Search

Slow-cycling/dormant cancer cells (SCCs) have pivotal roles in driving cancer relapse and drug resistance. A mechanistic explanation for cancer cell dormancy and therapeutic strategies targeting SCCs are necessary to improve patient prognosis, but are limited because of technical challenges to obtaining SCCs. Here, by applying proliferation-sensitive dyes and chemotherapeutics to non-small cell lung cancer (NSCLC) cell lines and patient-derived xenografts, we identified a distinct SCC subpopulation that resembled SCCs in patient tumors. These SCCs displayed major dormancy-like phenotypes and high survival capacity under hostile microenvironments through transcriptional upregulation of regulator of G protein signaling 2 (RGS2). Database analysis revealed RGS2 as a biomarker of retarded proliferation and poor prognosis in NSCLC. We showed that RGS2 caused prolonged translational arrest in SCCs through persistent eukaryotic initiation factor 2 (eIF2α) phosphorylation via proteasome-mediated degradation of activating transcription factor 4 (ATF4). Translational activation through RGS2 antagonism or the use of phosphodiesterase 5 inhibitors, including sildenafil (Viagra), promoted ER stress-induced apoptosis in SCCs in vitro and in vivo under stressed conditions, such as those induced by chemotherapy. Our results suggest that a low-dose chemotherapy and translation-instigating pharmacological intervention in combination is an effective strategy to prevent tumor progression in NSCLC patients after rigorous chemotherapy.

Background: Trastuzumab deruxtecan (T-DXd) has been approved in various types cancer and recently expanded its indication to cancers with various HER2 expressions, such as HER2-low breast cancer. Since the effect of T-DXd is based on toxin payload, topoisomerase I inhibitor, the emergence of toxin-resistant cancer cells is unavoidable. Clinical cases of Primary, or secondary resistance to T-DXd have been reported. Therefore, it is necessary to develop novel drugs that can effectively exhibit antitumor effects against T-DXd-resistant carcinoma. Interferon beta (IFN-β), a promising potent cytokine, has been attracting attention for treatment of cancer. It is one of the few cytokines that induce tumor cell killing directly. Importantly, it could be an alternative to conventional drug-resistant cancer, because it regulates tumor growth in a way different from cytotoxic agents. Here, GNP101, an IFN-β mutein-based immunocytokine, exhibits antitumor properties against HER2-positive cancers, including T-DXd-resistant cancer. Method: A panel of human HER2-positive cancer cell lines derived from breast, stomach and lung were treated with GNP101 or HER2 therapeutics (trastuzumab, T-DM1, T-DXd) to compare direct antitumor effects. To generate a T-DXd-resistant cells, HCC1954, NCI-N87 and Calu-3 were exposed to the IC50, IC80 or IC90 of T-DXd by cyclic treatment. After 4 months, HCC1954-R cells were established and IFN-β signaling was assessed by STAT1-phosphorylation. The direct antitumor effect of GNP101 in vivo was tested in HER2-positive cancer xenograft models. Result: GNP101 showed superior potency compared to existing HER2 therapies (trastuzumab, T-DM1, and T-DXd) in all HER2-positive cancers regardless of tissue origin. To investigate the antitumor effect of GNP101 against T-DXd resistant cancer, we first generated T-DXd-resistant cell line (HCC1954-R). These HCC1954-R cells were strongly resistant to T-DXd, and showed no antitumor effect even at 100 nM of T-DXd. However, they were sensitive to GNP101, suggesting that GNP101 maintained its antitumor effect in T-DXd resistant cells. In addition, there was no difference in IFN-β-induced STAT1 phosphorylation between the parent cell and HCC1954-R. Taken together, these results indicate that HCC1954-R cells maintain IFN-β signaling and are selectively resistant to T-DXd. The activities of GNP101, T-DM1 and T-DXd on HER2-positive cancer cells were compared in vivo. GNP101 inhibited the growth of tumor by 90%, with no difference from that of T-DXd or T-DM1. Conclusion: We have evaluated the antitumor properties of GNP101, a trastuzumab-IFN-β mutein in HER2-expressing cancer. Given that GNP101 retains its antitumor properties in T-DXd-resistant cancer cells, the study suggests that anti-HER2-IFN-β mutein is a promising candidate for the treatment of HER2-positive and T-DXd resistant cancers. Citation Format: Chan Gyu Lee, Minkyung Park, Hyun Kyung Lee, Na Young Kim, Jun Young Choi, Sungyoul Hong, Young Kee Shin, Hae Min Jeong. GNP101, anti-HER2-IFN-β mutein immunocytokine, for the treatment of Trastuzumab deruxtecan-resistant HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1803.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, 20 μL of the week 24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international unit (IU) of a reference equine antitoxin. Furthermore, 20 μL of equine serum and 100 μg of purified equine F(ab')2 showed 100% neutralization of 10,000 LD50 in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.

The tight junctions of epithelial tissues, that most malignant tumors are derived from, are associated in cell-cell interactions. Among the tight junction proteins, claudin-3 (CLDN3) is overexpressed in many types of solid cancers, such as breast, ovarian, colorectal, and gastric cancers. Although CLDN3 can be potential therapeutic target due to the overexpression in various types of cancers, high homology with other CLDN family members is a hurdle to develop antibodies specifically targeting CLDN3. In this study, human IgG1 monoclonal antibody (ABN501) against CLDN3 was developed by scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. It was confirmed that ABN501 specifically bound to human and mouse CLDN3 without cross-reactivity to other CLDN family members. Sub-nanomolar affinity in binding kinetics of ABN501 was measured in CLDN3 expressing cell lines. We observed the antibody-dependent cytotoxicity (ADCC) activity of ABN501 with human NK cells expressing CD16a (NK-92MI-CD16a), in various cancer cell lines according to CLDN3 expression levels. We also confirmed that ABN501 specifically targeted CLDN3-expressing tumors in biodistribution assay using fluorescence-conjugated ABN501. In addition, ABN501 showed anti-tumor effects when treated with NK-92MI-CD16a in xenograft mice bearing CLDN3 expressing tumors. Taken these results together, we suggest that ABN501, specifically recognizing to CLDN3, can be used as therapeutic agents for CLDN3 positive cancer, and developed in many different forms to treat cancers with its specificity for cancer diagnosis, antibody-drug conjugates, and chimeric antigen receptor (CAR) immunotherapy. Citation Format: Hong-Seok Choi, Euni Sim, Na Young Kim, Yong Jin Lee, Sae Hyung Lee, Hayeon Park, Hobin Yang, Jiwon Jo, Myeung-Ryun Seo, Heegeon Park, Ji-Hye Lee, Sungyoul Hong, Young Kee Shin, Jun-Young Choi. Development of cludin-3 specific human monoclonal antibody(ABN501) as therapeutic anti-tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 311.

Background: Many studies have suggested that MET protein overexpression or MET amplification plays a critical role in the progression of gastro-esophageal cancer (GC) and negatively affects survival in patients with GC. However, the criteria used to define overexpression of MET protein have differed among many studies, and in contrast to the initial phase II studies, the phase III trials failed to show any clinical benefit from anti-MET therapies in GCs, even in patients with MET-positive disease. Several studies have used consistent criteria to define MET amplification on FISH, and recent MET inhibitor trials for MET amplified cancers have been successful. Furthermore, hyperactivation of MET pathway are known to reduce the growth-inhibitory effect of trastuzumab in HER2 positive GC. Currently, there is no FDA approved MET inhibitors for GC. Capmatinib and savolitinib, which are selective MET inhibitors, has been evaluated for various MET positive cancers. The aim of this study was to test these MET inhibitors on MET amplified gastro-esophageal cancer (GC) cells. Material and Methods: Among a total of 37 GC cells in our lab, the following cells were positive for MET by copy number amplification (CNV > 10): SNU-620, ESO51, MKN-45, SNU-5 and OE-33. We assessed cytotoxic response of these GC cells to capmatinib or savolitinib alone by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. Western blots were performed to assess the cellular activity of capmatinib or savolitinib in MET signal pathway. Xenograft studies were performed to evaluate in vivo therapeutic efficacy of savolitinib with MKN-45 cells. Results: Savolitinib and capmatinib showed anti-proliferative activity against MET amplified GC cell lines in dose-dependent manner. There was no inhibitory effect on GC cells without MET amplification, such as SNU-216 and SNU-1750. Savolitinib inhibited phosphorylation of MET and down-stream signaling pathway, such as AKT or ERK, in MET amplified GC cells. We observed that treatment with savolitinib induced a significant decrease in the number of colonies formed in soft agar. In addition, Savolitinib exhibited dose-responsive anti-tumor efficacy in xenograft model with MKN-45 GC cells. Furthermore, the combination of Trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in OE-33 cells, which is positive for both HER2 and MET, and resistant to trastuzumab alone. Conclusion: Collectively, these data support that targeting MET with Savolitinib and capmatinib efficiently suppresses the growth of MET amplified GC cells. Moreover, MET inhibitor could synergize with trastuzumab to induce the therapeutic effect on the HER2 and MET amplified GC cells. Further studies are warranted to test this concept in clinical setting. Citation Format: Jin-Soo Kim, Sungyoul Hong, Mi Young Kim. Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6376.

Type I interferon (IFN) has been approved as an anticancer agent to treat some malignancies. However, IFNs have a short in vivo half-life, systemic toxicity, and poor biophysical properties, which prevent it from being widely used for cancer therapy. This study aimed to construct recombinant IFN-β-1a mutein immunocytokines that comprise a human epidermal growth factor receptor 2 (HER2)-targeting antibody and IFN-β muteins with an additional glycosylation, which can overcome the limitation of the cytokine itself. Hence, the molecular design aims to 1) enhance productivity and biophysical properties by adding secondary glycosylation in IFN-β, 2) increase the therapeutic index of IFN-β therapy by preferential retention at the tumor by possessing high affinity for HER2-expressing cancer cells, and 3) improve the pharmacokinetics and, thus, the convenience of IFN-β administration. The yield of trastuzumab-IFN-β mutein was higher than that of trastuzumab-wild-type IFN-β in the mammalian cell culture system. Trastuzumab-IFN-β mutein showed similar IFN activity and HER2-targeting ability equivalent to that of IFN-β mutein and trastuzumab, respectively. Trastuzumab-IFN-β mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-β mutein alone. Trastuzumab-IFN-β mutein and IFN-β mutein displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-β mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Tumor-targeting effect of trastuzumab-IFN-β mutein was analyzed using in vivo fluorescence imaging. The accumulation of trastuzumab-IFN-β mutein was observed in HER2-positive tumors rather than other tissues except the liver. To evaluate the both direct tumor growth inhibition effect and indirect immune cell-mediated antitumor effect, we tested the effect of trastuzumab-IFN-β mutein in HER2-positive cancer xenograft models using nude mice or humanized mice. Trastuzumab-IFN-β mutein could significantly enhance tumor regression when compared with trastuzumab or IFN-β mutein. In addition, an increase in tumor-infiltrating lymphocytes was observed in the trastuzumab-IFN-β mutein-treated group, implying that the tumor-targeting IFN-β may have an enhanced antitumor effect through increased immune response. Therefore, targeting IFN-β with an anti-HER2 monoclonal antibody makes the immunocytokine more potent than either agent alone. These novel findings suggest that trastuzumab-IFN-β mutein merits clinical evaluation as a new candidate of anticancer therapeutics.

Slow-cycling cancer cells (SCCs) with a quiescence-like phenotype are believed to perpetrate cancer relapse and progression. However, the mechanisms that mediate SCC-derived tumor recurrence are poorly understood. Here, we investigated the mechanisms underlying cancer recurrence after chemotherapy, focusing on the interplay between SCCs and the tumor microenvironment. We established a preclinical model of SCCs by exposing non-small-cell lung cancer (NSCLC) cells to either the proliferation-dependent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) or chemotherapeutic drugs. An RNA sequencing analysis revealed that the established SCCs exhibited the upregulation of a group of genes, especially epidermal growth factor (EGF). Increases in the number of vascular endothelial growth factor receptor (VEGFR)-positive vascular endothelial cells and epidermal growth factor receptor (EGFR) activation were found in NSCLC cell line- and patient-derived xenograft tumors that progressed upon chemotherapy. EGFR tyrosine kinase inhibitors effectively suppressed the migration and tube formation of vascular endothelial cells. Furthermore, activating transcription factor 6 (ATF6) induced the upregulation of EGF, and its antagonism effectively suppressed these SCC-mediated events and inhibited tumor recurrence after chemotherapy. These results suggest that the ATF6-EGF signaling axis in SCCs functions to trigger the angiogenesis switch in residual tumors after chemotherapy and is thus a driving force for the switch from SCCs to actively cycling cancer cells, leading to tumor recurrence.

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell&ndash, cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of Fc&gamma, RIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.

Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.

Background: Interferon beta (IFN-β), a promising potent cytokine, has been attracting attention for treatment of cancer. The pleiotropic antitumor effects of IFN-β have been studied, with specific reference to their direct role on cancer cells and indirect action through the immune effector cells. However, its systemic toxicities and poor biophysical properties prevent IFN-β from being widely used for cancer therapy. Since the potential of cytokine therapies are impaired due to dose-limiting systemic toxicities, novel treatment methods should be developed to safely deliver effective drug quantities at the tumor sites. Like antibody-drug conjugates (ADCs), immunocytokine can be attempted to induce cytokine's organ-targeting and alleviate its systemic side effects. Here, we designed recombinant IFN-β mutein immunocytokines that comprise a HER2-targeting antibody and IFN-β mutein, and evaluate the antitumor properties against HER2-positive cancers.Method: A panel of human gastric cancer cell lines was treated with trastuzumab-IFN-β mutein to evaluate direct antitumor effect. In addition, to test the immune cell-mediated antitumor effect, cancer cells were co-cultured with effector cells (e.g. PBMC) in the absence or drug. The antitumor efficacy of trastuzumab-IFN-β mutein in vivo was tested in HER2-positive cancer xenograft models using nude mice or humanized mice. Result: Trastuzumab-IFN-β mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-β mutein alone. Trastuzumab-IFN-β mutein also displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-β mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Moreover, trastuzumab-IFN-β mutein significantly suppress tumor growth in HER2-positive cancer xenograft models. Tumor-infiltration of lymphocytes was enhanced by trastuzumab-IFN-β mutein, implying that the tumor-targeting IFN-β may have an antitumor effect through increased immune response. Conclusion: We have characterized and evaluated the antitumor properties of trastuzumab-IFN-β mutein in HER2-expressing cancers. Since IFN-β activates antitumor immune responses, it is expected to be administered in combination with immunotherapeutic drugs. Therefore, the study suggests that trastuzumab-IFN-β mutein is a promising candidate for the treatment of HER2-positive carcinoma. Citation Format: Chan Gyu Lee, Tae Eun Kim, Sungyoul Hong, Jongwan Chu, Ju Eun Kang, Hae Min Jeong, Young Kee Shin. HER2-targeted interferon-beta-1a mutein, a potent immunocytokine for the treatment of HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB170.

// Mi Jeong Kwon 1, 2, * , Ryong Nam Kim 3, 4, * , Kyoung Song 5 , Sinyoung Jeon 3 , Hae Min Jeong 3 , Joo Seok Kim 3 , Jinil Han 6 , Sungyoul Hong 3 , Ensel Oh 7 , Jong-Sun Choi 8 , Jungsuk An 9 , Jonathan R. Pollack 10 , Yoon-La Choi 7, 11, 12 , Cheol-Keun Park 11 and Young Kee Shin 3, 4, 8, 13 1 College of Pharmacy, Kyungpook National University, Daegu, Korea 2 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu, Korea 3 Laboratory of Molecular Pathology and Cancer Genomics, College of Pharmacy, Seoul National University, Seoul, Korea 4 Tumor Microenvironment Global Core Research Center, Seoul National University, Seoul, Korea 5 R&D center, ABION Inc., Guro-gu, Seoul, Korea 6 Gencurix, Inc., Guro-gu, Seoul, Korea 7 Laboratory of Cancer Genomics and Molecular Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 8 The Center for Anti-cancer Companion Diagnostics, Institutes of Entrepreneurial BioConvergence, Seoul National University, Seoul, Korea 9 Department of Pathology, Gachon University Gil Medical Center, Incheon, Korea 10 Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America 11 Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 12 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Korea 13 Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Korea * These authors have contributed equally to this study Correspondence to: Cheol-Keun Park, email: ckpark@skku.edu Young Kee Shin, email: ykeeshin@snu.ac.kr Keywords: gastric cancer, DNA copy number alterations, potential cancer-promoting genes, ERBB2, MET Received: August 05, 2016 Accepted: August 27, 2017 Published: September 21, 2017 ABSTRACT Gastric cancer (GC), one of the most common cancers worldwide, has a high mortality rate due to limited treatment options. Identifying novel and promising molecular targets is a major challenge that must be overcome if treatment of advanced GC is to be successful. Here, we used comparative genomic hybridization and gene expression microarrays to examine genome-wide DNA copy number alterations (CNAs) and global gene expression in 38 GC samples from old and young patients. We identified frequent CNAs, which included copy number gains on chromosomes 3q, 7p, 8q, 20p, and 20q and copy number losses on chromosomes 19p and 21p. The most frequently gained region was 7p21.1 (55%), whereas the most frequently deleted region was 21p11.1 (50%). Recurrent highly amplified regions 17q12 and 7q31.1-7q31.31 harbored two well-known oncogenes: ERBB2 and MET . Correlation analysis of CNAs and gene expression levels identified CAPZA2 (co-amplified with MET ) and genes GRB7 , MIEN1 , PGAP3 , and STARD3 (co-amplified with ERBB2 ) as potential candidate cancer-promoting genes (CPGs). Public dataset analysis confirmed co-amplification of these genes with MET or ERBB2 in GC tissue samples, and revealed that high expression (except for PGAP3 ) was significantly associated with shorter overall survival. Knockdown of these genes using small interfering RNA led to significant suppression of GC cell proliferation and migration. Reduced GC cell proliferation mediated by CAPZA2 knockdown was attributable to attenuated cell cycle progression and increased apoptosis. This study identified novel candidate CPGs co-amplified with MET or ERBB2 , and suggests that they play a functional role in GC.

Development of tungsten brazing technology for the upgraded Korea superconducting tokamak advanced research divertor was launched in early 2013. ITER grade tungsten block was brazed on the CuCrZr alloy in vacuum at 980 °C for 30 min using silver free brazing alloy. An OFHC-copper was used as an interlayer between tungsten and the CuCuZr. The brazing alloy is a 0.05 mm thickness of plate of which component is a Ni–Cu–Mn. It is found that the optimal loading on mock-up is about 20 kPa. Ultrasonic test and shear strength test were carried out to check the strength between tungsten and substrate material. And the joint conditions were analyzed by scanning electron microscopy. Tungsten-brazed mock-ups with a cooling tube were tested at the electron beam facility, Korea heat load test facility-electron beam in Korea Atomic Energy Research Institute. The high heat flux test was performed for tungsten-brazed mock-ups under heat flux of about 5 MW/ $\text{m}^{2}$ with more than 2000 cycles. All the tungsten-brazed mock-ups met the requirements and there was no delamination or failure at the bonding joints.

// Tae-Eun Kim 1 , Sungyoul Hong 1 , Kyoung Song 2 , Sang-Ho Park 2, 3 , Young Kee Shin 1 1 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea 2 Abion Inc., R&D Center, Seoul 08394, Republic of Korea 3 GE Healthcare Korea, R&D Center, Incheon 21988, Republic of Korea Correspondence to: Young Kee Shin, email: ykeeshin@snu.ac.kr Keywords: IFN-β resistance, R27T, cFLIP, sensitization, anti-cancer therapy Received: October 29, 2016 Accepted: December 27, 2016 Published: January 10, 2017 ABSTRACT In this study, we examined the molecular mechanism underlying the resistance of cancer cells to R27T, a glycoengineered version of recombinant human interferon (IFN)-β1a, and sought to overcome R27T resistance through combination therapy. R27T has been shown to induce anti-proliferation and apoptosis in human OVCAR-3 and MCF-7 cells, but not in HeLa cells. R27T treatment increased caspase-8 activity and the consequent cleavage of caspase-8 and -3 in R27T-sensitive OVCAR-3 cells, but not in R27T-resistant HeLa cells. Conversely, R27T increased the expression of cellular FLICE-like inhibitory protein (cFLIP) in HeLa cells, but not in OVCAR-3 cells. The sensitization of HeLa cells with cFLIP small interfering RNA or 4,5,6,7-tetrabromobenzotriazole (TBB, an inhibitor of casein kinase-2) facilitated R27T-induced caspase activation, and consequently apoptosis. In OVCAR-3-xenografted mice, intraperitoneal administration of R27T showed 2.1-fold higher anti-tumor efficacy than did the control vehicle. The combined administration of R27T and TBB showed the greatest anti-tumor effect in HeLa tumor-bearing mice, reducing the relative tumor volume by 35.7% compared to that in R27T-treated mice. Taken together, our results suggest that R27T has potential as an anti-cancer drug, and combination therapy with cFLIP inhibitors may be an effective strategy for overcoming R27T resistance.

Cordyceps militaris is used widely as a traditional medicine in East Asia. Although a few studies have attempted to elucidate the anticancer activities of C. militaris, the precise mechanism of C. militaris therapeutic effects is not fully understood. We examined the anticancer activities of C. militaris ethanolic extract (Cm-EE) and its cellular and molecular mechanisms. For this purpose, a xenograft mouse model bearing murine T cell lymphoma (RMA) cell-derived cancers was established to investigate in vivo anticancer mechanisms. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, immunoblotting analysis, and flow cytometric assay were employed to check in vitro cytotoxicity, molecular targets, and proapoptotic action of Cm-EE. Interestingly, cancer sizes and mass were reduced in a C. militaris-administered group. Levels of the phosphorylated forms of p85 and AKT were clearly decreased in the group administered with Cm-EE. This result indicated that levels of phosphoglycogen synthase kinase 3β (p-GSK3β) and cleaved caspase-3 were increased with orally administered Cm-EE. In addition, Cm-EE directly inhibited the viability of cultured RMA cells and C6 glioma cells. The number of proapoptotic cells was significantly increased in a Cm-EE treated group compared with a control group. Our results suggested that C. militaris might be able to inhibit cancer growth through regulation of p85/AKT-dependent or GSK3β-related caspase-3-dependent apoptosis.

SMAD4 is a tumor suppressor that is frequently inactivated in many types of cancer. The role of abnormal expression of SMAD4 has been reported in developmental processes and the progression of various human cancers. The expression level of SMAD4 has been related to the survival rate in gastric cancer patients. However, the molecular mechanism underlying transcriptional regulation of SMAD4 remains largely unknown. In the present study, we characterized the promoter region of SMAD4 and identified myeloid zinc finger 1 (MZF1), as a putative transcription factor. MZF1 directly bound to a core region of the SMAD4 promoter and stimulated transcriptional activity. We also found that the expression of MZF1 influences the migration ability of gastric adenocarcinoma cells. Collectively, our results showed that MZF1 has a role in cellular migration of gastric cancer cells via promoting an increase in intracellular SMAD4 levels. This study might provide new evidence for the molecular basis of the tumor suppressive effect of the MZF1-SMAD4 axis, a new therapeutic target in advanced human gastric cancer.

Selective bioactive compounds have emerged as major players in chemical biology for their potential in disrupting diverse biological pathways with minimal adverse effects. Using phenotypic screening, we identified an anti-cancer agent, SB2001, with a highly specific cytotoxicity toward HeLa human cervical cancer cells. The subsequent mechanistic study revealed that SB2001 induced apoptotic cell death through restoring p53 function and suppressed the human papillomavirus (HPV)-mediated oncoprotein signaling pathway via oxidative damage in HeLa cells. SB2001 also selectively induced HeLa-specific tumor regression without any adverse effects in an in vivo tumor xenograft model, demonstrating its potential as a promising chemical probe.

// Yu Jin Kim 1 , Sungyoul Hong 2 , Minjung Sung 1 , Min Jeong Park 2 , Kyungsoo Jung 1, 3 , Ka-Won Noh 1, 3 , Doo-Yi Oh 1, 3 , Mi-Sook Lee 1, 3 , Ensel Oh 1, 3 , Young Kee Shin 2, 4 , Yoon-La Choi 1, 3, 5 1 Laboratory of Cancer Genomics and Molecular Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2 Laboratory of Molecular Pathology and Cancer Genomics, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea 3 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Korea 4 The Center for Anti-cancer Companion Diagnostics, Bio-MAX/N-Bio, Seoul National University, Seoul, Korea 5 Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Correspondence to: Yoon-La Choi, email: ylachoi@skku.edu Young Kee Shin, email: ykeeshin@snu.ac.kr Keywords: LYN, dasatinib, lung adenocarcinoma subgroup, SRC, YES Received: March 16, 2016 Accepted: October 01, 2016 Published: October 14, 2016 ABSTRACT Therapies targeting SRC family kinases (SFKs) have shown efficacy in treating non-small cell lung cancer (NSCLC). However, recent clinical trials have found that the SFK inhibitor dasatinib is ineffective in some patient cohorts. Regardless, dasatinib treatment may benefit some NSCLC patient subgroups. Here, we investigated whether expression of LYN, a member of the SFK family, is associated with patient survival, the efficacy of dasatinib, and/or NSCLC cell viability. LYN expression was associated with poor overall survival in a multivariate analysis, and this association was strongest in non-smoker female patients with adenocarcinoma (ADC). In lung ADC cells, LYN expression enhanced cell proliferation, migration, and invasion. Dasatinib inhibited LYN activity and decreased cell viability in LYN-positive ADC cell lines and xenografts. Additionally, we identified the SFKs SRC and YES as candidate dasatinib targets in LYN-negative ADC cell lines. Our findings suggest that LYN is a useful prognostic marker and a selective target of dasatinib therapy in the lung ADC subpopulation especially in female non-smokers with lung ADC.

Tungsten (W) monoblock that consists of W according to ITER specifications, oxygen-free high conductive copper (Cu-OFHC), and CuCrZr has been designed in preparation for KSTAR upgrade for steady-state long-pulse operation with 27.5-MW heating power. Finite-element analysis has been performed by ANSYS Workbench. The hydraulic thermomechanical analysis is performed to investigate the optimal design parameters and the expected fatigue lifetime under the operating conditions with 5.4-MW/ $\text{m}^{2}$ incident heat flux at the divertor. For a single W monoblock, the optimization gives a W thickness of 5 mm, a side thickness of 2.5 mm, a thickness in the back part of 6 mm, and a Cu-OFHC interlayer thickness of 0.5 mm. For a mock-up consisting of five optimized W monoblocks, the thermal fatigue analysis is performed. As a result, the maximum value of equivalent total strain in the CuCrZr tube is found to be 0.266%, which corresponds to for a fatigue lifetime of 23 000 cycles.

We aimed to study the role of protein L-isoaspartyl methyltransferase (PIMT) in neuronal differentiation using basic fibroblast growth factor (bFGF)-induced neuronal differentiation, characterized by cell-body shrinkage, long neurite outgrowth, and expression of neuronal differentiation markers light and medium neurofilaments (NF). The bFGF-mediated neuronal differentiation of PC12 cells was induced through activation of mitogen-activated protein kinase (MAPK) signaling molecules [MAPK kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p90RSK], and phosphatidylinositide 3-kinase (PI3K)/Akt signaling molecules PI3Kp110β, PI3Kp110γ, Akt, and mTOR. Inhibitors (adenosine dialdehyde and S-adenosylhomocysteine) of protein methylation suppressed bFGF-mediated neuronal differentiation of PC12 cells. PIMT-eficiency caused by PIMT-specific siRNA inhibited neuronal differentiation of PC12 cells by suppressing phosphorylation of MEK1/2 and ERK1/2 in the MAPK signaling pathway and Akt and mTOR in the PI3K/Akt signaling pathway. Therefore, these results suggested that PIMT was critical for bFGF-mediated neuronal differentiation of PC12 cells and regulated the MAPK and Akt signaling pathways. [BMB Reports 2016; 49(8): 437-442]

Background Although Korean Red Ginseng (KRG) has been traditionally used for a long time, its anti-inflammatory role and underlying molecular and cellular mechanisms have been poorly understood. In this study, the anti-inflammatory roles of KRG-derived components, namely, water extract (KRG-WE), saponin fraction (KRG-SF), and nonsaponin fraction (KRG-NSF), were investigated. Methods To check saponin levels in the test fractions, KRG-WE, KRG-NSF, and KRG-SF were analyzed using high-performance liquid chromatography. The anti-inflammatory roles and underlying cellular and molecular mechanisms of these components were investigated using a macrophage-like cell line (RAW264.7 cells) and an acute gastritis model in mice. Results Of the tested fractions, KGR-SF (but not KRG-NSF and KRG-WE) markedly inhibited the viability of RAW264.7 cells, and splenocytes at more than 500 μg/mL significantly suppressed NO production at 100 μg/mL, diminished mRNA expression of inflammatory genes such as inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, and interferon-β at 200 μg/mL, and completely blocked phagocytic uptake by RAW264.7 cells. All three fractions suppressed luciferase activity triggered by interferon regulatory factor 3 (IRF3), but not that triggered by activator protein-1 and nuclear factor-kappa B. Phospho-IRF3 and phospho-TBK1 were simultaneously decreased in KRG-SF. Interestingly, all these fractions, when orally administered, clearly ameliorated the symptoms of gastric ulcer in HCl/ethanol-induced gastritis mice. Conclusion These results suggest that KRG-WE, KRG-NSF, and KRG-SF might have anti-inflammatory properties, mostly because of the suppression of the IRF3 pathway.

Protein arginine methyltransferases (PRMTs) mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I–IV). Although most PRMTs do not require posttranslational modification (PTM) to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6) in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses.

This paper proposes a thread-type magnetic core as a new structured magnetic core made of iron threads and a surrounding insulator. The thread-type core has the advantages of lower eddy current loss, simple production, and low cost. The eddy current loss of the thread-type core is compared with that of bulk- and lamination-type cores. The losses are numerically analyzed using the finite-element method and are measured with an experimental test. The effect of eddy current on copper loss is also addressed. The numerical and experimental results show the usefulness of the thread-type core. Further numerical results show the size effect of the thread core, whereby the smaller size thread results in less eddy current loss.

Staphylococcus aureus (S. aureus) bacteria, the gram-positive coccus and occurring irregular clusters, have the power to produce many types of enterotoxin. It is known as a foodborne poisoning agent as well as being a potential inhaled bioterrorism agent. Previously we confirmed the structure of modified-S. aureus enterotoxin B (SEB) vaccine candidate (ABN701) that can induce neutralization of antigen activity and also elicit humoral and cellular immune response. In this study, we studied the immunization efficacy and toxicity of ABN701 in various animal models. ABN701 was immunized in a dose dependent manner to BalB/C mouse and rabbit and about 20 ug ABN701 can induce an immune response in these models. Through the adjuvant tests, aluminum-composed adjuvant triggered more antibody inducement in mice and rabbit. The immunized BalB/C mouse were challenged with the SEB toxin and survived against 20LD50 SEB toxin treatment. Emesis is one of the most important evaluation factors for SEB vaccine development because emesis is related to SEB toxin symptom. House musk shrew mouse (Suncus Murinus) is widely known to be sensitive to various emetic drug evaluation and a new experimental animal model for SEB toxin related emesis. We confirmed that ABN701 did not cause emesis while SEB toxin cause emesis in this model. We also immunized ABN701 to monkey and showed no emesis symptom, but induced an effective immune response. In this study, we identified that ABN701 can induce an immune response in mouse, rabbit and monkey and ABN701 do not have toxicity related to emesis in the House musk shrew mouse model and monkey model.

Smallpox, caused by variola virus, could be a bio-terrorism agent although eradicated from the WHO program in the 1980s. DNA vaccine is a third generation vaccine system which contains DNA sequence with specific antigen codes that activates the immune system. DNA vaccines show great potential in inducing immune response with strong triggered-humoral, and cellular immunity. DNA vaccines have emerged as an attractive approach for infectious disease because of rapid manufacture, fast adaptation to newly emerging pathogens and high stability at ambient temperatures. However, since the potency of naked DNA vaccines is limited to immune efficacy, adjuvant incorporation could be a good option to increase the immune response. Several different compound-composed adjuvants are developed to help DNA vaccine efficacy. In other aspects, for inducing a more effective immune response, global vaccine manufacturer focused on targeting dendritic cell (DC) through the DC-specific surface protein, DEC-205. In this study, with plasmid construction modification, we developed a DNA vaccine vector that have effective antigen expression and confirmed immunogenicity against smallpox. For enhancing in vivo immune responses, we screened various adjuvant strategies and observed effective DNA vaccination response with various adjuvant studies and time-pointed reaction. Also, we tested the enhancement of immune response using DNA vaccine containing the smallpox antigen fused with DEC-205 and revealed a th1–th2 cellular immune response. Our results propose how to regulate modified-DNA immunization efficacy within DC targeting stimulation and adjuvant strategy against DNA vaccine development.

c-Met is a receptor tyrosine kinase with no commercially available product despite being a pivotal target in cancer progression. Unlike other c-Met inhibitors that fail clinically, ABN401 is a newly synthesized c-Met inhibitor that is not potentially degraded by aldehyde oxidase (AO) in human liver cytosol. This study aimed to determine the physicochemical stability, pharmacokinetics in beagle dogs, and therapeutic effect of ABN401 in a c-Met-amplified non-small-cell lung cancer (NSCLC) patient-derived xenograft (PDX) model. ABN401 was found to be a weak basic compound, with pKa and log P values of 7.49 and 2.46, respectively. It is poorly water-soluble but soluble at acidic pH. The accelerated storage stability is dependent on temperature, but the purity remains at over 97% after 6 months. The bioavailability is approximately 30% in dogs and it is highly efficient in the PDX model, achieving around 90% tumor growth inhibition in combination with erlotinib. These observations indicate that the compound is acceptable for the next phase of trials.

Makgeolli, a traditional Korean alcoholic beverage, includes many kinds of lactic acid bacteria (LAB). In this study, 17 different LAB strains were isolated from makgeolli and their functional effects were examined. The 17 LAB were identified as eight Pediococcus acidilactici strains, six P. pentosaceus strains, two Lactobacillus curvatus strains, and one L. curstorum strain. Evaluation of nitric oxide (NO) production showed that all of the LAB strains except P. acidilactici 4 (PA4) produced higher amounts of NO than L. rhamnosus GG, indicating that they may have immunomodulatory effects. In addition, P. acidilactici 5 ( PA5), P. pentosaceus 5 (PP5), L. curvatus 1, and L. curstorum 1 exhibited high acid tolerance properties. PA5 and PP5 also exhibited good bile acid tolerance. Lastly, Caco-2 cell adhesion experiments revealed that PA5 had a high cell adhesion rate of 19-43%. In conclusion, PA5 may be useful as a probiotic for further food industry applications.

Lenalidomide modulates the host immune response against myeloma via multiple actions. Although these effects have been elucidated in vitro, the central action of lenalidomide-mediated anti-myeloma immune response in vivo is not clear. To investigate its immune action in vivo, we selected the murine myeloma cell line 5TGM1, which is resistant to direct tumoricidal effects of lenalidomide in vitro and in immunodeficient mice, but sensitive to lenalidomide treatment in 5TGM1-bearing immunocompetent mice. Depletion of CD4+ T cells, but not NK cells, B cells, or CD8+ T cells, deprived lenalidomide of its therapeutic effects on 5TGM1-bearing immunocompetent mice. Lenalidomide significantly increased the numbers of IFN-γ-secreting CD4+ and CD8+ T cells but had no effects on NK cells and B cells in this mouse model. Lenalidomide slightly decreased the number of CD25+Foxp3+ T cells but increased perforin expression in CD8+ T cells in vivo. Using this mouse model for investigation of anti-tumor immune action of lenalidomide, we demonstrated that lenalidomide facilitated a type-1 anti-tumor immune response in vivo. The CD4+ T cell subset may play a critical role in the lenalidomide-mediated anti-myeloma immune response in vivo.

TANK-binding kinase 1 (TBK1) is a noncanonical IκB kinase family member that regulates the innate immune response. Misregulation of TBK1 activity can promote inflammatory disorders and oncogenesis; therefore, TBK1 inhibitors are considered a promising therapy for inflammation and cancer.In this review, the authors provide information on the role of TBK1 in human health and on recently developed inhibitors from patents granted from 2011 to 2014. The reader will gain an understanding of the mechanisms of TBK1 function as well as the structure and biological activity of recently developed TBK1 inhibitors. Google and NCBI search engines were used to find relevant patents and clinical information using "TBK1 inhibitor" as the search term.The role of TBK1 in various diseases has prompted the further investigation of significant targets. Although research on TBK1 inhibitors has increased over the last few years, only a few inhibitors of this kinase have been identified. In addition, almost all of the chemical inhibitors are modified from different scaffolds and/or chemotypes of pyrimidine. Specifically, compound BX795 is the representative one, which was first patented as a potent TBK1 inhibitor. Even though some compounds have displayed interesting potential inhibition and selectivity of TBK1 in vitro and in in vivo trials, the development of more efficient and selective TBK1 inhibitors is still required.