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The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.

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Activation of c-Met signaling is associated with an aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC); however, its contribution to organ preference in metastasis remains unclear. In this study, using a Lab on a Chip device, we defined the role of aberrant c-Met activation in regulating the extravasation and homing capacity of HCC cells. Our studies showed that (i) c-Met overexpression and activation direct HCC cells preferentially towards the hepatocytes-enriched microenvironment, and (ii) blockage of c-Met phosphorylation by a small molecule inhibitor attenuated extravasation and homing capacity of HCC cells. These results, thus, demonstrate the role of c-Met signaling in regulating the colonization of HCC cells preferentially in the liver.

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The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB2) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular).

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Introduction: The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Methods: Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). Results: These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). Discussion: EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.

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Hepatocellular carcinoma (HCC) is strongly associated with metabolic dysregulations/deregulations and hyperglycemia is a common metabolic disturbance in metabolic diseases. Hyperglycemia is defined to promote epithelial to mesenchymal transition (EMT) of cancer cells in various cancers but its molecular contribution to HCC progression and aggressiveness is relatively unclear. In this study, we analyzed the molecular mechanisms behind the hyperglycemia-induced EMT in HCC cell lines. Here, we report that high glucose promotes EMT through activating c-Met receptor tyrosine kinase via promoting its ligand-independent homodimerization. c-Met activation is critical for high glucose induced acquisition of mesenchymal phenotype, survival under high glucose stress and reprogramming of cellular metabolism by modulating glucose metabolism gene expression to promote aggressiveness in HCC cells. The crucial role of c-Met in high glucose induced EMT and aggressiveness may be the potential link between metabolic syndrome-related hepatocarcinogenesis and/or HCC progression. Considering c-Met inhibition in hyperglycemic patients would be an important complementary strategy for therapy that favors sensitization of HCC cells to therapeutics.

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BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) are both reversible processes, and regulation of phenotypical transition is very important for progression of several cancers including hepatocellular carcinoma (HCC). Recently, it is defined that cancer cells can attain a hybrid epithelial/mesenchymal (hybrid E/M) phenotype. Cells with hybrid E/M phenotype comprise mixed epithelial and mesenchymal properties, they can be more resistant to therapeutics and also more capable of initiating metastatic lesions. However, the mechanisms regulating hybrid E/M in HCC are not well described yet. In this study, we investigated the role of the potential crosstalk between lncRNA HOTAIR and c-Met receptor tyrosine kinase, which are two essential regulators of EMT and MET, in acquiring of hybrid E/M phenotype in HCC. METHODS: Expression of c-Met and lncRNA HOTAIR were defined in HCC cell lines and patient tissues through HCC progression. lncRNA HOTAIR was overexpressed in SNU-449 cells and its effects on c-Met signaling were analyzed. c-Met was overexpressed in SNU-398 cells and its effect on HOTAIR expression was analyzed. Biological significance of HOTAIR/c-Met interplay was defined in means of adhesion, proliferation, motility behavior, invasion, spheroid formation and metastatic ability. Effect of ectopic lncRNA HOTAIR expression on phenotype was defined with investigation of molecular epithelial and mesenchymal traits. RESULTS: In vitro and in vivo experiments verified the pivotal role of lncRNA HOTAIR in acquisition of hybrid E/M phenotype through modulating expression and activation of c-Met and its membrane co-localizing partner Caveolin-1, and membrane organization to cope with the rate limiting steps of metastasis such as survival in adhesion independent microenvironment, escaping from anoikis and resisting to fluidic shear stress (FSS) in HCC. CONCLUSIONS: Our work provides the first evidence suggesting a role for lncRNA HOTAIR in the modulation of c-Met to promote hybrid E/M phenotype. The balance between lncRNA HOTAIR and c-Met might be critical for cell fate decision and metastatic potential of HCC cells. Video Abstract.

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