Specific targeting of the KRAS mutational landscape in myeloma as a tool to unveil the elicited antitumor activity.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.

FGF-trapping hampers cancer stem-like cells in uveal melanoma.

BACKGROUND: Cancer stem-like cells (CSCs) are a subpopulation of tumor cells responsible for tumor initiation, metastasis, chemoresistance, and relapse. Recently, CSCs have been identified in Uveal Melanoma (UM), which represents the most common primary tumor of the eye. UM is highly resistant to systemic chemotherapy and effective therapies aimed at improving overall survival of patients are eagerly required. METHODS: Herein, taking advantage from a pan Fibroblast Growth Factor (FGF)-trap molecule, we singled out and analyzed a UM-CSC subset with marked stem-like properties. A hierarchical clustering of gene expression data publicly available on The Cancer Genome Atlas (TCGA) was performed to identify patients' clusters. RESULTS: By disrupting the FGF/FGF receptor (FGFR)-mediated signaling, we unmasked an FGF-sensitive UM population characterized by increased expression of numerous stemness-related transcription factors, enhanced aldehyde dehydrogenase (ALDH) activity, and tumor-sphere formation capacity. Moreover, FGF inhibition deeply affected UM-CSC survival in vivo in a chorioallantoic membrane (CAM) tumor graft assay, resulting in the reduction of tumor growth. At clinical level, hierarchical clustering of TCGA gene expression data revealed a strong correlation between FGFs/FGFRs and stemness-related genes, allowing the identification of three distinct clusters characterized by different clinical outcomes. CONCLUSIONS: Our findings support the evidence that the FGF/FGFR axis represents a master regulator of cancer stemness in primary UM tumors and point to anti-FGF treatments as a novel therapeutic strategy to hit the CSC component in UM.

Deficiency of AP1 Complex Ap1g1 in Zebrafish Model Led to Perturbation of Neurodevelopment, Female and Male Fertility; New Insight to Understand Adaptinopathies.

In vertebrates, two homologous heterotetrameric AP1 complexes regulate the intracellular protein sorting via vesicles. AP-1 complexes are ubiquitously expressed and are composed of four different subunits: γ, ß1, µ1 and σ1. Two different complexes are present in eukaryotic cells, AP1G1 (contains γ1 subunit) and AP1G2 (contains γ2 subunit); both are indispensable for development. One additional tissue-specific isoform exists for µ1A, the polarized epithelial cells specific to µ1B; two additional tissue-specific isoforms exist for σ1A: σ1B and σ1C. Both AP1 complexes fulfil specific functions at the trans-Golgi network and endosomes. The use of different animal models demonstrated their crucial role in the development of multicellular organisms and the specification of neuronal and epithelial cells. Ap1g1 (γ1) knockout mice cease development at the blastocyst stage, while Ap1m1 (µ1A) knockouts cease during mid-organogenesis. A growing number of human diseases have been associated with mutations in genes encoding for the subunits of adaptor protein complexes. Recently, a new class of neurocutaneous and neurometabolic disorders affecting intracellular vesicular traffic have been referred to as adaptinopathies. To better understand the functional role of AP1G1 in adaptinopathies, we generated a zebrafish ap1g1 knockout using CRISPR/Cas9 genome editing. Zebrafish ap1g1 knockout embryos cease their development at the blastula stage. Interestingly, heterozygous females and males have reduced fertility and showed morphological alterations in the brain, gonads and intestinal epithelium. An analysis of mRNA profiles of different marker proteins and altered tissue morphologies revealed dysregulated cadherin-mediated cell adhesion. These data demonstrate that the zebrafish model organism enables us to study the molecular details of adaptinopathies and thus also develop treatment strategies.

Binding to PI(4,5)P2 is indispensable for secretion of B-cell clonogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1-seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala-Ala) or 125 to 126 (Gly-Asn), respectively, are secreted from HIV-1-infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing.

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas.

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.

Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

VEGFR2 activation mediates the pro-angiogenic activity of BMP4.

The Bone Morphogenetic Protein 4 (BMP4) regulates multiple biological processes, including vascular development and angiogenesis. Here, we investigated the role of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in mediating the angiogenic activity of BMP4. BMP4 induces a rapid relocation and phosphorylation of VEGFR2 on the endothelial cell membrane. These effects occur in the absence of a direct interaction of BMP4 and/or BMP receptors with VEGFR2. At variance, BMP4, by interacting with the BMPRI-II hetero-complex, induces c-Src phosphorylation which, in turn, activates VEGFR2, leading to an angiogenic response. Accordingly, the BMPR inhibitor dorsomorphin prevents c-Src activation and specific inhibition of c-Src significantly reduces downstream VEGFR2 phosphorylation and the angiogenic activity exerted by BMP4 in a chick embryo chorioallantoic membrane assay. Together, our data indicate that the pro-angiogenic activity exerted by BMP4 in endothelial cells is mediated by a BMPR-mediated intracellular transactivation of VEGFR2 via c-Src.

ß-Galactosylceramidase Deficiency Causes Bone Marrow Vascular Defects in an Animal Model of Krabbe Disease.

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase ß-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31-/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.

3D endothelial cell spheroid/human vitreous humor assay for the characterization of anti-angiogenic inhibitors for the treatment of proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) represents a main cause of acquired blindness. Despite the recognition of the key role exerted by vascular endothelial growth factor (VEGF) in the pathogenesis of PDR, limitations to anti-VEGF therapies do exist. Thus, rapid and cost-effective angiogenesis assays are crucial for the screening of anti-angiogenic drug candidates for PDR therapy. In this context, evaluation of the angiogenic potential of PDR vitreous fluid may represent a valuable tool for preclinical assessment of angiostatic molecules. Here, vitreous fluid obtained from PDR patients after pars plana vitrectomy was used as a pro-angiogenic stimulus in a 3D endothelial cell spheroid/human vitreous assay. The results show that PDR vitreous is able to stimulate the sprouting of fibrin-embedded HUVEC spheroids in a time- and dose-dependent manner. A remarkable variability was observed among 40 individual vitreous fluid samples in terms of sprouting-inducing activity that was related, at least in part, to defined clinical features of the PDR patient. This activity was hampered by various extracellular and intracellular signaling pathway inhibitors, including the VEGF antagonist ranibizumab. When tested on 20 individual vitreous fluid samples, the inhibitory activity of ranibizumab ranged between 0 and 100% of the activity measured in the absence of the drug, reflecting a variable contribution of angiogenic mediators distinct from VEGF. In conclusion, the 3D endothelial cell spheroid/human vitreous assay represents a rapid and cost-effective experimental procedure suitable for the evaluation of the anti-angiogenic activity of novel extracellular and intracellular drug candidates, with possible implications for the therapy of PDR.

ß3 Integrin Promotes Long-Lasting Activation and Polarization of Vascular Endothelial Growth Factor Receptor 2 by Immobilized Ligand.

OBJECTIVE: During neovessel formation, angiogenic growth factors associate with the extracellular matrix. These immobilized factors represent a persistent stimulus for the otherwise quiescent endothelial cells (ECs), driving directional EC migration and proliferation and leading to new blood vessel growth. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main mediator of angiogenesis. Although VEGFR2 signaling has been deeply characterized, little is known about its subcellular localization during neovessel formation. Aim of this study was the characterization and molecular determinants of activated VEGFR2 localization in ECs during neovessel formation in response to matrix-immobilized ligand. APPROACH AND RESULTS: Here we demonstrate that ECs stimulated by extracellular matrix-associated gremlin, a noncanonical VEGFR2 ligand, are polarized and relocate the receptor in close contact with the angiogenic factor-enriched matrix both in vitro and in vivo. GM1 (monosialotetrahexosylganglioside)-positive planar lipid rafts, ß3 integrin receptors, and the intracellular signaling transducers focal adhesion kinase and RhoA (Ras homolog gene family, member A) cooperate to promote VEGFR2 long-term polarization and activation. CONCLUSIONS: A ligand anchored to the extracellular matrix induces VEGFR2 polarization in ECs. Long-lasting VEGFR2 relocation is closely dependent on lipid raft integrity and activation of ß3 integrin pathway. The study of the endothelial responses to immobilized growth factors may offer insights into the angiogenic process in physiological and pathological conditions, including cancer, and for a better engineering of synthetic tissue scaffolds to blend with the host vasculature.

Cyclic adenosine monophosphate-response element-binding protein mediates the proangiogenic or proinflammatory activity of gremlin.

OBJECTIVE: Angiogenesis and inflammation are closely related processes. Gremlin is a novel noncanonical vascular endothelial growth factor receptor-2 (VEGFR2) ligand that induces a proangiogenic response in endothelial cells (ECs). Here, we investigated the role of the cyclic adenosine monophosphate-response element (CRE)-binding protein (CREB) in mediating the proinflammatory and proangiogenic responses of ECs to gremlin. APPROACH AND RESULTS: Gremlin induces a proinflammatory response in ECs, leading to reactive oxygen species and cyclic adenosine monophosphate production and the upregulation of proinflammatory molecules involved in leukocyte extravasation, including chemokine (C-C motif) ligand-2 (Ccl2) and Ccl7, chemokine (C-X-C motif) ligand-1 (Cxcl1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Accordingly, gremlin induces the VEGFR2-dependent phosphorylation, nuclear translocation, and transactivating activity of CREB in ECs. CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to monocyte/macrophage adhesion to ECs and their extravasation. All these effects are inhibited by EC transfection with a dominant-negative CREB mutant or with a CREB-binding protein-CREB interaction inhibitor that competes for CREB/CRE binding. Also, both recombinant gremlin and gremlin-expressing tumor cells induce proinflammatory/proangiogenic responses in vivo that are suppressed by the anti-inflammatory drug hydrocortisone. Similar effects were induced by the canonical VEGFR2 ligand VEGF-A165. CONCLUSIONS: Together, the results underline the tight cross-talk between angiogenesis and inflammation and demonstrate a crucial role of CREB activation in the modulation of the VEGFR2-mediated proinflammatory/proangiogenic response of ECs to gremlin.

Correction: Alternative method to visualize receptor dynamics in cell membranes.

[This corrects the article DOI: 10.1371/journal.pone.0304172.].

Mutation in the Kinase Domain Alters the VEGFR2 Membrane Dynamics.

BACKGROUND: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. METHODS: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. RESULTS: Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. CONCLUSIONS: This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT.

The expression level of VEGFR2 regulates mechanotransduction, tumor growth and metastasis of high grade serous ovarian cancer cells.

Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.

Leucine-Rich Repeat Kinase-2 Controls the Differentiation and Maturation of Oligodendrocytes in Mice and Zebrafish.

Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson's disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of other brain cells and oligodendrocytes (OLs) in PD. Altogether, these observations suggest crucial function(s) of LRRK2 in OPCs/Ols, which would be interesting to explore. In this study, we investigated the role of LRRK2 in OLs. We showed that LRRK2 knock-out (KO) OPC cultures displayed defects in the transition of OPCs into OLs, suggesting a role of LRRK2 in OL differentiation. Consistently, we found an alteration of myelin basic protein (MBP) striosomes in LRRK2 KO mouse brains and reduced levels of oligodendrocyte transcription factor 2 (Olig2) and Mbp in olig2:EGFP and mbp:RFP transgenic zebrafish embryos injected with lrrk2 morpholino (MO). Moreover, lrrk2 knock-down zebrafish exhibited a lower amount of nerve growth factor (Ngf) compared to control embryos, which represents a potent regulator of oligodendrogenesis and myelination. Overall, our findings indicate that LRRK2 controls OL differentiation, affecting the number of mature OLs.

Involvement of αvß3 integrin in gremlin-induced angiogenesis.

α(v)ß(3) integrin modulates pro-angiogenic endothelial cell (EC) responses following vascular endothelial growth factor receptor-2 (VEGFR2) engagement. The bone morphogenic protein antagonist gremlin is a novel non-canonical VEGFR2 ligand that promotes the acquisition of a pro-angiogenic phenotype in ECs. Here we investigated the role of α(v)ß(3) and extracellular matrix components on EC activation induced by gremlin. Gremlin triggers VEGFR2 phosphorylation and cell motility in ECs adherent to the α(v)ß(3) ligand fibrinogen but not in ECs adherent to type-I collagen or fibronectin. Also, gremlin and VEGF-A stimulate the formation of VEGFR2/α(v)ß(3) integrin complexes as shown by co-immunoprecipitation experiments and fluorescence resonance energy transfer analysis of ß(3)-ECFP/VEGFR2-EYFP co-transfected ECs. Accordingly, anti-ß(3) antibodies block the angiogenic activity exerted by gremlin or VEGF-A in vitro, ex vivo and in vivo. The results demonstrate a non-redundant role for α(v)ß(3) in gremlin-induced angiogenesis and emphasize its contribution to the formation of functional multi-molecular VEGFR2 complexes responsible for the neovascularization events triggered by canonical and non-canonical pro-angiogenic VEGFR2 ligands.

Substrate-immobilized HIV-1 Tat drives VEGFR2/α(v)ß(3)-integrin complex formation and polarization in endothelial cells.

OBJECTIVE: The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation. METHODS AND RESULTS: Immobilized Tat induces actin cytoskeleton organization, formation of α(v)ß(3) integrin(+)focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/α(v)ß(3) coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of α(v)ß(3) integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK(1/2) activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK(1/2) pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs. CONCLUSIONS: Substrate-immobilized Tat causes VEGFR2/α(v)ß(3) complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK(1/2) phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk.

The impact of adipokines on vascular networks in adipose tissue.

Adipose tissue (AT) is a highly active and plastic endocrine organ. It secretes numerous soluble molecules known as adipokines, which act locally to AT control the remodel and homeostasis or exert pleiotropic functions in different peripheral organs. Aberrant production or loss of certain adipokines contributes to AT dysfunction associated with metabolic disorders, including obesity. The AT plasticity is strictly related to tissue vascularization. Angiogenesis supports the AT expansion, while regression of blood vessels is associated with AT hypoxia, which in turn mediates tissue inflammation, fibrosis and metabolic dysfunction. Several adipokines can regulate endothelial cell functions and are endowed with either pro- or anti-angiogenic properties. Here we address the role of adipokines in the regulation of angiogenesis. A better understanding of the link between adipokines and angiogenesis will open the way for novel therapeutic approaches to treat obesity and metabolic diseases.

Role of gremlin-1 in the pathophysiology of the adipose tissues.

Gremlin-1 is a secreted bone morphogenetic protein (BMP) antagonist playing a pivotal role in the regulation of tissue formation and embryonic development. Since its first identification in 1997, gremlin-1 has been shown to be a multifunctional factor involved in wound healing, inflammation, cancer and tissue fibrosis. Among others, the activity of gremlin-1 is mediated by its interaction with BMPs or with membrane receptors such as the vascular endothelial growth factor receptor 2 (VEGFR2) or heparan sulfate proteoglycans (HSPGs). Growing evidence has highlighted a central role of gremlin-1 in the homeostasis of the adipose tissue (AT). Of note, gremlin-1 is involved in AT dysfunction during type 2 diabetes, obesity and non-alcoholic fatty liver disease (NAFLD) metabolic disorders. In this review we discuss recent findings on gremlin-1 involvement in AT biology, with particular attention to its role in metabolic diseases, to highlight its potential as a prognostic marker and therapeutic target.

The D647N mutation of FGFR1 induces ligand-independent receptor activation.

The activation loop (A-loop) of kinases, a key regulatory region, is recurrently mutated in several kinase proteins in cancer resulting in dysregulated kinase activity and response to kinase inhibitors. FGFR1 receptor tyrosine kinase represents an important oncogene and therapeutic target for solid and hematological tumors. Here we investigate the biochemical and molecular effects of D647N mutation lying in the A-loop of FGFR1. When expressed in normal and tumoral in vitro cell models, FGFR1D647N is phosphorylated also in the absence of ligands, and this is accompanied by the activation of intracellular signaling. The expression of FGFR1D647N significantly increases single and collective migration of cancer cells in vitro and in vivo, when compared to FGFR1WT. FGFR1D647N expression exacerbates the aggressiveness of cancer cells, increasing their invasiveness in vitro and augmenting their pro-angiogenic capacity in vivo. Remarkably, the D647N mutation significantly increases the sensitivity of FGFR1 to the ATP-competitive inhibitor Erdafitinib suggesting the possibility that this mutation could become a specific target for the development of new inhibitors. Although further efforts are warranted for an exhaustive description of the activation mechanisms, for the identification of more specific inhibitors and for confirming the clinical significance of mutated FGFR1D647N, overall our data demonstrate that the D647N substitution of FGFR1 is a novel pro-oncogenic activating mutation of the receptor that, when found in cancer patients, may anticipate good response to erdafitinib treatment.