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PDF file, 80K, Level of EML4-ALK mRNA in NSCLC cells. Total RNA extracted from the indicated cell lines was subjected to RT and real-time PCR analysis for determination of the abundance of EML4-ALK mRNA. Data were normalized by the amount of GAPDH mRNA and then expressed relative to the corresponding value for H3122 cells, and they are means from three independent experiments.

Cox proportional hazard model for PFS

Effect of using a combination of targeting B7-H3 and PD-L1 on body weight in the murine tumor model

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Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting.Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry.Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction.Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

PDF file - 95KB, Supplementary Table S3-A. Human tumor pY proteome. S3-B. Comparison analysis of unique pTyr sites iden_fy from di_erent samples.

PDF file - 104KB, Supplementary Figure 1A-B: PC9GR cells acquired drug resistance to both reversible and irreversible EGFR-TKI. A: PC9 and PC9GR cells were treated for 72 h with increasing concentrations of erlotinib or CL387,785. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. B: PC9 and PC9GR cells were incubated for 1 hour in the absence or presence of erlotinib (1 �M) as indicated. Cell lysates were subjected to protein expression analysis with antibodies to pEGFR, pSrc, pAkt, and pErk along with antibodies to β-actin as a loading control. C-D: Gene status in PC9 and PC9GR cells. C: PC9GR cells acquired T790M while retaining exon 19 deletion as determined by PCR-invader assay. Values of red bars above zero represent positive results with the detection probe for indicated mutation sequences of EGFR, while values of light blue bars are quality controls showing good PCR quality, as previously described (1). The value of red bars = (fluorescence of the samples with detection probe for mutation sequences) - (fluorescence of the normal control with the same probe � 2). The value of light blue bars = (fluorescence of the samples with detection probe for wild type sequences) - (fluorescence of the normal control with the same probe � 0.8). DEL1 represents E746-A750del type1 (2235-2249del GGAATTAAGAGAAGC). DEL2 represents E746-A750del type2 (2236-2250del GAATTAAGAGAAGCA). DEL3 represents L747-P753del insS. D: FISH analysis (CEP7 (green)/D7S522 (red)) was done in PC9 and PC9GR cells as previously described (2). No additional copy of D7S522 compared to CEP 7 was found in these cells, explaining that neither PC9 nor PC9GR cells has MET amplification.

PDF file - 139KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non–small cell lung cancer positive for the echinoderm microtubule-associated protein–like 4 (EML4)–ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown.Experimental Design: We established lines of EML4-ALK–positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK–positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics.Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal–regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion.Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Clin Cancer Res; 18(22); 6219–26. ©2012 AACR.

PDF file - 54KB, A: Example of peak area (EIC) for EGFR pY 1172. B: Example of peak area (EIC) for MET pY 1234.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined.Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

PDF file - 91KB, A: Effects of dasatinib combined with afatinib on apoptosis in HCC4006-T790M and HCC827-T790M cells. Apoptosis assay was carried out using PE-conjugated caspase-3 antibody, following incubation of HCC4006-T790M or HCC827-T790M cells for 72 hours with DMSO, erlotinib (E), dasatinib (D), afatinib (A), and afatinib + dasatinib. Values are expressed as a percentage of caspase-3-positive cells. Determinations were done in triplicate. Bars, SD. *P < 0.001 versus DMSO or each single agent (Student's t-test). B: Validation of active pTyr sites from PC9 cell lines on lung cancer patient. Venn diagram was used to compare the pTyr sites identified from PC9 and human lung tumor tissue with positive EGFR mutation. Total 83 overlap active pTyr sites were obtained.

PDF file - 387KB, Effects of ligands exposure with or without EGFR-TKI in PC9 and PC9GR cells. A-F: PC9 (A, C, and E) or PC9GR cells (B, D, and F) were incubated for 1 hour in the absence or presence of 100 nM erlotinib (A, C, and E) or afatinib (B, D, and F) with indicated concentrations of HGF (A and B), IGF1 (C and D), or Gas6 (E and F). Cell lysates were subjected to protein expression analysis with antibodies to pMET (A and B), IGF1R (C and D), or pAXL (E and F) along with pEGFR, pAkt, pErk, and antibodies to β-actin as a loading control.

PDF file - 67KB, Supplementary Table S1: Quantitation of pY changes in between PC9 and PC9GR cell lines with and without erlotinib treatment. The cutoff was set as P value1.5; Supplementary Table S2: Cell viability IC50 in PC9GR cells and H1975 cells.

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PDF file - 303K, L704N or I941R cis mutation disrupts constitutive dimerization of mutant EGFR (S1); Tyrosine-phosphorylation is abrogated on L704N or I941R compound mutants of dimerization-dependent mutants (S2); Wild-type EGFR, but not Ex20Ins mutant are dependent on asymmetric dimerization for oncogenic transformation (S3); Tyrosine-phosphorylation on dimerization-independent mutants is significantly impaired by introduction of receiver-impairing or activator-impairing mutation (S4); Cell surface expression levels of various EGFR mutants in NIH-3T3 cells (S5); Activities of receiver monomers and activator monomers of wild-type EGFR (S6); Cell surface expression of mutant EGFR in stable cell lines (S7); Differential pharmacological effects of gefitinib and cetuximab against distinct mutant EGFR (S8); Generation and characterization of bitransgenic mice with inducible expression of EGFR Ex20Ins in lung compartments (S9); Cetuximab downregulates tyrosine phosphorylation on mutant EGFR in mouse tumors (S10).

Tumour burden (TB) is implicated in resistance to programmed cell death-1/PD-L1 inhibitor (immune checkpoint inhibitor [ICI]) therapy. However, whether TB contributes to such resistance in non-small-cell lung cancer (NSCLC) has remained unknown.A total of 260 treatment-naïve patients with advanced NSCLC who started ICI monotherapy (ICI cohort), platinum-doublet therapy (Chemo cohort) or ICI and platinum-doublet therapy (ICI+Chemo cohort) as first-line treatment were consecutively included. TB was estimated on the basis of the sum of the diameters of measurable target lesions as per Response Evaluation Criteria in Solid Tumours. Progression-free survival (PFS) in the ICI cohort was evaluated as per TB as a preplanned primary objective, with the analysis based on propensity score-weighted survival curves and estimation of restricted mean survival time (RMST). The Chemo cohort served as a control to determine whether TB is predictive of ICI treatment outcomes. The ICI+Chemo cohort was exploratory. The relation of TB to tumour immune status was assessed by immune-related gene expression profiling (irGEP) of pretreatment tumour tissue.In the ICI cohort, patients with a low TB showed a significantly longer PFS than did those with a high TB (median, 17.9 vs 4.3 months; weighted hazard ratio, 0.32 [95% confidence interval, 0.19-0.53]). No such difference was apparent in the other cohorts. A significant difference in overall survival was also observed only in the ICI cohort. RMST-based analysis confirmed these results. The irGEP analysis implicated M2-type macrophages, angiogenesis and transforming growth factor-β as well as protumourigenic signalling pathways in ICI resistance conferred by a high TB.A high TB was associated with a poor outcome of ICI therapy for advanced NSCLC as a result of immunosuppressive phenotypes. Development of combination or novel treatment strategies for such disease is thus warranted.

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction., Yoshiko Iwasaki-Takahashi et al. report the production of fertilization-capable eggs and sperm from rainbow trout spermatagonia, for application to endangered species preservation. Their method avoids the need for limited numbers of germline stem cells for transplantation, instead allowing in vitro expansion of these cells using a Sertoli cell-derived feeder layer.

Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting. Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry. Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction. Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

Several types of coumarins have been isolated from Peucedanum japonicum Thunb (PJT) and we previously reported the anti-obesity activity of pteryxin (PTX), as an active compound of PJT. However, during our research, we identified another variety of PJT, which does not contain any PTX. Here, we prepared concentrates from the two varieties of PJT and demonstrated that dietary PJT concentrates both with and without PTX significantly decreased the relative weights of white adipose tissue (WAT) and mRNA levels of lipogenic genes in epididymal WAT. Furthermore, we compared the inhibitory effects of dihydropyranocoumarins of PJT on lipid accumulation of differentiated 3T3-L1 adipocytes. All of the dihydropyranocoumarins used in this study significantly inhibited lipid accumulation and lipogenic-related gene expressions in 3T3-L1 adipocytes. However, the magnitudes of the effects were distinctly different. The present study demonstrated that several dihydropyranocoumarins in PJT possess anti-obesity activities, which mainly act by inhibiting lipogenesis in adipocytes.

The aims of this dose-escalating phase I study were to determine the maximum tolerable dose (MTD) and recommended dose (RD) of 5-fluorouracil (5-FU), docetaxel, and nedaplatin (UDON) combination therapy for future phase II studies, and to evaluate the safety and efficacy of this regimen in patients with untreated recurrent or metastatic esophageal cancer. Patients were administered 5-FU on days 1–5, docetaxel on days 1 and 15, and nedaplatin on day 1 at 4-week intervals. The dose levels of 5-FU/docetaxel/nedaplatin were escalated as follows (mg/m2): level 1, 800/30/80; level 2, 800/30/90; and level 3, 800/35/90. Toxicity was evaluated using Common Terminology Criteria for Adverse Events version 4.0. Overall, nine patients were enrolled in this study. All patients had an Eastern Cooperative Oncology Group performance status of 0 or 1 and were diagnosed with squamous cell carcinoma. No dose-limiting toxicity was observed at any level, and planned dose escalation was completed without reaching the MTD. No grade 4 or higher toxicity was observed in this study. The observed grade 3 hematological toxicities included neutropenia in five patients (55.6 %) and leukopenia in three patients (33.3 %). None of the patients developed febrile neutropenia, and no grade 3 or 4 non-hematological toxicities were observed. The overall response rate was 77.8 %, including two complete responses, and the disease control rate was 100 %. The RD of UDON was identified as level 3. The good tolerability and high antitumor efficacy of this regimen warrant further evaluation in this setting.

Linifanib is a potent, orally active, and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor kinase activities with clinical efficacy in non-small cell lung cancer (NSCLC). This phase 1 dose-escalation study evaluated the pharmacokinetics, safety, and efficacy of linifanib in combination with carboplatin/paclitaxel in Japanese patients with advanced NSCLC. Carboplatin (AUC = 6 mg/mL/min) and paclitaxel (200 mg/m2) were administered on day 1 of each 21-day cycle up to a maximum of six cycles. Oral linifanib (7.5 mg) was given to six patients once daily throughout all cycles and escalated to 12.5 mg/day in a second cohort of six patients. Twelve patients received at least one dose of linifanib. The most common adverse events were hematologic and consistent with expected toxicities with carboplatin/paclitaxel. With 12.5 mg linifanib, grade 3/4 neutropenia, leukopenia, and thrombocytopenia occurred in 100, 83, and 83 % of patients, respectively. Dose-limiting grade 4 thrombocytopenia occurred in one patient at each dose level. Linifanib pharmacokinetics was similar to that in non-Japanese patients. At 12.5 mg, linifanib C max was 0.32 μg/mL and AUC24 was 4.29 μg h/mL. Linifanib C max occurred at 2–3 h with both doses and when given alone or in combination with carboplatin/paclitaxel. Exposure to linifanib appeared to be increased by carboplatin/paclitaxel, and exposure to paclitaxel appeared to be increased by linifanib. Partial responses were observed in nine patients. Linifanib added to carboplatin/paclitaxel is well tolerated in Japanese patients with advanced/metastatic NSCLC. The recommended dose of linifanib with carboplatin/paclitaxel is 12.5 mg, same as for US patients.

Kinase domain mutations of the EGF receptor (EGFR) are common oncogenic events in lung adenocarcinoma. Here, we explore the dependency upon asymmetric dimerization of the kinase domain for activation of lung cancer–derived EGFR mutants. We show that whereas wild-type EGFR and the L858R mutant require dimerization for activation and oncogenic transformation, the exon 19 deletion, exon 20 insertion, and L858R/T790M EGFR mutants do not require dimerization. In addition, treatment with the monoclonal antibody, cetuximab, shrinks mouse lung tumors induced by the dimerization-dependent L858R mutant, but exerts only a modest effect on tumors driven by dimerization-independent EGFR mutants. These data imply that different EGFR mutants show differential requirements for dimerization and that disruption of dimerization may be among the antitumor mechanisms of cetuximab. Cancer Res; 73(22); 6770–9. ©2013 AACR.

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non–small cell lung cancer positive for the echinoderm microtubule-associated protein–like 4 (EML4)–ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown. Experimental Design: We established lines of EML4-ALK–positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK–positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics. Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal–regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion. Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Clin Cancer Res; 18(22); 6219–26. ©2012 AACR.

The present study describes the effect of Peucedanum japonicum Thunb (PJT) intake on the expression of obesity-related genes in mice fed a high-fat diet. To explore the mechanism underlying the effect of PJT, This study focused on the expression of genes, especially those related to obesity and metabolism syndrome, in the liver and adipose tissues. In agreement with our previous observations, intake of 10 % PJT for 4 weeks significantly reduced serum triglyceride (TG), leptin, abdominal fat, and adipocyte size. PJT also significantly increased fecal excretion of TG, decreased that of bile acid, and tended to increase the fecal excretion of total cholesterol. Microarray analysis was used to monitor changes in 324 metabolic syndrome-related genes in the liver. Statistically significant upregulation of PPP1R10, RORC, and PBEF1 genes and downregulation of DUSP1, INSIG2, and SERPINA12 genes were noted and confirmed by real-time RT-PCR. These changes were indicative of increased fatty acid oxidation in the maintenance of lipid homeostasis and insulin sensitivity in the livers of PJT-fed mice. PJT increased the expression of PPARγ, FXRα, DGAT1, and ATGL genes, suggesting an enhancement of adipocyte differentiation and normalization of functionality of adipose tissue.

Porous diaphragm syndrome describes a defect in the diaphragm in which substances pass from the peritoneal cavity to the pleural space. Defects may be congenital or acquired. Acquired defects are caused by the thinning and eventual splitting of collagen fibres in the tendinous part of the diaphragm. We report a case of porous diaphragm syndrome with recurrent thymoma that presented with massive ascites. Increasing intra-abdominal pressure by ascites and diaphragmatic thinning due to malnutrition by malignancies resulted in the formation of an artificial hole. Thoracentesis changed the balance of hydrostatic pressure, which initiated the influx of a large volume of ascites to the pleural cavity through a hole in the diaphragm.

Nitidine (NTD) cytotoxicity is highly specific for A549 human lung adenocarcinoma cells. We hypothesized that this cytotoxicity involved the accumulation of NTD in intracellular organelles. However, there have been no reports of NTD transporting factors. In this study, we screened for an NTD transporter and evaluated its association with NTD cytotoxicity.Gene expression analyses were done for A549 and human fetal lung normal diploid fibroblast (WI-38) cells. We screened for ABC transporter, multi-drug resistance-associated genes. Gene expressions of ATP-binding cassette transporter A1 (ABCA1) were confirmed in 8 cell lines by quantitative PCR. The involvement of ABCA1 in NTD cytotoxicity was evaluated using siRNA-mediated ABCA1 gene silencing.Gene expression analysis indicated that A549 cells expressed higher levels of ABCC1, ABCC2, ABCC3, and ABCG2 and a lower level of ABCA1 compared to WI-38 cells. NTD resistant cell lines uniformly showed higher ABCA1 expression levels. Gene silencing experiments showed that the down-regulation of ABCA1 resulted in increased sensitivity to NTD.These results indicated that NTD efflux is controlled by ABCA1 activity, suggesting that ABCA1 transports molecules other than lipids. Thus, there is a possibility that ABCA1 acts as a drug resistance transporter involved in the cytotoxicity of NTD derivatives. This also suggested that the expression level of the ABCA1 gene may be an indicator for the efficiency of NTD treatment.

To develop a novel anti-cancer drug of low side effect against lung adenocarcinoma, the authors screened the bioresources of Okinawa Island, Japan. The medicinal plant Toddalia asiatica Lam. contained three benzo[c]phenanthridine derivatives: dihydronitidine (DHN), nitidine (NTD) and demethylnitidine (DMN). Of the three derivatives, DHN had been shown to selectively inhibit the growth of cancer cells in our previous study. Because of similar molecular topology of NTD or DMN to DHN, it can be expected that NTD and DMN also show selective cytotoxicity. The aim of the present study was therefore to examine the selective cytotoxicity of these two compounds in vitro and in vivo. Benzo[c]phenanthridine derivatives were isolated from T. asiatica Lam., and their chemical structures were identified by interpretation of NMR and MS spectrum. Of the isolated compounds, NTD and DMN were evaluated for cytotoxicity in vitro or in vivo. NTD as well as DHN selectively reduced the growth of murine and human lung adenocarcinoma in vitro with selective intracellular accumulation. NTD has also been proven to be highly effective in vivo to inhibit the growth of both murine and human lung adenocarcinoma in a subcutaneous xenograft model without any deteriorating side effect. In contrast, DMN had no selective cytotoxicity suggesting that 8-methoxy group of NTD is the critical structural feature for the selective cytotoxicity. This study thus proves the effectiveness of benzo[c]phenanthridine derivatives as anti-cancer agent in vivo for the first time, and discusses the mechanisms responsible for the selective cytotoxicity.

Purpose: Most non–small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib and erlotinib, but they almost invariably develop resistance to these drugs. A secondary mutation in EGFR (T790M) and amplification of the MET proto-oncogene have been identified as mechanisms of such acquired resistance to EGFR-TKIs. We have now investigated whether addition of the oral fluoropyrimidine derivative S-1 to gefitinib might overcome gefitinib resistance in NSCLC cell lines.Experimental Design: The effects of gefitinib on EGFR signaling and on the expression both of thymidylate synthase and of the transcription factor E2F-1 in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of S-1 (or 5-fluorouracil) and gefitinib on the growth of NSCLC cells were examined in vitro as well as in nude mice.Results: Gefitinib induced down-regulation of thymidylate synthase and E2F-1 in gefitinib-resistant NSCLC cells with MET amplification but not in those harboring the T790M mutation of EGFR. The combination of 5-fluorouracil and gefitinib synergistically inhibited the proliferation of cells with MET amplification, but not that of those with the T790M mutation of EGFR, in vitro. Similarly, the combination of S-1 and gefitinib synergistically inhibited the growth only of NSCLC xenografts with MET amplification.Conclusions: Our results suggest that the addition of S-1 to EGFR-TKIs is a promising strategy to overcome EGFR-TKI resistance in NSCLC with MET amplification.

This study describes the structure-cytotoxicity relationship of beta-glucosylceramide (beta-GluCer) and its antitumor activity in vivo. Unglycosylated ceramide had no selective cytotoxicity which demonstrated that the sugar moiety plays a critical role for the expression of selective cytotoxicity by beta-GluCer. beta-Galactosylceramide also showed tumor specific cytotoxicity suggesting that the chemical structure of sugar group is not a factor for the selective toxicity. Similarly, unglycosylated ceramides of short acyl chain also selectively inhibited the growth of cancer cells. These findings in concert point to the importance of the hydrophilicity of the ceramide molecule rather than the chemical structure for the cyto-selectivity. Treatment of the cells with beta-GluCer increased the concentration of reactive oxygen species leading to cell cycle arrest and necrosis. Intraperitoneal administration of beta-GluCer significantly suppressed the growth of tumor implanted to the back of mice. beta-GluCer also induced antitumor immunity via the activation of NKT cells in vivo, and decreased the tumor metastasis of lymphoma cells. The present study thus demonstrated the antitumor activity of beta-GluCer in vivo, and discussed the mechanisms responsible for the growth inhibition.

In order to realize highly sensitive image comparison, a method for robust image matching, which is not affected by the form or the density of the patterns contained in the images, is required. Thus, a method that utilizes the characteristics of a normalized correlation coefficient is proposed in this study. Using this method, an object image obtained under active illumination control is subdivided into multiple smaller images, and the correlation coefficient maps are individually computed. The matching position is subsequently determined by integrating the maps. The effectiveness of the method is confirmed through experiments.

Kokuto, a traditional cane sugar of Okinawa, has been reported to have antioxidative and lipid-lowering properties. In this experiment, we investigated the effect of three different kinds of Kokuto (KA, KB, and KC) on atherosclerosis in two different animal models : Japanese quail and apolipoprotein E deficient (apoE-/-) mice. Ingestion of Kokuto had no significant effect on the serum and liver lipid levels of Japanese quail. Dietary intake of atherogenic diet (AD) with KA and KB decreased the liver triglyceride level and body weight in apoE-/- mice. Quail fed on AD with KA developed less extent of lipid-containing aortic intimal thickening lesions than those fed on AD with sucrose. Dietary intake of AD with Kokuto or sucrose induced aortic atheromatous lesions in mice, but the extent of atheromatous lesions was roughly comparable between these dietary groups of apoE-/- mice. The present study suggests that Kokuto prevents lipid-containing aortic intimal thickening lesions in Japanese quail.

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.

Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI–resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined. Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. ©2014 AACR.

Supplementation of the diet with Peucedanum japonicum Thunb (PJT) powder inhibits high-fat diet-induced obesity in mice. Either the fiber component or other bioactive components in the PJT powder may inhibit obesity. This study, therefore, was an attempt to identify the components, fiber or other phytochemicals of PJT that were responsible for the anti-obesity activity, and also studied the modulation of obesity-related gene expression in C57BL/6 mice. Animals were fed a modified-AIN76 diet supplemented with PJT powder or extracts of PJT in water, 50 % ethanol, or ethanol. Body weight gain, tissue weight, serum biochemical parameters, liver lipid concentrations, and gene expression in tissues were compared between the control and treatment groups. Of the extracts, the ethanol extract of PJT decreased fat accumulation and adipocyte size, reduced serum and liver triglyceride concentrations, and inhibited obesity. This finding clearly demonstrates the presence of anti-obesity phytochemicals in PJT. Ethanol extract of PJT inhibited lipase activity in vitro. Modulation of gene expression by PJT ethanol extract was largely similar to that by PJT powder in the hepatic and adipose tissues: RORC and PBEF1 were upregulated and DUSP1, INSIG2, and SERPINA12 were downregulated in the liver; FXRα and PPARγ were upregulated and PEG1/MEST, the size-marker of adipocytes, was downregulated in the adipose tissue. Furthermore, PJT ethanol extract increased the expression of the UCP3 gene in muscle. These results suggest that the anti-obesity phytochemicals in PJT lower lipid levels by inhibiting fat absorption and by modulating obesity-related gene expression in the liver, adipose tissue, and muscle.

This study examined the antiobese activity of Peucedanum japonicum Thunb (PJT) in mice. In the first experiment, 4-week-old C57BL/6 mice were fed seven different diets containing 15% corn oil and 0-20% PJT powder for 4 weeks. Feeding the 10% and 20% PJT diet suppressed the body weight gain and the accumulation of abdominal and subcutaneous fats. PJT reduced serum and liver levels of triglyceride and serum levels of leptin in a dose-dependent manner. PJT intake decreased the proportion of saturated fatty acids and increased polyunsaturated and n-3 fatty acids in the liver. To obtain more insight into the antiobese activity of PJT, its effect on lipid absorption and enzyme activities related to lipid metabolism was studied in the second experiment. There was an increased faecal excretion of triglyceride in mice fed 5% and 10% PJT diets. Fatty acid synthase activity was decreased while carnitine palmitoyltransferase activity was increased by 10% PJT intake. These findings pointed to the usefulness of PJT for the development of a safe natural agent to reduce obesity or body weight for the first time. The rationale for the lipid lowering mechanism of PJT and the candidate compound responsible for the observations have also been discussed.

To optimize the adequate atherogenic diet composition for nutritional atherosclerosis studies utilizing B6.KOR-Apoe(shl) mice, we investigated the effect of dietary cholesterol, AIN formula, and dietary fats on the development of atherosclerosis. Cholesterol supplementation (0.15-2%) for 12 weeks resulted in a dose-dependent increase in the development of atherosclerosis. Furthermore, there were no significant differences in the degree of atherosclerosis between B6.KOR-Apoe(shl) mice fed a modified-AIN-76 diet and those fed a modified-AIN-93M diet containing corn oil or soybean oil for 10 and 12 weeks. The present experiment indicates that the adequate dietary level of cholesterol was 0.15%, and that further studies are necessary to determine the optimal level of various types of dietary oils for nutritional atherosclerosis experiments in B6.KOR-Apoe(shl) mice.

In the present study, we investigated the effect of phenolic compounds (PCs) and policosanol of Kokuto, Okinawan noncentrifugal cane sugar, on the development of atherosclerosis. A total of 67 male Japanese quail were divided into eight dietary groups in trial 1. The dietary groups were fed the atherosclerotic diet (AD) containing 5% corn oil, 2% cholesterol, and 30% sucrose or seven different types of Kokuto. Dietary intakes of Kokuto notably prevented the development of atherosclerosis. Furthermore, there was a significant negative correlation between the serum radical scavenging activity and the degree of atherosclerosis in the dietary groups. In trial 2, a total of 63 Japanese quail were fed AD with sucrose, Kokuto, PC extracts from Kokuto, wax extracts from sugar cane, octacosanol, vitamin C, and vitamin E. As a result, the supplementation of the diet with Kokuto and PCs significantly reduced the development of atherosclerosis as compared with the ingestion of AD with sucrose. In conclusion, these findings suggest that, among various components of Kokuto, PCs play a central role for the prevention of experimental atherosclerosis in Japanese quail.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the therapeutic response to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced non-small cell lung cancer (NSCLC). The response rate to these drugs remains low, however, in NSCLC patients with wild-type EGFR alleles. Combination therapies with EGFR-TKIs and cytotoxic agents are considered a therapeutic option for patients with NSCLC expressing wild-type EGFR. We investigated the antiproliferative effect of the combination of the oral fluorouracil S-1 and the EGFR-TKI gefitinib in NSCLC cells of differing EGFR status. The combination of 5-fluorouracil and gefitinib showed a synergistic antiproliferative effect in vitro in all NSCLC cell lines tested. Combination chemotherapy with S-1 and gefitinib in vivo also had a synergistic antitumor effect on NSCLC xenografts regardless of the absence or presence of EGFR mutations. Gefitinib inhibited the expression of the transcription factor E2F-1, resulting in the down-regulation of thymidylate synthase at the mRNA and protein levels. These observations suggest that gefitinib-induced down-regulation of thymidylate synthase is responsible, at least in part, for the synergistic antitumor effect of combined treatment with S-1 and gefitinib and provide a basis for clinical evaluation of combination chemotherapy with S-1 and EGFR-TKIs in patients with solid tumors. [Mol Cancer Ther 2008;7(3):599–606]

Molecular inhibition of the epidermal growth factor receptor (EGFR) is a promising anticancer strategy, and monoclonal antibodies (mAbs) to EGFR are undergoing extensive evaluation in preclinical and clinical trials. However, the effects of anti-EGFR mAbs on EGFR signaling have remained unclear. We have now examined the effects of 2 anti-EGFR mAbs, matuzumab (EMD72000) and cetuximab (Erbitux), both of which are currently under assessment for treatment of various cancers, on EGFR signal transduction and cell survival in nonsmall cell lung cancer cell lines. Similar to EGF, matuzumab and cetuximab each induced phosphorylation of EGFR at several tyrosine phosphorylation sites as a result of receptor dimerization and activation of the receptor tyrosine kinase. In contrast to the effects of EGF, however, EGFR activation induced by these antibodies was not accompanied by receptor turnover or by activation of downstream signaling pathways that are mediated by Akt and Erk and are important for regulation of cell proliferation and survival. In addition, clonogenic survival assays revealed that matuzumab and cetuximab reduced the survival rate of H292 cells, in which they also inhibited the EGF-induced activation of Akt and Erk. Although we have examined only a few cell lines, our results indicate that the antitumor effects of matuzumab and cetuximab depend on inhibition of EGFR downstream signaling mediated by Akt or Erk rather than on inhibition of EGFR itself.

The identification of somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in patients with non–small cell lung cancer (NSCLC) and the association of such mutations with the clinical response to EGFR tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, have had a substantial effect on the treatment of this disease. EGFR gene amplification has also been associated with an increased therapeutic response to EGFR-TKIs. The effects of these two types of EGFR alteration on EGFR function have remained unclear, however. We have now examined 16 NSCLC cell lines, including eight newly established lines from Japanese NSCLC patients, for the presence of EGFR mutations and amplification. Four of the six cell lines that harbor EGFR mutations were found to be positive for EGFR amplification, whereas none of the 10 cell lines negative for EGFR mutation manifested EGFR amplification, suggesting that these two types of EGFR alteration are closely associated. Endogenous EGFRs expressed in NSCLC cell lines positive for both EGFR mutation and amplification were found to be constitutively activated as a result of ligand-independent dimerization. Furthermore, the patterns of both EGFR amplification and EGFR autophosphorylation were shown to differ between cell lines harboring the two most common types of EGFR mutation (exon 19 deletion and L858R point mutation in exon 21). These results reveal distinct biochemical properties of endogenous mutant forms of EGFR expressed in NSCLC cell lines and may have implications for treatment of this condition. [Cancer Res 2007;67(5):2046–53]

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown. Experimental Design: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristic. Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of the epidermal growth factor receptor (EGFR), HER2, and HER3 was up-regulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion. Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Citation Format: Junko Tanizaki, Isamu Okamoto, Takafumi Okabe, Kazuko Sakai, Kaoru Tanaka, Hidetoshi Hayashi, Hiroyasu Kaneda, Ken Takezawa, Kazuto Nishio, Kazuhiko Nakagawa. Activation of HER Family signaling as a mechanism of acquired resistance to ALK Inhibitors in EML4-ALK-positive Non-Small Cell Lung Cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4446. doi:10.1158/1538-7445.AM2013-4446

Background A secondary mutation of EGFR (T790M) accounts for 50% of all cases of acquired resistance to EGFR-TKIs in NSCLC patients with EGFR mutation. Although irreversible EGFR-TKIs are thought to be one of the strategies to overcome T790M, the efficacy of these drugs appears limited. Methods We generated EGFR-TKI resistant PC9GR cells with T790M by long-term gefitinib exposure of PC9 cells with exon 19 deletion. To visualize their molecular networks related to T790M, we carried out immunoaffinity purification of tyrosine-phosphorylated peptides by anti-phospho tyrosine antibodies (pTyr100) and subsequent LC-MS/MS identification in PC9 and PC9GR cells with or without erlotinib treatment. Results Our phosphoproteomics detected a total of 403 unique phosphosites on 266 proteins. Compared with non-treated PC9 cells, 32 pY sites (24 proteins) are decreased and 45 pY sites (33 proteins) are increased in non-treated PC9GR cells. In parental PC9 cells, 24 pY sites (19 proteins) are decreased and 12 pY sites (12 proteins) are increased by erlotinib treatment. In resistant PC9GR cells, 5 pY sites (5 proteins) are decreased and 6 pY sites (6 proteins) are increased by erlotinib treatment. The detailed analyses including different response to erlotinib between PC9 and PC9GR cells will be presented at the meeting. As a preliminary validation, we found that PC9GR cells have persistent Src kinase levels even with erlotinib treatment in our phosphoproteomics. Western blot assays also demonstrated that the irreversible EGFR-TKI BIBW-2992 (afatinib) did not alter the activation of Src family kinases in PC9GR cells. Src inhibitor dasatinib combined with afatinib abolished this Src phosphorylation along with complete suppression of downstream phospho Akt and Erk. Dasatinib enhances anti-tumor activity of afatinib in both cell proliferation assay and caspase-3 apoptosis assay in these cells. This combination showed significant in vivo tumor regression in PC9GR xenograft studies. Conclusions Applying global phosphoproteomics effectively visualizes the molecular networks of EGFR-TKI-resistant NSCLC related to T790M gate keeper mutation. Our preliminary validation supports further studies of irreversible EGFR inhibitors combined with dasatinib in NSCLC patients who acquired resistance to EGFR-TKI by T790M.