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Purpose: Rearranged during transfection (RET) aberrations represent a targetable oncogene in several tumor types, with RET inhibitors displaying marked efficacy. However, some patients with RET-aberrant cancer are insensitive to RET tyrosine kinase inhibitors (TKIs). Recently, drug-tolerant mechanisms have attracted attention as targets for initial therapies to overcome drug resistance. The underlying mechanisms of drug-tolerant cell emergence treated with RET-TKIs derived from RET-aberrant cancer cells remain unknown. This study investigated the role of YAP-mediated HER3 signaling in the underlying mechanisms of adaptive resistance to RET-TKIs in RET-aberrant cancer cells., Experimental Design: Four RET-aberrant cancer cell lines were used to assess sensitivity to the RET-TKIs selpercatinib and pralsetinib and to elucidate molecular mechanisms underlying adaptive resistance using RNA sequencing, phospho-RTK antibody arrays, chromatin immunoprecipitation assay, and luciferase reporter assays. Clinical specimens from patients with RET-fusion-positive lung cancer were analyzed for pre-treatment YAP expression and correlated with treatment outcomes., Results: In high YAP-expressing RET-aberrant cancer cells, YAP-mediated HER3 signaling activation maintained cell survival and induced the emergence of cells tolerant to the RET-TKIs selpercatinib and pralsetinib. The pan-ErBB inhibitor afatinib and YAP/TEAD inhibitors verteporfin and K-975 sensitized YAP-expressing RET-aberrant cancer cells to the RET-TKIs selpercatinib and pralsetinib. Pre-treatment YAP expression in clinical specimens obtained from patients with RET-fusion-positive lung cancer was associated with poor RET-TKI treatment outcomes., Conclusion: The YAP-HER3 axis is crucial for the survival and adaptive resistance of high YAP-expressing RET-aberrant cancer cells treated with RET-TKIs. Combining YAP/HER3 inhibition with RET-TKIs represents a highly potent strategy for initial treatment.

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

We previously reported that combined therapy with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib and AXL inhibitor ONO-7475 is effective in preventing the survival of drug-tolerant cells in high-AXL-expressing EGFR-mutated non-small cell lung cancer (NSCLC) cells. Nevertheless, certain residual cells are anticipated to eventually develop acquired resistance to this combination therapy. In this study, we attempted to establish a multidrug combination therapy from the first-line setting to overcome resistance to this combination therapy in high-AXL-expressing EGFR-mutated NSCLC. siRNA screening assay showed that fibroblast growth factor receptor 1 (FGFR1) knockdown induced pronounced inhibition of cell viability in the presence of the osimertinib-ONO-7475 combination, which activates FGFR1 by upregulating FGF2 via the c-Myc pathway. Cell-based assays showed that triple therapy with osimertinib, ONO-7475, and the FGFR inhibitor BGJ398 significantly increased apoptosis by increasing expression of proapoptotic factor Bim and reduced cell viability compared with that observed for the osimertinib-ONO-7475 therapy. Xenograft models showed that triple therapy considerably suppressed tumor regrowth. A novel therapeutic strategy of additional initial FGFR1 inhibition may be highly effective in suppressing the emergence of osimertinib- and ONO-7475-resistant cells., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T. Yamada received commercial research grants from Ono Pharmaceutical, Janssen Pharmaceutical K.K., AstraZeneca, and Takeda Pharmaceutical Company Limited and speaking honoraria from Eli Lilly and Chugai-Roche. H. Kawachi received personal fees from Ono Pharmaceutical, Bristol-Myers Squibb, Chugai Pharmaceutical, AstraZeneca, Taiho Pharmaceutical, Eli Lilly Japan, and MSD outside the purview of the submitted work. A. Osoegawa received consultant honoraria from AstraZeneca and speaking honoraria from AstraZeneca, MSD Japan, Chugai Pharmaceutical, Bristol Myers Squibb, and Ono Pharmaceutical. T. Sakai received research grants from Otsuka Pharmaceutical, Taiho Pharmaceutical, and Oncolys BioPharma and a patent fee from JT Pharmaceutical. T. Yasuhiro and R. Kozaki are paid employees of Ono Pharmaceutical. S. Yano received research grants from Chugai-Roche and Boehringer-Ingelheim and speaking honoraria from Amgen, Chugai-Roche, Boehringer-Ingelheim, Novartis, and Pfizer. K. Takayama received research grants from Chugai-Roche and Ono Pharmaceutical and personal fees from AstraZeneca, Chugai-Roche, MSD-Merck, Eli Lilly, Boehringer-Ingelheim, and Daiichi-Sankyo. The other authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Recent therapeutic strategies for KRAS-mutated cancers that inhibit the MAPK pathway have attracted considerable attention. The RAF/MEK clamp avutometinib (VS-6766/CH5126766/RO5126766/CKI27) is promising for patients with KRAS-mutated cancers. Although avutometinib monotherapy has shown clinical activity in patients with KRAS-mutated cancers, effective combination strategies will be important to develop., Methods: Using a phosphorylation kinase array kit, we explored the feedback mechanism of avutometinib in KRAS-mutated NSCLC cells, and investigated the efficacy of combining avutometinib with inhibitors of the feedback signal using in vitro and in vivo experiments. Moreover, we searched for a biomarker for the efficacy of combination therapy through an in vitro study and analysis using the The Cancer Genome Atlas Programme dataset., Results: Focal adhesion kinase (FAK) phosphorylation/activation was increased after avutometinib treatment and synergy between avutometinib and FAK inhibitor, defactinib, was observed in KRAS-mutated NSCLC cells with an epithelial rather than mesenchymal phenotype. Combination therapy with avutometinib and defactinib induced apoptosis with upregulation of Bim in cancer cells with an epithelial phenotype in an in vitro and in vivo study., Conclusions: These results demonstrate that the epithelial-mesenchymal transition status may be a promising biomarker for the efficacy of combination therapy with avutometinib and defactinib in KRAS-mutated NSCLC., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Recently, novel Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitors have been clinically developed to treat KRAS G12C-mutated non-small cell lung cancer (NSCLC) patients. However, achieving complete tumor remission is challenging. Therefore, the optimal combined therapeutic intervention with KRAS G12C inhibitors has a potentially crucial role in the clinical outcomes of patients. We investigated the underlying molecular mechanisms of adaptive resistance to KRAS G12C inhibitors in KRAS G12C-mutated NSCLC cells to devise a strategy preventing drug-tolerant cell emergence. We demonstrate that AXL signaling led to the adaptive resistance to KRAS G12C inhibitors in KRAS G12C-mutated NSCLC, activation of which is induced by GAS6 production via YAP. AXL inhibition reduced the viability of AXL-overexpressing KRAS G12C-mutated lung cancer cells by enhancing KRAS G12C inhibition-induced apoptosis. In xenograft models of AXL-overexpressing KRAS G12C-mutated lung cancer treated with KRAS G12C inhibitors, initial combination therapy with AXL inhibitor markedly delayed tumor regrowth compared with KRAS G12C inhibitor alone or with the combination after acquired resistance to KRAS G12C inhibitor. These results indicated pivotal roles for the YAP-GAS6-AXL axis and its inhibition in the intrinsic resistance to KRAS G12C inhibitor., Competing Interests: Declaration of competing interest T. Yamada received commercial research grants from Pfizer, Ono Pharmaceutical, Janssen Pharmaceutical K.K., AstraZeneca, and Takeda Pharmaceutical Company Limited and has received speaking honoraria from Eli Lilly. H. Kawachi received personal fees from Ono Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., AstraZeneca KK, Taiho Pharmaceutical Co. Ltd., Eli Lilly Japan KK, and MSD KK, outside the purview of the submitted work. N. Furuya received personal fees from AstraZeneca, Chugai, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, MSD, Pfizer, Taiho, and Novartis. S. Watanabe received grants and personal fees from Boehringer Ingelheim and Nippon Kayaku. Personal fees from Lilly, Pfizer, Novartis Pharma, AstraZeneca, Chugai Pharma, Bristol-Myers, Ono Pharmaceutical, Daiichi Sankyo, and Taiho Pharmaceutical. S. Yano received research grants from Chugai Pharmaceutical and Boehringer-Ingelheim and has received speaking honoraria from Amgen, Chugai Pharmaceutical, Boehringer-Ingelheim, Novartis, and Pfizer. T. Sakai received research grants from Otsuka Pharmaceutical, Taiho Pharmaceutical and Oncolys BioPharma, and a patent fee from JT Pharmaceutical. K. Takayama reports receiving research grants from Chugai-Roche Co., and Ono Pharmaceutical Co., and personal fees from AstraZeneca Co., Chugai-Roche Co., MSD-Merck Co., Eli Lilly Co., Boehringer-Ingelheim Co., and Daiichi-Sankyo Co.No potential conflicts of interest were disclosed by the other authors., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Small molecule-based selective cancer cell-targeting can be a desirable anticancer therapeutic strategy. Aiming to discover such small molecules, we previously developed phenylcyclopropylamine (PCPA)-drug conjugates (PDCs) that selectively release anticancer agents in cancer cells where lysine-specific demethylase 1 (LSD1) is overexpressed. In this work, we designed PCPA-entinostat conjugates for selective cancer cell targeting. PCPA-entinostat conjugate 12 with a 4-oxybenzyl group linker released entinostat in the presence of LSD1 in in vitro assays and selectively inhibited the growth of cancer cells in preference to normal cells, suggesting the potential of PCPA-entinostat conjugates as novel anticancer drug delivery small molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Few treatment options exist for pleural mesothelioma (PM), which is a progressive malignant tumor. However, the efficacy of molecular-targeted monotherapy is limited, and further therapeutic strategies are warranted to treat PM. Recently, the cancer cell-cycle checkpoint inhibitors have attracted attention because they disrupt cell-cycle regulation. Here, we aimed to establish a novel combinational therapeutic strategy to inhibit the cell-cycle checkpoint kinase, ATR in PM cells. The siRNA screening assay showed that anexelekto (AXL) knockdown enhanced cell growth inhibition when exposed to ATR inhibitors, demonstrating the synergistic effects of the ATR and AXL combination in some PM cells. The AXL and ATR inhibitor combination increased cell apoptosis via the Bim protein and suppressed cell migration when compared with each monotherapy. The combined therapeutic targeting of AXL and ATR significantly delayed regrowth compared with monotherapy. Thus, optimal AXL and ATR inhibition may potentially improve the PM outcome., (©2023 The Authors; Published by the American Association for Cancer Research.)

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation., (© 2023. The Author(s).)

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors rarely elicit complete responses in patients with advanced ALK-rearranged non-small cell lung cancer (NSCLC), as a small population of tumor cells survives due to adaptive resistance. Therefore, we focused on the mechanisms underlying adaptive resistance to lorlatinib and therapeutic strategies required to overcome them. We found that epidermal growth factor receptor (EGFR) signaling was involved in the adaptive resistance to lorlatinib in ALK-rearranged NSCLC, activation of which was induced by heparin-binding EGF-like growth factor production via c-Jun activation. EGFR inhibition halted ALK-rearranged lung cancer cell proliferation by enhancing ALK inhibition-induced apoptosis via suppression of Bcl-xL. Xenograft models showed that the combination of EGFR inhibitor and lorlatinib considerably suppressed tumor regrowth following cessation of these treatments. This study provides new insights regarding tumor evolution due to EGFR signaling after lorlatinib treatment and the development of combined therapeutic strategies for ALK-rearranged lung cancer., (© 2023. The Author(s).)

Background: Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired., Methods: We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo., Results: Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs., Conclusions: RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

Colorectal cancer is a significant cause of morbidity and represents a serious public health issue in many countries. The development of a breakthrough preventive method for colorectal cancer is urgently needed. Aspirin has recently been attracting attention as a cancer preventive drug, and its inhibitory effects on the development of various cancers have been reported in several large prospective studies. However, the underlying molecular mechanisms have not yet been elucidated in detail. In the present study, we attempted to identify the target proteins of aspirin using a chemical biology technique with salicylic acid, the main metabolite of aspirin. We generated salicylic acid-presenting FG beads and purified salicylic acid-binding proteins from human colorectal cancer HT-29 cells. The results obtained showed the potential of ribosomal protein S3 (RPS3) as one of the target proteins of salicylic acid. The depletion of RPS3 by siRNA reduced CDK4 expression and induced G1 phase arrest in human colorectal cancer cells. These results were consistent with the effects induced by the treatment with sodium salicylate, suggesting that salicylic acid negatively regulates the function of RPS3. Collectively, the present results show the potential of RPS3 as a novel target for salicylic acid in the protective effects of aspirin against colorectal cancer, thereby supporting RPS3 as a target molecule for cancer prevention., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Toshiyuki Sakai reports financial support was provided by Japan Society for the Promotion of Science., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Anticancer drug delivery by small molecules offers a number of advantages over conventional macromolecular drug delivery systems. We previously developed phenylcyclopropylamine (PCPA)-drug conjugates (PDCs) as small-molecule-based drug delivery vehicles for targeting lysine-specific demethylase 1 (LSD1)-overexpressing cancers. In this study, we applied this PDC strategy to the HDAC-inhibitory anticancer agent vorinostat. Among three synthesized PCPA or arylcyclopropylamine (ACPA)-vorinostat conjugates 1 , 9 , and 32 , conjugate 32 with a 4-oxybenzyl linker showed sufficient stability in buffer solutions, potent LSD1 inhibition, efficient LSD1-dependent vorinostat release, and potent and selective antiproliferative activity toward LSD1-expressing human breast cancer and small-cell lung cancer cell lines. These results indicate that the conjugate selectively releases vorinostat in cancer cells. A similar strategy may be applicable to other anticancer drugs., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Aspirin has gained great attention as a cancer preventive agent. Our previous study revealed that the low-dose aspirin prevents colorectal tumor recurrence in Japanese patients with colorectal adenomas and/or adenocarcinomas, whereas aspirin increases risks in smokers and has no effects on regular drinkers. Our recent study revealed that aspirin reduces polyp growth in Japanese patients with familial adenomatous polyposis (FAP). In this study, we have studied the association of genotypes of alcohol metabolizing enzymes (ADH1B and ALDH2) on aspirin's efficacy of suppressing polyp growth (≥5 mm) in a total of 81 Japanese patients with FAP. Our study revealed that aspirin showed significant preventive effects for patients with ADH1B -AA and AA+GA types [OR = 0.21; 95% confidence interval (CI), 0.05-0.95, and OR = 0.31; 95% CI, 0.10-0.95, respectively], and for patients with ALDH2 -GG and GG+GA types (OR = 0.10; 95% CI, 0.01-0.92, and OR = 0.29; 95% CI, 0.09-0.94, respectively), but not for patients with ADH1B -GG and GA+GG types, and ALDH2 -AA and GA+AA types. In addition, substantial preventive effects of aspirin were seen for patients with ADH1B -AA type who do not drink regularly (<3 times/week, OR = 0.11; 95% CI, 0.02-0.78), where a statistically significant interaction between aspirin and ADH1B was observed ( P interaction = 0.036). Results from this exploratory study strongly indicate that aspirin is beneficial in prevention of polyp growth for patients with FAP with ADH1B -AA and AA+GA types, and ALDH2 -GG and GG+GA types. Taken together, we propose ADH1B and ALDH2 as candidate markers for the personalized prevention by aspirin., Significance: Aspirin is beneficial to patients with FAP with ADH1B -AA and AA+GA types or ALDH2 -GG and GG+GA types. ADH1B and ALDH2 genotypes can be the markers for the personalized prevention of colorectal cancer by aspirin., Competing Interests: K. Mure, H. Ishikawa, M. Mutoh, K. Wakabayashi, T. Sakai, and S. Tanaka report a grant from Japan Agency for Medical Research and Development during the conduct of the study. Y. Takeuchi reports personal fees from Olympus, Boston Scientific Japan, Daiichi-Sankyo, Miyarisan Pharmaceutical, Asuka Pharmacoceutical, AstraZeneca, EA Pharma, Zeria Pharmaceutical, Fujifilm, Kaneka Medical, and Kyorin Pharmaceutical outside the submitted work. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L. -derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L .-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L .-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L. -derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L .-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L .-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Multiple myeloma (MM) is characterized by remarkable cytogenetic/molecular heterogeneity among patients and intraclonal diversity even in a single patient. We previously demonstrated that PDPK1, the master kinase of series of AGC kinases, is universally active in MM, and plays pivotal roles in cell proliferation and cell survival of myeloma cells regardless of the profiles of cytogenetic and genetic abnormalities. This study investigated the therapeutic efficacy and mechanism of action of dual blockade of two major PDPK1 substrates, RSK2 and AKT, in MM. The combinatory treatment of BI-D1870, an inhibitor for N-terminal kinase domain (NTKD) of RSK2, and ipatasertib, an inhibitor for AKT, showed the additive to synergistic anti-tumor effect on human MM-derived cell lines (HMCLs) with active RSK2-NTKD and AKT, by enhancing apoptotic induction with BIM and BID activation. Moreover, the dual blockade of RSK2 and AKT exerted robust molecular effects on critical gene sets associated with myeloma pathophysiologies, such as those with MYC, mTOR, STK33, ribosomal biogenesis, or cell-extrinsic stimuli of soluble factors, in HMCLs. These results provide the biological and molecular rationales for the dual-targeting strategy for RSK2 and AKT, which may overcome the therapeutic difficulty due to cytogenetic/molecular heterogeneity in MM.

As colon cancer is one of the most common cancers in the world, practical prevention strategies for colon cancer are needed. Recently, treatment with aspirin and/or 5-aminosalicylic acid-related agents was reported to reduce the number of intestinal polyps in patients with familial adenomatous polyposis. To evaluate the mechanism of aspirin and 5-aminosalicylic acid for suppressing the colon polyp growth, single and combined effects of 5-aminosalicylic acid and sodium salicylate (metabolite of aspirin) were tested in the two human colon cancer cells with different cyclooxygenase-2 expression levels and intestinal polyp-derived cells from familial adenomatous polyposis model mouse. The combination induced cell-cycle arrest at the G1 phase along with inhibition of cell growth and colony-forming ability in these cells. The combination reduced cyclin D1 via proteasomal degradation and activated retinoblastoma protein. The combination inhibited the colony-forming ability of mouse colonic mucosa cells by about 50% and the colony-forming ability of mouse intestinal polyp-derived cells by about 90%. The expression level of cyclin D1 in colon mucosa cells was lower than that in intestinal polyp-derived cells. These results suggest that this combination may be more effective in inhibiting cell growth of intestinal polyps through cyclin D1 down-regulation., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2022 JCBN.)

EGFR-T790M mutation is a major mechanism underlying acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung cancer with mutated EGFR. However, differences in the biological characteristics of T790M tumors based on treatment regimens with each generation of EGFR-TKI are not fully understood. We established cell lines with acquired resistance harboring EGFR-T790M mutation derived from xenograft tumors treated with each generation of EGFR-TKI and examined their biological characteristics with respect to third-generation EGFR-TKI osimertinib sensitivity. Second-generation EGFR-TKI dacomitinib-resistant cells with T790M-exhibited higher sensitivity to osimertinib than first-generation EGFR-TKI gefitinib-resistant cells with T790M via inhibition of AKT and ERK signaling and promotion of apoptosis. Furthermore, gefitinib-resistant cells showed enhanced intratumor heterogeneity accompanied by genomic instability and activation of alternative resistance mechanisms compared with dacomitinib-resistant cells; this suggests that the maintenance of EGFR dependency after acquiring resistance might depend on the type of EGFR-TKI. Our results demonstrate that the progression of tumor heterogeneity via both genetic and non-genetic mechanisms might affect osimertinib sensitivity in tumors with acquired resistance harboring EGFR-T790M mutation., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Cellular senescence is a state of irreversible cell growth arrest that functions as a biological defense mechanism against severe DNA damage. Senescent cells with DNA damage produce pro-inflammatory cytokines, such as IL-6 and IL-8, and this phenomenon is called the senescence-associated secretory phenotype (SASP). SASP factors have been implicated in various disorders, including cancer. We performed a screening assay and identified oridonin as a candidate SASP inhibitor. Oridonin is an active diterpenoid that is isolated from Isodon plants and has been reported to exhibit anti-inflammatory, antibacterial, antioxidant, and antitumor activities. It reduced the secretion of IL-6 and IL-8 in senescent cells at the protein and mRNA levels. Oridonin also inhibited p65 subunit of NF-κB activity. However, oridonin did not affect SA β-gal activity and enhanced the expression of p21. The expression and phosphorylation of p38 were down-regulated by oridonin. The p38 inhibitor SB203580 inhibited the secretion of IL-8, slightly inhibited the secretion of IL-6, and did not affect NF-κB activity. Therefore, the NF-κB and p38 pathways may contribute to the inhibition of SASP by oridonin. Oridonin has potential as a therapeutic agent for SASP-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

Anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) have shown dramatic efficacy in patients with ALK-rearranged lung cancer; however, complete response in these patients is rare. Here, we investigated the molecular mechanisms underlying the emergence and maintenance of drug-tolerant cells in ALK-rearranged lung cancer. Cell based-assays demonstrated that HER3 activation and mesenchymal-to-epithelial transition, mediated through ZEB1 proteins, help maintain cell survival and induce the emergence of ALK-TKI-tolerant cells. Compared with ALK-TKIs alone, cotreatment with pan-HER inhibitor afatinib and ALK-TKIs prevented tumor regrowth, leading to the eradication of tumors in ALK-rearranged tumors with mesenchymal features. Moreover, pre-treatment vimentin expression in clinical specimens obtained from patients with ALK-rearranged lung cancer was associated with poor ALK-TKI treatment outcomes. These results demonstrated that HER3 activation plays a pivotal role in the emergence of ALK-TKI-tolerant cells. Furthermore, the inhibition of HER3 signals combined with ALK-TKIs dramatically improves treatment outcomes for ALK-rearranged lung cancer with mesenchymal features., (© 2022. The Author(s).)

Metabolic reprogramming leading to aerobic glycolysis, termed the "Warburg effect," is a critical property of cancer cells. However, the precise mechanisms underlying this phenomenon are not fully understood. A growing body of evidence indicates that γ-glutamylcyclotransferase (GGCT), an enzyme involved in glutathione homeostasis that is highly expressed in many types of cancer, represents a promising therapeutic target. In this study, we identified GGCT as a novel regulator of hypoxia-inducible factor-1α (HIF-1α), a transcription factor that plays a role in hypoxia adaptation promoting aerobic glycolysis. In multiple human cancer cell lines, depletion of GGCT downregulated HIF-1α at the mRNA and protein levels. Conversely, in NIH3T3 mouse fibroblasts, overexpression of GGCT upregulated HIF-1α under normoxia. Moreover, depletion of GGCT downregulated HIF-1α downstream target genes involved in glycolysis, whereas overexpression of GGCT upregulated those genes. Metabolomic analysis revealed that modulation of GGCT expression induced a metabolic switch from the citric acid cycle to glycolysis under normoxia. In addition, we found that GGCT regulates expression of HIF-1α protein via the AMPK-mTORC1-4E-BP1 pathway in PC3 cells. Thus GGCT regulates the expression of HIF-1α in cancer cells, causing a switch to glycolysis., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc. part of Springer Nature.)

Anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) have improved clinical outcomes in non-small cell lung cancer (NSCLC) harboring ALK- rearrangements. However, a small population of tumor cells survives due to adaptive resistance under drug pressure and ultimately acquires drug resistance. Thus, it is necessary to elucidate the mechanisms underlying the prevention of drug resistance to improve the prognosis of patients with ALK-rearranged NSCLC. We identified novel adaptive resistance, generated through c-Jun N-terminal kinase (JNK)/c-Jun signaling, to initial ALK-TKIs-alectinib and brigatinib-in ALK-rearranged NSCLC. Inhibition of JNK/c-Jun axis showed suppression of growth and promotion of apoptosis induced by ALK-TKIs in drug-tolerant cells. JNK inhibition, in combination with the use of ALK-TKIs, increased cell apoptosis through repression of the Bcl-xL proteins, compared with ALK-TKI monotherapy. Importantly, combination therapy targeting JNK and ALK significantly delayed the regrowth following cessation of these treatments. Together, our results demonstrated that JNK pathway activation plays a pivotal role in the intrinsic resistance to ALK-TKIs and the emergence of ALK-TKI-tolerant cells in ALK-rearranged NSCLC, thus indicating that optimal inhibition of tolerant signals combined with ALK-TKIs may potentially improve the outcome of ALK-rearranged NSCLC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Various molecular-targeting drugs have markedly improved the treatment of patients with breast cancer. As yet, therapies for triple-negative breast cancer are mainly cytotoxic agents. To investigate the novel therapy for triple-negative breast cancer, we herein examined the effects of a new combination therapy comprising a RAF/MEK inhibitor CH5126766, also known as VS-6766, which we originally discovered, and eribulin. The combination of CH5126766 and eribulin potently inhibited cell growth in the triple-negative breast cancer cell lines tested. The underlying mechanism in the efficacy of this combination treatment in vitro and in vivo was due to enhanced apoptosis through the suppression of survivin and Bcl-2 family proteins. We also showed the suppressed expression of programmed cell death ligand 1 (PD-L1) in combination therapy in vivo. We found that combination therapy with eribulin and CH5126766 for triple-negative breast cancer inhibited cell growth by apoptosis and raised a possibility that immune responses through suppression of PD-L1 might partially contribute to inhibition of tumor growth, indicating the potential of this combination as a novel strategy for triple-negative breast cancer., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Cigarette smoking and alcohol consumption are major risk factors for lifestyle-related diseases. Although it has been reported that the combination of these habits worsens risks, the underlying mechanism remains elusive. Reactive carbonyl species (RCS) cause chemical modifications of biological molecules, leading to alterations in cellular signaling pathways, and total RCS levels have been used as a lipid peroxidation marker linked to lifestyle-related diseases. In this study, at least 41 types of RCS were identified in the lipophilic fraction of plasma samples from 40 subjects using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Higher levels of 10 alkanals, 5 trans -2-alkenals, 1 cis -4-alkenal, and 3 alkadienals were detected in the smoking/drinking group ( N = 10) as compared to those with either habit ( N = 10 each) or without both habits ( N = 10) in the analysis of covariances adjusted for age and BMI. The levels of 3 alkanals, 1 trans -2-alkenal, 1 alkadienal, and 1 4-hydroxy-2-alkenal in the smoking/drinking group were significantly higher than those in the no-smoking/drinking and no-smoking/no-drinking groups. These results strongly indicate that the combination of cigarette smoking and alcohol drinking synergistically increases the level and variety of RCS in the circulating blood, and may further jeopardize cellular function.

Purpose: To evaluate the presence of ocular surface mucin in patients with atopic and vernal keratoconjunctivitis (AKC/VKC), we investigated the mRNA expression levels of SAM-pointed domain-containing ETS-like factor ( SPDEF ) and mucin-related genes on the ocular surface., Methods: Nineteen patients with AKC or VKC were divided into two groups based on the severity of the disease as determined by their clinical scores for AKC/VKC: the stable group and the active group. Impression cytology was performed in all patients using filter paper, and the expression levels of SPDEF , MUC1 , MUC4 , MUC5AC , MUC16 , and eotaxin-2 mRNA were determined by real-time reverse-transcription polymerase chain reaction., Results: The results showed that the expression levels of SPDEF and MUC5AC mRNA in the active group were significantly decreased compared with those in the stable group. Furthermore, clinical scores were significantly negatively correlated with the expression levels of SPDEF mRNA and significantly positively correlated with the expression levels of eotaxin-2, which is a biomarker for eosinophilic inflammation on the ocular surface. Cluster analysis classified the patients with AKC/VKC into three clusters, and the stable group was divided into two clusters according to the condition of ocular surface mucin., Conclusions: Ocular surface mucin in patients with AKC/VKC is altered in accordance with the clinical severity of the disease., Competing Interests: J. S. has previously received honoraria from Santen Pharmaceutical Co., Ltd., Senju Pharmaceutical Co., Ltd., and Alcon Pharmaceuticals, outside the submitted work. S. Y., N. I., Y. T., A. T., and M. H. declare that they have no conflicts of interest., (Copyright © 2021 Mariko Horinaka et al.)

Purpose: Currently, an optimal therapeutic strategy comprising molecularly targeted agents for treating EGFR -mutated non-small cell lung cancer (NSCLC) patients with acquired resistance to osimertinib is not available. Therefore, the initial therapeutic intervention is crucial for the prolonged survival of these patients. The activation of anexelekto (AXL) signaling is known to be associated with intrinsic and acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study, we investigated the best therapeutic strategy to combat AXL-induced tolerance to EGFR-TKIs using the novel AXL inhibitor ONO-7475., Experimental Design: We examined the efficacy of ONO-7475 in combination with EGFR-TKIs in EGFR -mutated NSCLC cells using in vitro and in vivo experiments. We investigated the correlation between AXL expression in tumors and clinical outcomes with osimertinib for EGFR -mutated NSCLC patients with acquired resistance to initial EGFR-TKIs., Results: ONO-7475 sensitized AXL-overexpressing EGFR -mutant NSCLC cells to the EGFR-TKIs osimertinib and dacomitinib. In addition, ONO-7475 suppressed the emergence and maintenance of EGFR-TKI-tolerant cells. In the cell line-derived xenograft models of AXL-overexpressing EGFR -mutated lung cancer treated with osimertinib, initial combination therapy of ONO-7475 and osimertinib markedly regressed tumors and delayed tumor regrowth compared with osimertinib alone or the combination after acquired resistance to osimertinib. AXL expression in EGFR-TKI refractory tumors did not correlate with the sensitivity of osimertinib., Conclusions: These results demonstrate that ONO-7475 suppresses the emergence and maintenance of tolerant cells to the initial EGFR-TKIs, osimertinib or dacomitinib, in AXL-overexpressing EGFR -mutated NSCLC cells, suggesting that ONO-7475 and osimertinib is a highly potent combination for initial treatment., (©2020 American Association for Cancer Research.)

Squamous cell lung carcinoma (SQCLC) is an aggressive type of lung cancer. In contrast with the marked advances that have been achieved in the treatment of lung adenocarcinoma, there are currently no effective targeted therapies for SQCLC, for with cytotoxic drugs are still the main treatment strategy. Therefore, the present study aimed to develop novel combination therapies for SQCLC. The results demonstrated that a combined treatment with the potent histone deacetylase (HDAC) inhibitor OBP‑801 and the third‑generation anthracycline amrubicin synergistically inhibited the viability of SQCLC cell lines by inducing apoptosis signal‑regulating kinase 1 (ASK1)‑dependent, as well as JNK‑ and p38 mitogen‑activated protein kinase (MAPK)‑independent apoptosis. OBP‑801 treatment strongly induced the protein expression levels of thioredoxin‑interacting protein (TXNIP), and amrubicin treatment increased the levels of intracellular reactive oxygen species (ROS), which suggested that this combination oxidized and dissociated thioredoxin 2 (Trx2) from mitochondrial ASK1 and activated ASK1. Moreover, mouse xenograft experiments using human H520 SQCLC cells revealed that the co‑treatment potently suppressed tumor growth in vivo. These results suggested that a combined treatment with OBP‑801 and amrubicin may have potential as a therapeutic strategy for SQCLC.

Purpose: Myxofibrosarcoma is characterized by a high rate of recurrence after surgery. Since myxofibrosarcoma is refractory to conventional cytotoxic chemotherapy, the established radical treatment is primary wide resection. The effects of histone deacetylase (HDAC) inhibitors on myxofibrosarcoma have not yet been investigated. Therefore, the main purpose of the present study was to examine the effects of a HDAC inhibitor on myxofibrosarcoma., Methods: The effects of the HDAC inhibitor OBP-801 on human myxofibrosarcoma cells were examined using cell viability assay, flow cytometric analysis of the cell cycle and apoptosis, and Western blotting. The effects of combinations of OBP-801 with pazopanib or Akt-mTOR inhibitors were also investigated using cell viability assay., Results: OBP-801 inhibited the growth of myxofibrosarcoma NMFH-1 and NMFH-2 cells. It also induced cell cycle arrest at the G2 phase and apoptosis in both cell lines. The inhibitory effects of pazopanib and Akt-mTOR inhibitors on the growth of myxofibrosarcoma cells were enhanced by the combination with OBP-801., Conclusions: The present results demonstrated that OBP-801 exerted therapeutic effects in myxofibrosarcoma in both single and concomitant administrations. Therefore, OBP-801 has potential as a novel treatment for myxofibrosarcoma.

Sulforaphane and Lactobacilli induce apoptosis in several cancer cells. Sulforaphane, a dietary isothiocyanate, is an attractive agent due to its potent anticancer effects. Sulforaphane suppresses the proliferation of various cancer cells in vitro and in vivo . The present study investigated the effect of sulforaphane and a co-culture with Lactobacillus -treated peripheral blood mononuclear cells (PBMCs) in human colon cancer cells. The combination markedly induced apoptosis in human colon cancer HCT116 and SW480 cells. A pan-caspase inhibitor markedly inhibited apoptosis, and a tumor necrosis factor (TNF) receptor/Fc chimera partially inhibited apoptosis in both cells. The amount of TNFα secretion in the culture supernatant was significantly increased by co-culture with Lactobacillus -treated normal human PBMCs. On the other hand, the expression of cellular inhibitor of apoptosis-2 (cIAP-2), an anti-apoptotic protein, was increased by co-culture with Lactobacillus -treated PBMCs in colon cancer cells, but sulforaphane treatment significantly suppressed the induction of cIAP-2. The present results revealed that sulforaphane enhances apoptosis in human colon cancer cells under co-culture with Lactobacillus -treated PBMCs via the TNFα signaling pathway., (Copyright © 2019, Spandidos Publications.)

Sulindac sulfone is a metabolite of sulindac, a non-steroidal anti-inflammatory drug (NSAID), without anti-inflammatory ability. However, sulindac sulfone has been reported to significantly reduce polyps in patients with colorectal adenomatous polyposis in clinical trials. Thus, sulindac sulfone is expected to be useful for the chemoprevention of neoplasia with few side effects related to anti-inflammatory ability. To date, the molecular targets of sulindac sulfone have not yet fully investigated. Therefore, in order to newly identify sulindac sulfone-binding proteins, we generated sulindac sulfone-fixed FG beads and purified sulindac sulfone-binding proteins from human colon cancer HT-29 cells. we identified mitochondrial outer membrane proteins voltage-dependent anion channel (VDAC) 1 and VDAC2 as novel molecular targets of sulindac sulfone, and sulindac sulfone directly bound to both VDAC1 and VDAC2. Double knockdown of VDAC1 and VDAC2 by siRNA inhibited growth and arrested the cell cycle at G1 phase in HT-29 cells. Depletion of VDAC1 and VDAC2 also inhibited the mTORC1 pathway with a reduction in cyclin D1. Interestingly, these effects were consistent with those of sulindac sulfone against human colon cancer cells, suggesting that sulindac sulfone negatively regulates the function of VDAC1 and VDAC2. In the present study, our data suggested that VDAC1 and VDAC2 are direct targets of sulindac sulfone which suppresses the mTORC1 pathway and induces G1 arrest., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Eribulin was approved for the treatment of metastatic breast cancer through the EMBRACE trial, and a subgroup analysis in this clinical trial indicated the efficacy of eribulin in patients with TNBC. However, the prognosis of patients with TNBC is still poor due to various molecular characteristics. Therefore, there is an urgent need for a more effective treatment for the management of TNBC., Methods: We investigated the synergistic effect of a novel histone deacetylase (HDAC) inhibitor, OBP-801, and eribulin in TNBC cell lines because OBP-801 has been known to enhance the anti-tumor activities of other chemotherapeutic agents. The cell growth was analyzed, and the flow cytometry analysis was conducted to evaluate the effects on cell cycle and the induction of apoptosis. The mechanism underlying the enhancement of inhibition of TNBC cell growth was investigated through Western blot analyses., Results: The combination treatment of OBP-801 with eribulin showed the synergistic inhibition of the growth in TNBC cells, involved with the enhancement of apoptosis. We, for the first time, found that eribulin upregulated survivin and also that OBP-801 could remarkably suppress the upregulation of survivin by eribulin. Moreover, this combination potently suppressed Bcl-xL and the MAPK pathway compared with either agent alone., Conclusion: We found that the combination of OBP-801 and eribulin synergistically inhibited the growth with apoptosis in TNBC cells, suggesting that this combination might be a promising novel strategy for treating TNBC patients.

In advanced bladder cancer, cisplatin-based chemotherapy has been the standard treatment for many years, but there are many problems in terms of side-effects. Recently, a number of clinical trials using molecular-targeted agents have been conducted, and new therapies are expected that could replace conventional cytotoxic chemotherapy. We herein report that concurrent treatment with fibroblast growth factor receptor (FGFR) inhibitor BGJ398 and the novel histone deacetylase (HDAC) inhibitor OBP-801/YM753/spiruchostatin A synergistically inhibited cell growth and markedly induced apoptosis in high-grade bladder cancer cells. This combination activated caspase-3, -8 and -9, and the pan-caspase inhibitor zVAD-fmk significantly reduced the apoptotic response to the combined treatment. The combination upregulated the expression of Bim, one of the pro-apoptotic molecules. In the present study, Bim siRNA efficiently reduced apoptosis induced by the co-treatment of BGJ398 and OBP-801. Therefore, the apoptosis induced by the combination was shown to be at least partially dependent on Bim. Taken together, these results suggest that the combination of BGJ398 and OBP-801 is a novel high potential therapeutic strategy for muscle-invasive bladder cancer.

Keloids are fibroproliferative diseases characterized by the accumulation of an extracellular matrix including collagen. Various growth factors, or cytokines, and their receptors are overexpressed in keloids, and they are expected to be therapy targets. Sulforaphane, a dietary isothiocyanate, has recently shown anti-tumor, anti-inflammatory, and anti-fibrotic properties. In this study, we found that sulforaphane inhibited cell growth and reduced collagen at the mRNA and protein levels in keloid fibroblasts. Moreover, sulforaphane markedly suppressed the expression of IL-6 and α-SMA and inhibited Stat3 and Smad3 signaling pathways in keloid fibroblast KF112 cells. Sulforaphane induced G2/M cell-cycle arrest with the induction of p21 in KF112 cells. In addition, sulforaphane inhibited cell growth and suppressed the expression of collagen in keloid fibroblasts under a coculture with peripheral blood mononuclear cells. Furthermore, sulforaphane suppressed IL-6, Stat3, and Smad3 signaling in the coculture system. This study suggests that sulforaphane may be a novel keloid treatment., (© 2017 by the Wound Healing Society.)

The prognosis of muscle-invasive bladder cancer with metastasis is poor. There have been no therapeutic improvements for many years, and an innovative therapy for muscle-invasive bladder cancer has been awaited to replace the conventional cytotoxic chemotherapy. Here, we show a candidate method for the treatment of bladder cancer. The combined treatment with a novel histone deacetylase (HDAC) inhibitor, OBP-801, and celecoxib synergistically inhibited cell growth and markedly induced apoptosis through the caspase-dependent pathway in high-grade bladder cancer cells. Furthermore, the combined treatment induced expression of death receptor 5 (DR5). We identified that knockdown of DR5 by small interfering RNA (siRNA) significantly suppressed apoptosis by the combined treatment. Therefore, we conjectured that the apoptosis induced by OBP-801 and celecoxib is at least partially dependent on DR5. However, it was interesting that the combined treatment drastically suppressed expression of DR5 ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). These data suggest that there is no involvement of TRAIL in the induction of apoptosis by the combination, regardless of the dependence of DR5. Moreover, xenograft studies using human bladder cancer cells showed that the combined therapy suppressed tumor growth by upregulating expressions of DR5 and Bim. The inhibition of tumor growth was significantly more potent than that of each agent alone, without significant weight loss. This combination therapy provided a greater benefit than monotherapy in vitro and in vivo These data show that the combination therapy with OBP-801 and celecoxib is a potential novel therapeutic strategy for patients with muscle-invasive bladder cancer. Mol Cancer Ther; 15(9); 2066-75. ©2016 AACR., (©2016 American Association for Cancer Research.)

Although many chemotherapeutic strategies against cancer have been developed, pancreatic cancer is one of the most aggressive and intractable types of malignancies. Therefore, new strategies and anti-cancer agents are necessary to treat this disease. Metformin is a widely used drug for type-2 diabetes, and is also known as a promising candidate anti-cancer agent from recent studies in vitro and in vivo. However, the mechanisms of metformin's anti-cancer effects have not been elucidated. We demonstrated that metformin suppressed the expression of miR-221, one of the most well-known oncogenic microRNAs, in human pancreatic cancer PANC-1 cells. Moreover, we showed that the down-regulation of miR-221 by metformin caused G1-phase arrest via the up-regulation of p27, one of the direct targets of miR-221. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also a promising agent for cancer treatment. While recent studies showed that treatment with only TRAIL was not effective against pancreatic cancer cells, the present data showed that metformin sensitized p53-mutated pancreatic cancer cells to TRAIL. Metformin induced the expressions of death receptor 5 (DR5), a receptor for TRAIL, and Bim with a pro-apoptotic function in the downstream of TRAIL-DR5 pathway. We suggest that the up-regulation of these proteins may contribute to sensitization of TRAIL-induced apoptosis. The combination therapy of metformin and TRAIL could therefore be effective in the treatment of pancreatic cancer.

Background: Angiosarcoma is a rare and aggressive malignant neoplasm of endothelial cells. Recent studies have shown that the mTOR pathway is also aberrantly activated in cutaneous angiosarcoma. New therapeutic strategies are required because the prognosis of this disease is still poor., Objective: The aim of the present study was to determine the driver gene of angiosarcoma useful for the screening of small molecule inhibitors., Methods: We investigated the sensitivity of inhibitors for the PI3K/AKT/mTOR pathway in ISOS-1 and ISO-HAS cutaneous angiosarcoma cell lines. Flow cytometric analysis was conducted to evaluate cell-cycle progression and apoptosis. Western blot analyses were performed to elucidate the possible underlying mechanisms of growth inhibition. The colony formation assay was conducted to evaluate the clonogenic potential. We used the siRNA for PDK1 to examine the role of PDK1 on the growth of angiosarcoma cells., Results: The PI3K inhibitor and mTOR inhibitor inhibited the growth of both cell lines in a dose-dependent manner. The PI3K inhibitor more effectively induced cell-cycle arrest at the G1 phase with the downregulated expression of cyclin D in ISOS-1 cells than the mTOR inhibitor. The PI3K inhibitor and mTOR inhibitor weakly but significantly induced G1 cell cycle arrest at the same degree in ISO-HAS cells. The expression of cyclin D was downregulated by the treatment with siRNA for PDK1, but not by the AKT inhibitor in ISOS-1 and ISO-HAS cells. The knock down of PDK1 with siRNA was more effective at reducing colony numbers than the mTOR inhibitor in ISOS-1 cells., Conclusion: These data showed that PDK1 played a pivotal role in the growth of angiosarcoma cells. Therefore, inhibition of PDK1, but not AKT, may be a more appropriate strategy than that of mTORC1 for the treatment of cutaneous angiosarcoma; the PDK1 inhibitor is promising as a therapeutic agent., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.

A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the -301 to -253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to cause apoptosis in several types of malignant tumor cells through its interaction with the death domain-containing receptor, death receptor 5 (DR5). In the present study, we showed that co-treatment with troglitazone (TGZ), a synthetic ligand of peroxisome proliferator-activated receptor γ (PPARγ), and TRAIL synergistically induced apoptosis through DR5 upregulation in human colon cancer DLD-1 cells. TGZ elevated DR5 expression at the promoter level through the CCAAT/enhancer-binding protein homologous protein (CHOP) binding site. These results suggest that combined treatment with TGZ and TRAIL may be promising as a new therapy against malignant tumors.

The development of methods of cancer prevention is a particularly important issue. We have proposed a strategy termed "combination-oriented molecular-targeting prevention" of cancer. As the molecular target of our model, we focused on the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor. TRAIL is a cytokine from immune cells and is important for immune surveillance and defense mechanisms against cancer cells. First, we searched for food factors inducing the expression of death receptor 5 (DR5), one of the TRAIL receptors. DR5 is specifically expressed on the membrane of cancer cells, not on that of normal cells. We found that many food factors markedly induced DR5 expression, enhancing TRAIL sensitivity against cancer cells. We next found that lactic acid bacteria induced endogenous TRAIL production in human peripheral blood mononuclear cells (PBMCs). Importantly, lactic acid bacteria increased the NK activity of PBMCs against cancer cells. Moreover, we found that butyric acid bacterium, one of the antiflatulents, induced the release of endogenous TRAIL from polymorphonuclear neutrophils. The butyric acid bacterium was proven effective against cancer cells by inducing apoptosis in vivo as well as in vitro. Taken together, these results strongly suggest that the strategy of "combination of DR5-inducing food factors and TRAIL-inducing food factors" as molecular-targeting prevention of cancer will be a practical approach.

Numerous human chemoprevention studies have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) possess chemopreventive effects against a variety of malignant tumors. However, there have been many clinical studies on aspirin, but not ibuprofen, even though ibuprofen is one of the most clinically and safely used NSAIDs showing potent anti-inflammatory effects. Moreover, we reported that many chemopreventive agents enhance the apoptosis-inducing effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is known to be crucial for cancer prevention. We, therefore, investigated whether ibuprofen enhances the cytocidal effect of TRAIL and found that ibuprofen markedly stimulated the apoptosis-inducing efficacy of TRAIL against human colon cancer HCT116 cells. As detected by western blot analysis and real-time RT-PCR, ibuprofen upregulated the expression of death receptor 5 (DR5), a TRAIL receptor. TRAIL-induced apoptosis enhanced by ibuprofen was effectively decreased by a caspase inhibitor and dominant-negative DR5. Noteworthy, co-treatment of ibuprofen with TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMCs). These results demonstrated that ibuprofen and TRAIL synergistically induced apoptosis in human colon cancer HCT116 cells but not in normal PBMCs, raising the possibility that ibuprofen may be promising as a safe chemopreventive agent against colon cancer.