C-met Gene Research Articles

Expression and Clinical Significance of <i>PIK3CA</i>, <i>c-MET</i> and <i>c-KIT</i> Mutations in Saudi Breast Cancer Patients

Objective: PIK3CA is the most common pathway affected by mutations in breast cancer. PIK3CA/PTEN pathway is under intense investigation as a possible target for molecular therapy. Dysregulation PIK3CA/PTEN pathway is a substantial mechanism for the development of resistance to anti-HER2 therapy. Therefore, we aimed to study the PIK3CA/PTEN in breast cancer patients in Saudi population. Methods: We applied PTEN immunohistochemistry on 98 patients. Then, we applied next-generation sequencing to determine the genetic variations associated with the development of breast cancer and their correlations with clinicopathological variables. Results: PTEN expression was significantly correlated with lymph node metastasis (LNM), tumor stage, lymphovascular invasion (LVI) and triple negative breast cancer (TNBC). The prevalence of the PIK3CA mutation was 33.3% of cases and it was significantly associated with LNM, tumor stage, and with PTEN expression. c-MET mutation was identified in 41.7% of cases and it was associated with tumor stage and with TNBC, while c-KIT mutation was detected in 20.8% of cases, and it was significantly associated with TNBC only. Patients with positive PTEN expression had a significantly better overall survival (OS); on the contrary, patients with PIK3CA and c-MET had a significantly worse OS. Conclusion: Our study confirms the importance of PIK3CA/PTEN pathway in breast cancer patients. A high frequency of PIK3CA and c-MET mutations was detected and was associated with poor prognosis. Both c-MET and c-KIT genes have significant roles in developing TNBC. These findings should be expanded to a larger group study to improve the clinical outcomes and individualizing treatment.

Oncogenic gene transcripts detection by FISH on liquid-based cytology slides of 338 advanced lung cancer patients

Oncogenic gene transcripts in advanced lung cancer are a strong indication for targeted therapy. Cytology specimens are often the only materials available for oncogenic fusion analysis. This prospective study is to evaluate the feasibility of anaplastic lymphoma kinase (ALK) gene rearrangements, ROS oncogene 1 (ROS-1), and c-mesenchymal-epidermal transformation (c-MET) detected by fluorescence in situ hybridization (FISH) using liquid-based cytology (LBC) slides. Consecutive cytology specimens including fine-needle aspiration biopsy (FNAB) and serous effusions from 338 advanced lung cancer patients were collected between March 1, 2015, and July 6, 2016. The correlation between ALK, ROS-1, c-MET, and other common driver gene abnormalities and the therapeutic response to crizotinib in ALK-positive patients were also evaluated. ALK fusion transcripts were detected in 31 of 338 patients (9.17%). Twenty-two of the 31 ALK-positive patients were treated with crizotinib at our institution (2 were lost to follow-up), and the overall response rate was 75.0 % (15 of 20); disease control rate was 90.0% (18 of 20). FISH analyses for ROS-1 and c-MET were performed on 75 and 73 patients, respectively, and showed 3 patients positive for ROS-1 and 3 positive for c-MET. These positive cases were all ALK-negative. For patients with advanced lung cancer, LBC slides are suitable for detecting oncogenic gene transcripts, and the results can provide a reliable guideline for targeted therapy.

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.

HDAC1-Smad3-mSin3A complex is required for Smad3-induced transcriptional inhibition of hepatocyte growth factor receptor in human lung cancers.

c-Met hyperactivity has been observed in numerous neoplasms. Several researchers have shown that the abnormal activation of c-Met is mainly caused by transcriptional activation. However, the molecular mechanism behind this transcriptional regulation is poorly understood. Here, we suggest that Smad3 negatively regulates the expression and activation of c-Met via a transcriptional mechanism. We explore the molecular mechanisms that underlie Smad3-induced c-Met transcription inhibition. We found in contrast to the high expression of c-Met, Smad3 showed low protein and mRNA levels. Smad3 and c-Met expressions were inconsistent between lung cancer tissues and cell lines. We also found that Smad3 overexpression suppresses whereas Smad3 knockdown significantly promotes Epithelial-Mesenchymal Transition and production of the angiogenic factors VEGF, CTGF and COX-2 through the ERK1/2 pathway. In addition, Smad3 overexpression decreases whereas Smad3 knockdown significantly increases protein and mRNA levels of invasion-related β-catenin and FAK through the PI3K/Akt pathway. Furthermore, using the chromatin immunoprecipitation analysis method, we demonstrate that a transcriptional regulatory complex consisting of HDAC1, Smad3 and mSin3A binds to the promoter of the c-Met gene. By either silencing endogenous mSin3A expression with siRNA or by pretreating cells with a specific HDAC1 inhibitor (MS-275), Smad3-induced transcriptional suppression of c-Met could be effectively attenuated. These results demonstrate that Smad3-induced inhibition of c-Met transcription depends on of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the c-Met promoter. Collectively, our findings reveal a new regulatory mechanism of c-Met signaling, and suggest a potential molecular target for the development of anticancer drugs.

Chinese herbal formula QHF inhibits hepatocellular carcinoma metastasis via HGF/c-Met signaling pathway

Hepatocellular Carcinoma (HCC) is one of the most common malignant tumors, and high recurrence and metastasis are the major obstacles to successful treatment of HCC. Traditional Chinese medicine has little known and unique advantages in the treatment of HCC. Previous studies have confirmed that Chinese herbal formula Qingrejiedu (clears away heat and toxins), Huoxuehuayu (promotes blood flow to remove stasis) and Fuzhengguben (strengthens healthy qi and root) (QHF) has a significant effect on patients with advanced HCC, improves the quality of life and prolongs the survival time of patients significantly. In this study, we investigated the effect of QHF on proliferation, migration and invasion of human high metastatic hepatocellular carcinoma cell line HCCLM3 and its underlying mechanism. The results from our in vitro experiments showed that QHF has the ability to inhibit the proliferation by inducing G2/M phase cell cycle arrest and induce apoptosis. Moreover, QHF can also inhibit migration and invasion of HCCLM3 cells and the expression of the p-c-Met protein in HCCLM3 cells was down-regulated. c-Met is closely related to the metastasis of HCC, then we constructed a stable transfected cell line HepG2-met with high expression of c-Met by transfection. Further study in vivo revealed that c-Met gene will promote the growth of tumors and lung metastases in nude mice, and QHF intervention can reduce tumor lung metastases by inhibiting the HGF/c-Met signaling pathway. In conclusion, our study reveals that QHF can inhibit the proliferation, migration and invasion of HCCLM3, and this effect may be related to inhibiting HGF/c-Met signaling pathway.

Tivantinib Decreases Hepatocyte Growth Factor-Induced BCRP Expression in Hepatocellular Carcinoma HepG2 Cells.

Tivantinib, a mesenchymal-epithelial transition factor (cMET) inhibitor, is a molecular targeting drug that kills hepatocellular carcinoma (HCC) cells. Tivantinib alone does not affect the overall survival of patients with HCC, and combination treatment with tivantinib and other therapies has not been evaluated. This study was conducted to clarify the effect of the tivantinib in regulating breast cancer therapy-resistant protein (BCRP), a key transporter of 5-fluorouracil (5-FU), and dihydropyridine dehydrogenase (DPYD), a major metabolic enzyme of 5-FU. To this end, cMET gene expression was determined by RT-PCR in HepG2 (human hepatoma) cells. The transcriptional start sites of BCRP were determined by 5'-rapid amplification of cDNA ends (5'-RACE). BCRP and DPYD mRNA levels were determined by real-time RT-PCR, and promoter activities were measured by dual-luciferase assays. Results show that hepatocyte growth factor (HGF) upregulated the mRNA level of BCRP, but not DPYD, in HepG2 cells. The upregulation of BCRP expression by HGF was down-regulated by tivantinib. We also identified two transcriptional start sites (E1α, E1β) in BCRP by 5'-RACE. The transcriptional activity of the region -287 to E1α of BCRP was upregulated by HGF, which was decreased by tivantinib, whereas activity of the region -297 to E1βo f BCRP was not affected by tivantinib. Therefore, tivantinib regulates BCRP expression upstream of exon 1α. Combination treatment of tivantinib and 5-FU should be further evaluated for HCC therapy.

Abstract 3044: The novel bi-specific antibody CKD-702 is a potential agent for NSCLC patients with aberrant cMET and EGFR signaling

Abstract Development of drug resistance is one of the main causes of poor prognosis and therapeutic failure in NSCLC. Mutual synergism between cMET and EGFR is known to promote acquired drug resistance. In NSCLC, cMET gene amplification is reported in 2-4% previously untreated patients and in 5-20% of patients with EGFR mutation and acquired resistance to EGFR TKIs. Because of this crosstalk between cMET and EGFR pathways, dual inhibition of these targets holds promise as a treatment for NSCLC patients, especially those with acquired cMET activation. CKD-702 is a bispecific antibody designed to dually target cMET and EGFR. It is tetravalent IgG1-like bispecific antibody, in which single-chain variable fragment specific to EGFR is genetically fused to the c-terminus of a conventional IgG1 of a cMET. The antibody binds to extracellular domains of cMET and EGFR with high affinity and inhibits signaling by blocking ligand binding. CKD-702 also induces internalization and degradation of both receptors. There are various types of cMET activation in NSCLC patients, including point mutations and amplification. An emerging aberrant cMET activation is through a splice site mutation of cMET that leads to skipping of exon 14, inducing prolonged signaling and oncogenic activity. In this study, we carried out genetic analysis of cMET activation in cancer cell line as well as PDX model with or without EGFR mutation, and the efficacy of CKD-702 in each model for NSCLC was determined. CKD-702 showed complete tumor regression as monotherapy in tumor models that have primary/secondary activating EGFR mutations and cMET amplification. Also, CKD-702 resulted in complete tumor regression in models where cMET is activated by either skipping of exon 14 or amplification. Interestingly, unlike other previously developed EGFR-targeting agents, CKD-702 showed limited skin rash in GLP toxicity study. In summary, CKD-702 is confirmed to inhibit tumors with activated cMET pathway such as cMET amplification and exon 14 skipping as well as with EGFR-activating mutations. This profile of preclinical data supports the clinical trial of CKD-702 as monotherapy in selected lung cancer patients with aberrant cMET and EGFR signaling. A Phase I study of CKD-702 is planned to initiate in 2020. Citation Format: kyu jin park, Eun-Ju Jeon, Ji-Hye Choi, Kwan-Woo Lee, Kyung-Woo Lee, Gun-Woo Park, Hae-Jin Hong, In-Chang Hwang, Seung-Kee Moon, Yeo-Wook Koh. The novel bi-specific antibody CKD-702 is a potential agent for NSCLC patients with aberrant cMET and EGFR signaling [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 3044.

A phase Ib study of a novel c-MET, AXL and VEGFR-2 inhibitor ningetinib and gefitinib combination therapy in Chinese EGFR-TKI resistant NSCLC with T790M negative.

9583 Background: Ningetinib is a novel tyrosine kinase inhibitor, targeted at c-Met, Axl, VEGFR-2, Mer and Flt3. This phase Ib trial (NCT03758287) evaluated the safety, determined the recommended phase II dose (RP2D), and further explored the pharmacokinetic and efficacy of Ningetinib + Gefitinib in EGFR-TKIs acquired resistant NSCLC patients (pts) with T790M negative. Methods: Chinese Pts with advanced or metastatic NSCLC, acquired resistant to at least one EGFR-TKI, T790M negative were enrolled. Pts received Ningetinib 30, 40, 60 mg + Gefitinib 250mg orally once daily in dose-escalation (n = 12) by a Fibonacci 3+3 design. Expansion phase (n = 74, enrollment is ongoing) started at tolerated dosage. Safety, RP2D were primary endpoints; PK, antitumor activity were secondary endpoints. Non-mandatory tumor samples at baseline were collected for exploratory objectives. Results: Totally, 86 eligible pts were enrolled between Nov 2016 and Dec 2019, and received treatment (Ningetinib 30 mg, n = 36; 40 mg, n = 46; 60 mg, n = 4), with median age 56.7 years, 36% with baseline brain metastasis, 66%/33%/1% prior 1/2/3 lines EGFR-TKI treatment, respectively. Treatment-related adverse events (TRAEs) occurred in 82 (95%) pts, grade 3/4 in 32 pts (37%). Most common TRAEs (≥30%) were myocardial enzyme elevation (all grade 74.4%; grade 3-4 0%), transaminase elevation (73.3%; 2.3%), skin rash (60.5%; 3.5%), albuminuria (44.2%; 0%), coagulation abnormalities (mostly asymptomatic Fbp decrease; 37.2%; 15.1%), diarrhea (33.7%; 2.3%) and hypertension (32.6%; 11.6%). Two Dose limited toxicities were observed at 60 mg dosage (both were grade 3 Fbg decrease), RP2D was decided at 40 mg. Of 84 efficacy evaluable pts, ORR was 19.1% (16 PR), DCR was 91.7% (61 SD, 7 PD). Totally, 65 (75.6%) progression events occurred at data cut-off (9 Jan 2020), the median PFS for all pts was 4.4 months (95%CI 3.7-4.6). No PFS differences were found between pts grouped by 3rd TKIs history or brain metastasis. C-Met gene amplification by FISH was conducted in 72 pts (83.7%). Pts with higher gene copy number (GCN) responded better in treatment, ORR in the GCN ≥6 (n = 11), GCN ≥5 (n = 16) and GCN ≥3 (n = 37) subgroups was 36.4%, 25.0% and 21.6% respectively. Conclusions: Ningetinib was well tolerated at 30 mg and 40 mg dosage with Gefitinib 250 mg, the RP2D for Ningetinb was 40 mg. This combination therapy showed promising anti-tumor activity in prior EGFR-TKIs acquired resistant NSCLC pts with T790M negative. C-Met GCN was the potential efficacy biomarker. Clinical trial information: NCT03758287 .

Tivantinib inhibits the VEGF signaling pathway and induces apoptosis in gastric cancer cells with c-MET or VEGFA amplification.

Tivantinib has been described as a selective inhibitor of c-Met and is being studied in various types of cancer. In this study, we evaluated the effects of tivantinib on the suppression of gastric cancer (GC) cell migration and apoptosis. We also examined the mechanism of action of tivantinib by oncogenic pathway analysis. We applied an RNA-sequencing approach in 34 GC patients to identify oncogenes that are differentially expressed in GC tissues. To examine the inhibitory effect of tivantinib on GC cells, we conducted apoptosis analysis using an annexin V-APC/PI apoptosis detection kit and trans-well migration assay with human GC cell lines. For oncogenic pathway analysis, Western blot and quantitative real-time PCR analysis were used to detect the expression of proteins and genes before and after tivantinib exposure. In the RNA-sequencing analysis of 34 GC patients, c-Met and VEGFA genes were expressed and positively correlated with each other. Cell migration and apoptosis analysis demonstrated that tivantinib induced the best inhibition effect in SNU620, MKN45 (carries VEGFB mutation), AGS, and MKN28 cells, but not in KATO III (carries VEGFB and VEGFC mutations) cells. Oncogenic pathway analysis showed that tivantinib, in addition to c-Met signaling pathway inhibition, also inhibits VEGF signaling and MYC expression in VEGFA-expressing GC cells. We found that tivantinib has anti-cancer activity not only in GC cells overexpressing c-Met but also in non-c-Met GC cells by inhibition of the VEGF signaling pathway.

C-MET in intrahepatic cholangiocarcinoma: High-Frequency amplification predicts protein expression and a unique molecular subtype

As an increasing number of gene alterations have been discovered in intrahepatic cholangiocarcinoma (ICC), molecular targets are promising for the diagnosis and treatment of distinct subpopulations carrying unique molecular signatures. C-MET amplification is associated with a variety of tumors, including ICC; however, the characteristics of this alteration have not been assessed in ICC. By determining the ratios of C-MET/chromosome enumeration probe (CEP) 7 double-colour probes, we evaluated the presence of C-MET amplification in a cohort of 133 ICC tumors by fluorescence in situ hybridization (FISH). We further determined the levels of MET protein expression by immunohistochemistry (IHC) and analyzed clinicopathologic records. Of the samples, 21 (15.8 %) had high-frequency and 41 (30.8 %) had low-frequency C-MET genetic amplification, and 71 (53.4 %) had a normal C-MET gene. There were significant differences in gross classification (p = 0.045), microscopic cholangitis (p = 0.030), mucus level in tumors (p = 0.012) and T stage (p = 0.007) between the three groups. When we combined high-frequency and low-frequency amplifications of C-MET into one group, only microscopic cholangitis (p = 0.010) and stage (p = 0.016) showed significant differences compared to normal C-MET gene expression. However, when we combined the low-frequency C-MET amplification group with the normal C-MET group and compared this combined group with the high-frequency C-MET amplification group, the high-frequency group had more younger patients (p = 0.047), had more non-mass-forming (MF)-type cases according to gross classification (p = 0.015), secreted more mucus (p = 0.002) and appeared to have a higher T stage (p = 0.031) than the combined group. For IHC results, although only cluster C-MET amplification predicted protein overexpression, high-frequency amplification was associated with more protein expression than the other genetic statuses (p = 0.000). As low-frequency C-MET amplification exhibited similar biology to that of the normal gene, we regarded high-frequency amplification of C-MET as a unique molecular subtype. It may play important roles in tumor progression and may be used as a prognostic marker for targeted therapy.

A novel neuronal organoid model mimicking glioblastoma (GBM) features from induced pluripotent stem cells (iPSC)

BackgroundCurrent experimental models using either human or mouse cell lines, are not representative of the complex features of GBM. In particular, there is no model to study patient-derived iPSCs to generate a GBM model. Overexpression of c-met gene is one of the molecular features of GBM leading to increased signaling via STAT3 phosphorylation. We generated an iPSC line from a patient with c-met mutation and we asked whether we could use it to generate neuronal-like organoids mimicking features of GBM. MethodsWe have generated iPSC-aggregates differentiating towards organoids. We analyzed them by gene expression profiling, immunostaining and transmission electronic microscopy analyses (TEM). ResultsHerein we describe that c-met-mutated iPSC aggregates spontaneously differentiate into dopaminergic neurons more rapidly than control iPSC aggregates in culture. Gene expression profiling of c-met-mutated iPSC aggregates at day +90 showed neuronal- and GBM-related genes, reproducing a genomic network described in primary human GBM. Comparative TEM analyses confirmed the enrichment of these structures in intermediate filaments and abnormal cilia, a feature described in human GBM. The c-met-mutated iPSC-derived organoids, as compared to controls expressed high levels of glial fibrillary acidic protein (GFAP), which is a typical marker of human GBM, as well as high levels of phospho-MET and phospho-STAT3. The use of temozolomide (TMZ) showed a preferential cytotoxicity of this drug in c-met-mutated neuronal-like organoids. General significanceThis study shows the feasibility of generating “off-the shelf” neuronal-like organoid model mimicking GBM using c-met-mutated iPSC aggregates and its potential future use in research.

Endogenous and artificial miRNAs explore a rich variety of conformations: a potential relationship between secondary structure and biological functionality

Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.

Exploring the Molecular Crosstalk between Pancreatic Bud and Mesenchyme in Embryogenesis: Novel Signals Involved.

Pancreatic organogenesis is a multistep process that requires the cooperation of several signaling pathways. In this context, the role of pancreatic mesenchyme is important to define the epithelium development; nevertheless, the precise space–temporal signaling activation still needs to be clarified. This study reports a dissection of the pancreatic embryogenesis, highlighting the molecular network surrounding the epithelium–mesenchyme interaction. To investigate this crosstalk, pancreatic epithelium and surrounding mesenchyme, at embryonic day 10.5, were collected through laser capture microdissection (LCM) and characterized based on their global gene expression. We performed a bioinformatic analysis to hypothesize crosstalk interactions, validating the most promising genes and verifying the precise localization of their expression in the compartments, by RNA in situ hybridization (ISH). Our analyses pointed out also the c-Met gene, a very well-known factor involved in stimulating motility, morphogenesis, and organ regeneration. We also highlighted the potential crosstalk between Versican (Vcan) and Syndecan4 (Sdc4) since these genes are involved in pancreatic tissue repair, strengthening the concept that the same signaling pathways required during pancreatic embryogenesis are also involved in tissue repair. This finding leads to novel strategies for obtaining functional pancreatic stem cells for cell replacement therapies.

Apoptotic Effects of Mucin1 Aptamer-Conjugated Nanoparticles Containing Docetaxel and c-Met siRNA on SKBR3 Human Metastatic Breast Cancer Cells

Abstract Background: Apoptosis is a crucial process in the failure of cancer progression. However, the occurrence of resistance to chemotherapy in cancer cells may prevent apoptosis via induction of the expression of the anti-apoptotic genes (Bcl-2) and/or reduction of the expression of the apoptotic caspases. Objectives: The current study aimed at investigating the apoptotic effects of targeted co-delivery of docetaxel and cMet siRNA (siMet) through mucin1 aptamer-conjugated chitosan nanoparticles on mucin1 + metastatic breast cancer cells (SKBR3). Methods: Characterization of nano-drugs,Western blotting assay, annexin V/ propidium iodide staining assay, and gene expression studies were evaluated based on metastatic breast cancer cells. Results: Characterization showed the appropriate size (110.5  3.9 nm), zeta potential (11.6  0.8 mV), and spherical shape of nanoparticles. Loading efficiency of 90.7% and 88.3% were gained for siRNA and docetaxel, respectively. The siRNA entrapment onto nanoparticles and conjugation of aptamers to nanoparticles were confirmed by gel electrophoresis. Gene knockdown assay represented the effectiveness of siMet on cMet gene silencing. According to the flow cytometry results, targeted co-delivery was successful in leading tumor cells to apoptosis (94.9%). Also, targeted co-delivery could reduce the expression of Bcl-2 gene (P < 0.0001) and increase the expression of caspase-8 and caspase-9 genes (P < 0.0001). Conclusions: Combination treatment of metastatic breast cancer cells with aptamer-conjugated nanoparticles containing docetaxel and cMet siRNA significantly increased apoptosis.

Recent Progress in the Development of Small Molecule c-Met Inhibitors.

C-Met, also referred to as Hepatocyte Growth Factor Receptor (HGFR), is a heterodimeric receptor tyrosine kinase. It has been determined that c-Met gene mutations, overexpression, and amplification also occur in a variety of human tumor types, and these events are closely related to the aberrant activation of the HGF/c-Met signaling pathway. Meanwhile, high c-Met expression is closely associated with poor prognosis in cancer patients. The c-Met kinase has emerged as an attractive target for developing antitumor agents. In this review, we cover the recent advances on the small molecule c-Met inhibitors discovered from 2018 until now, with a main focus on the rational design, synthesis and structureactivity relationship analysis.

Validation of molecular biomarkers for preoperative diagnostics of human papillary thyroid carcinoma in fine needle aspirates.

Despite substantial efforts, reliable preoperative diagnostic for human thyroid malignancies in case of cytologically indeterminate nodules is still missing, resulting in high number of unnecessary thyroidectomies. In an attempt to increase precision of existing preoperative diagnostics, we aimed at validating the panel of molecular biomarkers predictive for papillary thyroid carcinoma (PTC) in preoperative fine needle aspirate (FNA) samples. In this prospective study conducted in preoperative thyroid FNA from 44 thyroid nodules, expression levels of 11 molecular biomarkers previously validated on the postoperative samples of PTCs were measured by Cell-to-CT and QuantiGene Plex methods and correlated with final diagnosis. The QuantiGene Plex resulted in reliable gene expression measurements for FNA and core-needle biopsy (CNB) samples, however this method was less sensitive than pre-amplification based Cell-to-CT. Measurements conducted on the same samples by the two methods significantly correlated for most of the genes. Expression levels of TIMP1, c-MET and ARNTL were upregulated in PTC nodules as compared to benign counterparts, supporting previous post-operative studies. Strong correlation was observed between these biomarker alterations in the same samples. Within the sub-group of 15 indeterminate nodules (Bethesda II-V), TIMP1 had 100% specificity and 83% sensitivity for PTC cases. Assessment of TIMP1, c-MET and core-clock gene ARNTL expression levels by QuantiGene Plex assay in FNA samples holds promise as an ancillary method to the cytological preoperative diagnostics.

Abstract 1121: Role of c-Met in colorectal cancer lung metastasis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; systemic metastasis to the lungs occurs in approximately 10-20% of patients with CRC. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. The present study was designed to determine the role of c-Met in growth and metastasis of CRC cells to the lung. We evaluated patient-derived xenografts (PDX) tumors and CRC cells for c-Met activation (phospho-Met [Tyr1234/1235]), established PDX cell lines with high phospho-Met (Tyr1234/1235) expression from lung or liver metastases, and investigated the role of c-Met expression in CRC lung metastasis. Methods. (1) PDX tumors were implanted into NOD-scid IL2Rgammanull mice; PDX cell lines were established from F3 tumor generation. (2) The protein expression of phospho-Met (Tyr1234/1235) was observed by western blotting and immunohistochemistry. (3). HT29 LungM3 cell line was derived from the human CRC line HT29 following multiple rounds of in vivo selection for lung metastasis in immunodeficient mice. HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene. HT29LungM3 cells were injected into SCID mice subcutaneously to measure tumor growth or intravenously to examine the role of c-Met in CRC lung metastasis. Results. (1) High phospho-Met (Tyr1234/1235) expression was identified in 6 out of 20 CRC PDX models. Two PDX cell lines with high phospho-Met (Tyr1234/1235) expression, 2377LM (from liver metastasis) and 2387 (from lung metastasis), were established. Metastatic potential of the 2377LM cell line to the liver was confirmed after intrasplenic injection of cancer cells into SCID mice. The metastatic potential of 2387 cells to the lung was confirmed after intravenous injection of cancer cells into SCID mice. (2) Western blot analysis demonstrated high phospho-Met (Tyr1234/1235) expression in HT29LungM3, 2387LM and 2387 cell lines, compared to HCT116 (human CRC), LS174T (human CRC), Caco-2 (human CRC), SK-OV-3 (human ovarian cancer) and JAR (placenta choriocarcinoma) cell lines. (3) HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene with c-Met knockdown of at least 90%. Surprisingly, c-Met inhibition in the HT29LungM3 cell line had no effect on subcutaneous tumor growth or lung metastasis. Conclusions. Aberrant expression of c-Met is associated with the progression of multiple human malignancies and has been identified as a novel target for the treatment of malignant tumors. Here, we examined expression of phospho-Met (Ty1234/1235) in CRC PDX cell samples and determined that c-Met knockdown does not decrease CRC lung metastasis. These results are important for understanding the mechanism of CRC lung metastasis and proper strategy selection for prevention and treatment of patients with advanced disease. Citation Format: Piotr G. Rychahou, Nick Roller, Eun Y. Lee, Nicole Rychagov, Matthew Melton, Carrigan Wasilchenko, B. Mark Evers. Role of c-Met in colorectal cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1121.

Effect of Fibroblast Growth Factor-2 and its Receptor Gene Polymorphisms on the Survival of Patients With Hepatitis B Virus-associated Hepatocellular Carcinoma.

Fibroblast growth factor (FGF), vascular endothelial growth factor, and hepatocyte growth factor play a critical role in the pathogenesis of hepatocellular carcinoma (HCC). We assessed nine single nucleotide polymorphisms (SNPs) in the FGF1, FGF2, FGF receptor (FGFR)-2, Flt-1, and c-MET genes in 245 HCC patients and 483 chronic hepatitis B virus (HBV) carriers without HCC. Kaplan-Meier analysis showed that patients with the FGF2 rs308447 TT genotype had shorter overall survival than patients with the CC or CT genotype (p=0.016) and that FGF2 rs308379 A allele carriers had shorter overall survival than patients with the TT genotype (p=0.020). Multivariate Cox proportional analysis revealed that the FGF2 rs308379 A allele (hazard ratio(HR)=1.663, p=0.004) and advanced tumor stage (HR=3.430, p<0.001) were independent prognostic factors for overall survival in patients with HCC.

EFFECT OF BEE AND SCORPION VENOMS ON PROSTATE CANCER IN VITRO STUDY

Prostate cancer (PCa) is the commonest diagnosed visceral malignancy among males worldwide. Recent studies have shown that bee venom target the cancer cells without effect on the normal cells by activating PC3 with oxidative substances against prostate cancer. The induction of the apoptotic cell death through several cancer cell death mechanisms, includes activation of up regulation of c-myc, and c-met genes and down regulation of Casp-7, that are important to induce anticancer. Scorpion venom is a potential bio-source and therapeutic tool to design potent drugs against variety of diseases. It has been used as medicinal and therapeutic tool since ancient times in China. Scorpion venom consists of neurotoxins, salts, low molecular weight peptides and different enzymes with high molecular activities. These activities make them novel therapeutic agents. The results revealed low cytotoxicity of bee venom and satusporin while scorpion venom showed the highest level of cytotoxicity. On the other hand, apoptosis was shown in all tested subjects at pre G1, while cell cycle arrest was different, which was at G1 by scorpion venom and at G2M by both bee venom and satusporin. Apoptosis was at late stage by scorpion venom and satusporin drug while at an early stage by bee venom as Casp-7, C-myc, C-met showed up regulation, the highest level shown in scorpion venom. This study explains that bee venom and scorpion have a potencial therapeutic effect on prostate cancer.

Differential Expression of Epidermal Growth Factor Receptor, Hepatocyte Growth Factor Receptor and Hypoxia‐Inducible Factor‐1‐Alpha in Aged Rat Livers

Epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (cMET) and the transcription factor, hypoxia‐inducible factor 1‐ alpha (HIF1‐α) are highly important in angiogenesis, mitogenesis and cell proliferation which are all vital processes required for mammalian liver regeneration. In as much as aging is characterised with general decline in maintenance and function of certain body organs, this is not exactly the same with the liver in regeneration. Aging could result to reduction in the response of the liver to certain extrinsic stimuli such as initiation of stress proteins as a result of injury. To study the effects of age on the expression of these molecules, EGFR, cMET and HIF1‐α gene expression in aged rat liver samples (18months old) vs. young animals (3 months old) was studied through semi and relative quantitative quantification of gene specific mRNA coding for these genes. β‐Actin was used as a reference/housekeeping gene for normalisation of the EGFR, cMET and HIF1‐α genes, the Livak method was used to quantify the relative expression of these genes in relation to the ages of the animals. The results show high mRNA expression of EGFR and cMET in 3 months old samples (young). Analysis of EGFR showed an mRNA fold decrease of 0.56 gene expression and 2.3 for cMET gene express in the 18 months liver. HIF1‐α analysis showed a 5.16 increase in the mRNA gene expression in the 18 month old when compared with the 3 months old. Data from this study show that decrease in expression of epidermal growth factor receptor in the liver with age accelerates decrease in regeneration rate. The fact that EGFR and cMET were expressed at significantly lower levels in older samples reflects the differential decrease in regeneration ability and physiologic activities such proliferation, mitogenesis and angiogenesis in the liver with age. This result in reduction in the ability of the liver to regenerate, thus making the aged liver more susceptible to infections.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.