Presence Of Hepatocyte Growth Factor Research Articles

Abstract 4555: c-Met/AXL crosstalk in mediating therapeutic resistance in renal cell carcinoma

Abstract Receptor Tyrosine Kinase (RTK), c-Met, is overexpressed in renal cancer and correlates with a decreased survival rate. Upon binding with its specific ligand, Hepatocyte Growth Factor (HGF), c-Met activates pro-tumorigenic signaling. c-Met/RTK inhibitors are used for the treatment of advanced renal cell carcinoma (RCC). However, acquired therapeutic resistance is a major clinical problem and the underlying mechanisms are largely unknown. Hence, novel therapeutic targets are needed to overcome the clinical hurdles; and the RTK, AXL, can be a potential target. AXL is overexpressed and a poor prognostic marker in RCC. AXL overexpression drives wide-ranging processes, including epithelial-to-mesenchymal transition (EMT), tumor angiogenesis, and resistance to chemotherapeutics. Cabozantinib (XL184/Cabo), is approved for advanced-stage RCC and can inhibit c-Met and VEGFR2 activation (including AXL and a few other RTKs); however, it is not a specific/potent inhibitor of AXL. Hence, inhibiting c-Met alone may not be sufficient when AXL remains active. Therefore, the pro-tumorigenic role of AXL and its cross-talk with c-Met in renal cancer need to be thoroughly investigated. We compared the expression of c-Met and AXL in TCGA and found significantly high expression of both c-Met and AXL in primary renal tumor tissues compared to normal tissues. Our data showed that AXL forms a complex with c-Met and may have a significant cross-talk which can be involved in therapeutic resistance. We have also found that prolonged treatment of c-Met inhibitor Cabo, (including Crizotinib and PF-4217903) induced c-Met and AXL overexpression in RCC cells. Interestingly, c-Met inhibitor(s)-induced overexpressed c-Met can also be increasingly phosphorylated (at low concentrations) in the presence of HGF, which may cause enhanced downstream tumor-promoting signaling. To understand the role of AXL in tumor-promoting pathways and therapeutic resistance, we have generated CRISPR/Cas9-mediated AXL knock-out cells (AXL-KO) and found that silencing AXL can prevent c-Met-inhibitor(s)-mediated c-Met overexpression. Furthermore, to identify the effect of AXL-KO on pro-tumorigenic signaling, transcriptional profiles of control clones and AXL-KO cells were compared. The gene set enrichment analysis (GSEA) showed that genes involved in ‘EMT’, ‘Hypoxia’, and ‘K-Ras’ signaling pathways were significantly downregulated. We have also utilized an AXL-specific small molecule inhibitor, TP-0903, in combination with Cabo and validated our data. We have also generated a Cabo-resistant cell line (Caki-1) and found high expression of c-Met and AXL in these cells. Furthermore, either silencing of AXL (using siRNA) or inhibiting AXL (using TP0903) induced significant apoptosis in these Cabo-resistant cells. Together, our data suggest that inhibition of AXL along with c-Met can be beneficial in preventing acquired therapeutic resistance in RCC. Citation Format: Akash Sabarwal, Laxminarayan Rawat, Johannes Wedel, Murugabaskar Balan, Gwo-Shu Mary Lee, Toni K. Choueiri, Soumitro Pal. c-Met/AXL crosstalk in mediating therapeutic resistance in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4555.

C-Met+ Cytotoxic T Lymphocytes Exhibit Enhanced Cytotoxicity in Mice and Humans In Vitro Tumor Models.

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met+ CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met+ CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met+ CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell-cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met+) and compared it to c-Met- CTL clones. Our results indicated that, similar to their murine counterparts, c-Met+ human CTL clones exhibited increased cytolytic activity compared to c-Met- CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer.

Effect of Antiviral Therapy for HCV Treatment in the Presence of Hepatocyte Growth Factor

The effect of antiviral therapy during Hepatitis C Virus (HCV) infection is the focus of this study. HCV infection destroys healthy hepatocyte cells in the human liver, causing cirrhosis and hepatocellular carcinoma. We introduce a cell-population model representing the long-term dynamics of HCV infection in response to antiviral drug therapies. The proliferation of existing cells can create hepatocyte cells in the system. Such models are based on the dynamics of susceptible hepatocytes, infected hepatocytes and HCV with interactive dynamics, which can give a complete understanding of the host dynamics of the system in the presence of antiviral drug therapy. Infection-free equilibrium and endemic equilibrium are two equilibrium states in the absence of drugs. The existence and stability conditions for both systems are presented. We also construct an optimal control system to find the optimal control strategy. Numerical results show that the effects of the proliferation rate and infection rate are critical for the changes in the dynamics of the model. The impact of different weight factors on the optimal control problem is analysed through numerical simulation.

Discovery of anticancer agents with c-Met inhibitory potential by virtual and experimental screening of a chemical library

c-Met receptor tyrosine kinase has recently emerged as an important target with therapeutic implications in pancreatic cancer. In this study, we carried out a docking virtual screening on an in-house library of 441 synthesized compounds and selected the compounds with the best interactions with the c-Met protein to be subjected to experimental tests. Ten compounds belonging to 3 different classes of chemical structures were selected for this purpose and their antiproliferative effects were studied against 4 pancreatic ductal adenocarcinoma (PDAC) cell lines including AsPC-1, Suit-2, Panc-1 and Mia-Paca-2 cells, primary PDAC cells and also c-Met amplified EBC-1 cell line by sulforhodamine-B assay. Apoptosis induction was examined by Hoechst 33258 staining and annexin V-FITC/propidium iodide flow cytometric assay. The best compound was also assayed in three-dimensional cultures of AsPC-1 cells and its c-Met inhibitory potential was studied by immunoblotting and a homogenous time resolved fluorescence (HTRF) assay. The compound with a phenanthrotriazine hydrazinyl scaffold bearing nitrophenyl pendant (PhTH) was the most active derivative, with IC50 values in the range of 5–8 μM. This compound exerted antiproliferative effect against AsPC-1 cells also in the presence of hepatocyte growth factor (HGF). PhTH induced apoptosis, dose-dependently inhibited spheroid growth, inhibited c-Met activity in cell-free HTRF assay and also inhibited the phosphorylation of c-Met and its downstream effector ERK1/2 in AsPC-1 cells. Molecular docking and dynamics simulation and MM-PBSA analysis confirmed close interactions of PhTH with c-Met kinase domain. Some of the tested compounds in this study seem to be potential c-Met inhibitors with promising activities against PDAC cells.

Abstract 5138: Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer

Abstract Background: Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the therapeutic implications of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancer (CRC). Methods: Clinical outcomes were analyzed according to the plasma HGF levels in patients with metastatic CRC (mCRC) receiving palliative first-line cetuximab or bevacizumab + FOLFIRI chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition in the HGF-high population. Results: From March 2015 to September 2019, a total of 80 patients were enrolled. The median age was 63 (range, 24-85) years. Of these, 45 patients (56.2%) were treated with bevacizumab + FOLFIRI and 35 patients (43.8%) were treated with cetuximab + FOLFIRI. Among patients with evaluable disease (43 patients (95.6%) in the bevacizumab group and 34 patients (97.1%) in the cetuximab group), the objective response rate was 60.0% and 37.8%, respectively (p=0.162). The median serum HGF level was 374.75 (range, 9.43-30,644.44) pg/mL, and the optimal cut-off value of HGF levels was 12.70 pg/mL by the maximal chi-square method. During a median follow-up of 29.3 (range, 1.2-71.3) months, both progression-free survival (PFS) and overall survival (OS) were significantly shorter in the high HGF group compared with the low HGF group: median 11.8 (95% confidence interval [CI], 10.5-13.1) months vs. 24.7 (95% CI, 23.4-25.9) months for PFS (p=0.009), and median 21.1 (95% CI, 17.9-24.3) months vs. not reached for OS (p=0.018). The difference in PFS and OS was statistically significant in the cetuximab group (p=0.003 for PFS and p=0.035 for OS), but not in the bevacizumab group. In five RAS/RAF wild-type CRC cell lines (Caco-2, COLO 320DM, KM12C, SNU-C1, and SNU-C4), the addition of HGF activated ERK1/2 and AKT via MET phosphorylation. In the cytotoxicity assays, Caco-2 and SNU-C4 were relatively sensitive to cetuximab compared with the others and, in the presence of HGF, cetuximab sensitivity was significantly decreased in the two cells. Moreover, capmatinib, a MET inhibitor, abrogated the effect of HGF on common downstream signaling pathways of EGFR and MET and thus the cetuximab resistance was significantly overcome in vitro. Conclusions: Among patients with mCRC receiving cetuximab + FOLFIRI, those with high plasma HGF levels had significantly worse PFS and OS. HGF induced cetuximab resistance by AKT and ERK activation while capmatinib, a MET inhibitor, significantly increased the anti-tumor effects of cetuximab in the presence of HGF in vitro. Our data may provide evidence of future clinical trials of dual EGFR and MET targeting strategies in patients with HGF-high, RAS/RAF wild-type mCRC. Citation Format: Sang-A Kim, Hyejoo Park, Kui-Jin Kim, Ji-Won Kim, Ji Hea Sung, Milang Nam, Ju Hyun Lee, Eun Hee Jung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jeong-Ok Lee, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Soo-Mee Bang, Jong Seok Lee, Keun-Wook Lee. Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5138.

Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancers.

Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the counter effect of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancers (CRCs). Plasma HGF levels were analyzed with clinical outcomes of patients with metastatic CRC (mCRC) receiving palliative first-line chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition by capmatinib. A total of 80 patients were included: cetuximab + FOLFIRI (n = 35) and bevacizumab + FOLFIRI (n = 45). Both progression-free survival (PFS) and overall survival (OS) were significantly lesser in the high vs low HGF group: median 11.8 vs. 24.7months, respectively, for PFS (p = 0.009), and median 21.1months vs. not reached, respectively, for OS (p = 0.018). The difference was significantly evident in the cetuximab group. In five RAS/RAF wild-type CRC cells, the addition of HGF activated ERK1/2 and AKT via MET phosphorylation, resulting in cetuximab resistance in vitro. In cetuximab-sensitive Caco-2 and SNU-C4 cells, capmatinib overcame cetuximab resistance in the presence of HGF by attenuating HGF-induced MET signaling activation. Patients with mCRC receiving cetuximab + FOLFIRI who presented with high plasma HGF levels had significantly worse PFS and OS. Cetuximab resistance induced by HGF was mediated by AKT and ERK activation and overcome by MET inhibition in vitro.

Growth factor-eluting hydrogels for management of corneal defects

With 1.5–2.0 million new cases annually worldwide, corneal injury represents a common cause of vision loss, often from irreversible scarring due to surface corneal defects. In this study, we assessed the use of hepatocyte growth factor (HGF) loaded into an in situ photopolymerizable transparent gelatin-based hydrogel for the management of corneal defects. In vitro release kinetics showed that, in regard to the total amount of HGF released over a month, 55 ± 11% was released during the first 24 h, followed by a slow release profile for up to one month. The effect of HGF was assessed using an ex vivo model of pig corneal defect. After three days of organ culture, epithelial defects were found to be completely healed for 89% of the corneas treated with HGF, compared to only 11% of the corneas that had fully re-epithelialized when treated with the hydrogel without HGF. The thickness of the epithelial layer was found to be significantly higher for the HGF-treated group compared to the group treated with hydrogel without HGF (p = 0.0012). Finally, histological and immunostaining assessments demonstrated a better stratification and adhesion of the epithelial layer in the presence of HGF. These results suggest that the HGF-loaded hydrogel system represents a promising solution for the treatment of persistent corneal defects at risk of scarring.

Detection of hepatocyte growth factor in oral rinses using water for possible periodontal diagnosis.

The aim of this study is to analyze the relationship between Hepatocyte Growth Factor (HGF) levels in oral rinses using water and clinical parameters of periodontitis; and furthermore, to evaluate the potential of a prototype HGF immunochromatographic paper test strip (HGF-TS) for screening of periodontitis, in comparison with a commercially-available occult blood (hemoglobin) test strip (Hb-TS). Clinical periodontal parameters were recorded, and oral rinses were collected, from 125 subjects. Then, the presence of HGF, and hemoglobin (Hb), in each sample was detected using a prototype HGF-TS and an Hb-TS. In addition, the concentrations of HGF and Hb were also determined in each sample is necessary HGF concentrations in oral rinses showed significant correlations with clinical parameters of periodontitis. The positive rate and read value on HGF-TS showed significantly high values in cases of severe periodontitis compared to healthy subjects. Hb-TS showed generally higher positive rates than HGF-TS; however, it showed false positive results in healthy subjects. The concentration of HGF in oral rinses showed close association with the severity of periodontitis, suggesting that the prototype HGF-TS has potential for use in the diagnosis of periodontitis, although further refinement of the test strip is required to increase the sensitivity.

Myeloma-derived extracellular vesicles mediate HGF/c-Met signaling in osteoblast-like cells

Multiple myeloma is an incurable cancer of antibody-producing plasma cells. Hepatocyte growth factor (HGF), a cytokine aberrantly expressed in half of myeloma patients, is involved in myeloma pathogenesis by enhancing myeloma growth and invasiveness, and may play a role in myeloma bone disease by inhibiting osteoblastogenesis. In this study, we investigated whether extracellular vesicles (EVs) may play a role in HGF signaling between myeloma cells and osteoblast-like target cells. EVs from the HGF-positive cell line JJN-3 and the HGF-negative cell line INA-6, and from bone marrow plasma and primary human myeloma cells, were isolated using sequential centrifugation techniques and the presence of HGF on the EV-surface was investigated with ELISA. EVs from both cell lines were added to an established bioassay where HGF is known to induce interleukin-11 secretion in osteoblast-like cells. Our results show that HGF was bound to the surface of JJN-3-derived EVs, while INA-6-derived EVs were negative for HGF. Only JJN-3-derived EVs induced IL-11 secretion in osteoblast-like recipient cells. When osteoblast-like cells were preincubated with a specific HGF-receptor (c-Met) inhibitor, no induction of interleukin-11 was observed. Downstream c-Met phosphorylation was demonstrated by immunoblotting. EVs isolated from bone marrow plasma and primary myeloma cells were HGF-positive for a subset of myeloma patients. Taken together, this work shows for the first time that HGF bound on the surface of myeloma-derived EVs can effectuate HGF/c-Met signaling in osteoblast-like cells. Myeloma-derived EVs may play a role in myeloma bone disease by induction of the osteoclast-activating cytokine interleukin-11 in osteoblasts.

Enhancement of HGF-induced tubulogenesis by endothelial cell-derived GDNF.

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.

Clonal expansion of hepatic progenitor cells and differentiation into hepatocyte-like cells.

Hepatic progenitor cells (HPCs) in adult liver are promising for treatment of liver diseases. A biliary-derived HPC population in adult mice has been characterized by co-expression of stem cell marker Sry (sex determining region Y)-box 9 (SOX9) and biliary marker cytokeratin 7 (CK7). However, isolation of these HPCs in adult healthy liver without any selection procedures remains a big challenge in this field. Here, by establishing a simple and efficient method to isolate and expand the CK7+ SOX9+ HPCs in vitro as clones, we acquired a stable and largely scalable cell source. The CK7+ SOX9+ progenitor cells were then further induced to differentiate into hepatocyte-like cells with expression of mature hepatocyte markers albumin (Alb) and hepatocyte nuclear factor 4 alpha (HNF4α), both in vitro and in vivo in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor 9 (FGF9). Furthermore, we found that the HPCs are highly responsive to transforming growth factor-beta (TGF-β) signals. Collectively, we identified and harvested a CK7+ SOX9+ progenitor cell population from adult mouse liver by a simple and efficient approach. The exploration of this HPC population offers an alternative strategy of generating hepatocyte-like cells for cell-based therapies of acute and chronic liver disorders.

Zebularine Promotes Hepatic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells by Interfering with p38 MAPK Signaling.

Demethylating agent zebularine is reported to be capable of inducing differentiation of stem cells by activation of methylated genes, though its function in hepatocyte differentiation is unclear. p38 signal pathway is involved in differentiation of hepatocytes and regulating of DNA methyltransferases 1 (DNMT1) expression. However, little is known about the impact of zebularine on bone marrow mesenchymal stem cells (BMMSCs) and p38 signaling during hepatic differentiation. The present study investigated the effects of zebularine on hepatic differentiation of rabbit BMMSCs, as well as the role of p38 on DNMT1 and hepatic differentiation, with the aim of developing a novel strategy for improving derivation of hepatocytes. BMMSCs were treated with zebularine at concentrations of 10, 20, 50, and 100 μM in the presence of hepatocyte growth factor; changes in the levels of hepatic-specific alpha-fetoprotein and albumin were detected and determined by RT-PCR, WB, and immunofluorescence staining. Expression of DNMT1 and phosphorylated p38 as well as urea production and ICG metabolism was also analyzed. Zebularine at concentrations of 10, 20, and 50 μM could not affect cell viability after 48 h. Zebularine treatment leads to an inhibition of DNMT activity and increase of hepatic-specific proteins alpha-fetoprotein and albumin in BMMSCs in vitro; zebularine addition also induced expression of urea production of and ICG metabolism. p38 signal was activated in BMMSCs simulated with HGF; inhibition of p38 facilitated the synthesis of DNMT1 and albumin in cells. Zebularine restrained DNMT1 and phosphorylated p38 which were induced by HGF. Therefore, this study demonstrated that treatment with zebularine exhibited terminal hepatic differentiation of BMMSCs in vitro in association with hepatocyte growth factor; p38 pathway at least partially participates in zebularine-induced hepatic differentiation of rabbit BMMSCs.

Effective Timing of Surgical Resection of Colorectal Cancer Liver Metastases During Chemotherapy.

The aim of the present study was to further develop our previous study on c-Met expression in colorectal cancer and epithelial-mesenchymal transition (EMT) induced by hepatocyte growth factor (HGF), to investigate EMT in the process of liver metastases, and evaluate the effects of chemotherapy on EMT cells as a therapeutic strategy for colorectal liver metastasis. CT26 colon cancer cells were treated with 5-FU and oxaliplatin with or without HGF. The signaling pathway was evaluated by western blotting analysis, and drug resistance was evaluated by the MTT (3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide) assay. Under pretreatment with HGF for 96 h, 5 μM and 10 μM of 5-FU mediated significant growth inhibition by 72.5±3.9% and 76.2±2.4%, respectively, compared to HGF alone, and by 105.1±2.8% and 103.5±2.9%, respectively, without HGF. The expression of E2F1 was decreased significantly to 50.5±3.8% after 24 hours by HGF with a reduction of both cyclin D1 to 52.1±7.0% and E to 73.7±3.8%. Thymidylate synthase was also decreased in a time-dependent manner to 80.6±2.0% after 24 h and to 52.7±1.5% after 96 h. The presence of HGF was found to increase the 5-FU-induced death signal, JNK pathway, and inhibition of cell growth. As its mechanism, HGF was shown to decrease E2F-1 by reducing cyclin D or E by cell-cycle activation, resulting in inactivation of thymidylate synthase. The chemotherapeutic effect of 5-FU was increased in HGF- but not TGF-β-induced EMT.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway

BackgroundThe hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration.MethodsWe evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting.ResultsWe demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin.ConclusionsWe demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

In vitro tubulogenesis of Madin–Darby canine kidney (MDCK) spheroids occurs depending on constituent cell number and scaffold gel concentration

In vitro tubulogenesis has been employed as an experimental model system to study tissue morphogenesis of internal organs. It has been previously shown that Madin–Darby canine kidney (MDCK) cells form tubes in the presence of hepatocyte growth factor in 3D cultures. Although these cells are expected to form tube structures in some microenvironments independent of chemical stimulation, little is known about the cellular mechanisms in organizing such an anisotropic multicellular structure. Here, we report 3D culture conditions that induce MDCK tubulogenesis without growth factor stimulation. We found that the cells spontaneously form elongated tube structures through aggregation processes in a specific range of both constituent cell number and scaffold gel concentration, while they form spherical aggregates in other conditions. We then examined cellular activities affecting tubulogenesis and showed that cell proliferation is not required for the tube elongation. Furthermore, we revealed that cells in the tube tips generate traction forces and pull the surrounding scaffold gel to migrate, resulting in the tube elongation. Our results suggest that the constituent cells during the aggregation process interact each other via mechanical forces transmitted in the scaffold gel, leading to the spontaneous tube formation.

Abstract 290: A novel function of HGF in the activation of Chk1 phosphorylation in colon cancer cells

Abstract ATR/Chk1 pathway plays an essential role in modulation of DNA damage response (DDR) and homologous recombination (HR). Particularly, Chk1 phosphorylation is involved in cancer prognosis and therapeutic resistance. Although some receptor tyrosine kinases (RTKs) participate in the regulation of Chk1 phosphorylation, the effect of hepatocyte growth factor (HGF) on Chk1 phosphorylation is unknown. In the present study, Western blotting and subcellular fractionation were performed to identify the expression and the location of Chk1 phosphorylation in the presence of HGF. With small interfering RNA transfections, co-immunoprecipitations were performed to detect the interaction between TopBP1 and ATR complex. Our results demonstrated that HGF moderately activated Chk1 phosphorylation in colon cancer cells via upregulation of TopBP1, RAD51, and complex formation of TopBP1 and ATR. Furthermore, AKT activation promoted by HGF served as an important intermediator linking HGF/MET signaling and Chk1 phosphorylation. Depletion of AKT activity attenuated expression of basal line p-Chk1 and induced Chk1 activation due to HGF stimulation. Meantime, activation of AKT directly regulated expression of TopBP1 and RAD51. Suppression of AKT restored HGF-induced upregulation of TopBP1, RAD51, and enhancement of TopBP1/ATR complex as well. Our studies showed that HGF was implicated in regulating Chk1 phosphorylation and further demonstrated that AKT activity was responsible for HGF-induced Chk1 phosphorylation, which might potentially result in management of prognosis and therapeutic sensitivity in cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Na Song, Xiaofang Che, Xiujuan Qu, Lu Xu, Kezuo Hou, Yunpeng Liu. A novel function of HGF in the activation of Chk1 phosphorylation in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 290. doi:10.1158/1538-7445.AM2017-290

Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer

Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.

Preclinical Evaluation of MET Inhibitor INC-280 With or Without the Epidermal Growth Factor Receptor Inhibitor Erlotinib in Non–Small-Cell Lung Cancer

BackgroundAlthough the epidermal growth factor receptor (EGFR) inhibitor erlotinib is initially effective in non–small-cell lung cancer (NSCLC) patients with tumors harboring activating mutations of EGFR, most subsequently develop acquired resistance. One recognized resistance mechanism occurs through activation of bypass signaling via the hepatocyte growth factor (HGF)-MET pathway. INC-280 is a small molecule kinase inhibitor of MET. We sought to demonstrate the activity of INC-280 on select NSCLC cell lines both as a single agent and in combination with erlotinib using exogenous HGF to simulate MET up-regulation. MethodsFour NSCLC cell lines (HCC827, PC9, H1666, and H358) were treated with either single-agent INC-280 or in combination with erlotinib with or without HGF. The activity of the drug treatments was measured by cell viability assays. Immunoblotting was used to monitor expression of EGFR/pEGFR, MET/pMET, GAB1/pGAB1, AKT/pAKT, and ERK/pERK as well as markers of apoptosis (PARP and capase-3 cleavage) in H1666, HCC827, and PC9. ResultsAs a single agent, INC-280 showed minimal cytotoxicity despite potent inhibition of MET kinase activity at concentrations as low as 10 nM. Addition of HGF prevented erlotinib-induced cell death. The addition of INC280 to HGF-mediated erlotinib-resistant models restored erlotinib sensitivity for all cell lines tested, associated with cleavage of both PARP and caspase-3. In these models, INC-280 treatment was sufficient to restore erlotinib-induced inhibition of MET, GAB1, AKT, and ERK in the presence of HGF. ConclusionAlthough the MET inhibitor INC-280 alone had no discernible effect on cell growth, it was able to restore sensitivity to erlotinib and promote apoptosis in NSCLC models rendered erlotinib resistant by HGF. These data provide a preclinical rationale for an ongoing phase 1 clinical trial of erlotinib plus INC-280 in EGFR-mutated NSCLC.

Monitoring of ovarian cancer cell invasion in real time with frequency-dependent impedance measurement.

The conventional approach to assessing cancer invasion is primarily for end-point analysis, which does not provide temporal information on the invasion process or any information on the interactions between invading cells and the underlying adherent cells. To alleviate these limitations, the present study exploited electric cell-substrate impedance sensing (ECIS) to monitor the invasion of ovarian cancer cells (SKOV-3) through an adherent monolayer of human umbilical vein endothelial cells (HUVECs). Impedance was measured at 4 kHz of AC voltage or was measured as a function of AC frequency (25 Hz to 60 kHz). By measuring impedance at 4-kHz AC, we found that the invasion of SKOV-3 cells through the HUVEC monolayer was manifested as a rapid decrease in transendothelial electrical resistance in real time. The invasion was augmented in the presence of hepatocyte growth factor (HGF). The enhancing effect of HGF was attenuated by c-Met inhibitor (SU11274). By measuring the frequency-dependent impedance of SKOV-3 cells over time, we found that HGF-enhanced SKOV-3 cell invasion was accomplished with reduced junctional resistance (Rb), increased average cell-substrate separation (h), and increased micromotion. SU11274 attenuated the effects of HGF on Rb, h, and micromotion in the SKOV-3 monolayer. SU11274 also increased the barrier function of the HUVEC monolayer by increasing Rb and decreasing h In conclusion, this study demonstrated an improved method for monitoring and studying the interactions between cancer cells and the underlying adherent cells during invasion in real time. Alterations in cellular biophysical properties (Rb, h) associated with cancer transendothelial invasion were detected.

Bufalin reverses hepatocyte growth factor-induced resistance to afatinib in H1975 lung cancer cells

To investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism. The afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot. The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly. Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.