerging as an integrative node in many signaling networks. Moreover, changes in GRK2 abundance and activity have been identified in several inflammatory, cardiovascular disease, and tumor contexts, suggesting that those alterations may contribute to the initiation or development of pathologies. GRKs were initially identified as key players in the desensitization and internalization of multiple G protein-coupled receptors (GPCRs), but GRK2 also phosphorylates several non-GPCR substrates and dynamically associates with a variety of proteins related to signal transduction. Ongoing research in our laboratory is aimed at understanding how specific GRK2 interactomes are orchestrated in a stimulus-, context-, or cell type-specific manner. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6) that modulates cell spreading and motility. HDAC6 is a major cytoplasmic a-tubulin deacetylase that is involved in cell motility and adhesion. GRK2 dynamically and directly associates with and phosphorylates HDAC6 to stimulate its a-tubulin deacetylase activity at specific cellular localizations, such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. GRK2-HDAC6-mediated regulation of tubulin acetylation also modulates cellular spreading. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to epithelial cell migration.

Multiple scaffolding functions of {beta}-arrestins in the degradation of G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that ß-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse ß-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, ß-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, ß-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined ß-arrestin-recruited ligase in the absence of GPCR activation. The ability of ß-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts.