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PDF file - 114K, Chemical structures of investigated molecules in this article

PDF file - 135K, Treatment of saracatinib in LS174T and SKBR3 cells

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PDF file - 173K, Induction of Bim by saracatinib treatment

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

PDF file - 96K, Downregulation of thymidylate synthase (TS) by saracatinib

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PDF file - 3250K, Figure S1 Olaparib has variable levels of anti-tumor activity in human cancer cell lines. Figure S2 Olaparib sensitive cell lines have different patterns of gene expression compared to insensitive cell lines. Figure S3 PTEN expression does not affect olaparib sensitivity. Figure S4 RAD51C expression affects olaparib sensitivity in human cancer cell lines. Figure S5 RAD51C over-expression decreases sensitivity to olaparib in the BT-549 cell line. Figure S6 Olaparib induces G2/M cell cycle arrest and apoptosis in sensitive cell lines. Figure S7 RAD51C depletion increases the accumulation of DNA damage. Figure S8 RAD51C expression decreases in tumor tissues. Figure S9 RAD51C expression is down-regulated in gastric tumor tissues via DNA methylation. Table S1. Sequences of the primers for PCR and real-time PCR analyses. Table S2. BRCA1 and BRCA2 mutations in the gastric cancer cell lines.

PDF file - 215K, mRNA and protein expression level of integrin �8 in gastric cancer cell lines

EGFR tyrosine kinase inhibitors have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, tyrosine kinase inhibitors have also proven ineffective against most tumors with EGFR wild-type (WT) alleles. Although some genetic changes, including the KRAS mutation, have been shown to confer resistance to tyrosine kinase inhibitors, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. The principal objective of this study was to improve our current understanding of drug interactions between EGFR and MAP/ERK kinase (MEK) inhibitors in an effort to gain insight into a novel therapeutic strategy against EGFR WT tumors. Using a panel of human EGFR WT gastric cancer cell lines, we showed that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phosphoprotein arrays showed that, in MEK inhibitor resistant cell lines, AKT was activated through the EGFR/HER3/PI3K pathway following AZD6244 (ARRY-142886) treatment. Blockade of this feedback mechanism through the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines in vitro and in vivo. Our results provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. [Mol Cancer Ther 2009;8(9):2526–36]

Supplementary Data from Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells

BACKGROUND Early detection or notification of adverse event (AE) occurrences during clinical trials is essential to ensure patient safety. Clinical trials take advantage of innovative strategies, clinical designs, and state-of-the-art technologies to evaluate efficacy and safety, however, early awareness of AE occurrences by investigators still needs to be systematically improved. OBJECTIVE This study aimed to build a system to promptly inform investigators when clinical trial participants make unscheduled visits to the emergency room or other departments within the hospital. METHODS We developed the Adverse Event Awareness System (AEAS), which promptly informs investigators and study coordinators of AE occurrences by automatically sending text messages when study participants make unscheduled visits to the emergency department or other clinics at our center. We established the AEAS in July 2015 in the clinical trial management system. We compared the AE reporting timeline data of 305 AE occurrences from 74 clinical trials between the preinitiative period (December 2014-June 2015) and the postinitiative period (July 2015-June 2016) in terms of three AE awareness performance indicators: onset to awareness, awareness to reporting, and onset to reporting. RESULTS A total of 305 initial AE reports from 74 clinical trials were included. All three AE awareness performance indicators were significantly lower in the postinitiative period. Specifically, the onset-to-reporting times were significantly shorter in the postinitiative period (median 1 day [IQR 0-1], mean rank 140.04 [SD 75.35]) than in the preinitiative period (median 1 day [IQR 0-4], mean rank 173.82 [SD 91.07], P≤.001). In the phase subgroup analysis, the awareness-to-reporting and onset-to-reporting indicators of phase 1 studies were significantly lower in the postinitiative than in the preinitiative period (preinitiative: median 1 day, mean rank of awareness to reporting 47.94, vs postinitiative: median 0 days, mean rank of awareness to reporting 35.75, P=.01; and preinitiative: median 1 day, mean rank of onset to reporting 47.4, vs postinitiative: median 1 day, mean rank of onset to reporting 35.99, P=.03). The risk-level subgroup analysis found that the onset-to-reporting time for low- and high-risk studies significantly decreased postinitiative (preinitiative: median 4 days, mean rank of low-risk studies 18.73, vs postinitiative: median 1 day, mean rank of low-risk studies 11.76, P=.02; and preinitiative: median 1 day, mean rank of high-risk studies 117.36, vs postinitiative: median 1 day, mean rank of high-risk studies 97.27, P=.01). In particular, onset to reporting was reduced more in the low-risk trial than in the high-risk trial (low-risk: median 4-0 days, vs high-risk: median 1-1 day). CONCLUSIONS We demonstrated that a real-time automatic alert system can effectively improve safety reporting timelines. The improvements were prominent in phase 1 and in low- and high-risk clinical trials. These findings suggest that an information technology-driven automatic alert system effectively improves safety reporting timelines, which may enhance patient safety.

Pemetrexed and platinum (PP) combination chemotherapy is the current standard first-line therapy for treatment of malignant mesothelioma (MM). However, a useful predictive biomarker for PP therapy is yet to be found. Here, we performed targeted exome sequencing to profile somatic mutations and copy number variations in 12 MM patients treated with PP therapy. We identified 187 somatic mutations in 12 patients (65 synonymous, 102 missense, 2 nonsense, 5 splice site, and 13 small coding insertions/deletions). We identified somatic mutations in 23 genes including BAP1, TP53, NRAS, and EGFR. Interestingly, rare NRAS p.Q61K and EGFR exon 19 deletions were observed in 2 patients. We also found somatic chromosomal copy number deletions in CDKN2A and CDKN2B genes. Genetic alteration related to response after PP therapy was not found. Somatic mutation profiling in MM patients receiving PP therapy revealed genetic alterations in potential therapeutic targets such as NRAS and EGFR. No alterations in genes with potential predictive role for PP therapy were found.

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

This paper presents a novel approach to the (generation maintenance scheduling) GMS problem in electricity markets. The main contribution of this study is the modeling of a coordination procedure for an (independent system operator) ISO, based on a game-theoretic framework for the GMS problem. The GMS process of generation companies (Gencos) is designed as a non-cooperative dynamic game, and the Gencos' optimal strategy profile is determined by the Nash equilibrium of the game. The coordination procedure performed by the ISO is characterized by the use of a reliability assessment and a so-called ‘rescheduling signal’. A numerical example for a three-Genco system is used to demonstrate the applicability of the proposed scheme to the GMS problem. The results obtained indicate that the GMS of a profit-oriented Genco can be modified to satisfy the reliability requirements of the ISO.

In the present work, detailed experimental investigations have been carried out to understand the role of various spray parameters, such as fuel injection pressure, droplet size distribution, and velocity field on the stabilization of lifted spray flames with kerosene fuel. Six full cone pressure swirl nozzles N1-N6 (fuel flow rate, m(f) = 1.7-7.08 kg/h at P-inj = 9 bar) are considered for experiments with fuel injection pressure varying from 4-9 bar. The Sauter mean diameter (SMD) has been observed to decrease (58-34 mu m) with an increase in fuel injection pressure (4-9 bar). The flame liftoff height of the spray flame increases with an increase in the fuel injection pressure, even though the spray SMD decreases. To understand the role of the various important spray parameters on stabilization of lifted spray flames, particle image velocimetry measurements have been carried out near the flame stabilization zone of the spray. It has been observed that a zone of high velocity is formed near the nozzle exit. The size of this zone varies with injection pressure and this has been attributed to increased fluctuations in a lifted spray flame along with the increased momentum of the droplets emanating from the spray at higher injection pressures.

This paper reports the measurement of laminar burning velocities of DME (dimethyl ether)-air mixtures at higher mixture temperatures using planar flames stabilized in a controlled temperature mesoscale diverging channel. The reliability of this technique for flame speed measurement at high mixture temperatures has been established through various numerical studies using detailed 3-D computational model for DME-air mixtures. The distribution of fuel -air mass flux, reaction zone, and flame shape indicate that a planar flame is indeed formed in both transverse and depth directions of the channel. The stabilized flame is independent of any stretch effects except mild hydrodynamic strain due to channel divergence (30-50 s(-1)), and measured values of laminar burning velocities are within 5% of the actual value. The experiments were carried out at various equivalence ratios (phi = 0.7-1.4) and elevated mixture temperatures (350-640 K). The burning velocities and temperature exponents were determined using the planar flames stabilized at different mixture inlet velocities and temperatures. Slightly rich mixtures (phi = 1.1) point to the maximum burning velocity, in good agreement with recent literature at ambient conditions. Temperature exponents for different equivalence ratios increase to both sides of 4) = 1.1. Numerically calculated laminar burning velocities with different chemical kinetic schemes compared well with the measured burning velocities at higher mixture temperatures. (C) 2017 Elsevier Ltd. All rights reserved.

Src is a nonreceptor tyrosine kinase involved in the cross-talk and mediation of many signaling pathways that promote cell proliferation, adhesion, invasion, migration, and tumorigenesis. Increased Src activity has been reported in many types of human cancer, including gastric cancer. Therefore, this factor has been identified as a promising therapeutic target for cancer treatments, and targeting Src in gastric cancer is predicted to have potent effects. We evaluated the antitumor effect of a c-Src/Abl kinase inhibitor, saracatinib (AZD0530), alone or combined with chemotherapeutic agents in gastric cancer cell lines and a NCI-N87 xenograft model. Among 10 gastric cancer cell lines, saracatinib specifically inhibited the growth and migration/invasion of SNU216 and NCI-N87 cells. Saracatinib blocked the Src/FAK, HER family, and oncogenic signaling pathways, and it induced G1 arrest and apoptosis in SNU216 and NCI-N87 cells. Apoptosis required induction of the proapoptotic BCL2 family member Bim. Knockdown of Bim using siRNA decreased apoptosis induced by treatment with saracatinib, suggesting that Bim has an important role in saracatinib-induced apoptosis. Saracatinib enhanced the effects of lapatinib, an EGFR/HER2 dual inhibitor, in SNU216 and NCI-N87 cells. Furthermore, combined treatment with saracatinib and 5-fluorouracil (5-FU) or cisplatin exerted synergistic effects in both saracatinib-sensitive and saracatinib-resistant cells. Consistent with our in vitro findings, cotreatment with saracatinib and 5-FU resulted in enhanced antitumor activity in the NCI-N87 xenografts. These data indicate that the inhibition of Src kinase activity by saracatinib alone or in combination with other agents can be a strategy to target gastric cancer. Mol Cancer Ther; 12(1); 16–26. ©2013 AACR.

Background Cetuximab has shown clinical benefit in patients with metastatic colorectal cancer (mCRC) harboring wild-type RAS . Human epidermal growth factor receptor 2 ( HER2 ) amplification may be a mechanism of cetuximab resistance. We evaluated the association between HER2 amplification and cetuximab efficacy in patients with mCRC harboring wild-type RAS and BRAF . Patients and Methods Between December 2003 and June 2013, we identified 142 patients with mCRC whose tumors harbored both wild-type exons 2, 3, and 4 in KRAS and NRAS , and wild-type exon 15 in BRAF using high throughput sequencing (OncoMap version 4.0). All patients received cetuximab after oxaliplatin, irinotecan, and fluoropyrimidine failure. HER2 status was determined using immunohistochemistry and silver in situ hybridization (SISH) and correlated with cetuximab efficacy. Results Of 142 RAS and BRAF wild-type tumors, we observed 7 cases (4.9%) of HER2 amplification by SISH. After a median follow-up of 13.2 months (range, 1.4-78.1 months), median progression-free survival (PFS) was significantly different according to HER2 status: 3.1 months in patients with HER2 amplification compared with 5.6 months in those with non-amplified HER2 (hazard ratio, 2.73; 95% confidence interval, 1.18-6.31; P = .019). Overall survival (OS) was not significantly different between groups, although there was a tendency towards shorter OS in patients with HER2 -amplified tumors (hazard ratio, 1.31; 95% confidence interval, 0.61-2.82; 10.1 vs. 13.5 months; P = .488). Conclusions HER2 amplification is predictive of shorter PFS after cetuximab treatment in patients with mCRC harboring wild-type RAS and BRAF . Further study is warranted for this patient population.

Epiregulin (EREG) induces cell growth by binding to the epidermal growth factor receptor (EGFR). Expression of EREG affects sensitivity to cetuximab a chimeric monoclonal antibody that inhibits the EGFR signaling pathway. The mechanism through which EREG is regulated is largely unknown, but a methyl-array study previously performed by our group revealed that EREG is methylated in gastric cancer cells. In this study, we found that EREG gene expression was low in 7 out of 11 gastric cancer cells and this downregulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated CpG sites in the EREG promoter. Compared with DNA methyltransferase 1 (DNMT1), knock-down of DNA methyltransferase 3b (DNMT3b) significantly increased the expression of EREG and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates CpG methylation and silencing of the EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues we evaluated. In addition to DNA methylation, results from a chromatin immunoprecipitation assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone marks (H3K4me3 and AcH3) and of a repressive mark (H3K27me2). Treatment with 5-aza-CdR dynamically increased the low occupancy of H3K4me3 and AcH3, while decreasing the high enrichment of H3K27me2, indicating that dynamic histone modifications contribute to EREG regulation in addition to DNA methylation. Finally, the combination of 5-aza-CdR and cetuximab exerted a synergistic anti-proliferative effect on gastric cancer cells. Taken together, the results of our study showed for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification.

Previously, we found that KRAS mutant cancer cells showed variable response to AZD6244, a MEK inhibitor through differential activation of EGFR/AKT. To investigate its mechanism, we performed cDNA microarray using four KRAS mutant cancer cells. We found that treatment with AZD6244 reduced the expression of mitogen-inducible gene 6 (MIG6), a negative feedback regulator for EGFR, in AZD6244-resistant cells, while activity of EGFR and AKT was increased in these cells. Reconstitution or knockdown of MIG6 expression affected cancer cell responses to AZD6244. Treatment with a combination of EGFR inhibitor and AZD6244 inhibited cell proliferation synergistically without activation of AKT in AZD6244-resistant cells. Our study provides a mechanism of differential response to MEK inhibition in KRAS mutant cancer.

EGFR tyrosine kinase inhibitors have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, tyrosine kinase inhibitors have also proven ineffective against most tumors with EGFR wild-type (WT) alleles. Although some genetic changes, including the KRAS mutation, have been shown to confer resistance to tyrosine kinase inhibitors, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. The principal objective of this study was to improve our current understanding of drug interactions between EGFR and MAP/ERK kinase (MEK) inhibitors in an effort to gain insight into a novel therapeutic strategy against EGFR WT tumors. Using a panel of human EGFR WT gastric cancer cell lines, we showed that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phosphoprotein arrays showed that, in MEK inhibitor resistant cell lines, AKT was activated through the EGFR/HER3/PI3K pathway following AZD6244 (ARRY-142886) treatment. Blockade of this feedback mechanism through the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines in vitro and in vivo. Our results provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. [Mol Cancer Ther 2009;8(9):2526–36]

HER2 overexpression is observed in 5-25% of gastric cancers. Lapatinib is a dual inhibitor of the epidermal growth factor receptor and HER2 tyrosine kinase. We examined the antitumor effect of lapatinib in gastric cancer cell lines. Lapatinib induced selective and potent growth inhibition in two HER2-amplified gastric cancer cell lines (SNU-216 and NCI-N87). Lapatinib inhibited the phosphorylation of HER2, EGFR and downstream signaling proteins, resulting in G1 arrest in both cell lines with down-regulation of cMyc and induction of p27kip1. Lapatinib also induced apoptosis in NCI-N87 which has high HER2 amplification ratio. Lapatinib combined with 5-fluorouracil, cisplatin, oxaliplatin or paclitaxel showed an additive or synergistic effect. These results provide a rationale for the future clinical trials of lapatinib combined with cytotoxic drugs in the treatment of HER2-positive gastric cancer.

HM781-36B is an orally administered pan-human epidermal growth factor receptor (HER) inhibitor. To explore the role of pan-HER inhibitor in breast cancer, we investigated the antitumor effect and mechanisms of HM781-36B in breast cancer cell lines. Six breast cancer cell lines (BT474, MDA-MB-453, SK-BR-3, T47D, MCF-7, and MDA-MB-231) were tested. The growth inhibitory effect was assessed using the tetrazolium bromide [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide] assay. The cell cycle at various concentrations of HM781-36B was analyzed by flow cytometry, and analysis of downstream molecules was performed by western blot analysis. Interaction of HM781-36B with cytotoxic chemotherapeutic agents was analyzed by combination index using CalcuSyn. The HER2-amplified cells (SK-BR-3, BT474, and MDA-MB-453) were sensitive to HM781-36B (IC50=0.001 μmol/l, 0.0012 μmol/l, and 0.0095 μmol/l, respectively). HM781-36B induced G1 arrest and resulted in apoptosis. It reduced the level of p-HER2, p-AKT, p-ERK, and p-STAT3. HM781-36B combined with 5-fluorouracil, cisplatin, paclitaxel, or gemcitabine showed a synergistic inhibitory effect on the HER2-amplified and on some of the HER2-nonamplified breast cancer cells. HM781-36B could be a promising treatment for HER2-amplified breast cancer as a single agent or in combination with cytotoxic agents and can be a candidate for treatment of HER2-nonamplified breast cancer in combination with cytotoxic agents.

An increase in intraocular pressure after intravitreal steroid injection facilitates reduction of macular edema

We evaluated the effects of sunitinib monotherapy and in combination with cisplatin in human gastric cancer cell lines. Sunitinib showed antiproliferative effect in gastric cancer cells line with high PDGFRA expression. Knockdown of PDGFRA showed that sunitinib sensitivity was correlated with the basal expression of PDGFRA. Synergistic growth inhibitory activity in combination with cisplatin was identified. We further explored how sunitinib potentiated the activity of cisplatin. We found that sunitinib treatment resulted in the down-regulation of ERCC1 expression via the modulation of PDGFRA expression in gastric cancer cells. The effect was verified via SNU484 xenograft model. Our data support the rationale of clinical trial using sunitinib in combination of cisplatin in gastric cancer.

Trastuzumab, a HER2 directed treatment has shown clinical benefit in HER2 amplified gastric cancer. This study demonstrated the potent antitumor activity of HM781-36B, a quinazoline-based irreversible pan-HER inhibitor, in HER2 amplified gastric cancer cells (SNU216 and N87) in vitro and in vivo. HM781-36B inhibited phosphorylation of HER family and downstream signaling molecules, and induced apoptosis and G1 arrest. Furthermore, HM781-36B exerted synergistic effects with chemotherapeutic agents in both HER2 amplified and HER2 non-amplified gastric cancer cells. Therefore, HM781-36B may be useful for the treatment of HER2 amplified gastric cancer alone or in combination with chemotherapeutic agents.

KRAS is frequently mutated in nonsmall cell lung cancer (NSCLC), resulting in the activation of the MAPK/ERK kinase (MEK)/ERK pathway. High-throughput mutation profile has shown that lung cancer frequently harbors comutation of cancer-related genes. Therefore, given that cancer cells have multiple genetic alterations, combinatorial therapeutic strategy is demanded for effective cancer therapy. To address this, we first characterized MEK dependence in four NSCLC cells. Two cells (H358, A549) carried KRAS mutation only, and the other two (H23, H157) harbored comutation of KRAS/PTEN. H358 cells with KRAS mutation only were sensitive to MEK inhibition. However, the other KRAS mutant A549 cells were resistant to MEK inhibition. Previously, we have shown that dual inhibition of EGFR and MEK signaling shows a synergistic effect on KRAS mutant gastric cancer cells by suppressing compensatory activation of AKT. Here we also observed that this combination was effective in KRAS mutant A549 cells. However, the combination was ineffective in H23 and 157 cells with comutation of KRAS/PTEN. Compared to KRAS mutant/PTEN wild-type cells, signal transducer and activator of transcription 3 (STAT3) was significantly activated following MEK inhibition in KRAS/PTEN comutant cells. Combined STAT3 inhibition by a JAK2 inhibitor or gene knockdown with MEK inhibition blocked STAT3 activation, synergistically suppressed cell growth, and induced apoptosis in comutant cells. Taken together, our study provides molecular insights that help explain the heterogeneous response to MEK inhibition in KRAS mutant lung cancers, and presents a rationale for the clinical investigation of combination of MEK and EGFR inhibitor or MEK and JAK2 inhibitor depending on PTEN status.

We evaluated RAD001, an inhibitor of the mammalian target of rapamycin (mTOR) in human gastric cancer cell lines and determined the molecular mechanisms. RAD001 has marked growth inhibitory activity against the SNU-1 and SNU-216 cells. It inhibited phosphorylation of mTOR and S6 K, and induced G1 cell cycle arrest. Synergistic growth-inhibitory effects in combination with 5-fluorouracil (5-FU) was identified. Furthermore, RAD001 conferred sensitivity to 5-FU-resistant cell lines by downregulating thymidylate synthase (TS). In conclusion, RAD001 showed growth inhibitory activity against gastric cancer cells and acted synergistically with cytotoxic agents such as 5-FU by downregulating TS.

To compare the effectiveness and safety of 25- and 23-gauge sutureless microincision vitrectomy surgery (MIVS) in the management of various vitreoretinal diseases.Eighty-five consecutive patients undergoing sutureless vitrectomy during January to April 2008 were randomized to either 25- or 23-gauge MIVS. Data collected prospectively included best-corrected visual acuity (BCVA), operation time, postoperative visual recovery, postoperative anterior segment change, and complications.The most common indications for MIVS were macular hole, macular pucker, vitreous haemorrhage, and diabetic traction retinal detachment. Mean operation times of the 25-gauge (n=38) and 23-gauge groups (n=47) were 33.68 and 34.47 min, respectively (P=0.942). Mean BCVA improved significantly in both the 25- and 23-gauge groups when measured 3-month postoperatively. There was no between-group difference in either the degree or the rate of postoperative visual recovery. Seven patients in the 23-gauge group, compared with three in the 25-gauge group, required suturing of sclerotomy at the end of the surgery. No patients in either group developed postoperative wound leakage or endophthalmitis.Our prospective study suggests that, within the limited indications, both 25- and 23-gauge MIVS are equally effective, with similar safety profiles. Gauge selection thus may be made according to a surgeon's preference and the availability of appropriate instruments.

Background: Src kinases have been involved in cell proliferation, invasion, and metastasis and is one of the highly expressed oncogenes in breast cancer. Therefore, it could be a reasonable target for therapeutic strategy. Although several Src inhibitors, targeting the ATP binding site, were developed, none of them have shown remarkable responses as monotherapeutic agents in breast cancer clinical trials. KX-01 (KX2-391) is a novel peptidomimetic agent, and a non ATP-competitive Src inhibitor, which has potent efficacy for inhibiting both Src as well as tubulin polymerization. Its dual inhibition effects could potentially overcome the limitations of the Src inhibitors that have been evaluated previously. Materials & Methods: We determined the anti-tumor effects of KX-01 on breast cancer cell lines using a cytotoxicity assay, cell cycle analysis, a wound healing assay and western blotting following KX-01 treatment. Results: KX-01 effectively inhibited cell growth in most breast cancer cells including ER/PR/HER2 negative breast cancer cells. Short exposure of KX-01 down-regulated the expression level of Src phosphorylation. Phosphorylated FAK, ERK, AKT and STAT3 were also down-regulated by KX-01 treatment in MDA-MB-231, MDA-MB-468 and BT-549 cells. By wound healing assay, we confirmed migration inhibitory effect of KX-01 in BT-549 cells. Increase of G2/M cell cycle arrest was observed in dose dependent manner. In addition, mitotic catastrophes were also observed in KX-01 sensitive cell lines. Consistent with induction of mitotic catastrophe, the increased levels of aneuploidy in sensitive cell lines were examined following the treatment of KX-01. Conclusion: Inhibition of cell growth and migration, as well as an induction of mitotic catastrophy, were observed in triple negative breast cancer cell lines treated with KX-01. Our data strongly supports utilizing KX-01 as a new therapeutic agents for treating triple negative breast cancer. Citation Format: Seongyeong Kim, Seock-Ah Im, Ahrum Min, Miso Lee, Heymin Jang, Kyung-Hun Lee, Hee-Jun Kim, Tae-Yong Kim, Sae-Won Han, Do-Youn Oh, Tae-You Kim, Young-Kwang Yoon, David Hangauer, Johnson Lau, Yung-Jue Bang. Antitumor effect of KX-01, a novel Src and tubulin inhibitor, in triple negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5479. doi:10.1158/1538-7445.AM2014-5479

Background: The poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, has been found to have a therapeutic potential for treating cancers that have an impaired DNA repair ability. RAD51C has been reported to play an essential role in DNA repair mediated by homologous recombination. In addition, RAD51C is a gene that affects human cancer susceptibility, similar to BRCA1 and BRCA2. RAD51C-defective cancers can therefore be potentially treated with olaparib because DNA damage induced by olaparib cannot be effectively repaired by HR since RAD51C deficiency interferes with RAD51-mediated HR. Materials and Methods: We studied the growth inhibitory effects of olaparib on human cancer cell lines using clonogenic survival assays. Cell cycle analysis and molecular changed induced by olaparib were also performed. DNA methylation status in cancer cells and tumor tissue samples was also determined by using bisulfate sequencing. Results: Olaparib is effective to RAD51C-defective cells. Olaparib leads to the accumulation of unrepaired DSBs due to dysfunctional RAD51 foci formation along with increased caspase 3-dependent cell death and G2/M arrest in RAD51C-defective cells. Moreover, the growth of RAD51C-deficient SNU601 tumor cells in a xenograft model decreased significantly following treatment with olaparib. Furthermore, RAD51C expression was significantly decreased in cancer and the lack of RAD51C was attributed to DNA methylation and histone modification. Our findings showed that the loss of RAD51C expression due to epigenetic silencing leads to olaparib sensitivity in cancer cells. Conclusion: The loss of RAD51C expression was frequently associated with human cancers. We used a novel synthetic lethal approach using PARP inhibitors and observed that RAD51C was a participant in the DNA repair pathway. Our findings have potential clinical application for treating cancers with RAD51C deficiencies. Furthermore, we determined that RAD51C may serve as a novel biomarker for identifying olaparib-sensitive patients, thereby allowing physicians to select the most effective modalities for treating these individuals. Citation Format: Ahrum Min, Seongyeong Kim, Seock-Ah Im, Young-Kwang Yoon, Sang-Hyun Song, Hyun-Jin Nam, Hyung-Seok Hur, Kyung-Hun Lee, Sae-Won Han, Do-Youn Oh, Tae-You Kim, Mark J. O`Connor, Woo-Ho Kim, Yung-Jue Bang. Olaparib increases antitumor effects on epigenetically RAD51C-deficient human cancer cells. [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 3442. doi:10.1158/1538-7445.AM2013-3442

Cancer is a result of complex genetic alterations including mutations, copy number alterations, and gene fusions. Recent advance in sequencing technology enables comprehensive genome-wide study of the complex genetic alterations. Better understanding of the genetic alteration of cancer will ultimately lead to better treatment options and improved survival of the patients. We have established a set of cancer-related genes comprised of 184 genes for targeted sequencing using next generation sequencing technology. Targeted genome sequencing of the coding regions in the gene set was performed using the Illumina Genome Analyzer IIx. We analyzed the gene set in 13 gastric cancer cell lines. Average coverage depth was 140X. A total of 237 somatic sequence variations were found in 111 genes. The frequently mutated genes were ATM in 7, MSH6 in 6, TP53 in 5, and KRAS in 4 gastic cancer cells. Using the coverage data, we could also analyze copy number variations. Copy number gain (>2.0 fold) was found in 5 genes, whereas copy number loss ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5096. doi:1538-7445.AM2012-5096

Background: Src is a nonreceptor tyrosine kinase involved in the crosstalk and mediation of many signaling pathways that promote cell proliferation, adhesion, invasion, migration, metastasis, angiogenesis, and tumorigenesis. Elevation of Src activity has been reported in many types of human cancers. Therefore, Src has been promising therapeutic target for the treatment of cancers. However, the mechanism of Src inhibition in gastric cancers has not been fully understood yet. Saracatinib is an orally active small molecule c-Src/abl kinase inhibitor currently in phase II clinical trials. The purpose of this study is to evaluate the therapeutic potential of saracatinib alone or in combination with chemotherapeutic agents for the treatment of gastric cancers by testing its in vitro and in vivo efficacy. Methods: Experiments were conducted in human gastric cancer cell lines from the Korea Cell Line Bank or the American Type Culture Collection. MTT assay was used for determining growth inhibitory effect of saracatinib alone or in combination with chemotherapeutic agents (5-FU, cisplatin). Cell cycle distribution was analyzed by FACS Calibur flow cytometer and apoptotic effect was measured by annexin V assay. Western blot analysis was performed to verify molecular mechanism of this compound. The methods described by Chou and Talalay were used to determine if a synergistic effect existed. The analysis of the median effect was conducted using the Calcusyn software to determine a combination index value. (CI>1: antagonistic effect, CI=1:additive effect, CI Results: Saracatinib specifically inhibited the growth of SNU216 and NCI N87 cells among 10 gastric cancer cell lines. We found that saracatinib induces G1 arrest and apoptosis in sensitive cell lines. The apoptotic process required induction of the proapoptotic BH3-only BCL2 family member, BIM, and saracatinib led to increase in the BIM level in SNU216 and N87 cells. Knockdown of BIM using siRNA decreased the apoptotic cell numbers induced by treatment of saracatinib suggesting important role for BIM in saracatinib induced apoptosis pathway. The sensitivity to saracatinib in SNU216 and N87 cell lines was correlated with inhibiton of AKT and ERK pathways and blockade of HER family kinases. Furthermore, the combined treatment of saracatinib with 5-FU or cisplatin exerted synergistic effects in gastric cancer cell lines including NCI N87 cells. Consistent with these in vitro findings, saracatinib alone or in combination with 5-FU showed antitumor activity in a NCI-N87 xenograft model. Conclusion: Taken together, these preclinical evaluations support that Src is a potential therapeutic target in gastric cancer and clinical development of saracatinib alone or in combination with chemotherapy in gastric cancer could be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3588. doi:10.1158/1538-7445.AM2011-3588

Insulin-like Growth factor I receptor (IGF-IR) is highly expressed in many human cancers and plays a major role in proliferation, survival, and metastasis. Accordingly, abundant IGF-1R targeting drugs, especially monoclonal antibody drugs, have been focused and developed due to their safety and efficacy. In order to define biomarkers correlating with the drug sensitivity, we screened 10 gastric and 10 hepatocellular cancer cell lines, for in vitro response to CP-751,871, anti-insulin like growth factor type 1 receptor antibody. Our results show that CP-751,871 inhibits IGF-1 ligand-dependent IGF-1R phosphorylation and downstream signaling of the two major IGF-1R pathways, mitogen-activated protein kinase and phosphatidylinositol 3′-kinase/AKt. Moreover, long-term exposure to CP-751,871 also caused to decrease phospho-stat 3 as well as phospho-Akt level and induced G1 phase cell cycle arrest in dose-dependent manner preferentially in the sensitive cells. Surprisingly, among these 20 cell lines, cells with IGF-IR and insulin receptor (IR) were significantly inhibited by CP-751,871 compared to cells with IGF-IR or IR overexpression only. Considering their highly homologous structure, we thereby determined if the effect of CP-751,871 had relevance to the level of IR/IGF1R hybrid receptors through co-immunoprecipitation with either CP-751,871 or IGF1R antibody. Its immunoblot analysis revealed that not only did sensitive cell lines, such as SNU719, SNU368, and HepG2, show relatively higher levels of IR/IGF1R hybrid, but also superior ability of CP-751,871 to recognize their IR/IGF1R heterodimer complexes. Furthermore, we could observe that the hybrid forms were recruited by IGF1 ligands but this IGF-I ligand dependent formation was blockaded by CP-751,871. Lastly, the treatment of these sensitive cell lines with CP-751,871 caused dimer dissociation. Therefore, our data suggest that the antiproliferative effect by CP-751,871 requires the co-expression of IGF-1R and IR and may be related with the expression level of hybrid Insulin/IGF-1 receptors in gastric and hepatocellular carcinoma cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2427.

Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Olaparib (KU-0059436; AZD2281) is a potent oral inhibitor of PARP currently in clinical development, which has selective antitumor activity in cancers, associated with BRCA1 and BRCA2 mutations. Gastric cancer is the most common cancer and the second leading cause of cancer deaths in Korea. New therapies including molecular targeting agents are eagerly awaited, and treatment strategies to overcome chemoresistance are also needed in the treatment of gastric cancer. Materials and Methods We studied the growth inhibitory effects of Olaparib on gastric cancer (GC) cells using clonogenic survival assays and studied the effect of combination with chemotherapeutic agents using MTS assays. Cell cycle analysis and molecular changes induced by olaparib were also performed. Results Olaparib showed marked growth inhibitory activity against the SNU 601 cell line, with IC50 value of 0.015 μM. In addition, 5 out of 11 GC cells (45%) were sensitive to olaparib with IC50s ≤ 2μM. Olaprib induced G2/M cell cycle arrest and apoptosis in sensitive GC cells. We identified synergistic growth inhibitory effects of Olaparib in combination with clinically relevant cytotoxic agents (5-FU, cisplatin, and oxaliplatin). Furthermore, Olaparib-induced apoptosis was associated with PARP cleavage and caspase-3 activation. Conclusion: Olaparib showed growth inhibitory activity against GC cells as a single agent and acted synergistically with cytotoxic agents. These results provide a rationale for the future clinical trials of olaparib combined with cytotoxic drugs in the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1775.

Background: HM781-36B is a novel quinazoline-based irreversible pan-HER inhibitor. Although the effects of many other pan-HER inhibitors (CI-1033, PF00299804, BMS-599626, BMS-690514 and JNJ-28871063) have been studied in various types of cancer, data regarding the use of pan-HER inhibitors to treat gastric cancers are limited. In this study, we evaluated the therapeutic potential of HM781-36B for the treatment of gastric cancers by testing its in vitro and in vivo efficacy. In addition, we further characterized its activity when administered in combination with chemotherapeutic agents. Methods: Experiments were conducted in gastric cancer cell lines. Enzyme activity assay was conducted to determine the IC50 values of HM781-36B for kinase inhibition and MTT assay was used for determining growth inhibitory effect. Cell cycle distribution was analyzed by flow cytometry and apoptotic effect was measured by annexin V assay. And N87 xenograft model was used to see tumor regression effect. Western blot analysis was used to verify mechanism of this compound. Results: We found that HM781-36B suppressed the proliferation of HER2-amplified gastric cancer cell lines (SNU216 and N87) potently when compared to the effects of other HER family tyrosine kinase inhibitors (gefitinib, lapatinib, BIBW-2992 and CI-1033). Furthermore, we found that HM781-36B inhibited the phosphorylation of HER family and their downstream signaling proteins, which resulted in apoptosis through the caspase-mediated mitochondrial pathway and G1 arrest. The apoptotic process requires the induction of the proapoptotic BH3-only BCL2 family member, BIM, and HM781-36B led to a dramatic increase in the BIM level in SNU216 and N87 cells. Consistent with these in vitro findings, HM781-36B showed exceptional anti-tumor activity in a xenograft model of N87 cells. Furthermore, HM781-36B exerted synergistic effects when administered in combination with 5-fluorouracil or cisplatin in both HER2 amplified cells and HER2 non-amplified gastric cancer cells. Conclusion: Taken together, these data suggest that HM781-36B may be useful as a targeted therapy for the treatment of HER2 amplified gastric cancer as well as a chemosensitizer of cytotoxic chemotherapeutic agents used to treat gastric cancer, even in HER2 non-amplified gastric cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B260.

KRAS is frequently mutated in non-small cell lung cancer (NSCLC), resulting in the activation of the MEK/ERK pathway. High-throughput mutation profile has shown that lung cancer frequently harbors co-mutation of cancer related genes. Therefore, given that cancer cells have multiple genetic alterations, combinatorial therapeutic strategy is demanded for effective cancer therapy. To address this, we first characterized MEK dependence in four NSCLC cells. Two cells (H358, A549) carried KRAS mutation only, and the other two (H23, H157) harbored co-mutation of KRAS/PTEN. H358 cells with KRAS mutation only were sensitive to MEK inhibition. However, the other KRAS mutant A549 cells were resistant to MEK inhibition. Previously, we have shown that dual inhibition of EGFR and MEK signaling shows a synergistic effect on KRAS mutant gastric cancer cells by suppressing compensatory activation of AKT. Here we also observed that this combination was effective in KRAS mutant A549 cells. However, the combination was ineffective in H23 and 157 cells with co-mutation of KRAS/PTEN. Compared to KRAS mutant/PTEN wild-type cells, STAT3 was significantly activated following MEK inhibition in KRAS/PTEN co-mutant cells. Combined STAT3 inhibition by a JAK2 inhibitor or gene knockdown with MEK inhibition blocked STAT3 activation, synergistically suppressed cell growth, and induced apoptosis in co-mutant cells. Taken together, our study provides molecular insights that help explain the heterogeneous response to MEK inhibition in KRAS mutant lung cancers, and presents a rationale for the clinical investigation of combination of MEK and EGFR inhibitor or MEK and STAT3 inhibitor depending on PTEN status. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A252.