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Background: Epithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progression in many cancer types, including colorectal cancer. While metastasis is one of the main causes of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer. Methods: Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was validated via a tail vein injection method for the metastasis and orthotopic colon cancer models. Results: This study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelial-to-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active β-catenin and Snail proteins by decreasing GSK3β activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer. Conclusion: Our findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression. [ABSTRACT FROM AUTHOR]

Based on the obtained SARs, further structural optimisation of compound BC2021-104511-15i was conducted in this investigation, and totally ten novel quinoline derivates were designed, synthesised and optimised for biological activity. Among them, compound 10a displayed significant in vitro anticancer activity against COLO 205 cells with an IC50 value of 0.11 µM which was over 90-fold more potent than that of Regorafenib (IC50 > 10.0 µM) and Fruquintinib (IC50>10.0iM). Furthermore, compound 10a exhibited over 90-fold selectivity towards COLO 205 relative to human normal colorectal mucosa epithelial cell FHC cells. Flow cytometry study demonstrated that compound 10a could induce apoptosis in COLO 205 cells, however, it could not induce cell cycle arrest in COLO 205 cells. The results of preliminary kinase profile study showed that compound 10a was a potential HGFR and MST1R dual inhibitor, with IC50 values of 0.11 µM and 0.045 µM, respectively. [ABSTRACT FROM AUTHOR]

1 Targeted proteomic signatures differentiate molecular classes of uveal melanoma: a The vitreous contains native vitreous proteins, systemic protein biomarkers, and tumor biomarkers that can be sampled through proteomic analysis. Our research has demonstrated that retinal proteins leak into the vitreous, and that proteomic analysis of vitreous biopsies can detect molecular changes in the adjacent retina, uncover biomarkers, and identify protein targets for adjuvant therapy [[6]]. Keywords: Proteomics; Vitreous; Liquid biopsy; Uveal melanoma; Cancer; Biomarker; SCFR; HGFR; STAT3 EN Proteomics Vitreous Liquid biopsy Uveal melanoma Cancer Biomarker SCFR HGFR STAT3 1 5 5 03/02/21 20210224 NES 210224 Supplementary Information The online version contains supplementary material available at https://doi.org/10.1186/s12943-021-01336-4. [Extracted from the article]

Summary: Among various solid tumours, gastric cancer (GC) is one of the leading causes of cancer‐related deaths worldwide. Expansion into the peritoneal cavity, which results from dissemination of diffuse cancer cells, is the main cause of mortality in gastric adenocarcinoma patients. Therefore, investigation of putative biomarkers involved in metastasis is prerequisite for GC management. In an effort to discover potential tumour markers associated with peritoneal metastasis of GC, a semi‐synthetic human scFv library (Tomlinson I) was used to isolate novel antibody fragments recognizing MKN‐45, a poorly differentiated diffuse gastric adenocarcinoma cell line. Four rounds of subtractive selection each consisting of extensive pre‐absorption of phage library with NIH‐3T3 murine embryonic fibroblasts and AGS (a well‐differentiated intestinal gastric adenocarcinoma) cell line were carried out prior to positive selection on MKN‐45 target cells. ELISA‐based screening of 192 phage‐displayed scFv clones indicated 21 high‐affinity binders with specific staining of MKN‐45 compared with AGS cells. Diversity analysis of the selected phage‐scFvs resulted in five distinct sequences with multiple frequency. Further analysis by ELISA and flow cytometry verified three clones that specifically recognized MKN‐45 cells. Liquid chromatography‐mass spectrometry analysis of the scFv‐immunoprecipitated proteins has led to identification of c‐Met, HSP90 α and HSP90 β as candidate biomarkers associated with diffuse GC. Immunohistochemistry revealed the capability of purified scFvs to differentiate diffuse and intestinal gastric adenocarcinoma. Taken together, the isolated MKN‐45‐specific scFv fragments and their cognate antigens would be beneficial in screening and management as well as targeting and therapy of the diffuse gastric adenocarcinoma. [ABSTRACT FROM AUTHOR]

Litchi chinensis Sonn. (Litchi) seed, a traditional Chinese medicine, is habitually used in the clinical treatment of prostate cancer (PCa)-induced bone pain. In our previous study, flavonoids have been identified as the active ingredient of litchi seed against PCa. However, its anti-tumor activities in bone and associated molecular mechanisms are still unclear. To investigate the effects and underlying mechanisms of total flavonoids of litchi seed (TFLS) on the growth of PCa in bone. The effect of TFLS on the growth of PCa in bone was observed using a mouse model constructed with tibial injection of luciferase-expressing RM1−luc cells. Conditioned medium (CM) from bone marrow stromal cells OP9 and CM treated with TFLS (T-CM) was used to investigate the effect on the proliferation, colony formation, and apoptosis of PCa cells (LNCaP, PC3, RM1). An antibody microarray was performed to detect cytokine expression in the supernatant fraction of OP9 cell cultures treated with TFLS or left untreated. Western blot assay was employed to determine the expression and activity of HGFR and its key downstream proteins, Akt, mTOR, NF-κB, and Erk, in PCa cells. The potential target was further verified using immunofluorescence and immunohistochemistry assays. Treatment with TFLS (80 mg/kg, 24 days) significantly suppressed the growth of RM1 cells in bone. CM from bone marrow stromal cells OP9 stimulated the proliferation and colony formation of the PCa cells as well as inhibited the apoptosis of PC3 cells, while T-CM reversed the effects mediated by OP9 cells in vitro. In an antibody array assay, TFLS regulated the majority of cytokines in OP9 cell culture supernatant, among which HGF, HGFR, IGF-1R, and PDGF-AA showed the greatest fold changes. Mechanistically, CM upregulated HGFR and promoted phosphorylation of NF-κB while T-CM induced reduction of HGFR and dephosphorylation of NF-κB in PC3 cells. Moreover, T-CM inhibited NF-κB entry into PC3 cell nuclei. Data from in vivo experiments further confirmed the inhibitory effects of TFLS on NF-κB. TFLS suppresses the growth of PCa in bone through regulating bone microenvironment and the underlying mechanism potentially involves attenuation of the HGFR/NF-κB signaling axis. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners. Highlights • SHIP2-SH2 consists of two α-helices and six β-strands adopting a typical SH2 fold. • SHIP2-SH2 surface contains a positive-charged pocket for binding phosphotyrosine. • The pocket and its surrounding regions are for binding Y1356-phosphorylated c-MET. • The SHIP2-SH2 affinity for Y1356-phosphorylated c-MET is at low micromolar level. • R28, S49 and R70 of SHIP2-SH2 are crucial for binding Y1356-phosphorylated c-MET. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies. [ABSTRACT FROM AUTHOR]

Highlights: [•] The metastasis suppressor CD82 exerts its activity together with glycosphingolipids (GSLs). [•] The synergistic inhibitory effects of CD82 and GSLs on cell migration through EGFR and cMet activation. [•] CD82 expression selectively attenuated EGFR and cMet signalling via PI3K/Akt signalling pathway. [Copyright &y& Elsevier]

Abstract: An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner. [Copyright &y& Elsevier]

Objective. The risk of development of colorectal carcinoma is elevated in chronic, long-standing ulcerative colitis (UC). The changes in regenerative and immortalizing pathways caused by the inflammatory process, and that have been proved to be carcinogenic in other human tissues, have not been fully and uniformly described. We assayed the expression alterations of regenerative signal receptors and cell-aging inhibitory systems within colonic crypts by considering the histological activity of the disease. Methods. I-type insulin-like growth factor receptor (IGF1R), hepatocyte growth factor receptor (HGFR), telomerase reverse transcriptase (TERT) and telomerase associated protein (TP-1) expression were evaluated immunohistochemically on biopsy specimens from 11 mild, 11 moderate and 12 severe active inflammation of UC cases and from 10 normal colonic tissue cases. Independent colonic biopsies from 5 healthy and 7 severe UC cases were used for TaqMan real-time RT-PCR validation. Results. In mild inflammation, all observed parameters showed significantly elevated epithelial protein expression (IGF1R: 22.3±9.46%; HGFR: 35.3±22.8%; TERT/TP-1: 2.1±1.87%/2±1.32%) compared to normal (p<0.005). In moderately active inflammation, only IGF1R expression was significantly higher (50.2±8.6%) compared to normal and mild inflammation (p<0.005). In severe inflammation, all parameters showed decreased epithelial expression; IGF1R showed decreased mRNA expression, while HGFR was overexpressed and TERT showed a decreased tendency. Conclusions. The epithelial expression of IGF1R, HGFR and TERT/TP-1 is elevated in mildly active UC. This phenomenon may allow the epithelial cells that collected genetic defects during severe inflammatory episodes pathologically to survive and proliferate. [ABSTRACT FROM AUTHOR]

Abstract: For reliable results from quantitative RT-PCR, the starting quantity of total RNA and other parameters need to be controlled. Most studies do this by normalising their results to a single reference gene. This study quantified the mRNA expression of three putative reference genes (ubiquitin C, cyclophilin E, and porphobilinogen deaminase) and the target gene hepatocyte growth factor receptor (HGFR) in matched colorectal tumour and normal mucosa samples. Each of the putative reference genes was found to be significantly over-expressed in the tumour samples compared to the normal samples. When HGFR expression was normalised to each of these reference genes using the method of relative quantification, the number of tumour samples in which HGFR was found to be over-expressed varied from 30% to 63% depending on the reference gene chosen for normalisation. This shows that normalising to a single reference gene without prior validation is inappropriate. [Copyright &y& Elsevier]

Increased hepatocyte growth factor receptor (HGFR) signaling correlates closely with neoplastic invasion and metastatic potential of many human cancers. Hepatocyte growth factor receptor signaling is initiated by binding the physiological ligand HGF or the internalin B (InlB) protein ofListeria monocytogenes. Subsequent degradation of endocytosed HGFR terminates receptor signaling. Previously reported discrepancies in InlB and HGF-induced HGFR signaling could reflect differences in receptor internalization and degradation in response to these distinct ligands. We report that soluble InlB and HGF are mechanistically equivalent in triggering clathrin-dependent endocytosis and lysosomal degradation of HGFR. After internalization, InlB and HGF colocalize with Rab5, EEA1 and the transferrin receptor in classical early endosomes. Hepatocyte growth factor receptor internalization was prevented by overexpression of dominant negative mutants of dynamin 1 and epidermal growth factor phosphorylation substrate 15, but not caveolin 1, the GTPase Arf6 or the cholesterol-chelating drug Nystatin. Thus, HGFR internalization is principally clathrin-mediated and is not regulated by clathrin- independent pathways. Phosphatidylinositol 3-kinase signaling and HGF-regulated tyrosine kinase substrate were not required for ligand-triggered internalization of HGFR but were essential for subsequent lysosomal degradation. Thus, soluble InlB and HGF induce HGFR endocytosis and degradation by indistinguishable mechanisms, suggesting that InlB may be exploited to regulate pathogenic HGFR signaling. [ABSTRACT FROM AUTHOR]

The zoonotic Old World mammarenavirus Lassa (LASV) causes severe hemorrhagic fever with high mortality and morbidity in humans in endemic regions. The development of effective strategies to combat LASV infections is of high priority, given the lack of a licensed vaccine and restriction on available treatment to off-label use of ribavirin. A better understanding of the fundamental aspects of the virus's life cycle would help to improve the development of novel therapeutic approaches. Host cell entry and restriction factors represent major barriers for emerging viruses and are promising targets for therapeutic intervention. In addition to the LASV main receptor, the extracellular matrix molecule dystroglycan (DG), the phosphatidylserine-binding receptors of the Tyro3/Axl/Mer (TAM), and T cell immunoglobulin and mucin receptor (TIM) families are potential alternative receptors of LASV infection. Therefore, the relative contributions of candidate receptors to LASV entry into a particular human cell type are a complex function of receptor expression and functional DG availability. Here, we describe the role of two receptor tyrosine kinases (RTKs), Axl and hepatocyte growth factor receptor (HGFR), in the presence and absence of glycosylated DG for LASV entry. We found that both RTKs participated in the macropinocytosis-related LASV entry and, regardless of the presence or absence of functional DG, their inhibition resulted in a significant antiviral effect. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT. [ABSTRACT FROM AUTHOR]