MET Signaling Pathway Research Articles

Characterization of MET Alterations in 37 Gastroesophageal Cancer Cell Lines for MET-Targeted Therapy.

Capmatinib and savolitinib, selective MET inhibitors, are widely used to treat various MET-positive cancers. In this study, we aimed to determine the effects of these inhibitors on MET-amplified gastric cancer (GC) cells. Methods: After screening 37 GC cell lines, the following cell lines were found to be MET-positive with copy number variation >10: SNU-620, ESO51, MKN-45, SNU-5, and OE33 cell lines. Next, we assessed the cytotoxic response of these cell lines to capmatinib or savolitinib alone using cell counting kit-8 and clonogenic cell survival assays. Western blotting was performed to assess the effects of capmatinib and savolitinib on the MET signaling pathway. Xenograft studies were performed to evaluate the in vivo therapeutic efficacy of savolitinib in MKN-45 cells. Savolitinib and capmatinib exerted anti-proliferative effects on MET-amplified GC cell lines in a dose-dependent manner. Savolitinib inhibited the phosphorylation of MET and downstream signaling pathways, such as the protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways, in MET-amplified GC cells. Additionally, savolitinib significantly decreased the number of colonies formed on the soft agar and exerted dose-dependent anti-tumor effects in an MKN-45 GC cell xenograft model. Furthermore, a combination of trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in human epidermal growth factor receptor-2 (HER2)- and MET-positive OE33 cells. Targeting MET with savolitinib and capmatinib efficiently suppressed the growth of MET-amplified GC cells. Moreover, these MET inhibitors exerted synergistic effects with trastuzumab on HER2- and MET-amplified GC cells.

Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition

The hepatocyte growth factor (HGF)/MET signaling pathway has been proposed to be involved in the resistance to radiotherapy of glioblastoma via proinvasive and DNA damage response pathways.Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models. We find that the murine glioma cell lines GL-261, SMA-497, SMA-540 and SMA-560 express HGF and its receptor MET and respond to exogenous HGF with MET phosphorylation. Glioma cell viability or proliferation are unaffected by genetic or pharmacological MET inhibition using tepotinib or CRISPR/Cas9-engineered Met gene knockout and MET inhibition fails to sensitize glioma cells to irradiation in vitro. In contrast, the combination of tepotinib with radiotherapy prolongs survival of orthotopic SMA-560 or GL-261 glioma-bearing mice compared with radiotherapy or tepotinib treatment alone. Synergy is lost when such experiments are conducted in immunodeficient Rag1−/− mice, and, importantly, also when Met gene expression is disrupted in the tumor cells. Combination therapy suppresses a set of pro-inflammatory mediators including matrix metalloproteases that are upregulated by radiotherapy alone and that have been linked to poor outcome in glioblastoma. Several of these mediators are positively regulated by transforming growth factor (TGF)-β, and pSMAD2 levels as a surrogate marker of TGF-β pathway activity are suppressed by combination treatment. We conclude that synergistic suppression of experimental syngeneic glioma growth by irradiation and MET inhibition requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined strategy in newly diagnosed glioblastoma is warranted.

MET Signaling Pathways, Resistance Mechanisms, and Opportunities for Target Therapies.

The MET gene, known as MET proto-oncogene receptor tyrosine kinase, was first identified to induce tumor cell migration, invasion, and proliferation/survival through canonical RAS-CDC42-PAK-Rho kinase, RAS-MAPK, PI3K-AKT-mTOR, and β-catenin signaling pathways, and its driver mutations, such as MET gene amplification (METamp) and the exon 14 skipping alterations (METex14), activate cell transformation, cancer progression, and worse patient prognosis, principally in lung cancer through the overactivation of their own oncogenic and MET parallel signaling pathways. Because of this, MET driver alterations have become of interest in lung adenocarcinomas since the FDA approval of target therapies for METamp and METex14 in 2020. However, after using MET target therapies, tumor cells develop adaptative changes, favoring tumor resistance to drugs, the main current challenge to precision medicine. Here, we review a link between the resistance mechanism and MET signaling pathways, which is not only limited to MET. The resistance impacts MET parallel tyrosine kinase receptors and signals shared hubs. Therefore, this information could be relevant in the patient's mutational profile evaluation before the first target therapy prescription and follow-up to reduce the risk of drug resistance. However, to develop a resistance mechanism to a MET inhibitor, patients must have access to the drugs. For instance, none of the FDA approved MET inhibitors are registered as such in Chile and other developing countries. Constant cross-feeding between basic and clinical research will thus be required to meet future challenges imposed by the acquired resistance to targeted therapies.

Abstract 6376: Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal cancer cells

Abstract Background: Many studies have suggested that MET protein overexpression or MET amplification plays a critical role in the progression of gastro-esophageal cancer (GC) and negatively affects survival in patients with GC. However, the criteria used to define overexpression of MET protein have differed among many studies, and in contrast to the initial phase II studies, the phase III trials failed to show any clinical benefit from anti-MET therapies in GCs, even in patients with MET-positive disease. Several studies have used consistent criteria to define MET amplification on FISH, and recent MET inhibitor trials for MET amplified cancers have been successful. Furthermore, hyperactivation of MET pathway are known to reduce the growth-inhibitory effect of trastuzumab in HER2 positive GC. Currently, there is no FDA approved MET inhibitors for GC. Capmatinib and savolitinib, which are selective MET inhibitors, has been evaluated for various MET positive cancers. The aim of this study was to test these MET inhibitors on MET amplified gastro-esophageal cancer (GC) cells. Material and Methods: Among a total of 37 GC cells in our lab, the following cells were positive for MET by copy number amplification (CNV > 10): SNU-620, ESO51, MKN-45, SNU-5 and OE-33. We assessed cytotoxic response of these GC cells to capmatinib or savolitinib alone by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. Western blots were performed to assess the cellular activity of capmatinib or savolitinib in MET signal pathway. Xenograft studies were performed to evaluate in vivo therapeutic efficacy of savolitinib with MKN-45 cells. Results: Savolitinib and capmatinib showed anti-proliferative activity against MET amplified GC cell lines in dose-dependent manner. There was no inhibitory effect on GC cells without MET amplification, such as SNU-216 and SNU-1750. Savolitinib inhibited phosphorylation of MET and down-stream signaling pathway, such as AKT or ERK, in MET amplified GC cells. We observed that treatment with savolitinib induced a significant decrease in the number of colonies formed in soft agar. In addition, Savolitinib exhibited dose-responsive anti-tumor efficacy in xenograft model with MKN-45 GC cells. Furthermore, the combination of Trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in OE-33 cells, which is positive for both HER2 and MET, and resistant to trastuzumab alone. Conclusion: Collectively, these data support that targeting MET with Savolitinib and capmatinib efficiently suppresses the growth of MET amplified GC cells. Moreover, MET inhibitor could synergize with trastuzumab to induce the therapeutic effect on the HER2 and MET amplified GC cells. Further studies are warranted to test this concept in clinical setting. Citation Format: Jin-Soo Kim, Sungyoul Hong, Mi Young Kim. Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal cancer cells [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 6376.

CPNE1 promotes non-small cell lung cancer progression by interacting with RACK1 via the MET signaling pathway

BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most lethal tumour worldwide. Copine 1 (CPNE1) was identified as a novel oncogene in NSCLC in our previous study. However, its specific function and relative mechanisms remain poorly understood.MethodsThe biological role of CPNE1 and RACK1 in NSCLC was investigated using gene expression knockdown and overexpression, cell proliferation assays, clonogenic assays, and Transwell assays. The expression levels of CPNE1, RACK1 and other proteins were determined by western blot analysis. The relationship between CPNE1 and RACK1 was predicted and investigated by mass spectrometry analysis, immunofluorescence staining, and coimmunoprecipitation. NSCLC cells were treated with a combination of a MET inhibitor and gefitinib in vitro and in vivo.ResultsWe found that CPNE1 facilitates tumorigenesis in NSCLC by interacting with RACK1, which further induces activation of MET signaling. CPNE1 overexpression promoted cell proliferation, migration, invasion and MET signaling in NSCLC cells, whereas CPNE1 knockdown produced the opposite effects. In addition, the suppression of the enhancing effect of CPNE1 overexpression on tumorigenesis and MET signaling by knockdown of RACK1 was verified. Moreover, compared to single-agent treatment, dual blockade of MET and EGFR resulted in enhanced reductions in the tumour volume and downstream signaling in vivo.ConclusionsOur findings show that CPNE1 promotes tumorigenesis by interacting with RACK1 and activating MET signaling. The combination of a MET inhibitor with an EGFR-TKI attenuated tumour growth more significantly than either single-drug treatment. These findings may provide new insights into the biological function of CPNE1 and the development of novel therapeutic strategies for NSCLC.3n8P5VYzQLauDz5o6kqwUEVideo

Association between the HGF/c‑MET signaling pathway and tumorigenesis, progression and prognosis of hepatocellular carcinoma (Review).

Hepatocellular carcinoma (HCC) is one of the most aggressive and lethal malignancies with a rising incidence, and is characterized by rapid progression, frequent metastasis, late diagnosis, high postoperative recurrence and poor prognosis. Therefore, novel treatment strategies for HCC, particularly advanced HCC, are urgently required. The hepatocyte growth factor(HGF)/c‑mesenchymal‑epithelial transition receptor (c‑MET) axis is a key signaling pathway in HCC and is strongly associated with its highly malignant features. Available treatments based on HGF/c‑MET inhibition may prolong the lifespan of patients with HCC; however, they do not achieve the desired therapeutic effects. The aim of the present article was to review the basic knowledge regarding the role of the HGF/c‑MET signaling pathway in HCC, and examine the association between the HGF/c‑MET signaling pathway and the tumorigenesis, progression and prognosis of HCC.

MET transcriptional regulator/serine peptidase inhibitor kunitz type 1 panel operating through HGF/c-MET axis as a prognostic signature in pan-cancer.

Dysregulations in transcription factors (TFs) and their genetic products play important roles in tumorigenesis, tumor progression and metastasis. However, prognostic value of the transcriptional regulatory networks in different cancers has not been investigated in depth. The purpose of our study was to identify and validate a potential predictive signature that combines TFs and their regulatory products in eight solid tumors. We used bioinformatics analysis to identify MET Transcriptional Regulator (MACC1) and Serine Peptidase Inhibitor Kunitz Type 1 (SPINT1) as candidate TFs with the respective downstream regulatory proteins for patient prognosis in pan‐cancer. Subsequent molecular analysis of clinical gastric cancer tissue samples further verified the negative correlation between MACC1 and SPINT1. Further, we showed that mechanistically, MACC1/SPINT1 mediated the pro‐HGF proteolysis and c‐Met phosphorylation in HGF/c‐MET signaling pathway. Kaplan‐Meier plots and receiver operating characteristics analysis revealed that the two‐gene signature combining MACC1 with SPINT1 was effective in predicting survival in all eight cancer cohorts tested. In conclusion, our study clarified the regulatory relationship between MACC1 and SPINT1 in the context of the HGF/c‐MET signaling pathway and determined MACC1/SPINT1 panel as a valuable signature for the prediction of prognosis in patients for multiple solid cancer types.

Relationship between IL10 and PD-L1 in Liver Hepatocellular Carcinoma Tissue and Cell Lines

Background Despite the large-scale clinical application of programmed death-ligand 1 (PD-L1) monoclonal antibody, reduction in its clinical response rate has become a gradual problem. As such, use of PD-L1 monoclonal antibody in combination with other anticarcinoma drugs has been the main strategy in improving its efficacy. Interleukin 10 (IL10) is a recognized inflammatory and immunosuppressive factor. Previous studies have suggested that there is a link between PD-L1 and IL10. Objective This study was aimed at clarifying the relationship between PD-L1 and IL10 in liver hepatocellular carcinoma (LIHC) and whether IL10 enhances the efficacy of PD-L1 inhibitor. Methods Expression levels of PD-L1 and IL10 in carcinoma and adjacent tissues were tested by immunochemistry, Western blotting, and RT-PCR. Survival duration and follow-up data of each patient were recorded. LIHC cell lines Bel7405 and MHCC 97-H were used for in vitro experiments. Exogenous IL10 and anti-IL10 were added to cell supernatant. Expression level of PD-L1 in the LIHC cell lines was determined using Western blotting and ELISA. CCK8 and transwell assays were adopted to examine the effect of PD-L1 combined with IL10 on proliferation, invasion, and metastasis of LIHC cells. Results The survival period of patients with low expression of IL10 was longer than that of patients with high expression (P = 0.01). Overexpression of PD-L1 increased the IL10 and Met levels in LIHC tissues and cell lines. IL10 downregulated the expression level of PD-L1 and enhanced the efficacy of crizotinib via the Met signaling pathway in the LIHC cells. Conclusions A combination of IL10 and PD-L1 inhibitor holds great promise as an effective treatment for LIHC.

Crizotinib inhibits activation of MET pathway caused by MET extracellular SEMA domain duplication

ObjectiveAberrant MET activation, which promotes cell proliferation and tumor metastasis, occurs in many types of cancer and results from multiple mechanisms. A novel MET duplication mutation was found in a non-small cell lung cancer (NSCLC) patient. The clinical response to crizotinib was investigated and the functional relevance was characterized in cellular models. Materials and MethodsNext-generation sequencing (NGS) was performed on the tumor tissue and circulating tumor DNA (ctDNA) of a patient with advanced NSCLC. In vitro studies including western blot, proliferation assays and colony formation assays were used to confirm the clinical observations. ResultsThe patient was identified to harbor a duplication of the MET SEMA domain. After a month of treatment, the patient showed a marked response to crizotinib, a multikinase inhibitor with potent activity against MET. Functional in vitro studies demonstrated that expression of MET SEMA duplication in NIH-3T3 cells stimulated the activation of MET signaling. Crizotinib treatment obviously repressed cell proliferation, colony formation, and MET signaling pathway. ConclusionCrizotinib treatment resulted in a clinical response in a patient with MET SEMA duplication. Results of cellular analyses together with the clinical data suggest that this novel alteration may represent an actionable target in NSCLC patients.

Relationship between IL-10 and PD-L1 in esophageal carcinoma tissues and IL-10 down-regulates PD-L1 expression via Met signaling pathway.

Programmed death-ligand 1 (PD-L1) plays a critical role in host immunity in the setting of cancer progression. Interleukin 10 (IL-10) is a multi-cellular, multi-functional cytokine that regulates cell growth and differentiation and participates in inflammatory and immune responses. The purpose of this study was to clarify the relationship between PD-L1 and IL-10 and their clinical importance in esophageal carcinoma (ESCA). ESCA patients (n=100) who underwent surgery with preoperative therapy were included in the study. By immuno-histochemical staining, PD-L1, IL-10 and CD8 positive cells were examined in resected specimens. The gene expression levels of PD-L1, IL-10 and Met were detected by qRT-PCR and Western blots, and differentially compared in cancer, adjacent and normal tissues. In cell experiments, the Eca109 and TE-1 cell-lines were incubated with IL-10 or anti-IL-10 antibody, and then PD-L1 and Met expression levels were compared by ELISA and Western blots. The effect of crizotinib and/or IL-10 on the proliferation, invasion and migration of esophageal squamous cell-lines was estimated by CCK8 and transwell assay. In tumor tissues, the mRNA and protein levels of PD-L1, IL-10 and Met were higher than those in adjacent tissues. The high expression levels of PD-L1 and IL-10 indicated a poor prognosis. IL-10 reduced the expression of PD-L1 in esophageal squamous cell-lines via Met signaling. Over-expression of PD-L1 in increased the levels of IL-10, and Met in in ESCA tissue and cell lines. The combination of crizotinib and IL-10 were more effective in inhibiting the proliferation, migration and invasion of esophageal squamous cell lines. The combination of IL-10 and PD-L1 monoclonal antibody may have therapeutic promise in treating ESCA.

Transformation of Receptor Tyrosine Kinases into Glutamate Receptors and Photoreceptors.

Receptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand-binding domain of metabotropic glutamate receptor type 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate-based photoswitch via a self-labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light-controlled human insulin receptor (LihIR) and light-controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photoreceptors.

Transformation of Receptor Tyrosine Kinases into Glutamate Receptors and Photoreceptors

AbstractReceptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand‐binding domain of metabotropic glutamate receptor type 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate‐based photoswitch via a self‐labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light‐controlled human insulin receptor (LihIR) and light‐controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photoreceptors.

Abstract P2-20-06: Comparative effect of Nerlynx & HER2+ positive & negative breast cancer cell lines on the barrier function of human brain endothelial cells

Abstract Background Brain Metastasis in Breast Cancer (BMBC) is a significant contributor to mortality & presents a challenge to modern treatment of advanced breast cancer. HER2+ breast cancers have a predilection for metastasis to the brain & therefore the HER2 inhibitor Nerlynx could exhibit efficacy in treating BMBC. Previous work suggests that Nerlynx can pass through the Blood-Brain Barrier (BBB) & may alter it during treatment of BMBC. Methods The BBB was modelled using hCMEC-D3 cells. BMBC was modelled using HER2- MDA-MB-231 cells or HER2+ SK-BR-3 cells. To evaluate changes in protein expression, hCMEC-D3 cells were treated with 50 nM Nerlynx ± SK-BR-3-conditioned medium & analysed using the Kinexus microarray service. Functional changes in electrical resistance, permeability, proliferation & migration were measured using Trans-Endothelial Resistance (TEER) & Paracellular Permeability (PCP) assays. Changes in TJ protein location were assessed using immunofluorescence (IF). Results Protein microarray analysis compared to the control, co-treatment with 50 nM Nerlynx & SK-BR-3-CM was associated with increased expression of migration-associated proteins (ROCK1, +166%) & cell cycle regulatory components (Rb, +114%; CDK6, +159%; PLK1, +168%) as well as the guanine exchange factor VAV1 (+123%), intracellular kinases RSK1 (+110%) & PKCβ (+107%), & the HGF receptor MET (+74%). In contrast, decreased expression of the transcription factor STAT2 (-98%), the cell cycle regulators CDK2 (-85%) & CDK1 (-75%), focal adhesion component Paxillin 1 (-74%) & the RTK Kit (- 59%) are features of co-treatment compared to control. Compared to treatment with SK-BR-3-CM alone, co-treatment with Nerlynx was associated with an increase in the expression of Paxillin 1 (+809%), AKT1 (+458%) & MET (+449%), as well as CDK6 (+310%), PDK1 (+215%), the RTK DDR1 (+212%), lymphangiogenic receptor VEGFR3 (+203%) & Rb (+183%). Compared to treatment with SK-BR-3-CM alone, co-treatment with Nerlynx was associated with a decrease in the expression of STAT2 (-98%), IRS1 (-92%), ROCK2 (-84%), CSK (-73%), Kit (-71%), STAT3 (-68%), JAK2 (-67%) & VEGFR2 (-64%) & an increase in the expression of ILK1 (+554%), Kit (+448%), AKT1 (+337%), Rb (+261%) & VAV1 (+184%). However, compared to treatment with Nerlynx alone, co-treatment with SK-BR-3 CM was associated with a decrease in the expression of IRS1 (-99%), STAT2 (-98%), Paxillin 1 (-92%), IKKα (-78%), CDK1 (- 65%) FAK (-63%) & STAT3 (-62%). hCMEC-D3 co-treated with MDA-MB231-CM, 100 nM Nerlynx treatment exhibited increased PCP, after 60 min post-treatment & a similar effect was seen after 5 nM Nerlynx treatment at 120 min. In hCMEC-D3 monolayers co-treated with SK-BR-3- CM, 100 nM Nerlynx there was a decrease in PCP by 120 min post-treatment. Conversely, treatment with 5 nM or 50 nM Nerlynx led to an increase in PCP by 60 min or 180 min, respectively. Nerlynx treatment inhibited the proliferation of hCMEC-D3 cells in a dose-dependent manner with larger effects seen upon the addition of medium conditioned by MDA-MB-231 cells or SK-BR-3 cells. There was a concurrent change in distribution of TJ proteins as assessed using IF. Discussion Nerlynx inhibits the proliferation of brain endothelial cells but its role in altering barrier function is unclear. Nerlynx exerts a greater effect on brain endothelial cells in the presence of medium conditioned by either HER2+ or HER2- cells, suggesting it may be efficacious in the treatment of BMBC. The MET signalling pathway is implicated as a potential regulator of TJ-mediated BBB permeability in BMBC. Further work including BBB co-culture models, alternative brain endothelial lines & evaluation at the gene & protein level are warranted. Citation Format: Tracey Amanda Martin, Isaac Meyers, Emily Appadurai, Wen G Jiang. Comparative effect of Nerlynx & HER2+ positive & negative breast cancer cell lines on the barrier function of human brain endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-06.

MicroRNA‑200a suppresses migration and invasion and enhances the radiosensitivity of NSCLC cells by inhibiting the HGF/c‑Met signaling pathway.

Hepatocyte growth factor (HGF), an activator of the c-Met signaling pathway, is involved in tumor invasiveness, metastasis and radiotherapy resistance. In the present study, a novel HGF regulatory pathway in lung cancer involving microRNAs (miRNAs/miR) is described. Immunohistochemical staining and western blot analyses demonstrated that HGF was upregulated and associated with miR-200a downregulation in non-small cell lung cancer (NSCLC) samples compared with normal lung tissues. The association between HGF and miR-200a was associated with the degree of tumor malignancy and cell migration and invasion. miR-200a negatively regulated HGF expression by targeting the 3′-untranslated region of the HGF mRNA. miR-200a overexpression induced HGF downregulation, decreased NSCLC cell migration and invasion, promoted apoptosis, and decreased cell survival in A549 and H1299 cells in response to ionizing radiation. The present results revealed a previously uncharacterized role of miRNA-200a in regulating tumor malignancy and radiosensitivity by suppressing HGF expression, a key factor in the HGF/c-Met pathway.

The effect of low-intensity ultrasound and met signaling on cellular motility and morphology

During the past decade, the potential of low-intensity ultrasound (LIUS) was demonstrated in medical research including microvascular remodeling, wound repair, blood flow restoration, angiogenesis and activation of mechanosensitive signaling pathways. However, the current clinical application of LIUS hasn’t been extended beyond physiotherapy. In cancer therapy, LIUS related research is focused on ultrasound-mediated techniques for chemotherapeutic drug activation, drug delivery enhancement and gene transfection improvement. While clinical trials show highly promising results, the ambiguity regarding the mechanism of ultrasound-cell interaction is a major limitation in the road to clinical applications. In 2011, the bilayer sonophore (BLS) model suggested that cell intramembrane cavitation is the underlying mechanism for ultrasound-induced bio-effects. According to the BLS model, ultrasound affects cell shape and functionality by inducing intramembrane gas bubbles that deform the cell membrane. This mechanical effect might involve alterations in the bio-chemical signaling pathways. Many biological processes such as cell motility, proliferation and angiogenesis are triggered by MET tyrosine kinase growth factor receptor and its ligand HGF/SF (Hepatocyte growth factor/Scatter Factor). The activation of MET receptor increases cancer cell motility via membrane alterations, leading to epithelial to mesenchymal transformation (EMT), invasion and metastasis. The aim of this work is to examine the crosstalk between MET-activation and LIUS under the assumption that both induce cell membrane alterations whereby affecting cell morphology and motility. In this paper, we present preliminary observations regarding the effect of MET-activation, LIUS exposure and their combined effect on living cells. Measurements of morphokinetic parameters using single cell time-lapse analysis are presented as well. The analysis demonstrates that LIUS inhibits the motility and modulates the morphology of MET-activated cells. These findings demonstrate the balance between the inner cellular processes (MET signaling pathways) and the external forces (LIUS exposure). Understanding the mechanism of LIUS on motile cells might improve the performance of existing LIUS-based cancer treatment modalities and help develop new ones.

Abstract 1816: Mechanism of resistance to single treatment with MET inhibitor in EGFR-mutant lung cancer cell secondarily acquiring c-MET amplification

Abstract Amplification in c-MET is the second most common mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI) in lung cancer harboring activated EGFR mutations. An acquired resistance mechanism to single treatment with MET kinase inhibitor remains unknown. In this study, we modeled acquired resistance to experimental type I MET kinase inhibitor PHA66572 in MET-amplified HCC827 lung cancer cell line, which was established under chronic exposure to gefitinib in our lab. We found that reactivation of the EGFR pathway emerges as a resistance mechanism in MET secondarily amplified EGFR-mutant lung cancer cells after prolonged exposure to PHA66572. Any gene alteration influencing sensitivity to the drugs was not observed. Long treatment with only MET inhibitor resulted in partial resensitization to EGFR-TKI in these resistant lung cancer cells. Thus, the combination of gefitinib and PHA66572 could completely suppress the cell growth. Our findings support an intimate relationship between the EGFR and MET signaling pathways in HCC827 lung cancer cell line. Citation Format: Youngjoo Lee, Yu-Ra Choi. Mechanism of resistance to single treatment with MET inhibitor in EGFR-mutant lung cancer cell secondarily acquiring c-MET amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1816.

Abstract 3625: MET signaling upregulates PD-L1 expression in human lung and gastric cancer

Abstract Background: Tyrosine kinase receptor, MET, plays an important role for the progression of tumor through diverse mechanisms. Cancer immunotherapy targeting PD-1/PD-L1 pathway shows clinical benefit in patient with PD-L1 expressing solid tumor. In our previous study on non-small cell lung cancer (NSCLC) using immunohistochemistry, MET overexpression was significantly associated with programmed cell death-ligand 1 (PD-L1) overexpression. Thus, we investigated if MET signaling pathway is involved in PD-L1 expression in tumor cells. Method and Result: Lung adenocarcinoma cell lines including H596 (harboring MET exon 14 skipping mutation), H1993 (harboring MET gene amplification), H23 and H522 (MET wild-type), and a gastric carcinoma cell line, Hs746T (harboring MET exon 14 skipping mutation and gene amplification) were used. Hs746T cells were treated with MET inhibitor or transfected with MET siRNA and subject to microarray analysis. MET inhibition or knockdown led to changes in expression level of variable immune-related molecules, of which PD-L1 was one of the most down-regulated molecules. Stimulation of H596, H23 and H522 cells with HGF, a MET ligand, increased PD-L1 expression at the mRNA and protein level. Treatment of H1993 and Hs746T cells with MET inhibitor or MET knockdown using siRNA resulted in down-regulation of PD-L1 at the mRNA, total protein, and surface expression levels. PI3K/Akt and MAPK/Erk signaling pathways were involved in MET-mediated PD-L1 up-regulation in H1993 and Hs746T cells. Finally, the Cancer Genome Atlas (TCGA) analysis showed that MET and PD-L1 expression are significantly positively correlates with each other in patients with lung cancer (in NSCLC, rho=0.219 [p<0.001]; in adenocarcinoma, r=0.401 [p<0.001]; in squamous cell carcinoma, r=0.144 [p=0.001]) and in patients with gastric cancer (r=0.137 [p=0.005]). Conclusion: This study demonstrates that MET signaling increases PD-L1 transcription and protein overexpression in lung and gastric cancer cells, which might contribute to immune escape of tumor via PD-1/PD-L1 pathway. MET-targeted therapy may be considered as combination therapy with PD-1/PD-L1 blockades. Citation Format: Hyunkyung Ahn, Sehui Kim, Ji Young Jang, Dohee Kwon, Jaemoon Koh, Young A Kim, Doo Hyung Chung, Yoon Kyung Jeon. MET signaling upregulates PD-L1 expression in human lung and gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3625.

Met is involved in TIGAR-regulated metastasis of non-small-cell lung cancer.

TIGAR is a p53 target gene that is known to protect cells from ROS-induced apoptosis by promoting the pentose phosphate pathway. The role of TIGAR in tumor cell invasion and metastasis remains elusive. Here we found that downregulation of TIGAR reduced the invasion and metastasis of NSCLC cells in vitro and in vivo. Immunohistochemical analysis of 72 NSCLC patients showed that TIGAR and Met protein expression was positively correlated with late stages of lung cancer. Besides, patients with high co-expression of TIGAR and Met presented a significantly worse survival. In addition, we found that Met signaling pathway is involved in TIGAR-induced invasion and metastasis. Our study indicates that TIGAR/Met pathway may be a novel target for NSCLC therapy. It is necessary to evaluate the expression of TIGAR before Met inhibitors are used for NSCLC treatment.

Foretinib (GSK1363089) induces p53-dependent apoptosis in endometrial cancer.

ObjectiveForetinib (GSK1363089 or XL880), which is an oral multikinase inhibitor developed to primarily target the hepatocyte growth factor (HGF)/Met signaling pathway, has shown anti-tumor effects against some cancers in preclinical and clinical studies.ResultsHGF/Met signaling in endometrial cancer cell lines was stimulated in an autocrine manner, and was essential for cell survival. Inhibiting the HGF/Met signaling with foretinib induced p53-dependent apoptosis in endometrial cancer cell lines in vitro. Foretinib also showed significant anti-cancer effects in vivo in experiments using cell tumor xenografts. p53 mutations were observed in 37 (10.8%) of 344 endometrial cancer specimens.ConclusionThe HGF/Met-MAPK/PI3K pathway in endometrial cancer is activated by HGF in an autocrine manner. Foretinib induces an anti-cancer effect through the anti-phosphorylation of Met, which results in the induction of p53-dependent apoptosis; foretinib was found to exert greater anti-cancer activity in endometrial cancer specimens with wild-type p53 than in specimens with p53 mutations. Our immunochemical analysis revealed that foretinib-induced p53-dependent apoptosis can be expected to have therapeutic potential in approximately 90% of endometrial cancer patients.MethodsWe evaluated the HGF/Met signaling pathway in endometrial cancer cell lines and assessed the anti-cancer effects of foretinib using in vitro and in vivo experimental models. Furthermore, endometrial cancer specimens were subjected to an immunohistochemical analysis.

Abstract 3201: Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) related mortality ranks second worldwide and stands one step behind lung cancer. Due to the asymptomatic nature of the cancer, early diagnosis is a major problem. Currently only liver transplantation is curative for early stage hepatocellular cancer. There are other multidisciplinary treatment options like radiation and chemotherapy available for advanced patients. However, chemotherapy resistance is a common problem in the treatment of liver cancer. Newer therapeutics are needed for better management of advanced HCC. Angiogenesis and lymphangiogenesis are processes that are vital for tumorigenesis and metastasis. HCC is known to be a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. We explored the therapeutic potential of the anti-angiogenic and anti-lymphangiogenic, biomimetic peptide SP2043 developed by our group in HCC. Hepatocellular carcinoma cell lines HuH-7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration following treatment. Furthermore, SP2043 was found to impair microvascular endothelial cell (MEC) tube formation induced by HepG2 tumor conditioned media and to significantly inhibit HepG2 tumor xenograft growth compared to untreated controls. The peptide drug was also found to improve the survival of autochthonous Myc induced HCC in a transgenic mouse model from two weeks to four weeks. We discovered the peptide to be a multi-modal anti-angiogenic drug that also inhibits IGF1R and MET signaling pathways in HCC cell lines. We also found that SP2043 treatment reduced the microvascular density in both subcutaneous and autochthonous liver tumors with reduced tumor cell proliferation and increased apoptosis. This study shows the therapeutic potential of SP2043 in the treatment of hepatocellular carcinoma. Note: This abstract was not presented at the meeting. Citation Format: Mustafa A. Barbhuiya, Adam C. Mirando, Brian W. Simons, Ghali Lemtiri-Chlieh, Jordan J. Green, Aleksander S. Popel, Niranjan B. Pandey, Phuoc T. Tran. Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma [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 3201. doi:10.1158/1538-7445.AM2017-3201