c-Met Protein Expression Research Articles

165P Consistency analysis of c-Met protein expression over time in patients with non-squamous non-small cell lung cancer

165P Consistency analysis of c-Met protein expression over time in patients with non-squamous non-small cell lung cancer

Retraction Note: Regulation of miRNAs on c-met protein expression in ovarian cancer and its implication.

The article "Regulation of miRNAs on c-met protein expression in ovarian cancer and its implication", by H. Liu, S.-R. Li, Q. Si, published in Eur Rev Med Pharmacol Sci 2017; 21 (15): 3353-3359-PMID: 28829508 has been retracted by the Editor in Chief. Following some concerns raised on PubPeer regarding a possible manipulation in Figure 5 (link: https://pubpeer.com/publications/7B6E6E6679990661654EBCAF472921), the Editor in Chief has started an investigation to assess the validity of the results as well as possible figure manipulation. The authors were informed about the journal's investigation but remained unresponsive and have not provided the manuscript's raw data. The journal's investigation revealed a figure overlap between panels pcDNA3.1-EGFP and pcDNA3.1-EGFP-204-up in Figure 5. Consequently, the Editor in Chief mistrusts the results presented and has decided to retract the article. This article has been retracted. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/13200.

Mesenchymal Stem Cell-Derived Exosomes Mitigate Acute Murine Liver Injury via Ets-1 and Heme Oxygenase-1 Up-regulation.

Mesenchymal stem cells (MSCs)-derived exosomes have been previously demonstrated to promote tissue regeneration in various animal disease models. This study investigated the protective effect of exosome treatment in carbon tetrachloride (CCl4)-induced acute liver injury and delineated possible underlying mechanism. Exosomes collected from conditioned media of previously characterized human umbilical cord-derived MSCs were intravenously administered into male CD-1 mice with CCl4-induced acute liver injury. Biochemical, histological and molecular parameters were used to evaluate the severity of liver injury. A rat hepatocyte cell line, Clone-9, was used to validate the molecular changes by exosome treatment. Exosome treatment significantly suppressed plasma levels of AST, ALT, and pro-inflammatory cytokines, including IL-6 and TNF-α, in the mice with CCl4-induced acute liver injury. Histological morphometry revealed a significant reduction in the necropoptic area in the injured livers following exosome therapy. Consistently, western blot analysis indicated marked elevations in hepatic expression of PCNA, c-Met, Ets-1, and HO-1 proteins after exosome treatment. Besides, the phosphorylation level of signaling mediator JNK was significantly increased, and that of p38 was restored by exosome therapy. Immunohistochemistry double staining confirmed nuclear Ets-1 expression and cytoplasmic localization of c-Met and HO-1 proteins. In vitro studies demonstrated that exosome treatment increased the proliferation of Clone-9 hepatocytes and protected them from CCl4-induced cytotoxicity. Kinase inhibition experiment indicated that the exosome-driven hepatoprotection might be mediated through the JNK pathway. Exosome therapy activates the JNK signaling activation pathway as well as up-regulates Ets-1 and HO-1 expression, thereby protecting hepatocytes against hepatotoxin-induced cell death.

Mesenchymal-epithelial transition factor amplification correlates with adverse pathological features and poor clinical outcome in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related mortality worldwide. Mesenchymal-epithelial transition factor (MET) gene participates in multiple tumor biology and shows clinical potential for pharmacological manipulation in tumor treatment. MET amplification has been reported in CRC, but data are very limited. Investigating pathological values of MET in CRC may provide new therapeutic and genetic screening options in future clinical practice. To determine the pathological significance of MET amplification in CRC and to propose a feasible screening strategy. A number of 205 newly diagnosed CRC patients undergoing surgical resection without any preoperative therapy at Shenzhen Cancer Hospital of Chinese Academy of Medical Sciences were recruited. All patients were without RAS/RAF mutation or microsatellite instability-high. MET amplification and c-MET protein expression were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Correlations between MET aberration and pathological features were detected using the chi-squared test. Progression free survival (PFS) during the two-year follow-up was detected using the Kaplan-Meier method and log rank test. The results of MET FISH and IHC were compared using one-way ANOVA. Polysomy-induced MET amplification was observed in 14.4% of cases, and focal MET amplification was not detected. Polysomy-induced MET amplification was associated with a higher frequency of lymph node metastasis (LNM) (P < 0.001) and higher tumor budding grade (P = 0.02). In the survival analysis, significant difference was detected between patients with amplified- and non-amplified MET in a two-year follow-up after the first diagnosis (P = 0.001). C-MET scores of 0, 1+, 2+, and 3+ were observed in 1.4%, 24.9%, 54.7%, and 19.0% of tumors, respectively. C-MET overexpression correlated with higher frequency of LNM (P = 0.002), but no significant difference of PFS was detected between patients with different protein levels. In terms of concordance between MET FISH and IHC results, MET copy number showed no difference in c-MET IHC 0/1+ (3.35 ± 0.18), 2+ (3.29 ± 0.11) and 3+ (3.58 ± 0.22) cohorts, and the MET-to-CEP7 ratio showed no difference in three groups (1.09 ± 0.02, 1.10 ± 0.01, and 1.09 ± 0.03). In CRC, focal MET amplification was a rare event. Polysomy-induced MET amplification correlated with adverse pathological characteristics and poor prognosis. IHC was a poor screening tool for MET amplification.

Topographic analysis of pancreatic cancer by TMA and digital spatial profiling reveals biological complexity with potential therapeutic implications

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies. Tissue microarrays (TMA) are an established method of high throughput biomarker interrogation in tissues but may not capture histological features of cancer with potential biological relevance. Topographic TMAs (T-TMAs) representing pathophysiological hallmarks of cancer were constructed from representative, retrospective PDAC diagnostic material, including 72 individual core tissue samples. The T-TMA was interrogated with tissue hybridization-based experiments to confirm the accuracy of the topographic sampling, expression of pro-tumourigenic and immune mediators of cancer, totalling more than 750 individual biomarker analyses. A custom designed Next Generation Sequencing (NGS) panel and a spatial distribution-specific transcriptomic evaluation were also employed. The morphological choice of the pathophysiological hallmarks of cancer was confirmed by protein-specific expression. Quantitative analysis identified topography-specific patterns of expression in the IDO/TGF-β axis; with a heterogeneous relationship of inflammation and desmoplasia across hallmark areas and a general but variable protein and gene expression of c-MET. NGS results highlighted underlying genetic heterogeneity within samples, which may have a confounding influence on the expression of a particular biomarker. T-TMAs, integrated with quantitative biomarker digital scoring, are useful tools to identify hallmark specific expression of biomarkers in pancreatic cancer.

Anti-hepatocellular carcinoma activity of Sorbaria sorbifolia by regulating VEGFR and c-Met/apoptotic pathway

Ethnopharmacological relevanceSorbaria sorbifolia (SS) is a traditional Chinese medicine (TCM) that has been employed anti-hepatocellular carcinoma (HCC) for over 2000 years; yet, its underlying mechanism is still not fully understood. Aim of the studyIn this study, we evaluated the anti-HCC effect on the freeze-dried powder of the water extract of SS (FDSS) by inhibiting tumor-induced neovascularization, and promoting apoptosis, and elucidated the underlying mechanisms. Materials and methodsHCC cell lines (HepG2 and Huh7 cells) and HepG2 xenograft tumors in zebrafish were employed as in vivo and in vitro models, respectively, to evaluate the anti- HCC-indued neovascularization and apoptosis. In HCC cell lines, CCK-8 assay, wound-healing assay, transwell assay, cell circle assay, apoptosis assay, transmission electron microscopy, and co-culture assay were performed in vitro; in HepG2 xenograft tumor-zebrafish, tumor growth inhibition assay, hematoxylin and eosin (HE) staining, xenograft tumor-zebrafish apoptosis assay, and HCC-indued neovascularization assay were performed to evaluate the effect of FDSS on biological behavior of tumor, HCC-indued neovascularization, and apoptosis. The expression of VEGFR and c-Met/apoptotic pathway-related proteins was detected by western blotting analysis. Assays for c-Met and VEGFR activation were conducted to assess the impact of FDSS in either agonistic or inhibitory roles on these receptor proteins. ResultsThe findings from our study revealed that FDSS effectively suppresses the proliferation, migration, and invasion of HepG2 and Huh7 cells, as well as inhibiting tumor growth in the HepG2 xenograft zebrafish model by downregulating the expression of p-Met and p-AKT proteins. FDSS decreased the tumor growth associated with promoting apoptosis, including arresting HepG2 and Huh7 cells cycle at G0/G1phase, increasing apoptotic cell numbers and apoptotic bodies in cancer cells, and increasing the apoptotic fluorescence of xenograft tumor zebrafish by downregulating Bcl-2 proteins and upregulating Bax, caspase-9, and caspase-3 levels. We also found that FDSS can inhibit HCC-induced neovascularization and regulate VEGFR. Using an agonist or inhibitor of c-Met and VEGFR in HepG2 cells, we discovered that FDSS can downregulate c-Met and VEGFR protein expression. ConclusionFDSS exerts an anti-HCC effect by inhibiting HCC-indued neovascularization and pro-apoptosis through the inhibition of the action of VEGFR and c-Met/apoptotic pathway.

Low co-expression of PD-L1 and oncogenic receptor tyrosine kinases HER2 and cMET in urothelial carcinoma is associated with discordant expression between primary and metastatic sites

Novel regimens targeting immune checkpoints and the cMET or HER2 pathways are under investigation in metastatic urothelial carcinoma (mUC) though co-expression of these molecular targets has not been defined. We sought to characterize the protein co-expression rates of PD-L1, cMET and HER2 in primary and metastatic mUC lesions and agreement rates in paired biopsies.We assessed PD-L1, cMET and HER2 protein expression by immunohistochemistry (IHC) in archival mUC samples identified from an institutional database (n = 143). Correlation of expression between primary and metastatic biopsies was performed in patients with available paired biopsies (n = 79). Protein expression levels by predefined thresholds were measured, and Cohen's kappa statistics (κ) were utilized to assess the agreement in expression between paired primary and metastatic samples.In primary tumors (n = 85), high expression of PD-L1, cMET, and HER2 was observed in 14.1%, 34.1%, and 12.9%, respectively. In metastatic samples (n = 143), high expression of PD-L1, cMET and HER2 was detected in 9.8%, 41.3%, and 9.8%, respectively. Expression agreement rates between paired specimens (n = 79) were PD-L1: 79.7% (κ = 0.09), cMET: 69.6% (κ = 0.35), HER2: 84.8% (κ = 0.17). High PD-L1/cMET co-expression was observed in only 5.1% (n = 4) of primary and 4.9% (n = 7) of metastatic specimens. High co-expression of PD-L1/HER2 occurred in 3.8% (n = 3) of primary samples and no metastatic samples. The overall co-expression agreement between paired samples was 55.7% (κ = 0.22) for PD-L1/cMET and 67.1% (κ = 0.06) for PD-L1/HER2, but agreement for high co-expression between paired samples was very low (2.5% for PD-L1/cMET and 0% for PD-L1/HER2).Tumor co-expression of high cMET or HER2 and PD-L1 is low in this cohort. Agreement of high co-expression between primary and metastatic sites is rare. Biomarker-based strategies used in selection of patients for contemporary trials testing combinations of immune checkpoint inhibitors with either cMET or HER2-targeted agents should account for discordant biomarker expression between primary and metastatic sites.

Identification of c-Met as a novel target of γ-glutamylcyclotransferase.

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation.

1175-P: The Alteration of Serum Level and Expression of c-MET in Gestational Diabetes Mellitus (GDM)

Objective: The aim of this study was to explore changes in serum levels and tissue expression of HGF and c-MET, and to determine how HGF/c-MET signaling contributes to the pathophysiology of GDM. Methods: A total of 44 pregnant women with normal glucose tolerance (NGT) and 32 pregnant women with GDM were studied. Blood samples were taken just before delivery, and placenta and visceral adipose tissue were taken immediately after cesarean delivery. Serum levels of HGF, soluble c-MET and insulin were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of HGF and c-MET was measured in the placenta and visceral adipose tissue. In addition, we investigated pregnancy of high fat diet (HFD) mice as an animal model of GDM. The glucose tolerance test (GTT) was performed at GD 10, and measured the serum levels of Hgf and soluble c-Met at GD 16. After scarifying mice, we measured the weight and expression of mRNA of Hgf and c-Met in WAT, placenta, liver and muscle. Results: Serum soluble c-MET levels were significantly higher in the GDM group (p &amp;lt;0.001). In multiple logistic regression analysis, soluble serum c-MET level was strongly associated with GDM (OR 9.063, p=0.027). There were more patients with increased mRNA expression of HGF and c-MET in placenta. Interestingly, the protein expression of HGF and c-MET in maternal visceral fat tissue was significantly higher in the GDM group. In mouse study, maternal serum levels of soluble c-Met in pregnant HFD (PH) mice were higher than in non-pregnant HFD (NPH) mice and pregnant chow diet (PC) mice. Relative mRNA expression of Hgf was significantly increased in eWAT of PH than PC mice. Relative c-Met mRNA expression was significantly increased in sWAT of PH than PC mice. Conclusion: We observed the differences in serum levels and expression of HGF and c-MET between GDM and control pregnant women in human and mice model. Our results suggest that HGF/c-MET signaling may be altered in GDM, which associated with insulin resistance and pathophysiology in GDM. Disclosure H.Kim: None. J.Lee: None. Y.Jung: None. M.Lee: None. J.Kim: None. K.Joung: None. B.Ku: Speaker's Bureau; AstraZeneca, Boehringer-Ingelheim, Eli Lilly and Company, Sanofi, Novo Nordisk, LG Chem. Funding National Research Foundation of Korea (2022R1F1A1074608)

Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Abstract 5087: Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of all breast cancer and has limited therapeutic option except for immunotherapy because ER, PR, and HER2 targeted for treatment are absent. It also tends to be more aggressive meaning more likely to metastasize faster to other organs such as lungs, liver, and bone, and its recurrence rate is high which leads to a poor prognosis. Immunotherapy is an option to treat TNBC such as programmed death ligand 1 (PD-L1) antibody. However, tumor cells use various immune evasion mechanisms in addition to PD-1/PD-L1 pathway to interrupt the effect of immunotherapy. Moreover, tumor microenvironment (TME) is one of the important factors for the effective anti-tumor immune responses from immunotherapy. Hypoxia is a typical event in TNBC. Hypoxia inducible factor 1-alpha (HIF1α) is a transcription factor that regulates angiogenesis by expressing VEGF, and in addition, upregulates the c-MET which increases the proliferation, migration, and survival of tumor cells. Moreover, HIF1α directly regulates the expression of CD47. CD47 is a transmembrane protein that binds to SIRPα of macrophages to inhibit the phagocytosis of macrophages. Therefore, the efficacy of immune checkpoint inhibitors decreases even if there is a higher degree of tumor-infiltrating lymphocytes (TILs). Combination therapy with immune checkpoint and tumor associated antigens (TAAs) derived peptide vaccine could inhibit metastasis. In present study, we found that HIF1α/c-MET peptide vaccination delayed metastasis in C3(1)Tag mouse. We also investigated whether combination therapy with immune checkpoint inhibitors and peptide vaccine inhibited metastasis. Next, we observed that combination strategy of HIF1α/c-MET peptide vaccine and treated anti-PD-L1 antibody or/and anti-CD47 antibody was more effective in systemically inoculated M6 cells growth in C3(1)Tag mice. Immunohistochemical analysis of the tumors showed that the protein expression of CD47, HIF1α, and c-MET were significantly decreased. On the other hand, CD4+ T cells, CD8+ T cells and M1 macrophages increased, but M2 macrophages decreased, resulting in delayed metastasis in combination therapy group compared to single therapy and control group. In addition, IFNγ ELISPOT showed that peptide vaccination significantly increased the HIF1α/c-MET specific IFNγ-secreting T cell response in splenocytes. We also assessed osteoclastogenesis in bones, which showed reduction of osteoclast in combination therapy group compared to single therapy and control group.Taken together, combination therapy with immune checkpoint inhibitor targeting PD-L1 and CD47 and peptide vaccine appears to be a promising strategy that inhibit metastasis by modulating tumor microenvironment in triple negative breast cancer. Citation Format: JinHwa Hong, Jimin Lee, Soon Young Lim, Ju Won Kim, Ah Reum Lim, Kyong Hwa Park. Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer. [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 5087.

C-Met Receptors Deficiency Was Involved in Absence Seizures Development in WAG/Rij Rats

Background: A variety of receptors may be involved in the pathogenesis of absence seizures. The c-Met receptors have a critical role in modulating the GABAergic interneurons and creating a balance between excitatory and inhibitory neurotransmission, sensorimotor gating, and normal synaptic plasticity. Objectives: This study aimed to assess the changes of the c-Met receptor during the appearance of absence attacks in the experimental model of absence epilepsy. Methods: A total of 48 animals were divided into four groups of two- and six-month-old WAG/Rij and Wistar rats. Epileptic WAG/Rij rats showing SWP in electrocorticogram (ECoG) were included in the epileptic group. The two-month-old WAG/Rij rats as well as two- and six-month-old Wistar rats not exhibiting SWP in ECoG were selected as the non-epileptic. Gene (RT-PCR) and protein expression (western blotting) of c-Met receptors as well as c-Met protein distribution (immunohistochemistry) in the somatosensory cortex and hippocampus were assessed during seizure development of the absence attacks. Results: According to the study findings, a lower c-Met gene and protein expression, as well as a lower protein distribution, were observed in the hippocampus (P &lt; 0.001, P &lt; 0.05, and P &lt; 0.001, respectively) and cortex (P &lt; 0.01, P &lt; 0.001 and P &lt; 0.001, respectively) of the two-month-old WAG/Rij rats compared to the same-age Wistar rats. Moreover, the data revealed a reduction of hippocampal and cortical c-Met protein expression (P &lt; 0.001, for both) in six-month-old WAG/Rij rats compared to two-month-old ones. Six-month-old WAG/Rij rats had a lower cortical c-Met gene (P &lt; 0.05) and protein expression (P &lt; 0.001) as well as lower hippocampal and cortical protein distribution (P &lt; 0.05 and P &lt; 0.001) than the same-age Wistar rats. Conclusions: In sum, the c-Met receptor was found to play a significant role in the development of absence epilepsy. This receptor, therefore, may have been considered as an effective goal for absence seizure inhibition.

Radiation modulates expression and related activities of c-Met protein in oral tongue squamous cell carcinoma cell lines

Objectivesc-Met, a receptor tyrosine kinase, is involved in the growth, invasion and metastasis of a variety of cancers. In a set of cell lines from several solid tumors, a five-fold increase in c-Met expression after irradiation has been reported. This study aimed to assess if c-Met is likewise abundantly expressed in oral tongue squamous cell carcinoma (OTSCC) upon exposure to irradiation, followed by a Met-induced biological response.Materials and methodsSix OTSCC cell lines were exposed to gamma radiation doses of 2, 4, and 6 Gray. The changes in c-Met protein levels were assessed by western blot and flow cytometry. c-Met gene expression, cell migration, proliferation and cell cycle assays were performed as phenotypic readouts.ResultsIrradiation resulted in upregulation of c.Met in all cell lines with different time kinetics. On average the cells displayed minimal c-Met expression on their surface ranging from 5 to 30% of total protein. Abrupt downregulation of c-Met surface expression occurred one hour after radiation but recovered 48 h post-radiation. Intracellularly, the highest level of expression was found on day 5 after radiation exposure. Irradiation induced aggressive invasive potential of the cells as determined in cell migration assays, particularly in cell lines with the highest c-Met expression.ConclusionsThese results provide novel insights into both intracellular and extracellular dynamics of c-Met expression profiles upon irradiation of OTSCC cells in vitro. It might also suggest that radiation enhances cell migration, indicative of invasiveness, through c-Met up-regulation, at least for certain types of OTSCC cells.

Overexpression of Metastasis-Associated in Colon Cancer 1-Antisense RNA 1 (MACC1-AS1) in Bone Marrow Mesenchymal Stem Cells (BMSCs) Inhibits miR-145-5P and Promotes Chemotherapy Resistance of Colorectal Cancer

BMSCs have the potential of multipotent differentiation. This study aimed to investigate the interaction between MACC1-AS1 and miR-145-5P in BMSCs and their effect on chemotherapy resistance in colorectal cancer (CRC). BMSCs extracted from mouse marrow were transfected with MACC1-AS1 mimic, or MACC1-AS1 NC (control group). CRC cells were treated wtih gemcitabine and then co-cultured with BMSCs to measure cell viability and invasiveness by MTT and Transwell assay, along with analysis of the expression of MACC1, miR-145-5P, HGF, C-met, P-gp, and MRP. Successful isolation of BMSCs was confirmed by flow cytometry with positive expression of CD44, CD105, and CD90 (purity &gt; 95%). Functionally, overexpression of MACC1-AS1 in BMSCs increased CRC cell viability and invasion, attenuated the inhibitory effect of gemcitabine (p &lt; 0.05). Up-regulation of MACC1-AS1 (9.23±1.21) as demonstrated by RT-qPCR, resulted in a decline of miR-145-5P expression (4.23±1.22) in CRC cells (p &lt; 0.05). In addition, overexpression of MACC1-AS1 increased the expression of HGF, C-met, and multidrug resistance-associated proteins (P-gp, and MRP). In conclusion, overexpression of MACC1-AS1 in BMSCs inhibits miR-145-5P expression to promote colorectal cancer cell progression possibly via activating HGF/C-met pathway and inducing resistance to chemotherapy.

MACC1 Promotes the Progression and Is a Novel Biomarker for Predicting Immunotherapy Response in Colorectal Cancer.

Aims As one of the most prevalent malignant diseases in the world, the mechanisms of metastasis in colon cancer are poorly understood. The aim of this study was to investigate the role of the HGF/c-MET axis in the proliferation and metastasis in colon cancer. Methods The effect of MACC1 on cell proliferation and metastasis was analyzed through a series of in vitro experiments. The role of MACC1 in cancer cells was demonstrated by overexpression and silencing of MACC1 in gain or loss function experiments. To investigate the relationship between MACC1 and c-MET/HGF, we detected c-MET protein expression by disrupting with or overexpressing MACC1. The bioinformatics analysis was used to investigate the correlation between MACC1 and c-MET, and the c-MET expression after the interference of HGF with MACC1 was determined. Subsequently, the function of c-MET was verified in colon cancer cells by a series of experiments. The mouse tumor transplantation model experiment is most suitable in vivo. Results The results indicated that the overexpression of MACC1 could accelerate proliferation and facilitate metastasis in colon cancer cell lines. Furthermore, c-MET was determined to be the downstream regulator of MACC1. The addition of HGF could stimulate the expression of MACC1. With further exploration, we proved that c-MET is downstream of MACC1 in colon cancer and that overexpression of c-MET in colon cancer enhances cell proliferation and migration capability. At last, MACC1 expression level negatively correlates with the infiltration levels and several immune checkpoint biomarkers. High MACC1 expression has a lower response rate with ICIs in COAD. Conclusions We found that, under the regulation of the MACC1/HGF/c-MET axis, the proliferation and metastasis of colorectal cancer are increased by MACC1, which can be a novel biomarker for predicting ICIs response in colorectal cancer. Our findings provide a new idea for the targeted treatment of colorectal cancer.

Alpha-fetoprotein can promote gastric cancer progression via upregulation of metastasis-associated colon cancer 1.

High serum alpha-fetoprotein (AFP) level is a predictor of poor prognosis in patients with gastric cancer (GC). AFP-producing GC (AFP-GC) is an aggressive subtype of GC characterized by a high incidence of liver metastasis and high c-Met expression. High expression of metastasis-associated colon cancer 1 (MACC1), which is the transcription activator of c-Met, also predicts a poor prognosis of GC. c-Met is known to be involved in tumor progression into malignant invasive phenotypes. Considering that high c-Met expression is simultaneously positively correlated with high AFP and MACC1 expression levels and that high expression of AFP or MACC1 predicts poor prognosis in GC, we hypothesized that an interaction may exist between AFP and MACC1. In the present study, GC cell lines with AFP-overexpression, MACC1-downregulation and the combination of both transfections were used as experimental models. The relative mRNA and protein expression of c-Met, AFP and MACC1 were analyzed using reverse transcription quantitative PCR and western blotting, respectively. Cell viability was evaluated using Cell Counting Kit-8 assay. Cell invasion and cell migration were examined using Transwell migration assay with and without Matrigel, respectively. The results demonstrated that, compared with the control group, the mRNA and protein expression of MACC1was significantly elevated in the AFP-overexpressed group and in the group with AFP overexpressed and MACC1 downregulated. Furthermore, a significantly enhanced cell viability, migration and invasion were observed in the AFP-overexpressing group, whereas opposite effects were found in the MACC1-downregulating group. In summary, the results from this study indicated that AFP may promote GC progression by stimulating MACC1. This finding may help illustrating the aggressive behaviors of GC in patients with high AFP serum level and AFP-GC.

Adiponectin negatively regulates pigmentation, Wnt/β-catenin and HGF/c-Met signalling within human scalp hair follicles ex vivo.

Adiponectin reportedly stimulates proliferation and elongation of human scalp hair follicles (HFs) ex vivo. In the current study, we investigated how adiponectin oligomers produced by perifollicular dermal white adipose tissue (dWAT), a potent source of adiponectin isoforms, influence human HF proliferation and pigmentation. To do so, we treated microdissected, organ-cultured HFs in the presence or absence of dWAT with a recombinant human adiponectin oligomer mix, or inhibited dWAT-derived adiponectin using a neutralizing antibody. Multiplex qPCR (Fluidigm) revealed that adiponectin oligomers downregulated pigmentation genes KITLG, PMEL and TYRP1 and Wnt genes AXIN2, LEF1 and WNT10B. In situ hybridization showed that adiponectin downregulated AXIN2 and LEF1, and up-regulated DKK1 within the dermal papilla (DP), a highly unusual transcriptional profile for a putative hair growth-promoting agent. Adiponectin oligomers also downregulated protein expression of the HGF receptor c-Met within the matrix and DP. However, adiponectin did not alter hair matrix keratinocyte proliferation within 48h ex vivo, irrespective of the presence/absence of dWAT; HF pigmentation (Masson-Fontana histochemistry, tyrosinase activity) was also unchanged. In contrast, neutralizing adiponectin isoforms within HF + dWAT increased proliferation, melanin content and tyrosinase activity but resulted in fewer melanocytes and melanocytic dendrites, as assessed by gp100 immunostaining. These seemingly contradictory effects suggest that adiponectin exerts complex effects upon human HF biology, likely in parallel with the pro-pigmentation effects of dWAT- and DP-derived HGF. Our data suggest that dWAT-derived ratios of adiponectin isoforms and the cleaved, globular version of adiponectin may in fact determine how adiponectin impacts upon follicular pigmentation and growth.

A novel fluorescent c-met targeted imaging agent for intra-operative colonic tumour mapping: Translation from the laboratory into a clinical trial

BackgroundThe c-Met protein is overexpressed in many gastrointestinal cancers. We explored EMI-137, a novel c-Met targeting fluorescent probe, for application in fluorescence-guided colon surgery, in HT-29 colorectal cancer (CRC) cell line and an in vivo murine model. MethodsHT-29 SiRNA transfection confirmed specificity of EMI-137 for c-Met. A HT-29 CRC xenograft model was developed in BALB/c mice, EMI-137 was injected and biodistribution analysed through in vivo fluorescent imaging. Nine patients, received a single intravenous EMI-137 bolus (0.13 mg/kg), 1–3 h before laparoscopic-assisted colon cancer surgery (NCT03360461). Tumour and LN fluorescence was assessed intraoperatively and correlated with c-Met expression in eight samples by immunohistochemistry. Findingsc-Met expression HT-29 cells was silenced and imaged with EMI-137. Strong EMI-137 uptake in tumour xenografts was observed up to 6 h post-administration. At clinical trial, no serious adverse events related to EMI-137 were reported. Marked background fluorescence was observed in all participants, 4/9 showed increased tumour fluorescence over background; 5/9 had histological LN metastases; no fluorescent LN were detected intraoperatively. All primary tumours (8/8) and malignant LN (15/15) exhibited high c-Met protein expression. InterpretationEMI-137, binds specifically to the human c-Met protein, is safe, and with further refinement, shows potential for application in fluorescence-guided surgery.

P60.12 Prevalence of c-Met overexpression (c-Met+) and Impact of Prior Lines of Treatment on c-Met Protein Expression in NSCLC

Teliso-V, an anti–c-Met antibody conjugated with monomethyl auristatin E, is currently being investigated as monotherapy in c-Met+ 2nd/3rd-line metastatic NSCLC in a phase 2 study (NCT03539536). Here we present prevalence of c-Met+ NSCLC based on histopathology and EGFR mutation status. c-Met expression change from archival (diagnostic (Dx)/post-Dx biopsy) vs fresh (tumor biopsy at time of study prescreening) is also presented for patient subsets. In this study, patients were prospectively selected for c-Met–positive expression by immunohistochemistry (central assay; SP44 antibody). Archival or fresh tumor tissue was eligible for c-Met expression testing and enrollment consideration. Per protocol, if archival tissue was c-Met negative, patients could provide fresh tumor tissue for reassessment of c-Met expression. As of December 2020, 841 patients evaluable for c-Met expression have been prescreened (Table 1). c-Met+ rates were lower in EGFR wild-type (25%) vs mutant (37%) non-squamous cohorts. Squamous histology had a 39% positivity rate. c-Met+ rates were higher in fresh vs archival tumor tissue. Similar trends were observed when EGFR wild-type and mutant cohorts were separated based on c-Met–high or –intermediate groups (Table 2). Sixteen patients (non-squamous cohorts: EGFR mutant, N=8; EGFR wild-type, N=8) initially reported c-Met–negative status based on archived tissue and submitted fresh tissue for c-Met reassessment. Tumors from EGFR mutant (50%) and wild-type (12.5%) cohorts had changes in c-Met status from negative to positive, potentially due to prior anti-EGFR or immuno-oncology/chemotherapy treatments, degradation of the epitope over time in the sample, or heterogeneity within tumor lesions.Table 1Patientsc-Met Positive, %a (n)NSQ EGFR MU24537 (90)NSQ EGFR WT44625 (122)Squamous15039 (58)aThe c-Met–positive cutoffs (by immunohistochemistry using SP44 antibody) were derived from retrospective receiver operating characteristic analysis of phase 1 (NCT02099058) Teliso-V monotherapy data. Cutoffs were established as membrane staining: non-squamous (≥25% 3+) and squamous (≥75% 1+) NSCLC. MU, mutant; NSQ, non-squamous; WT, wild-type. Open table in a new tab aThe c-Met–positive cutoffs (by immunohistochemistry using SP44 antibody) were derived from retrospective receiver operating characteristic analysis of phase 1 (NCT02099058) Teliso-V monotherapy data. Cutoffs were established as membrane staining: non-squamous (≥25% 3+) and squamous (≥75% 1+) NSCLC. MU, mutant; NSQ, non-squamous; WT, wild-type. Based on defined cutoffs, 25%–39% of NSCLC tumors were c-Met+. Consistent with prior observations, an increase in c-Met expression was observed in tumor tissue after anti-EGFR treatments. The increase in c-Met expression following immuno-oncology/chemotherapy treatments was a new observation. Additional data are needed to make conclusive remarks.

Correlation of c-MET expression with clinical characteristics and the prognosis of colorectal cancer

The proto-oncogene c-MET (mesenchymal-epithelial transition factor gene) plays a critical role in cellular proliferation, survival, migration, and invasion in cancers. The aim of this study is to explore the relationship between c-MET expression and the clinicopathological characteristics of colorectal cancer (CRC) patients. A total of 337 enrolled patients were collected in present study. Here, the c-MET and EGFR expression were detected by immunohistochemistry (IHC). The mutational statuses of KRAS in exons 2, 3, and 4, NRAS in exons 2, 3, and 4, and BRAF in exon 15 from formalin-fixed sections were detected by direct DNA sequencing. Our results showed that high c-MET expression was significantly associated with tumor perineural invasion (P=0.007) and gender (P=0.016). High level c-MET expression (c-MET-high) in the primary tumors was observed in 68.2% of patients. In the 337 enrolled patients, 43.2% of patients had KRAS mutations, 3.3% of patients had NRAS mutations, and 4.7% of patients had BRAF mutations. However, KRAS, NRAS, and BRAF gene mutations had no association with c-MET protein levels in primary tumors. Additionally, c-MET protein expression had a strong correlation with EGFR expression (P=0.002). The survival time was not significantly longer for patients with c-MET-high primary tumors than for those with c-MET-low primary tumors. c-MET immunohistochemistry was significantly higher in primary tumors with perineural invasion, female gender, and EGFR high expression. However, c-MET-high in the primary tumors was not significantly associated with longer survival compared with c-MET-low tumors. Further studies are required to investigate c-MET as potential molecular marker of progression and to test the possibility of its incorporation as a new therapeutic target.