PWWP2B promotes DNA end resection and homologous recombination.

Genome instability is one of the leading causes of gastric cancers. However, the mutational landscape of driver genes in gastric cancer is poorly understood. Here, we investigate somatic mutations in 25 Korean gastric adenocarcinoma patients using whole-exome sequencing and show that PWWP2B is one of the most frequently mutated genes. PWWP2B mutation correlates with lower cancer patient survival. We find that PWWP2B has a role in DNA double-strand break repair. As a nuclear protein, PWWP2B moves to sites of DNA damage through its interaction with UHRF1. Depletion of PWWP2B enhances cellular sensitivity to ionizing radiation (IR) and impairs IR-induced foci formation of RAD51. PWWP2B interacts with MRE11 and participates in homologous recombination via promoting DNA end-resection. Taken together, our data show that PWWP2B facilitates the recruitment of DNA repair machinery to sites of DNA damage and promotes HR-mediated DNA double-strand break repair. Impaired PWWP2B function might thus cause genome instability and promote gastric cancer development.

Comparison of Tepotinib, Paclitaxel, or Ramucirumab Efficacy According to the Copy Number or Phosphorylation Status of the MET Gene: Doublet Treatment versus Single Agent Treatment.

Although conventional combination chemotherapies for advanced gastric cancer (GC) increase survival, such therapies are associated with major adverse effects; more effective and less toxic treatments are required. Combinations of different anti-cancer drugs, for example, paclitaxel plus ramucirumab, have recently been used as second-line treatments for advanced GC. This study evaluated how copy number variations of the MET gene, MET mutations, and MET gene and protein expression levels in human GC cells modulate the susceptibility of such cells to single-agent (tepotinib, ramucirumab, or paclitaxel) and doublet (tepotinib-plus-paclitaxel or ramucirumab-plus-paclitaxel treatment regimens. Compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibited the growth of GC cells with MET exon 14 skipping mutations and those lacking MET amplification but containing phosphorylated MET; such inhibition was dose-dependent and associated with cell death. Tepotinib-plus-paclitaxel and ramucirumab-plus-paclitaxel similarly inhibited the growth of GC cells lacking MET amplification or MET phosphorylation, again in a dose-dependent manner, but without induction of cell death. However, tepotinib alone or tepotinib-plus-ramucirumab was more effective against c-MET-positive GC cells (>30 copy number variations) than was ramucirumab or paclitaxel alone or ramucirumab-plus-paclitaxel. These in vitro findings suggest that compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibits the growth of c-MET-positive GC cells, cells lacking MET amplification but containing phosphorylated MET, and cells containing MET mutations. Clinical studies are required to confirm the therapeutic effects of these regimens.

Entrectinib Induces Apoptosis and Inhibits the Epithelial-Mesenchymal Transition in Gastric Cancer with NTRK Overexpression.

Tropomyosin receptor kinase (TRK) and receptor tyrosine kinase (RTK class VII) expression are important in many human diseases, especially cancers, including colorectal, lung, and gastric cancer. Using RNA sequencing analysis, we evaluated the mRNA expression and mutation profiles of gastric cancer patients with neurotropic tropomyosin receptor kinase (NTRK) 1-3 overexpression (defined as a ≥2.0-fold change). Furthermore, we screened eight TRK inhibitors in NCI-N87, SNU16, MKN28, MKN7, and AGS cells. Among these inhibitors, entrectinib showed the highest inhibitory activity; therefore, this drug was selected for analysis of its therapeutic mechanisms in gastric cancer. Entrectinib treatment induced apoptosis in NTRK1-3-expressing and VEGFR2-expressing NCI-N87 and AGS cells, but it had no effect on NTRK1-3-, VEGFR2-, TGFBR1-, and CD274-expressing MKN7 cells. SNU16 and MKN28 cells with low NTRK1-3 expression were not affected by entrectinib. Therefore, a mechanistic study was conducted in NCI-N87 (high expression of NTRK1-3 but mutation of NTRK3), AGS (high expression of NTRK1-3) and MKN28 (low expression of NTRK1-3) gastric cancer cell lines. Entrectinib treatment significantly reduced expression levels of phosphorylated NFκB, AKT, ERK, and ß-catenin in NCI-N87 and AGS cells, whereas it upregulated the expression levels of ECAD in NCI-N87 cells. Together, these results suggest that entrectinib has anti-cancer activity not only in GC cells overexpressing pan NTRK but also in VEGFR2 GC cells via the inhibition of the pan NTRK and VEGFR signaling pathways.

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.

Tepotinib Inhibits the Epithelial-Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3ß, E-Cadherin, and Mucin 5AC and 6 Levels.

Aberrant expression of mucins (MUCs) can promote the epithelial-mesenchymal transition (EMT), which leads to enhanced tumorigenesis. Carcinogenesis-related pathways involving c-MET and ß-catenin are associated with MUCs. In this study, we characterized the expression of EMT-relevant proteins including MET, ß-catenin, and E-cadherin in human gastric cancer (GC) cell lines, and further characterized the differential susceptibility of these cell lines compared with the c-MET inhibitor tepotinib. We assessed the antitumor activity of tepotinib in GC cell lines. The effects of tepotinib on cell viability, apoptotic cell death, EMT, and c-MET and ß-catenin signaling were evaluated by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS), flow cytometry, Western blotting, and qRT-PCR. The antitumor efficacy was assessed in MKN45 xenograft mice. Tepotinib treatment induced apoptosis in c-MET-amplified SNU620, MKN45, and KATO III cells, but had no effect on c-MET-reduced MKN28 or AGS cells. Tepotinib treatment also significantly reduced the protein levels of phosphorylated and total c-MET, phosphorylated and total ERK, ß-catenin, and c-MYC in SNU620 and MKN45 cells. In contrast, this drug was only slightly active against KATO III cells. Notably, tepotinib significantly reduced the expression of EMT-promoting genes such as MMP7, COX-2, WNT1, MUC5B, and c-MYC in c-MET-amplified GC cells and increased the expression of EMT-suppressing genes such as MUC5AC, MUC6, GSK3ß, and E-cadherin. In a mouse model, tepotinib exhibited good antitumor growth activity along with increased E-cadherin and decreased phosphorylated c-MET (phospho-c-MET) protein levels. Collectively, these results suggest that tepotinib suppresses tumor growth and migration by negatively regulating c-MET-induced EMT. These findings provide new insights into the mechanism by which MUC5AC and MUC6 contribute to GC progression.

RNF43 R117fs mutant positively regulates Wnt/ß-catenin signaling by failing to internalize FZD expressed on the cell surface.

RING finger protein 43 (RNF43) encodes the transmembrane E3 ubiquitin ligase, which targets the Wnt receptor Frizzled (FZD). RNF43 mutations have been discovered in various human cancers including colon, pancreatic, stomach, ovarian, and liver cancers. Functional studies on RNF43 missense mutations have shown that they negatively regulate Wnt signaling; however, there are few functional studies on RNF43 frameshift mutations. In this study, we showed that R117fs and P441fs mutants enhanced Wnt/ß-catenin signaling, whereas Q409fs and G659fs mutants retained the ability to suppress Wnt/ß-catenin signaling. Specifically, R117fs was unable to ubiquitinate FZD5 due to lack of the RING domain, although it was able to interact with FZD5. Immunofluorescence showed that R117fs failed to internalize FZD5 expressed on the cell surface. We also showed that LGK974, a potent Wnt inhibitor, decreased the Wnt/ß-catenin activity by R117fs and P441fs mutations. Together, these results demonstrate that RNF43 frameshift mutations retain normal functionality; thus, targeted anti-cancer therapy can be developed according to the mutation type of RNF43.

Responses to the Tepotinib in Gastric Cancers with MET Amplification or MET Exon 14 Skipping Mutations and High Expression of Both PD-L1 and CD44.

Both MET exon 14 skipping mutation (METex14SM) and high copy-number variation (CNV) lead to enhanced carcinogenesis; additionally, programmed-death ligand 1 (PD-L1) is often upregulated in cancers. In this study, we characterized the expression of MET (including METex14SM), PD-L1, and CD44 in human gastric cancer (GC) cells as well as the differential susceptibility of these cells to tepotinib. Tepotinib treatments inhibited the growth of five GC cells in a dose-dependent manner with a concomitant induction of cell death. Tepotinib treatments also significantly reduced the expression of phospho-MET, total MET, c-Myc, VEGFR2, and Snail protein in SNU620, MKN45, and Hs746T cells. Notably, tepotinib significantly reduced the expression of CD44 and PD-L1 in METex14SM Hs746T cells. By contrast, tepotinib was only slightly active against SNU638 and KATO III cells. Migration was reduced to a greater extent in the tepotinib-treated group than in the control group. Tepotinib may have therapeutic effects on c-MET-amplified GC, a high expression of both PD-L1 and CD44, and METex14SM. Clinical studies are needed to confirm these therapeutic effects.

TRK inhibitors block NFKB and induce NRF2 in TRK fusion-positive colon cancer.

Tropomyosin receptor kinase (TRK) fusion is one of the oncogenic driver causes of colon cancer, and tropomyosin 3-neurotrophic receptor tyrosine kinase 1 (TPM3-NTRK1) fusion has been detected in the KM12SM cell line. In the present study, we investigated anticancer mechanisms in the KM12SM cell line using three different form of dovitinib (dovitinib (free base), dovitinib lactate (mono acid), and dovitinib dilactic acid (diacid)) and four TRK inhibitors (LOXO-101, entrectinib, regorafenib, and crizotinib). Exposure of TRK inhibitors at concentrations of 10 nM resulted in the apoptosis of KM12SM cells, whereas regorafenib had no effect. Treatment with all inhibitors except regorafenib also significantly increased the expression levels of the genes nuclear factor-erythroid 2-related factor 2 (NRF2) and glutamyl cysteine ligase catalytic subunit (GCLC) in KM12SM. These drugs significantly reduced expression of the phosphorylated proteins NFκB and COX-2 in the KM12SM cell line, and significantly attenuated KM12SM cell migration, according to a Transwell migration assay. Together, these results suggest that TRK inhibitors block products of carcinogenesis by negatively regulating the NFκB signaling pathway and positively regulating the antioxidant NRF2 signaling pathway.

RNF43 and PWWP2B inhibit cancer cell proliferation and are predictive or prognostic biomarker for FDA-approved drugs in patients with advanced gastric cancer.

Background: Abnormal regulation of genes has been closely related to gastric cancer. The characterization of gastric cancer has necessitated the development of new therapeutics as well as the identification of prognostic markers to predict the response to novel drugs. In our study, we used RNA sequencing analyses to show that on gastric cancer tissues to identification of gastric cancer prognostic markers. We specifically chose to study RNF43 because it inhibits gastric cancer-related Wnt/ß-catenin signaling by interacting with Wnt receptors. PWWP2B was chosen because it is a gene which is downregulated in gastric cancer. Methods: Utilizing RNA sequencing analysis, we evaluated the mRNA expression profile in gastric cancer patients. Also, we used HAP1 cells which is a human near-haploid cell line derived from the male chronic myelogenous leukemia cell line KBM-7. These cell line has one copy of each gene, ensuring the edited allele will not be masked by additional alleles. We investigated the screening of 1,449 FDA-approved drugs in HAP1, HAP1 RNF43 KO and HAP1 PWWP2B KO cells. RNA sequencing data reveals that RNF43 and PWWP2B expression were down-regulated in recurrence gastric cancer patients. Next, we investigated the anti-cancer effects of selected drugs in RNF43 and PWWP2B down-regulated MKN45 gastric cancer cells and xenograft model. Results: Among these FDA-approved drugs, three drugs (docetaxel trihydrate, pelitinib and uprosertib) showed strong inhibitory effects in RNF43 KO cells and PWWP2B KO cells. In MKN45 xenograft model, tumor volumes were significantly reduced in the docetaxel trihydrate, uprosertib or pelitinib-treated group. Our data demonstrated that RNF43 and PWWP2B are a biomarker that predict recurrence of gastric cancer. Conclusions: Our findings suggest that docetaxel trihydrate, uprosertib and pelitinib could be used as novel therapeutic agents for the prevention and treatment of gastric cancer with a decrease in RNF43 and PWWP2B expression.

Foretinib Inhibits Cancer Stemness and Gastric Cancer Cell Proliferation by Decreasing CD44 and c-MET Signaling.

PURPOSE: CD44 isoforms are highly expressed in cancer stem cells, initiating tumor growth and sustaining tumor self-renewal. Among these isoforms, CD44 variant 9 (CD44v9) is overexpressed in chronic inflammation-induced cancer. CD44 and the mesenchymal-to-epithelial transition (MET) receptor tyrosine kinase are coactivated in some gastric cancers (GCs). In this study, we characterized MET and CD44 expression and signaling in human GC cell lines and analyzed differences in the susceptibility of these lines to foretinib. PATIENTS AND METHODS: We analyzed cell viability and the rate of apoptotic cells using MTS assays and flow cytometry, respectively. Gene and protein expression were assessed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting, respectively. RESULTS: Foretinib treatment resulted in dose-dependent inhibition of growth in c-MET-amplified MKN45 and SNU620 cells with concomitant induction of apoptosis, but not in c-MET-reduced MKN28 and AGS cells. Foretinib treatment also significantly reduced phosphor-c-MET, phosphor-AKT, beta-catenin, and COX-2 protein expression in MKN45 and SNU620 cells. Interestingly, foretinib significantly reduced CD44, CD44v9, COX-2, OCT3/4, CCND1, c-MYC, VEGFA, and HIF-1a gene expression in CD44 and MET coactivated MKN45 cells and increased CD44s gene expression; in contrast, these drugs were only slightly active against SNU620 cells. CONCLUSION: The results of this study indicate that foretinib could be a therapeutic agent for the prevention or treatment of GCs positive for CD44v9 and c-MET.

INC280 inhibits Wnt/ß-catenin and EMT signaling pathways and its induce apoptosis in diffuse gastric cancer positive for c-MET amplification.

OBJECTIVE: Gastric cancer is more open related to genetic predisposition. In our RNA sequencing study on gastric cancer patients, Runt-related transcription factor-3 (RUNX3) expression was significantly down-regulated in gastric cancer. We showed that decreased levels of RUNX3 are significantly associated with c-MET (r = - 0.4216, P = 0.0130). In addition, c-MET expression is a candidate for targeted therapy in gastric cancer. Therefore, in the present study, the anti-cancer effects of the c-MET inhibitor on gastric cancer cells from positive or negative for c-MET amplification were evaluated. RESULTS: INC280 treatment inhibits growth of a c-MET-amplified MKN45 (RUNX3-positive) and SNU620 (RUNX3-negative) diffuse type cells. Then, INC280 showed the highest inhibition and apoptotic rates with the lowest IC50s in MKN45 cells but not in c-MET-reduced MKN28 (intestinal type) cells. We also showed that INC280 inhibits the WNT signaling pathway and SNAIL expression in MKN45 cells. The data indicate that INC280 could be used as therapeutic agents for the prevention or treatment of diffuse gastric cancer positive for c-MET amplification.

The effects of crizotinib in a transgenic Drosophila model expressing the human TPM4-ALK fusion gene or TPM4.

Anaplastic lymphoma kinase (ALK) fusion events lead to constitutive activation of the ALK kinase domain, thereby functioning as oncogenic drivers. These fusion proteins have been identified in numerous cancers. Crizotinib, a small molecule inhibitor of c-Met and ALK, is a Food and Drug Administration-approved drug with reported efficacy in the treatment of cancer. Tropomyosins (TPMs) are a family of actin filament-binding proteins. Altered TPM expression has been found in a variety of human tumors. Inhibitors of cancer-associated TPMs and actin-targeting compounds have been developed, but anti-actin agents have cardiac and respiratory muscle toxicities. In this study, we investigated the sensitivities of human TPM4 (hTPM4), human ALK (hALK), and their fusion gene (hTPM4-hALK) to crizotinib by measuring the lifespan of transgenic Drosophila Flies overexpressing hTPM4-hALK, hTPM4 and hALK showed decreased lifespans compared with controls. Although crizotinib is an inhibitor of ALK, treatment with crizotinib significantly extended the lifespans of Drosophila expressing hTPM4 and hTPM4-hALK but had no effect on hALK-expressing flies. Autophosphorylation of Tyr1278 is necessary for full activation of the ALK domain. We confirmed that hTPM4-hALK was phosphorylated at Tyr1278 in a ligand-independent manner, and hTPM4-hALK-expressing flies treated with crizotinib showed a decreased level of Tyr1278 phosphorylation compared with untreated hTPM4-hALK-expressing flies, with a greater decrease induced by 1 µM compared with 200 nM crizotinib. Taken together, the results suggest that crizotinib is effective for treating ALK-driven cancer and might be a new therapeutic drug, without cardiac or respiratory muscle toxic effects, for TPM4-expressing cancers.