c-Met Levels Research Articles

Combination therapy with pazopanib and tivantinib modulates VEGF and c-MET levels in refractory advanced solid tumors

SummaryThe vascular endothelial growth factor (VEGF)/VEGFR and hepatocyte growth factor (HGF)/c-MET signaling pathways act synergistically to promote angiogenesis. Studies indicate VEGF inhibition leads to increased levels of phosphorylated c-MET, bypassing VEGF-mediated angiogenesis and leading to chemoresistance. We conducted a phase 1 clinical trial with 32 patients with refractory solid tumors to evaluate the safety, pharmacokinetics, and pharmacodynamics of combinations of VEGF-targeting pazopanib and the putative c-MET inhibitor ARQ197 (tivantinib) at 5 dose levels (DLs). Patients either took pazopanib and tivantinib from treatment initiation (escalation phase) or pazopanib alone for 7 days, with paired tumor sampling, prior to starting combination treatment (expansion phase). Hypertension was the most common adverse event. No more than 1 dose limiting toxicity (DLT) occurred at any DL, so the maximum tolerated dose (MTD) was not determined; DL5 (800 mg pazopanib daily and 360 mg tivantinib BID) was used during the expansion phase. Twenty of 31 evaluable patients achieved stable disease lasting up to 22 cycles. Circulating VEGF, VEGFR2, HGF, and c-MET levels were assessed, and only VEGF levels increased. Tumor c-MET levels (total and phosphorylated) were determined in paired biopsies before and after 7 days of pazopanib treatment. Total intact c-MET decreased in 6 of 7 biopsy pairs, in contrast to previously reported c-MET elevation in response to VEGF inhibition. These results are discussed in the context of our previously reported analysis of epithelial-mesenchymal transition in these tumors.

CMet agonistic antibody prevents acute kidney injury to chronic kidney disease transition by suppressing Smurf1 and activating Smad7.

We aimed to investigate the role of cMet agonistic antibody (cMet Ab) in preventing kidney fibrosis during acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Additionally, we explored the effect of cMet Ab on TGF-β1/Smad pathway during the pathogenesis of kidney fibrosis. A unilateral ischemia-reperfusion injury (UIRI) mouse model was established to induce AKI-to-CKD transition. Furthermore, we incubated human proximal tubular epithelial cells (hPTECs) under hypoxic conditions as in vitro model of kidney fibrosis. We analyzed the soluble plasma cMet level in patients with AKI requiring dialysis. Patients who did not recover kidney function and progressed to CKD presented a higher increase in the cMet level. The kidneys of mice treated with cMet Ab showed fewer contractions and weighed more than the controls. The mice in the cMet Ab-treated group showed reduced fibrosis and significantly decreased expression of fibronectin and α-smooth muscle actin. cMet Ab treatment decreased inflammatory markers (MCP-1, TNF-α, and IL-1β) expression, reduced Smurf1 and Smad2/3 level, and increased Smad7 expressions. cMet Ab treatment increased cMet expression and reduced the hypoxia-induced increase in collagen-1 and ICAM-1 expression, thereby reducing apoptosis in the in vitro cell model. After cMet Ab treatment, hypoxia-induced expression of Smurf1, Smad2/3, and TGF-β1 was reduced, and suppressed Smad7 was activated. Down-regulation of Smurf1 resulted in suppression of hypoxia-induced fibronectin expression, whereas treatment with cMet Ab showed synergistic effects. cMet Ab can successfully prevent fibrosis response in UIRI models of kidney fibrosis by decreasing inflammatory response and inhibiting the TGF-β1/Smad pathway.

Circulating Levels of the Interferon-γ-Regulated Chemokines CXCL10/CXCL11, IL-6 and HGF Predict Outcome in Metastatic Renal Cell Carcinoma Patients Treated with Antiangiogenic Therapy.

Simple SummaryWe have studied blood levels of cytokines/chemokines in patients with metastatic renal cell carcinoma treated with sunitinib or pazopanib, with the goal of identifying biomarkers that can predict efficacy and survival. We have found that high levels of CXCL10, CXCL11, HGF and IL-6 before treatment associate with poor prognosis in these patients. Moreover, these factors are correlated in patients with renal carcinoma, suggesting a coordinated expression and secretion. We have developed a prognostic signature including these factors that predicts very accurately prognosis. Our results may help defining better the group of renal cell carcinoma patients who may benefit from sunitinib/pazopanib.Sunitinib and pazopanib are standard first-line treatments for patients with metastatic renal cell carcinoma (mRCC). Nonetheless, as the number of treatment options increases, there is a need to identify biomarkers that can predict drug efficacy and toxicity. In this prospective study we evaluated a set of biomarkers that had been previously identified within a secretory signature in mRCC patients. This set includes tumor expression of c-Met and serum levels of HGF, IL-6, IL-8, CXCL9, CXCL10 and CXCL11. Our cohort included 60 patients with mRCC from 10 different Spanish hospitals who received sunitinib (n = 51), pazopanib (n = 4) or both (n = 5). Levels of biomarkers were studied in relation to response rate, progression-free survival (PFS) and overall survival (OS). High tumor expression of c-Met and high basal serum levels of HGF, IL-6, CXCL11 and CXCL10 were significantly associated with reduced PFS and/or OS. In multivariable Cox regression analysis, CXCL11 was identified as an independent biomarker predictive of shorter PFS and OS, and HGF was an independent predictor of reduced PFS. Correlation analyses using our cohort of patients and patients from TCGA showed that HGF levels were significantly correlated with those of IL-6, CXCL11 and CXCL10. Bioinformatic protein–protein network analysis revealed a significant interaction between these proteins, all this suggesting a coordinated expression and secretion. We also developed a prognostic index that considers this group of biomarkers, where high values in mRCC patients can predict higher risk of relapse (HR 5.28 [2.32–12.0], p < 0.0001). In conclusion, high plasma HGF, CXCL11, CXCL10 and IL-6 levels are associated with worse outcome in mRCC patients treated with sunitinib or pazopanib. Our findings also suggest that these factors may constitute a secretory cluster that acts coordinately to promote tumor growth and resistance to antiangiogenic therapy.

Combined BRM270 and endostatin inhibit relapse of NSCLC while suppressing lung cancer stem cell proliferation induced by endostatin

Endostatin (ES, ENDO) has been reported to suppress the growth of tumors while inducing the proliferation of lung cancer stem cells (LCSCs), causing a poor prognosis for lung cancer. In this study, we aimed to clarify whether BRM270 can inhibit the proliferation of cancer stem cells (CSCs). Endostatin + BRM270 showed anti-tumor effects by reducing tumor volume and increasing survival. Administration of BRM270 reduced the number of aldehyde dehydrogenase-positive (ALDH+) cells and the level of ALDH1A1 expression in tumors by increasing the level of miR-128 while decreasing the levels of BMI-1, ABCC-5, E2F3, and c-MET. The luciferase activity of miR-128 promoter was increased by an increasing concentration of BRM270. In addition, BMI-1, ABCC-5, E2F3, and c-MET were identified as candidate targets of miR-128, and the overexpression of miR-128 significantly reduced mRNA/protein levels of BMI-1, ABCC-5, E2F3, and c-MET in A549 and H460 cells. Administration of BRM270 inhibited the expression of BMI-1, ABCC-5, E2F3, and c-MET in a dose-dependent manner. In this study, we showed for the first time that the combined administration of endostatin and BRM270 achieved anti-tumor effects while suppressing the proliferation of stem cells.

Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in colorectal cancer

Emerging evidences suggest that long noncoding RNA (lncRNA) plays a vital role in tumorigenesis and cancer progression. Here, the aim of this study is to investigate the biological function of long intervening noncoding RNA Linc00284 in colorectal cancer (CRC). The expression levels of Linc00284, miR-27a and c-Met were evaluated by qPCR and/or Western blotting. Immunohistochemistry was used to detect the expression of Ki67 and Phh3 in tumor tissues. The interaction between Linc00284, miR-27a and c-Met was validated by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Cell function experiments, including CCK-8, wound-healing and transwell invasion assays, were conducted. The in vivo studies were performed with the subcutaneous tumor xenograft mouse models. Our findings reveal that Linc00284 is upregulated in CRC tissues and colorectal cancer cell lines HCT116 and SW480 in comparison with corresponding para-carcinoma tissues and human fetal colonic mucosa cells FHC. High expression of Linc00284 in tumor tissues is associated with tumor metastasis and predicts a poor clinical outcome in CRC patients. Serum Linc00284 is increased, while miR-27a is decreased in CRC patients compared to healthy controls. ROC curve analysis indicates that serum Linc00284 and miR-27a produce the area under the curve (AUC) value of at 0.8151 and 0.7316 in patients with colorectal cancer compared to healthy individuals, respectively. Additionally, results in vitro and in vivo experiments suggest that Linc00284 silencing significantly suppresses CRC cell proliferation and/or invasion. Mechanistically, Linc00284 promotes c-Met expression by acting as miR-27a sponge, leading to the activation of downstream signaling pathways, thereby causing malignant phenotypes of CRC cells. Taken together, Linc00284 exhibits oncogenic function and the disturbance of Linc00284/miR-27a/c-Met regulatory axis contributes to CRC progression, providing new insight into the pathogenesis of colorectal cancer. Importantly, the expression levels of serum Linc00284 and miR-27a may serve as clinical biomarkers for CRC diagnosis.

Combined Treatment of Cinobufotalin and Gefitinib Exhibits Potent Efficacy against Lung Cancer.

This study aimed to evaluate the efficacy of cinobufotalin combined with gefitinib in the treatment of lung cancer. A549 cells were treated with gefitinib, cinobufotalin, or cinobufotalin plus gefitinib. MTT assay, annexin-V/PI staining and flow cytometry, TUNEL staining, DCFH-DA staining, Western blot, and real-time RT-PCR were performed to investigate the synergistic inhibitory effect of cinobufotalin combined with gefitinib on the growth of A549 cells. Results showed that cinobufotalin synergized with gefitinib displayed inhibited cell viability and enhanced apoptosis in the combination group. Cinobufotalin combined with gefitinib induced a significant enhancement in reactive oxygen species (ROS) production accompanied by cell cycle arrest in the S phase arrest, characterized by upregulation of p21 and downregulation of cyclin A, cyclin E, and CDK2. Besides, cinobufotalin plus gefitinib downregulated the levels of HGF and c-Met. In summary, cinobufotalin combined with gefitinib impedes viability and facilitates apoptosis of A549 cells, indicating that the combined therapy might be a new promising treatment for lung cancer patients who are resistant to gefitinib.

Lidocaine suppresses the malignant behavior of gastric cancer cells via the c-Met/c-Src pathway.

The present study was designed to investigate the role and mechanism of action behind the action of lidocaine in gastric cancer cells. Lidocaine was tested for its potential role in affecting the viability of cells using Cell Counting Kit-8 (CCK-8) assays. It was found that there was a decreased MKN45 cell viability upon lidocaine treatment in a dose-dependent manner. Phosphorylated c-Met, phosphorylated c-Src, c-Met and c-Src levels were detected using western blotting following lidocaine or hepatocyte growth factor (HGF) intervention. It was found that the phosphorylation levels of c-Met and c-Src were markedly reduced by lidocaine treatment, with this effect being further relieved by the addition of HGF. Subsequently, whether lidocaine repressed the malignant biological properties of gastric cancer cells through the c-Met/c-Src axis was further investigated through the detection of epithelial-mesenchymal transition markers (N-caderin and vimentin), wound healing and transwell assay analysis. In addition, cell apoptosis and the levels of apoptosis-related proteins were determined using TUNEL and western blot assays, respectively. The results demonstrated that the malignant behavior of cells were notably repressed upon lidocaine treatment, but the addition of HGF markedly reversed these effects, indicating that the effects of lidocaine on supressing the malignant behaviour of cells could be mediated through the c-Met/c-Src axis. Subsequently, whether lidocaine affected the sensitivity of cells to cisplatin or 5-FU was analyzed using a CCK-8 assay. Enhanced sensitivity of cells to cisplatin or 5-FU was observed when treated in combination with lidocaine. The present study concluded that the involvement of the c-Met/c-Src pathway in the biological behaviour of MKN45 cells was mediated by lidocaine. Therefore, lidocaine may have the potential to suppress the malignant behaviour and proliferation of gastric cancer cells.

The FAM3C locus that encodes interleukin-like EMT inducer (ILEI) is frequently co-amplified in MET-amplified cancers and contributes to invasiveness

BackgroundGene amplification of MET, which encodes for the receptor tyrosine kinase c-MET, occurs in a variety of human cancers. High c-MET levels often correlate with poor cancer prognosis. Interleukin-like EMT inducer (ILEI) is also overexpressed in many cancers and is associated with metastasis and poor survival. The gene for ILEI, FAM3C, is located close to MET on chromosome 7q31 in an amplification “hotspot”, but it is unclear whether FAMC3 amplification contributes to elevated ILEI expression in cancer. In this study we have investigated FAMC3 copy number gain in different cancers and its potential connection to MET amplifications.MethodsFAMC3 and MET copy numbers were investigated in various cancer samples and 200 cancer cell lines. Copy numbers of the two genes were correlated with mRNA levels, with relapse-free survival in lung cancer patient samples as well as with clinicopathological parameters in primary samples from 49 advanced stage colorectal cancer patients. ILEI knock-down and c-MET inhibition effects on proliferation and invasiveness of five cancer cell lines and growth of xenograft tumors in mice were then investigated.ResultsFAMC3 was amplified in strict association with MET amplification in several human cancers and cancer cell lines. Increased FAM3C and MET copy numbers were tightly linked and correlated with increased gene expression and poor survival in human lung cancer and with extramural invasion in colorectal carcinoma. Stable ILEI shRNA knock-down did not influence proliferation or sensitivity towards c-MET-inhibitor induced proliferation arrest in cancer cells, but impaired both c-MET-independent and -dependent cancer cell invasion. c-MET inhibition reduced ILEI secretion, and shRNA mediated ILEI knock-down prevented c-MET-signaling induced elevated expression and secretion of matrix metalloproteinase (MMP)-2 and MMP-9. Combination of ILEI knock-down and c-MET-inhibition significantly reduced the invasive outgrowth of NCI-H441 and NCI-H1993 lung tumor xenografts by inhibiting proliferation, MMP expression and E-cadherin membrane localization.ConclusionsThese novel findings suggest MET amplifications are often in reality MET-FAM3C co-amplifications with tight functional cooperation. Therefore, the clinical relevance of this frequent cancer amplification hotspot, so far dedicated purely to c-MET function, should be re-evaluated to include ILEI as a target in the therapy of c-MET-amplified human carcinomas.

Imidazopyridine hydrazone derivatives exert antiproliferative effect on lung and pancreatic cancer cells and potentially inhibit receptor tyrosine kinases including c-Met

Aberrant activation of c-Met signalling plays a prominent role in cancer development and progression. A series of 12 imidazo [1,2-α] pyridine derivatives bearing 1,2,3-triazole moiety were designed, synthesized and evaluated for c-Met inhibitory potential and anticancer effect. The inhibitory activity of all synthesized compounds against c-Met kinase was evaluated by a homogeneous time-resolved fluorescence (HTRF) assay at the concentration range of 5–25 µM. Derivatives 6d, 6e and 6f bearing methyl, tertiary butyl and dichloro-phenyl moieties on the triazole ring, respectively, were the compounds with the highest potential. They significantly inhibited c-Met by 55.3, 53.0 and 51.3%, respectively, at the concentration of 25 µM. Synthetic compounds showed antiproliferative effects against lung (EBC-1) and pancreatic cancer cells (AsPc-1, Suit-2 and Mia-PaCa-2) expressing different levels of c-Met, with IC50 values as low as 3.0 µM measured by sulforhodamine B assay. Active derivatives significantly blocked c-Met phosphorylation, inhibited cell growth in three-dimensional spheroid cultures and also induced apoptosis as revealed by Annexin V/propidium iodide flow cytometric assay in AsPc-1 cells. They also inhibited PDGFRA and FLT3 at 25 µM among a panel of 16 kinases. Molecular docking and dynamics simulation studies corroborated the experimental findings and revealed possible binding modes of the select derivatives with target receptor tyrosine kinases. The results of this study show that some imidazopyridine derivatives bearing 1,2,3-triazole moiety could be promising molecularly targeted anticancer agents against lung and pancreatic cancers.

High-Content Analysis-Based Sensitivity Prediction and Novel Therapeutics Screening for c-Met-Addicted Glioblastoma.

Simple SummaryReal-time ex vivo drug testing tailors individual therapeutics based on predicted drug responses. Most technologies to date rely on conventional drug screening that provides low confidence data. Here, we present high-content analysis-based drug testing of glioblastoma patients to identify the right glioblastoma patients for a given drug. This generates multi-parameter biomarker and phenotype readouts providing a better reliability of the assay. Additionally, we showed a high-content drug repurposing screen and defined a new c-Met-inhibiting function of the CDK4/6 inhibitor Abemaciclib. Large-scale high throughput screening results demonstrate that Abemaciclib sensitivity in glioblastoma patients is highly correlated with the c-Met inhibitors sensitivity, further supporting the accuracy of the platform and important new clinical implications regarding multiple functions of Abemaciclib.(1) Background: Recent advances in precision oncology research rely on indicating specific genetic alterations associated with treatment sensitivity. Developing ex vivo systems to identify cancer patients who will respond to a specific drug remains important. (2) Methods: cells from 12 patients with glioblastoma were isolated, cultured, and subjected to high-content screening. Multi-parameter analyses assessed the c-Met level, cell viability, apoptosis, cell motility, and migration. A drug repurposing screen and large-scale drug sensitivity screening data across 59 cancer cell lines and patient-derived cells were obtained from 125 glioblastoma samples. (3) Results: High-content analysis of patient-derived cells provided robust and accurate drug responses to c-Met-targeted agents. Only the cells of one glioblastoma patient (PDC6) showed elevated c-Met level and high susceptibility to the c-Met inhibitors. Multi-parameter image analysis also reflected a decreased c-Met expression and reduced cell growth and motility by a c-Met-targeting antibody. In addition, a drug repurposing screen identified Abemaciclib as a distinct CDK4/6 inhibitor with a potent c-Met-inhibitory function. Consistent with this, we present large-scale drug sensitivity screening data showing that the Abemaciclib response correlates with the response to c-Met inhibitors. (4) Conclusions: Our study provides a new insight into high-content screening platforms supporting drug sensitivity prediction and novel therapeutics screening.

Multi-protein spatial signatures in ductal carcinoma in situ (DCIS) of breast

BackgroundThere is limited knowledge about DCIS cellular composition and relationship with breast cancer events (BCE).MethodsImmunofluorescence multiplexing (MxIF) was used to image and quantify 32 cellular biomarkers in FFPE DCIS tissue microarrays. Over 75,000 DCIS cells from 51 patients (median 9 years follow-up for non-BCE cases) were analysed for profiles predictive of BCE. K-means clustering was used to evaluate cellular co-expression of epithelial markers with ER and HER2.ResultsOnly ER, PR and HER2 significantly correlated with BCE. Cluster analysis identified 6 distinct cell groups with different levels of ER, Her2, cMET and SLC7A5. Clusters 1 and 3 were not significant. Clusters 2 and 4 (high ER/low HER2 and SLC7A5/mixed cMET) significantly correlated with low BCE risk (P = 0.001 and P = 0.034), while cluster 6 (high HER2/low ER, cMET and SLC7A5) correlated with increased risk (P = 0.018). Cluster 5 (similar to cluster 6, except high SLC7A5) trended towards significance (P = 0.072). A continuous expression score (Escore) based on these 4 clusters predicted likelihood of BCE (AUC = 0.79, log-rank test P = 5E–05; LOOCV AUC = 0.74, log-rank test P = 0.006).ConclusionMultiplexed spatial analysis of limited tissue is a novel method for biomarker analysis and predicting BCEs. Further validation of Escore is needed in a larger cohort.

Human Telomerase Reverse Transcriptase Promotes the Epithelial to Mesenchymal Transition in Lung Cancer Cells by Enhancing c-MET Upregulation

Human telomerase reverse transcriptase (hTERT), the essential catalytic subunit of telomerase, is associated with telomere homeostasis to prevent replicative senescence and cellular aging. However, hTERT reactivation also has been linked to the acquisition of several hallmarks of cancer, although the underlying mechanism beyond telomere extension remains elusive. This study demonstrated that hTERT overexpression promotes, whereas its inhibition by shRNA suppresses, epithelial-mesenchymal transition (EMT) in lung cancer cells (A549 and H1299). We found that hTERT modulates the expression of EMT markers E-cadherin, vimentin, and cytokeratin-18a through upregulation of the c-MET. Ectopic expression of hTERT induces expression of c-MET, while hTERT-shRNA treatment significantly decreases the c-MET level in A549 and H1299 through differential expression of p53 and c-Myc. Reporter assay suggests the regulation of c-MET expression by hTERT to be at the promoter level. An increase in c-MET level significantly promotes the expression of mesenchymal markers, including vimentin and N-cadherin, while a notable increase in epithelial markers E-cadherin and cytokeratin-18a is observed after the c-MET knockdown in A549. Funding Information: Grants received from UGC (176), DST (2-DST-PURSE 2015-16), and ICMR. Declaration of Interests: The authors declare that there are no conflicts of interest.

ELTD1 as a multi-focal target for malignant gliomas: preclinical studies.

BackgroundGlioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults. These high-grade gliomas undergo unregulated vascular angiogenesis, migration and cell proliferation allowing the tumor cells to evade cell-cycle checkpoints and apoptotic pathways. The Epidermal growth factor, latrophilin, and seven transmembrane domain-containing 1 on chromosome 1 (ELTD1) is an angiogenic biomarker that is highly expressed in malignant gliomas. Novel treatments targeting ELTD1 with monovalent monoclonal (mmAb) and single chain variable fragment (scFv) antibodies were effective in increasing animal survival, decreasing tumor volume and normalizing the vasculature. Due to the success of our antibody treatments on angiogenesis, this study sought to determine if our anti-ELTD1 treatments affected other aspects of tumorigenesis (cell proliferation, migration, and apoptosis) in a G55 glioma xenograft preclinical mouse model.MethodsTumor tissue from untreated, mmAb and scFv anti-ELTD1 treated animals was used to quantify the positivity levels of human mitochondrial antibody, c-MET and Ki-67 for cellular proliferation, migratory markers CD44v6, TRPM8, and BMP2, and cleaved caspase 3 to assess apoptotic activity.ResultsThis approach demonstrated that our anti-ELTD1 treatments directly affected and decreased the human tumor cells within the tumor region. Additionally, there was a significant decrease in both cellular proliferation and migration due to anti-ETLD1 therapy. Lastly, anti-ELTD1 treatments successfully increased apoptotic activity within the tumor region.ConclusionOur data suggest that anti-ELTD1 therapies would be effective against malignant gliomas by having a multi-focal effect and targeting all four aspects of tumorigenesis.

Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas. The hepatocyte growth factor receptor (c-Met) has been considered as a potential therapeutic target for NSCLC. Proteasome inhibition induces cell apoptosis and has been used as a novel therapeutic approach for treating diseases including NSCLC; however, the effects of different proteasome inhibitors on NSCLC have not been fully investigated. The aim of this study is to determine a precise strategy for treating NSCLC by targeting c-Met using different proteasome inhibitors. Three proteasome inhibitors, bortezomib, MG132, and ONX 0914, were used in this study. Bortezomib (50 nM) significantly reduced c-Met levels and cell viability in H1299 and H441 cells, while similar effects were observed in H460 and A549 cells when a higher concentration (∼100 nM) was used. Bortezomib decreased c-Met gene expression in H1299 and H441 cells, but it had no effect in A549 and H460 cells. MG-132 at a low concentration (0.5 μM) diminished c-Met levels in H441 cells, while neither a low nor a high concentration (∼20 μM) altered c-Met levels in A549 and H460 cells. A higher concentration of MG-132 (5 μM) was required for decreasing c-Met levels in H1299 cells. Furthermore, MG-132 induced cell death in all four cell types. Among all the four cell lines, H441 cells expressed higher levels of c-Met and appeared to be the most susceptible to MG-132. MG-132 decreased c-Met mRNA levels in both H1299 and H441 cells. ONX 0914 reduced c-Met levels in H460, H1299, and H441 cells but not in A549 cells. c-Met levels were decreased the most in H441 cells treated with ONX 0914. ONX 0914 did not alter cell viability in H441; however, it did induce cell death among H460, A549, and H1299 cells. This study reveals that different proteasome inhibitors produce varied inhibitory effects in NSCLS cell lines.

C-Met/MAPK pathway promotes the malignant progression of residual hepatocellular carcinoma cells after insufficient radiofrequency ablation.

Radiofrequency ablation (RFA) is popularly used in the treatment of hepatocellular carcinoma (HCC). However, the accelerated malignant progression of residual HCC cells after RFA is the main obstacle for the application of this technology in HCC treatment. In the present study, HepG2 cells, an established human HCC cell line, experienced repeatedly with heat treatment, survived cells, HepG2-H cells, were used to simulate residual HCC cells after RFA. The abilities of proliferation, colony formation, and migration were compared between HepG2 and HepG2-H cells. Then, RNA sequencing was used to explore the difference in genes expression between two groups of cells. Subsequently, the level of c-Met, one of membranous receptors of MAPK signal pathway, was measured by RT-qPCR and western blot; the effect of c-Met inhibition on the malignant progression of HepG2-H cells was evaluated. The results showed that HepG2-H cells exhibited higher abilities in the proliferation, colony formation, and migration than that of HepG2 cells. Moreover, differentially expressed genes between two groups of cells were prominently enriched in MAPK signal pathway. The level of c-Met in HepG2-H cells was significantly higher than that in HepG2 cells, and the inhibition in the activity of c-Met could repress the malignant behaviors of HepG2-H cells. These results indicated that the accelerated malignant progression of residual HCC cells after RFA can be partly attributed to the overexpression of c-Met and the activation of MAPK signal pathway. Therefore, we proposed that RFA followed by c-Met inhibitor intake maybe is a better treatment protocol for HCC.

CMET promotes metastasis and epithelial-mesenchymal transition in colorectal carcinoma by repressing RKIP.

Increasing evidence indicates that c-mesenchymal-epithelial transition factor (cMET) plays an important role in the malignant progression of colorectal cancer (CRC). However, the underlying mechanism is not fully understood. As a metastasis suppressor, raf kinase inhibitory protein (RKIP) loss has been reported in many cancer types. In this study, the expression levels of cMET and RKIP in CRC tissues and cell lines were determined, and their crosstalk and potential biological effects were explored in vitro and in vivo. Our results showed that cMET was inversely correlated with RKIP. Both cMET upregulation and RKIP downregulation indicated poor clinical outcomes. Moreover, the MAPK/ERK signaling pathway was implicated in the regulation of cMET and RKIP. Overexpression of cMET promoted tumor cell epithelial-mesenchymal transition, invasion, migration, and chemoresistance, whereas the effects could be efficiently inhibited by increased RKIP. Notably, small hairpin RNA-mediated cMET knockdown dramatically suppressed cell proliferation, although no RKIP-induced influence on cell growth was observed in CRC. Altogether, cMET overexpression may contribute to tumor progression by inhibiting the antioncogene RKIP, providing preclinical justification for targeting RKIP to treat cMET-induced metastasis of CRC.

Paracrine HGF promotes EMT and mediates the effects of PSC on chemoresistance by activating c-Met/PI3K/Akt signaling in pancreatic cancer in vitro

BackgroundPancreatic stellate cells (PSCs) play key roles in the pancreatic tumor microenvironment and are considered to contribute to chemoresistance. PSCs can participate in malignant behaviors of pancreatic carcinoma (PC) by secreting hepatocyte growth factor (HGF). The objective of this research was to explore the potential molecular mechanism of HGF on gemcitabine (GEM) chemoresistance of PC. Materials and methodsHGF, c-Met, E-Cadherin and Vimentin levels were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The changes of HGF level were detected by ELISA. The half maximal inhibitory concentration, the growth inhibitions and apoptosis of pancreatic cancer cells (PCCs) were respectively assayed using CCK-8 and flow cytometry. Associated proteins were measured using western blot and cell immunofluorescence assay. Key findingsPSCs strongly expressed HGF, and its receptor c-Met was expressed in PCCs. PCCs exerted a positive regulative effect on HGF production. HGF neutralizing antibody AMG102 could effectively reduce the HGF level in PSC-conditioned medium (PSC-CM). PSC-CM promoted chemoresistance in PCCs. When exposed to PSC-CM, PCCs underwent epithelial-to-mesenchymal transition (EMT), and c-Met was also activated. Recombinant human HGF had the same protective effect. Blocking the HGF/c-Met axis with a c-Met inhibitor PHA665752 and AMG102 reduced the phosphorylation level of c-Met (p-c-Met) and attenuated EMT and chemoresistance. P-c-Met overexpression resulted in activation of the PI3K/Akt pathway, and inhibition of PI3K/Akt signaling with LY294002 reversed chemoresistance and EMT. SignificancePSCs can activate the c-Met/PI3K/Akt pathway in PCCs via paracrine HGF, induce EMT of PCCs and inhibit cancer cell apoptosis, thus enhance chemoresistance to Gem in PCCs.

CIRCR201-dPBD, a Novel Pyrrolobenzodiazepine Dimer-Containing Site-Specific Antibody-Drug Conjugate Targeting c-Met Overexpression Tumors.

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody–drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug–conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC–MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

Urinary cMet as a prognostic marker in immunoglobulin A nephropathy.

The prediction of prognosis in patients with immunoglobulin A nephropathy (IgAN) is challenging. We investigated the correlation between urinary cMet (ucMet) levels and clinical parameters and examined the effects of cMet agonistic antibody (cMet Ab) in an in vitro IgAN model. Patients diagnosed with IgAN (n = 194) were divided into three groups representing undetectable (Group 1), below‐median (Group 2) and above‐median (Group 3) levels of ucMet/creatinine (ucMet/Cr). Stained kidney biopsy samples were graded according to cMet intensity. Primary‐cultured human mesangial cells were stimulated with recombinant tumour necrosis factor (TNF)‐α and treated with cMet Ab. Our results showed that ucMet/Cr levels positively correlated with proteinuria (P < .001). During the follow‐up, patients in Group 3 showed a significantly lower probability of complete remission (CR; uPCr < 300 mg/g) than those in groups 1 and 2, after adjusting for blood pressure, estimated glomerular filtration rate, and proteinuria, which influence clinical prognosis (HR 0.60, P = .038); moreover, ucMet/Cr levels were also associated with glomerular cMet expression. After TNF‐α treatment, the proliferation of mesangial cells and increased interleukin‐8 and intercellular adhesion molecule‐1 expression were markedly reduced by cMet Ab in vitro. In conclusion, ucMet/Cr levels significantly correlated with proteinuria, glomerular cMet expression, and the probability of CR. Further, cMet Ab treatment alleviated the inflammation and proliferation of mesangial cells. Hence, ucMet could serve as a clinically significant marker for treating IgAN.

Abstract 4281: Overcoming preanalytical variabilities for peripheral blood mononuclear cells and exosome profiling in the IMMUcan multicentre study

Abstract IMMUcan is a joint public-private partnership to advance our understanding of how tumors and the immune system interact and the effect of therapeutic interventions. The project will collect tumor biopsies and peripheral blood from up to 3,000 patients across 13 European countries for multi-omic analyses. Here, we assessed the impact of sample collection and processing conditions on the quality of peripheral blood mononuclear cells (PBMCs). We also compared circulating exosomes obtained from plasma and serum to determine the ideal source for the study. We collected peripheral blood from 9 healthy donors across 3 centers in different countries. PBMC isolation and freezing were either performed locally at 3, 8, 24, 48 and 72 hours delays or alternatively shipped to a designated biobank for central processing at 8, 24, 48 and 72 hours delays. PBMCs were assessed for yield, viability and frequencies of immune cell populations by flow cytometry. Serum vs plasma derived exosomes were compared using nanoparticle tracking analysis for size distribution and immuno-dotblot for protein levels of HER1, HER2, HER3, cMET, P4HA1, S1000A9, PD-L1, and ALIX. Centralized processing yielded more consistent results with an overall improvement in PBMC numbers and viability. The delay between blood withdrawal and PBMC isolation had a noticeable impact on PBMC quality when it exceeded 24 hours. Between 24 and 48 hours, we observed a significant and donor dependent decrease in viability and a major increase in granulocytic contamination. Relative frequencies of NK cells were the most sensitive to processing delays during the first 24 hours. Despite a slight global decrease in viability at 24 hours, the relative proportions of other major immune subsets remained stable. Regarding exosomes, plasma-exosomes had a significantly higher modal size compared to serum-derived. Particle abundance was minimally higher in the serum-exosomes. There were no significant differences in the levels of most proteins except for HER3 and c-MET, which were significantly upregulated in plasma exosomes. We opted to centralize PBMC processing to one biobank to minimize variability. As it was logistically impossible to ensure shipments and processing within 24 hours for all recruitment sites, we limited sample inclusion up to 48 hours. This will be accounted for by tracking the number of granulocytes in future CyTOF measurements and including the delay durations in subsequent multi-variate analyses. Exosomes from serum and plasma will be included in the study as the data showed that both sources produced exosomes of adequate quality yet were different enough in protein levels to merit further investigation. Funding: IMI2 JU grant agreement 821558, supported by EU's Horizon 2020 and EFPIA. Citation Format: Rami Mustapha, Valentina Pomella, Jose Vicencio, Silvia Lopez-Lastra, Christelle Bahlawane, Wim Ammerlaan, Henoch S. Hong, Marie Morfouace, Caoimhe Vallely-Gilroy, Vassili Soumelis, Emanuela Romano, Sabine Tejpar, Tony Ng, Fay Betsou, IMMUcan consortium. Overcoming preanalytical variabilities for peripheral blood mononuclear cells and exosome profiling in the IMMUcan multicentre study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4281.