The Immune Suppressor IGSF1 as a Potential Target for Cancer Immunotherapy.

The development of first-generation immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 ushered in a new era in anticancer therapy. Although immune-checkpoint blockade therapies have shown clinical success, a substantial number of patients yet fail to benefit. Many studies are under way to discover next-generation immunotherapeutic targets. Immunoglobulin superfamily member 1 (IGSF1) is a membrane glycoprotein proposed to regulate thyroid function. Despite containing 12 immunoglobin domains, a possible role for IGSF1, in immune response, remains unknown. Here, our studies revealed that IGSF1 is predominantly expressed in tumors but not normal tissues, and increased expression is observed in PD-L1low non-small cell lung cancer (NSCLC) cells as compared with PD-L1high cells. Subsequently, we developed and characterized an IGSF1-specific human monoclonal antibody, WM-A1, that effectively promoted antitumor immunity and overcame the limitations of first-generation immune-checkpoint inhibitors, likely via a distinct mechanism of action. We further demonstrated high WM-A1 efficacy in humanized peripheral blood mononuclear cells (PBMC), and syngeneic mouse models, finding additive efficacy in combination with an anti-PD-1 (a well-characterized checkpoint inhibitor). These findings support IGSF1 as an immune target that might complement existing cancer immunotherapeutics.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

DGG-300273, a novel WNT/ß-catenin inhibitor, induces apoptotic cell death by activating ROS-BIM signaling in a Wnt-dependent manner in colon cancer cells.

Dysregulated Wnt signaling is associated with malignant oncogenic transformation, especially in colon cancer. Recently, numerous drugs have been developed based on tumorigenesis biomarkers, thus having high potential as drug targets. Likewise, WNT/ß-catenin pathway members are attractive therapeutic targets for colon cancer and are currently in various stages of development. However, although inhibitors of proteins regulating the WNT/ß-catenin signaling pathway have been extensively studied, they have yet to be clinically approved, and the underlying molecular mechanism(s) of their anticancer effects remain poorly understood. Herein, we show that a novel WNT/ß-catenin inhibitor, DGG-300273, inhibits colon cancer cell growth in a Wnt-dependent manner due to upregulation of the BCL2-family protein Bim and caspase-dependent apoptotic cell death. Additionally, DGG-300273-mediated cell death occurs by increased reactive oxygen species (ROS), as shown by abrogation of apoptotic cell death and ROS production following pretreatment with the antioxidant N-acetylcysteine. These results suggest that DGG-300273 represents a promising investigational drug for the treatment of Wnt-associated cancer, thus warranting further characterization and study.

CJ14939, a Novel JAK Inhibitor, Increases Oxaliplatin-induced Cell Death Through JAK/STAT Pathway in Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer is reported to have the highest mortality rate among human malignancies. Although many research results for the treatment of colorectal cancer have been reported, there is no suitable treatment when resistance has developed. Therefore, it is necessary to develop new therapeutic agents. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling plays an essential role in cell differentiation, proliferation, and survival. Abnormal activation of the JAK/STAT signaling pathway, by gene mutation or amplification, may induce cancer development, and sustained JAK/STAT activation is involved in chemoresistance. While many therapeutic agents have been developed to treat colon cancer, there remains no drug to overcome resistance to chemotherapies. The purpose of this study was to determine the potential of CJ14939 as a novel JAK inhibitor for the treatment of colorectal cancer. MATERIALS AND METHODS: In this study, cell culture, cell death assay, 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, colony formation assay, immunoblot analysis and tumor xenograft were applied. RESULTS: CJ14939 induced cell death, and inhibited phosphorylation of JAK1 and STAT3 in colorectal cancer cells. Furthermore, CJ14939 also promoted oxaliplatin-induced cell death, up-regulated expression of cleaved caspase-3, and down-regulated expression of phospho-JAK1 and phospho-STAT3. In vivo, co-treatment with CJ14939 and oxaliplatin notably reduced tumor growth when compared with CJ14939 or oxaliplatin treatment alone. CONCLUSION: This study identifies the important potential of CJ14939 in colorectal cancer treatment and suggests that combining CJ14939 with oxaliplatin might be a novel therapeutic strategy for patients with colorectal cancer.

Mutation SVCT2 promotes cell proliferation, invasion and migration in colorectal cancer.

The sodium-dependent vitamin C transporter 2 (SVCT2) surface glycoprotein regulates ascorbate accumulation in the plasma, often resulting in the induction of cancer cell death. Therefore, high expression of this gene associates with increased overall survival in several cancers. However, in colorectal cancer (CRC), high (likely mutated) SVCT2 expression relates to poor overall survival, and its functional significance has not been studied. Thus, we hypothesize that mutant SVCT2 expression could affect CRC patient survival. According to biological databases, SVCT2 has been found to be mutated frequently, and SVCT2 E264K has a particularly high pathogenic score (0.98), compared to other SVCT2 mutant sites, in CRC patients. Interestingly, our results reveal expression of SVCT2 E264K in many CRC tissues and cells. Also, we found wild-type SVCT2 expression to be largely localized to the cytoplasm and membrane, while SVCT2 E264K was restricted to the cytoplasm. We further found that SVCT2 E264K overexpression increases cell growth. By contrast, SVCT2 E264K knockdown significantly reduced cell proliferation and promoted cell apoptosis, resulting in inhibition of cell invasion and migration. Taken together, SVCT2 E264K plays a critical role in proliferation in CRC. Our results suggest that SVCT2 E264K could be a promising novel therapeutic target in CRC.

Role of p53 in transcriptional repression of SVCT2.

SVCT2, Sodium-dependent Vitamin C Transporter 2, uniquely transports ascorbic acid (also known as vitamin C and ascorbate) into all types of cells. Vitamin C is an essential nutrient that must be obtained through the diet and plasma levels are tightly regulated by transporter activity. Vitamin C plays an important role in antioxidant defenses and is a cofactor for many enzymes that enable hormone synthesis, oxygen sensing, collagen synthesis and epigenetic pathways. Although SVCT2 has various functions, regulation of its expression/activity remains poorly understood. We found a p53-binding site, within the SVCT2 promoter, using a transcription factor binding-site prediction tool. In this study, we show that p53 can directly repress SVCT2 transcription by binding a proximal- (~-185 to -171 bp) and a distal- (~-1800 to -1787 bp) p53-responsive element (PRE), Chromatin immunoprecipitation assays showed that PRE-bound p53 interacts with the corepressor-histone deacetylase 3 (HDAC3), resulting in deacetylation of histones Ac-H4, at the proximal promoter, resulting in transcriptional silencing of SVCT2. Overall, our data suggests that p53 is a potent transcriptional repressor of SVCT2, a critical transporter of diet-derived ascorbic acid, across the plasma membranes of numerous essential tissue cell types.

Inhibition of JAK1/2 can overcome EGFR-TKI resistance in human NSCLC.

Non-small lung cancer (NSCLC) is the most common cancer in the world. The epidermal growth factor receptor (EGFR) gene is mutated in approximately 10% of lung cancer cases in the US and 50% of lung cancer in Asia. The representative target therapeutic agent, erlotinib (EGFR tyrosine kinase inhibitor; EGFR TKI), is effective in inactivating EGFR in lung cancer patients. However, approximately 50-60% of patients are resistant to EGFR TKI. These populations are associated with the EGFR mutation. To overcome resistance to EGFR TKI, we discovered a JAK1 inhibitor, CJ14939. We investigated the efficacy of CJ14939 in human NSCLC cell lines in vitro and in vivo. Our results showed that CJ14939 induced the inhibition of cell growth. Moreover, we demonstrated that combination treatment with erlotinib and CJ14939 induced cell death in vitro and inhibited tumor growth in vivo. In addition, we confirmed the suppression of phosphorylated EGFR, JAK1, and Stat3 expression in erlotinib and CJ14939-treated human NSCLC cell lines. Our results provide evidence that JAK inhibition overcomes resistance to EGFR TKI in human NSCLCs.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Food poisoning associated with emetic-type of Bacillus cereus in Korea.

Bacillus cereus can cause diarrheal and emetic types of food poisoning but little study has been done on emetic type of food poisoning in Korea. The objective of this study was to report on the emetic type of food poisoning associated with B. cereus in Korea. The toxin gene profile, toxin production, and antibiotic resistance of B. cereus isolates were investigated in this study. B. cereus was detected in three out of four samples, while the other food poisoning bacteria were not detected. All isolates (KUGH 10, 11, and 12) presented nhe A, B, and C diarrheal toxin genes (755, 743, and 683 bp), detected using NHA, NHB, and NHC primers, and ces emetic toxin gene (1271 bp), detected using CES primer, and produced nonhemolytic enterotoxin and emetic toxin (cereulide), detected using immunochemical assay and high performance liquid chromotography/mass spectrometry (HPLC/MS) analysis. All emetic-associated isolates were resistant to beta-lactam antibiotics. Most important finding in this study was that the risk of emetic-type B. cereus food poisoning has existed in Korea. This suggested that the food poisoning caused by B. cereus producing emetic and diarrheal toxins should be constantly evaluated to prevent misdiagnosis between emetic and diarrheal types of food poisoning.