Hepatocyte Growth Factor Activator Research Articles

Hepsin as a potential therapeutic target for alleviating acetaminophen-induced hepatotoxicity via gap-junction regulation and oxidative stress modulation

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver damage, highlighting the limitations of current emergency treatments that primarily involve administering the glutathione precursor N-acetylcysteine and supportive therapy. This study highlights the essential protective role of the type II transmembrane serine protease (TTSP), hepsin, in mitigating acetaminophen-induced liver injury, particularly through its regulation of gap junction (GJ) abundance in response to reactive oxygen stress in the liver. We previously reported that reduced levels of activated hepatocyte growth factor and the c-Met receptor tyrosine kinase—both of which are vital for maintaining cellular redox balance—combined with increased expression of GJ proteins in hepsin-deficient mice. Here, we show that hepsin deficiency in mice exacerbates acetaminophen toxicity compared to wild-type mice, leading to more severe liver pathology, elevated oxidative stress, and greater mortality within 6 h after exposure. Administering hepsin had a protective effect in both mouse models, reducing hepatotoxicity by modulating GJ abundance. Additionally, transcriptome analysis and a functional GJ inhibitor have highlighted hepsin's mechanism for managing oxidative stress. Combining hepsin with relatively low doses of N-acetylcysteine had a synergistic effect that was more efficacious than high-dose N-acetylcysteine alone. Our results illustrate the crucial role of hepsin in modulating the abundance of hepatic GJs and reducing oxidative stress, thereby offering early protection against acetaminophen-induced hepatotoxicity and a new, combination approach. Emerging as a promising therapeutic target, hepsin holds potential for combination therapy with N-acetylcysteine, paving the way for novel approaches in managing drug-induced liver injury.Graphical 1. Hepsin−/− mice exhibit exacerbated APAP toxicity, resulting in more severe liver damage, elevated oxidative stress, and higher mortality.2. Hepsin is crucial in protecting against APAP-induced liver injury by regulating gap junctions and reducing oxidative stress.3. Combining hepsin with low doses of N-acetylcysteine provides greater protection against APAP-induced hepatotoxicity than high-dose NAC alone.

Hepatocyte growth factor-modified adipose-derived mesenchymal stem cells inhibit human hypertrophic scar fibroblast activation.

Nucleoside-modified messenger RNA (modRNA) holds the potential for facilitating genetic enhancement of stem cells. In this study, modRNA encoding hepatocyte growth factor (modHGF) was used to chemically modify adipose-derived mesenchymal stem cells (ADSCs) and the effect of modified ADSCs on the activation of hypertrophic scar fibroblasts (HSFs) was evaluated. CCK-8, wound healing, and transwell assays were utilized to evaluate the viability and migratory potential of modHGF-engineered ADSCs and their effect on HSF activation. Reverse transcription-polymerase chain reaction, western blot, and immunofluorescence staining were performed to detect the expression of collagen-I (Col I), collagen-III (Col III), alpha-smooth muscle actin (α-SMA), matrix metallopeptidase 1 (MMP-1), and MMP-3. Transfection of ADSCs with modHGF (HGF-ADSC) resulted in enhanced production of HGF. Meanwhile, modHGF modification enhanced the viability and migration of ADSCs. Notably, culture media from HGF-ADSCs exhibited a more potent inhibitory effect on the proliferation and migration of HSFs. In addition, culture media from HGF-ADSCs inhibited extracellular matrix synthesis of HSFs, as evidenced by reduced expression levels of Col I, Col III, and α-SMA, while increasing expression of MMP-1 and MMP-3. Conversely, neutralization experiments confirmed that these effects could be effectively alleviated by blocking HGF activity. modHGF modification optimizes the inhibitory effect of ADSCs on HSF activation, which provides a promising alternative for preventing and treating hyperplastic scars.

Spatiotemporal regulation of the hepatocyte growth factor receptor MET activity by sorting nexins 1/2 in HCT116 colorectal cancer cells.

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease.

Mechanisms of PAK1-Induced Skeletal Muscle Myokines in Improving Islet β-Cell Function

The critical role of skeletal muscle in regulating metabolic homeostasis is well-acknowledged. The p21-activated kinase (PAK1), a prominent signaling molecule in skeletal muscle, has garnered attention for its potential modulatory role in glucose and mitochondrial metabolism. By applying conditioned media derived from PAK1-enriched L6 Glut4 myc myotubes or myoblasts to INS-1 832/13 clonal β-cells, we confirmed that a muscle-derived circulating factor(s) could enhance β-cell function. Furthermore, our mass spectrometry analysis in the conditioned media (CM) collected from PAK1 enriched L6.GLUT4myc myotubes revealed elevated levels of one protein in particular, hepatocyte growth factor activator (HGFAC), which is categorized as a secreted myokine. HGFAC is secreted into the circulation, where, upon activation, it can act in a paracrine or endocrine fashion to proteolytically cleave and activate HGF. It has been reported that HGF has a vital role in islet β-cell mass and function and HNFα, a transcription factor that is expressed in pancreatic β-cells regulates β-cell mass or function by modulating HGFAC expression. Thus, we hypothesize that PAK1-enriched skeletal muscle releases HGFAC into the serum-free conditioned medium and the medium carrying HGFAC when applied to islet β-cells enhances insulin secretion. Elevated levels of HGFAC protein were detected in the cells and their associated CM from PAK1-enriched skeletal myotubes, as compared with that of GFP-transduced control myotubes, using sandwich ELISA to quantify HGFAC. Notably, these cells were incubated in a serum-free medium, a means to eliminate serum factors that might impact HGFAC levels or detection capacity. Given this, we tested the effect of this serum-free CM collected from PAK1-enriched L6.GLUT4myc myotubes application to species-matched (rat) clonal β-cells functional capacity. Indeed, CM from PAK1-enriched myotubes significantly enhanced glucose-stimulated insulin secretion (GSIS), as compared that of the control CM. The presence of elevated HGFAC levels in PAK1-enriched cells, and the beneficial impact of this HGFAC-enriched CM upon beta cell function, reveals a new mechanism by which PAK1-enrichment in skeletal muscle imparts improved overall whole-body glucose homeostasis. further hints at a possible intricate relationship between muscle-pancreas crosstalk in the regulation of islet β-cell insulin secretion. Future investigations into possible autocrine functions of PAK1-enriched skeletal muscle are underway. This research was funded by the National Institutes of Health to D.C.T. (DK067912, DK112917, and DK102233), and an American Heart Association Postdoctoral fellowship to R.B. (#905770). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lidocaine attenuates TMZ resistance and inhibits cell migration by modulating the MET pathway in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of malignant brain tumor. Currently, the predominant clinical treatment is the combination of surgical resection with concurrent radiotherapy and chemotherapy, using temozolomide (TMZ) as the primary chemotherapy drug. Lidocaine, a widely used amide‑based local anesthetic, has been found to have a significant anticancer effect. It has been reported that aberrant hepatocyte growth factor (HGF)/mesenchymal‑epithelial transition factor (MET) signaling plays a role in the progression of brain tumors. However, it remains unclear whether lidocaine can regulate the MET pathway in GBM. In the present study, the clinical importance of the HGF/MET pathway was analyzed using bioinformatics. By establishing TMZ‑resistant cell lines, the impact of combined treatment with lidocaine and TMZ was investigated. Additionally, the effects of lidocaine on cellular function were also examined and confirmed using knockdown techniques. The current findings revealed that the HGF/MET pathway played a key role in brain cancer, and its activation in GBM was associated with increased malignancy and poorer patient outcomes. Elevated HGF levels and activation of its receptor were found to be associated with TMZ resistance in GBM cells. Lidocaine effectively suppressed the HGF/MET pathway, thereby restoring TMZ sensitivity in TMZ‑resistant cells. Furthermore, lidocaine also inhibited cell migration. Overall, these results indicated that inhibiting the HGF/MET pathway using lidocaine can enhance the sensitivity of GBM cells to TMZ and reduce cell migration, providing a potential basis for developing novel therapeutic strategies for GBM.

Current Approach to Risk Factors and Biomarkers of Intestinal Fibrosis in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), especially Crohn's disease (CD), characterized by a chronic inflammatory process and progressive intestinal tissue damage, leads to the unrestrained proliferation of mesenchymal cells and the development of bowel strictures. Complications induced by fibrosis are related to high rates of morbidity and mortality and lead to a substantial number of hospitalizations and surgical procedures, generating high healthcare costs. The development of easily obtained, reliable fibrogenesis biomarkers is essential to provide an important complementary tool to existing diagnostic and prognostic methods in IBD management, guiding decisions on the intensification of pharmacotherapy, proceeding to surgical methods of treatment and monitoring the efficacy of anti-fibrotic therapy in the future. The most promising potential markers of fibrosis include cartilage oligomeric matrix protein (COMP), hepatocyte growth factor activator (HGFA), and fibronectin isoform- extra domain A (ED-A), as well as antibodies against granulocyte macrophage colony-stimulating factor (GM-CSF Ab), cathelicidin (LL-37), or circulatory miRNAs: miR-19a-3p and miR-19b-3p. This review summarizes the role of genetic predisposition, and risk factors and serological markers potentially contributing to the pathophysiology of fibrotic strictures in the course of IBD.

Androgen deprivation induces double-null prostate cancer via aberrant nuclear export and ribosomal biogenesis through HGF and Wnt activation

Androgen deprivation therapy (ADT) targeting androgen/androgen receptor (AR)- signaling pathways is the main therapy for advanced prostate cancer (PCa). However, ADT eventually fails in most patients who consequently develop castration-resistant prostate cancer (CRPC). While more potent AR antagonists and blockers for androgen synthesis were developed to improve clinical outcomes, they also show to induce more diverse CRPC phenotypes. Specifically, the AR- and neuroendocrine-null PCa, DNPC, occurs in abiraterone and enzalutamide-treated patients. Here, we uncover that current ADT induces aberrant HGF/MET signaling activation that further elevates Wnt/β-catenin signaling in human DNPC samples. Co-activation of HGF/MET and Wnt/β-catenin axes in mouse prostates induces DNPC-like lesions. Single-cell RNA sequencing analyses identify increased expression and activity of XPO1 and ribosomal proteins in mouse DNPC-like cells. Elevated expression of XPO1 and ribosomal proteins is also identified in clinical DNPC specimens. Inhibition of XPO1 and ribosomal pathways represses DNPC growth in both in vivo and ex vivo conditions, evidencing future therapeutic targets.

Proteomic analysis of pulmonary arteries and lung tissues from dogs affected with pulmonary hypertension secondary to degenerative mitral valve disease.

In dogs with degenerative mitral valve disease (DMVD), pulmonary hypertension (PH) is a common complication characterized by abnormally elevated pulmonary arterial pressure (PAP). Pulmonary arterial remodeling is the histopathological changes of pulmonary artery that has been recognized in PH. The underlying mechanisms that cause this arterial remodeling are poorly understood. This study aimed to perform shotgun proteomics to investigate changes in protein expression in pulmonary arteries and lung tissues of DMVD dogs with PH compared to normal control dogs and DMVD dogs without PH. Tissue samples were collected from the carcasses of 22 small-sized breed dogs and divided into three groups: control (n = 7), DMVD (n = 7) and DMVD+PH groups (n = 8). Differentially expressed proteins were identified, and top three upregulated and downregulated proteins in the pulmonary arteries of DMVD dogs with PH including SIK family kinase 3 (SIK3), Collagen type I alpha 1 chain (COL1A1), Transforming growth factor alpha (TGF-α), Apoptosis associated tyrosine kinase (AATYK), Hepatocyte growth factor activator (HGFA) and Tyrosine-protein phosphatase non-receptor type 13 (PTPN13) were chosen. Results showed that some of the identified proteins may play a role in the pathogenesis of pulmonary arterial remodeling. This study concluded shotgun proteomics has potential as a tool for exploring candidate proteins associated with the pathogenesis of PH secondary to DMVD in dogs.

MiR-4270 suppresses hepatocellular carcinoma progression by inhibiting DNMT3A-mediated methylation of HGFAC promoter

Background miR-4270 is a regulatory factor has been linked with the progression of various cancers, such as nasopharyngeal carcinoma, hepatocellular carcinoma (HCC), and gastric cancer. However, the underlying mechanisms through which miR-4270 modulates HCC development are not fully understood. Methods miR-4270 expression levels were analyzed in various HCC cell lines and tissue samples. An online bioinformatics tool was then utilized to predict the miR-4270 target gene. The binding relationship between miR-4270 and its target gene DNMT3A was verified using dual-luciferase reporter and Ago2–RIP assays. Then, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays were conducted to investigate the association between DNMT3A and the hepatocyte growth factor activator (HGFAC) promoter region. To assess the methylation level of the HGFAC promoter, methylation-specific PCR (MSP) was employed. Furthermore, rescue analyses were carried out to evaluate the functional relevance of miR-4270 and HGFAC in the modulation of the malignant properties of HCC cells. Finally, HepG2 cells overexpressing miR-4270 were subcutaneously injected into nude mice to estimate the impact of miR-4270 on the xenograft tumor growth of HCC. Results A substantial miR-4270 downregulation was revealed in HCC patient samples and cell lines. miR-4270 upregulation suppressed both cell proliferation and invasion while promoting apoptosis. At the molecular level, miR-4270 was found to bind to the 3′untranslated region (3′UTR) of DNMT3A, thereby inhibiting DNMT3A-mediated methylation of the HGFAC promoter. Functional assays indicated that inhibition of miR-4270 stimulated HCC cell growth, an effect counteracted by overexpression of HGFAC. In vivo assays further verified that miR-4270 effectively suppressed the progression of HCC xenograft tumors. Conclusions miR-4270 was found to mitigate the malignant characteristics of HCC by inhibiting DNMT3A-mediated methylation of the HGFAC promoter, suggesting a potential therapeutic avenue for the management of HCC.

Inhibition of MET Tyrosine Kinase Provides a Strategy for Targeting the Inflammatory Tumor Microenvironment

Background: In acute myeloid leukemia (AML), age-associated inflammation may contribute to leukemia progression and remodel the tumor microenvironment (TME). Macrophages, crucial components of the TME, play a critical role in modulating T cell immune response against leukemia cells. However, macrophage polarization to an M2 phenotype, known as tumor-associated macrophages (TAMs), have an immunosuppressive action and interplay with leukemia cells by promoting their survival, immune evasion, and chemotherapy resistance. MET, a hepatocyte growth factor (HGF) receptor, has been demonstrated to contribute to shifting macrophages to TAMs. Hence, we hypothesize that inhibiting Met and, consequently, HGF action and signaling could be a potential strategy to target the inflammatory TME and improve AML clinical outcomes. Aims: Here,we analyze the MET gene expression in leukemia patients mononuclear cells and the impact on clinical outcomes and we also assess the contribution of a specific MET inhibitor, B2000 (Brigham University), to leukemia progression by investigating the in vitro, ex vivo and in vivo effects with focus on the inflammatory TME. Method s: MET mRNA expression data were obtained from TCGA AML study, performed in cells isolated from 290 AML patients (median age 61 years [range:18-87]). Human peripheral blood and bone marrow (BM) mononuclear cells (BMNC/PBMC) (n=10) were obtained from healthy donors (HD). HD-PBMC were used for the isolation of monocytes. Human leukemia cell lines (OCI-AML3, MOLM-13) and BMNC from HD and AML patients (n=10) were treated with B2000 and apoptosis rates were evaluated. To obtain monocyte-derived macrophages (MDMs), monocytes were stimulated with GM-CSF/LPS/IFN-y or M-CSF/IL-4, for M1 or M2 polarization, respectively, and treated with B2000 in monocultures and cocultures. For in viv o analyses, BALB/c mice received a single intraperitoneal injection of lipopolysaccharide (LPS) to provoke an inflammatory shock and were treated with B2000. The analysis of the immune response and the characterization of inflammatory cells was performed by flow cytometry. Results: High MET expression in FAB M1, M3 and M7 AML patients ( P<.0001) was observed. B2000 was effective in killing leukemia cell lines ( P<.0001) and primary BMNCs from AML patients ( P<.005) without affecting HD-BMNC. HD-PBMC-derived monocytes treated with B2000 had increased classical monocyte population (CD14highCD16-) ( P<.001). B2000 also caused a significant apoptosis increase in leukemia cells cocultured with monocytes, without modulating the monocytes population ( P<.0001). To confirm whether monocytes could acquire a tumoricidal phenotype, monocytes were pre-treated with B2000 and, then, cocultured with leukemia cells. B2000-programmed monocytes enhanced leukemia cell apoptosis ( P<.001). The analysis of MDMs revealed a shorter population of M2 MDMs (HLA-DR-CD206+) and increased population of M1 MDMs (HLA-DR+CD80+) in B2000-treated MDMs when compared with the vehicle treatment ( P<.0001). To validate these findings, we evaluated B2000 action in the inflammatory TME by co-culturing leukemia cells with MDMs and results showed that apoptosis was increased in leukemia cells in the presence of B2000 compound ( P<.01). The in vivo analysis in an inflammatory mice model showed increased frequency of M1 monocytes (CD11b+F4/80+CD80+) and decreased M2 (CD11b+F4/80+CD206+) in B2000-treated ( P<.05). Accordingly, B2000 restored lymphocyte B ( P<.05) and T ( P<.05) levels in the BM, spleen and peripheral blood, decreasing the inflammatory burden. Conclusion: We demonstrate the pre-clinical efficacy of a MET inhibitor, B2000, in enhancing anti-tumor immunity in vitro, ex vivo and in vivo by promoting M1 macrophage polarization and tumoricidal phenotype, contributing to inhibition of leukemia cell expansion and inducing the specific leukemia apoptosis. B2000 also acted on restoring lymphocyte levels and, consequently, reducing inflammatory stress. Our findings highlight an important role of this novel MET-inhibitor onto the inflammatory TME, representing an important agent against hematological neoplasms with a novel immunomodulatory effect in directly reducing leukemia cell proliferation as well as promoting TAMs anti-tumoral effector functions. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

Genetic vulnerability to Crohn's disease reveals a spatially resolved epithelial restitution program.

Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.

Matriptase drives dissemination of ovarian cancer spheroids by a PAR-2/PI3K/Akt/MMP9 signaling axis.

The transmembrane serine protease matriptase is a key regulator of both barrier-disruptive and protective epithelial cell-cell interactions. Elevated matriptase is a consistent feature of epithelial ovarian cancers (OvCa), where multicellular spheroids shed from the primary tumor into the peritoneal cavity are critical drivers of metastasis. Dynamic cell-to-cell adhesive contacts are required for spheroid formation and maintenance. Here, we show that overactive matriptase, reflected in an increased ratio of matriptase to its inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), disrupts cell-cell contacts to produce loose prometastatic spheroids that display increased mesothelial cell adhesion and submesothelial invasion. We show that these activities are dependent on the matriptase activation of a protease-activated receptor-2 (PAR-2) signaling pathway involving PI3K/Akt and MMP9-induced disruption of cell-cell adhesion by the release of the soluble E-cadherin ectodomain. These data reveal a novel pathological connection between matriptase activation of PAR-2 and disruption of cell-cell adhesion, and support the clinical investigation of this signaling axis as a therapeutic strategy for aggressive metastatic OvCa.

Proteomic and Metabolomic Analysis of Bone Marrow and Plasma from Patients with Extramedullary Multiple Myeloma Identifies Distinct Protein and Metabolite Signatures.

Multiple myeloma (MM) is an incurable haematological malignancy of plasma cells in the bone marrow. In rare cases, an aggressive form of MM called extramedullary multiple myeloma (EMM) develops, where myeloma cells enter the bloodstream and colonise distal organs or soft tissues. This variant is associated with refractoriness to conventional therapies and a short overall survival. The molecular mechanisms associated with EMM are not yet fully understood. Here, we analysed the proteome of bone marrow mononuclear cells and blood plasma from eight patients (one serial sample) with EMM and eight patients without extramedullary spread. The patients with EMM had a significantly reduced overall survival with a median survival of 19 months. Label-free mass spectrometry revealed 225 proteins with a significant differential abundance between bone marrow mononuclear cells (BMNCs) isolated from patients with MM and EMM. This plasma proteomics analysis identified 22 proteins with a significant differential abundance. Three proteins, namely vascular cell adhesion molecule 1 (VCAM1), pigment epithelium derived factor (PEDF), and hepatocyte growth factor activator (HGFA), were verified as the promising markers of EMM, with the combined protein panel showing excellent accuracy in distinguishing EMM patients from MM patients. Metabolomic analysis revealed a distinct metabolite signature in EMM patient plasma compared to MM patient plasma. The results provide much needed insight into the phenotypic profile of EMM and in identifying promising plasma-derived markers of EMM that may inform novel drug development strategies.

O091 Predict factors of hepatocyte growth factor family and bone morphogenetic proteins for breast cancer nodal metastasis

Abstract Introduction Lymph node metastasis is essential for the outcome of patients with breast cancer. Bone morphogenetic proteins (BMP) and hepatocyte growth factor (HGF), key to angiogenesis and metastasis of cancers, were investigated for their roles in the predication of nodal metastasis from breast cancer. Methods Expression of BMP family ligands, BMP receptors, HGF, its receptor MET and regulatory factors of HGF activation including HGF activator (HGFA), HGFA inhibitor 1 (HAI1), HAI2, Matriptase-1 and Matriptase-2 were determined in breast cancers (n=127) using real time quantitative PCR. Transcript levels of these genes were analysed in the breast cancers for their implication in lymphatic involvement using Mann-Whitney test. Predictive potential of those genes for nodal metastasis of these molecules was evaluated using both binary logistic regression and receiver operating characteristic (ROC) with SPSS (Version 27). Results With median as a cut-off value, logistic regression analyses showed that MET, Matriptase-1 and BMP15 were positively associated with nodal metastasis whereas Matriptase-2, BMP3 and HAI1 were inversely correlated with nodal involvement. After integrating these six prospective factors, ROC model returned with a significant value against nodal status (RUC=0.657, p=0.001). With the optimal ROC cut-off value to dichotomise patients, the integrated expression produced a significant prediction of nodal metastasis (p=0.006, HR=2.929). MET and HAI1 was correlated with the lymphangiogenesis marks of LYVE1, podo, prox1. Matriptase-1 was inversely correlated with podo, whilst Matriptase-2 and BMP15 showed a positive correlation with prox1. Conclusion The MET/Matriptase-1/BMP15 and the inversed Matriptase-2/BMP3/HAI1 are connected significantly with lymph node metastasis in breast cancer.

Hepatocyte growth factor mimetic confers protection from aminoglycoside-induced hair cell death in vitro

Loss of sensory hair cells from exposure to certain licit drugs, such as aminoglycoside antibiotics, can result in permanent hearing damage. Exogenous application of the neurotrophic molecule hepatocyte growth factor (HGF) promotes neuronal cell survival in a variety of contexts, including protecting hair cells from aminoglycoside ototoxicity. HGF itself is not an ideal therapeutic due to a short half-life and limited blood-brain barrier permeability. MM-201 is a chemically stable, blood-brain barrier permeable, synthetic HGF mimetic that serves as a functional ligand to activate the HGF receptor and its downstream signaling cascade. We previously demonstrated that MM-201 robustly protects zebrafish lateral line hair cells from aminoglycoside ototoxicity. Here, we examined the ability of MM-201 to protect mammalian sensory hair cells from aminoglycoside damage to further evaluate MM-201’s clinical potential. We found that MM-201 exhibited dose-dependent protection from neomycin and gentamicin ototoxicity in mature mouse utricular explants. MM-201’s protection was reduced following inhibition of mTOR, a downstream target of HGF receptor activation, implicating the activation of endogenous intracellular substrates by MM-201 as critical for the observed protection. We then asked if MM-201 altered the bactericidal properties of aminoglycosides. Using either plate or liquid growth assays we found that MM-201 did not alter the bactericidal efficacy of aminoglycoside antibiotics at therapeutically relevant concentrations. We therefore assessed the protective capacity of MM-201 in an in vivo mouse model of kanamycin ototoxicity. In contrast to our in vitro data, MM-201 did not attenuate kanamycin ototoxicity in vivo. Further, we found that MM-201 was ototoxic to mice across the dose range tested here. These data suggest species- and tissue-specific differences in otoprotective capacity. Next generation HGF mimetics are in clinical trials for neurodegenerative diseases and show excellent safety profiles, but neither preclinical studies nor clinical trials have examined hearing loss as a potential consequence of pharmaceutical HGF activation. Further research is needed to determine the consequences of systemic MM-201 application on the auditory system.

ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma

BackgroundSustained activation of hepatocyte growth factor (HGF)/c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression, but underlying mechanism is unclear. ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 2 (ASAP2) can reportedly activate GTPases and promote receptor tyrosine kinase signaling. However, the exact role of ASAP2 in HCC, especially for c-MET activation, also remains elusive.MethodsASAP2 expression levels in HCC tissues and cells were quantified using qRT-PCR, western blot (WB) analysis, and immunohistochemistry staining. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell proliferation rates. Flow cytometry assays were conducted to assess apoptosis rates. Wound healing and Transwell assays were performed to determine cell migration and invasion capacities. Epithelial-mesenchymal transition (EMT)-related marker expression levels were also examined. Subcutaneous implantation and tail vein injection models were applied for in vivo growth and metastasis evaluations, respectively. Bioinformatics analyses of The Cancer Genome Atlas and STRING datasets were performed to explore ASAP2 downstream signaling. Co-immunoprecipitation and Cycloheximide chasing experiments were performed to assess protein–protein interactions and protein half-life, respectively.ResultsASAP2 had higher expression levels in HCC tissues than in normal liver, and also predicted poor prognosis. Knocking down ASAP2 significantly impaired cell proliferation, migration, and invasion capacities, but promoted apoptosis in HCC cells in vitro. However, overexpression of ASAP2 achieved the opposite effects. In vivo experiments confirmed that ASAP2 could promote HCC cell growth and facilitate lung metastasis. Interestingly, ASAP2 was essential for triggering EMT. Gene Set Enrichment Analysis demonstrated that c-MET signaling was greatly enriched in ASAP2-high HCC cases. Additionally, c-MET signaling activity was significantly decreased following ASAP knockdown, evidenced by reduced c-MET, p-AKT, and p-ERK1/2 protein levels. Importantly, ASAP2 knockdown effectively attenuated HGF/c-MET signaling-induced malignant phenotypes. c-MET and ASAP2 expression levels were positively correlated in our cohort. Mechanistically, ASAP2 can directly bind to CIN85, thereby disrupting its interaction with c-MET, and can thus antagonize CIN85-induced c-MET internalization and lysosome-mediated degradation. Notably, knocking down CIN85 can rescue the observed inhibitory effects caused by ASAP2 knockdown.ConclusionsThis study highlights the importance of ASAP2 in sustaining c-MET signaling, which can facilitate HCC progression.

Abstract 2584: Overcoming cetuximab resistance in colorectal cancer through inhibition of HGF processing

Abstract Cetuximab is a monoclonal antibody against epidermal growth factor receptor (EGFR) and can prolong survival in metastatic colorectal cancer (CRC) patients. However, therapeutic resistance can limit its efficiency and use. Our lab has previously identified that the activation of receptor tyrosine kinases (RTKs) MET and RON contributed to cetuximab resistance. In the absence of activating mutations of MET and RON, the activity of these RTKs depends on the availability of their ligands, hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HGFL), respectively. To become biologically active, the inactive precursors of HGF and HGFL must be processed by serine proteases (HGFA, matriptase, and hepsin). A survey of TCGA datasets indicated that MET and HGF were overexpressed in several CRC CMS subtypes. However, we did not observe any significant overexpression of the serine proteases. Hepatocyte growth factor activator inhibitor type 1 and 2 (HAI-1/2) are two Kunitz-type serine protease inhibitors that displayed reduced expression in TCGA datasets. The addition of recombinant human HGF conferred cetuximab resistance to cetuximab-sensitive cells and cetuximab-sensitive cells engineered to overexpress HGF also exhibited cetuximab-resistant phenotype. Cetuximab resistance induced by HGF addition or overexpression could be overcome by downstream inhibition with crizotinib, a multi-targeted RTK inhibitor. ZFH7116 is a small molecule inhibitor of the serine proteases that mimics HAI-1 activity. ZFH7116 suppressed HGF cleavage and maturation in our cetuximab-resistant SC cells. We observed cooperative effect of ZFH7116 with cetuximab to reduce SC cell growth in 3D collagen cultures. We are currently testing if upstream inhibition with recombinant human HAI-1 addition can suppress HGF cleavage and overcome cetuximab resistance induced by HGF overexpression. Collectively, our findings suggest that inhibiting HGF cleavage and processing may be a unique strategy for overcoming EGFR-targeted therapy resistance in colorectal cancer. Citation Format: Vivian Truong Jones, Ramona Graves-Deal, Galina Bogatcheva, Zheng Cao, James Higginbotham, Sarah Harmych, Gregory Ayers, Marisol Ramirez, Qi Liu, James Janetka, Bhuminder Singh. Overcoming cetuximab resistance in colorectal cancer through inhibition of HGF processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2584.

Therapeutic targets for inflammatory bowel disease: proteome-wide Mendelian randomization and colocalization analyses.

Identifying new drug targets for inflammatory bowel disease (IBD) is urgently needed. The proteome is a major source of therapeutic targets. We conducted a proteome-wide Mendelian randomization (MR) and colocalization analyses to identify possible targets for IBD. Summary-level data of 4907 circulating protein levels were extracted from a large-scale protein quantitative trait loci study including 35,559 individuals. Genetic associations with IBD and its subtypes were obtained from the Inflammatory Bowel Disease Genetics Consortium (25,024 cases and 34,915 controls), the FinnGen study (7206 cases and 253,199 controls), and the UK Biobank study (7045 cases and 449,282 controls). MR analysis was conducted to estimate the associations between protein and IBD risk. The colocalization analysis was used to examine whether the identified proteins and IBD shared casual variants. Genetically predicted levels of 3, and 5 circulating proteins were associated with IBD and ulcerative colitis (UC), respectively. With high supporting evidence of colocalization, genetically predicted MST1 (macrophage stimulating 1) and HGFAC (hepatocyte growth factor activator) levels were inversely associated with IBD risks. The associations of STAT3 (signal transducer and activator of transcription 3), MST1, CXCL5 (C-X-C motif chemokine ligand 5), and ITPKA (inositol-trisphosphate 3-kinase A) with the risk of UC were supported by colocalization analysis. The proteome-wide MR investigation identified many proteins associated with the risk of IBD. MST1, HGFAC, STAT3, ITPKA, and CXCL5 deserve further investigation as potential therapeutic targets for IBD. SCL is supported by research grants from the Swedish Research Council for Health, Working Life and Welfare (Forte; grant no. 2018-00123) and the Swedish Research Council (Vetenskapsrådet; grant no. 2019-00977). XYW is supported by research grants from the National Natural Science Foundation of China (81970494) and Key Project of Research and Development Plan of Hunan Province (2019SK2041). XL is supported by research grants from the Natural Science Fund for Distinguished Young Scholars of Zhejiang Province (LR22H260001).

Abstract P3-05-42: Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis

Abstract Introduction. The CUB domain containing protein 1, also known as TRASK (Transmembrane and associated with Src kinases) or CD318, has been suggested as a biomarker for stem cells or stem like cells. The transmembrane CDCP1 protein is known to activate the Src kinase and has recently shown to play a key part in the aggressiveness of breast cancer cells including migration, invasiveness and possibly growth induced by the motogenic Hepatocyte Growth Factor (HGF) and its receptor MET (Kawase et al 2022), a process requires Rho-GEF, PI3K and STAT5. This appears to allow identification of a subset of cancer cells that are aggressive in their biological behaviours. Here, we examined the expression profile of CDCP1, SRC, the HGF/HGF receptor complex and HGF activation regulators in a cohort of breast cancer and attempt to establish if such protein axis is able to identify patients with high risk of poor clinical outcome. Methods. Using an establish breast cancer cohort which is comprised of both normal mammary tissues and breast cancer tissues freshly obtained after surgery, we quantified the gene transcripts of CDCP1/CD318 and examined its clinical and pathological relevance. Integrated analysis was conducted for CDCP1 and the expression profile of SRC, PI3K, HGF, the HGF receptor cMET, HGF activator (HGFA), HGF activation regulators including HAI-1, HAI2, matriptase-1 and matriptase-2, and STAT family members. This was done against the clinical outcome of the patients as well as the clinical and pathological factors. Results. Breast cancer tissues expressed high levels of CDCP1 compared with normal mammary tissues. CDCP1 itself had a weak yet significant value in predicting the overall survival of the patients (p=0.047). The expression levels of these CDCP1 related molecules aren’t significantly correlated with CDCP1. However, integrated analyses revealed that CDCP1, together with its pathway regulators SRC, HGF, the HGF receptor (MET) and the HGF activation regulators matriptases form a power prognostic indicator for the clinical outcome of the patients (p< 0.0001, HR=1.8 for overall survival (OS) and p=0.002, HR=1.5 for disease-free survival (DFS)). This integrated profile has also identified subgroups of patients with highly favourable and very poor clinical outcomes over a ten-year follow-up period, with the respective survival at 100% and 36% respectively. The predictive power for OS and DFS is highly independent of other clinical and pathological factors in a multivariate analysis (p=0.003 and p=0.017 respectively). The predictive power is applicable to ER negative and HER2 positive tumours. Discussion. CDCP1/CD318, a factor known to stimulate cancer cell aggressiveness, together with its pathway kinase SRC and newly identified extracellular activator HGF and the HGF regulators forms a significant independent prognostic factor. It identifies subgroups of patients with favourable and poor prognosis, allowing consideration of targeted therapies for the patients. Reference Kawase N, Sugihara A, Kajiwara K, Hiroshima M, Akamatsu K, Nada S, Matsumoto K, Ueda M, Okada M. SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells. J Biol Chem, 2022, 298:101630. doi: 10.1016/j.jbc.2022.101630 Citation Format: Yiming Yang, Xuefei Dong, Tracey A. Martin, Lin Ye, Jane Lane, Andrew J. Sanders, QingPing Dou, Eleri Davies, Wen G. Jiang. Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-42.

Prediction of outcome after ligation or thin film banding of extrahepatic shunts, based on plasma albumin concentration and hematologic expression of 8 target genes in 85 dogs.

In dogs with a congenital extrahepatic portosystemic shunt (EHPSS), outcome after surgical attenuation is difficult to predict. Develop a minimally invasive test to predict outcome after surgical EHPSS attenuation and establish risk factors for postattenuation seizures (PAS). Eighty-five client-owned dogs referred for surgical attenuation of a single EHPSS. mRNA expression of 8 genes was measured in preoperatively collected venous blood samples. Outcome was determined at a median of 92 days (range, 26-208) postoperatively by evaluating clinical performance, blood test results and abdominal ultrasonography. Multivariable logistic regression was used to construct models predicting clinical and complete recovery. The associations between putative predictors and PAS were studied using univariable analyses. Five of 85 dogs developed PAS. Risk factors were age, white blood cell (WBC) count and expression of hepatocyte growth factor activator and LysM and putative peptidoglycan-binding domain-containing protein 2. Clinical recovery was observed in 72 of 85 dogs and complete recovery in 51 of 80 dogs (median follow-up, 92 days). The model predicting clinical recovery included albumin, WBC count, and methionine adenosyltransferase 2 alpha (MAT2α) expression, whereas the model predicting complete recovery included albumin, and connective tissue growth factor precursor and MAT2α expression. The areas under the receiver operating characteristic curves were 0.886 (95% confidence interval [CI]: 0.783, 0.990) and 0.794 (95% CI: 0.686, 0.902), respectively. Two models were constructed for predicting outcome after EHPSS attenuation using venous blood samples. The model predicting clinical recovery showed the best diagnostic properties. Clinical application requires further validation.