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Background/aim LY2835219 (LY), a novel CDK4/6 inhibitor, prevents cell proliferation through G1 arrest. Docetaxel (DTX) and paclitaxel (PTX) are cytotoxic drugs targeting tubulin-mediated apoptotic cell death via G2/M arrest. We evaluated the antitumor effects of DTX/PTX and LY individually and in combination in lung adenocarcinoma cells with or without KRAS mutations and xenograft mice harboring KRAS mutations. Materials and methods We investigated in vitro/in vivo changes in signaling molecules and analyzed cell proliferation, cycle, and apoptosis via flow cytometry and western blotting. Results LY cytotoxicity was dose-dependent and varied with KRAS mutation status. DTX→LY showed synergistic cytotoxicity regardless of KRAS mutation. Furthermore, the synergistic effect of PTX→LY was significantly greater than that of PTX+LY. DTX→LY remarkably reduced the number of G0/G1 cells and increased the number of G2/M arrested cells, resulting in an increase in apoptosis and subG1 cells. Conclusion DTX→LY has synergistic antitumor effect in lung cancer cells and xenograft mice regardless of KRAS mutation.

Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (mEGFR) well respond to EGFR TKIs, but the majority of patients eventually acquire drug resistance, leading to treatment failure. Recently, intracellular potential autophagy in addition to primary genetic alterations implicates the acquired drug resistance to targeted drugs in various carcinomas such as melanoma, pancreas, and breast cancer. We aimed to identify autophagy flux-related signal molecules which may be closely linked to acquired resistance in mEGFR NSCLC. Methods: We constructed the TMA composed of 579 surgically resected tumor tissues from lung adenocarcinoma patients and evaluated protein expression of autophagy flux-related signal molecules including CAGE, p-Beclin1, LC3B, p-AMPK, ULK1, p62, and ATG5 using immunohistochemistry (IHC). We established H1975OSI (166-folds) and PC9ERL (3,650-folds) resistant sub cell lines through the prolonged exposure of Osimertinib and Erlotinib to H1975 (L858R/T790M) and PC9 (Del19) cells, respectively. We also explored genes that induce autophagy flux activity using mRNA seq and qRT-PCR. Results: H-score of CAGE ( 95.6±7.9, 70±5.5), p-AMPK (73.4±7.8, 32±5.6) and p-Beclin1 (44.4±16.1, 19.7±5.5), ULK1( 57.4±4.9, 41.9±4.9) were significantly higher in mEGFR, compared to those of wild type EGFR NSCLC (P Conclusion: Taken together, our results strongly support the evidence that autophagy flux-related signal molecules as well as known genetic alterations are closely connected to the acquired resistance during period of EGFR TKI treatment in mEGFR NSCLC patients. Now, we are undergoing additional RNA profiling and function study of identified novel autophagy-related genes to examine a possibility as potential target to overcome EGFR TKI resistance. Citation Format: Jeong-Oh Kim, Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Seoree Kim, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Kyoungjune Lee, Youn Soo Lee, Jin-Hyoung Kang. The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2232.

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is the standard of care as 1st-line treatment for NSCLC patients with activating EGFR mutations (exon 19 deletion or L858R mutation). However, EGFR-TKI therapy eventually leads to acquired drug resistance approximately 1-2 years from the beginning of treatment. Multiple acquired resistance mechanisms including on-target EGFR mutations, phenotype transformation, and activation of bypass signaling molecules have been identified, but over 35% still remains unknown. CAGE, a cancer/testis antigen, was originally isolated from the sera of patients with gastric cancer and is known to regulate the autophagic flux in acquired resistance to EGFR TKI in NSCLC. Herein, the expression of CAGE was found in more than 50% of NSCLC patients using immunohistochemistry (IHC). Interestingly, the expression level was increased especially for the patients acquiring resistance to EGFR-TKIs. In addition, both PC-9/ER (Erlotinib-resistance cells) and H1975/OR (Osimertinib-resistant cells) cells demonstrated increased CAGE expression and autophagic flux, when compared with their parental cells. Thus, we developed the peptide drugs targeting the DEAD box domain of CAGE. This novel peptide effectively inhibited tumor cell proliferation in EGFR-TKI resistant cell line. Moreover, in Erlotinib-resistant patient-derived xenograft (PDX) mice, the peptide drug retarded tumor growth and enhanced anti-tumor efficacy. Collectively, our data suggest that CAGE plays a crucial role in the tumor progression and acquired resistance of EGFR TKI by the modulation of autophagic flux. Furthermore, we provided evidence that the inhibition of autophagy through CAGE- derived peptide could overcome the acquired resistance to EGFR TKIs in advanced/metastatic NSCLC patients. Citation Format: Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Jaemoon Koh, Dooil Jeoung, Jin Hyoung Kang. Inhibition of autophagy by CAGE targeting peptide retard tumor growth in non-small cell lung cancer of EGFR TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1582.

BackgroundHypoxic tumors are known to be highly resistant to radiotherapy and cause poor prognosis in non-small cell lung cancer (NSCLC) patients. CKD-516, a novel vascular disrupting agent (VDA), mainly affects blood vessels in the central area of the tumor and blocks tubulin polymerization, thereby destroying the aberrant tumor vasculature with a rapid decrease in blood, resulting in rapid tumor cell death. Therefore, we evaluated the anti-tumor efficacy of CKD-516 in combination with irradiation (IR) and examined tumor necrosis, delayed tumor growth, and expression of proteins involved in hypoxia and angiogenesis in this study.MethodsA xenograft mouse model of lung squamous cell carcinoma was established, and the tumor was exposed to IR 5 days per week. CKD-516 was administered with two treatment schedules (day 1 or days 1 and 5) 1 h after IR. After treatment, tumor tissues were stained with hematoxylin and eosin, and pimonidazole. HIF-1α, Glut-1, VEGF, CD31, and Ki-67 expression levels were evaluated using immunohistochemical staining.ResultsShort-term treatment with IR alone and CKD-516 + IR (d1) significantly reduced tumor volume (p = 0.006 andp = 0.048, respectively). Treatment with CKD-516 + IR (d1 and d1, 5) resulted in a marked reduction in the number of blood vessels (p p = 0.02) and decreased HIF-1α, Glut-1, VEGF, and Ki-67 expression. Long-term administration of CKD-516 + IR reduced tumor volume and delayed tumor growth. This combination also greatly reduced the number of blood vessels (p = 0.0006) and significantly enhanced tumor necrosis (p = 0.004). CKD-516 + IR significantly increased HIF-1α expression (p = 0.0047), but significantly reduced VEGF expression (p = 0.0046).ConclusionsTaken together, our data show that when used in combination, CKD-516 and IR can significantly enhance anti-tumor efficacy compared to monotherapy in lung cancer xenograft mice.

Epidermal growth factor receptor (EGFR) mutation testing is essential for individualized treatment using tyrosine kinase inhibitors. We evaluated two EGFR mutation tests, cobas v2 and PANAMutyper, for detection of EGFR activating mutations Ex19del, L858R, and T790M in tumor tissue and plasma from 244 non-small cell lung cancer (NSCLC) patients. The Kappa coefficient (95% CI) between the tests was 0.82 (0.74−0.92) in tumor samples (suggesting almost perfect agreement) and 0.69 (0.54−0.84) in plasma (suggesting substantial agreement). In plasma samples, both tests showed low to moderate sensitivity depending on disease stage but high diagnostic precision (86%−100%) in all disease stages (sensitivity: percentage of mutations in tumors that are also detected in plasma; precision: percentage of mutations in plasma which are also detected in tumors). Among the 244 patients, those previously diagnosed as T790M carriers who received osimertinib treatment showed dramatically better clinical outcomes than T790M carriers without osimertinib treatment. Taken together, our study supports interchangeable use of cobas v2 and PANAMutyper in tumor and plasma EGFR testing. Both tests have high diagnostic precision in plasma but are particularly valuable in late-stage disease. Our clinical data in T790M carriers strongly support the clinical benefits of osimertinib treatment guided by both EGFR mutation tests.

Additional file 2. Toxicity of low- or high-dose IR in BALB/c nude mice. (A) The shielding device was made of 4 mm thick lead. The anesthetized mice were fixed in a 50 mL tube and irradiated with a lead shield. (B) Tumor growth and (C) body weight according to radiation dose. (D) Tumor growth during long-term treatment with 4 Gy of IR.

Additional file 1. Short-term and long-term drug administration schedules.

Additional file 3. IHC staining with HIF-1α, VEGF, Glut-1, and Ki-67 antibodies in all five treatment groups (72 h after the end of treatment). Mice were divided into five groups according to the dosing and treatment schedule: vehicle (PBS), IR alone (4 Gy/day), CKD-516 alone (3 mg/kg), and CKD-516 (3 mg/kg, day 1 or days 1 and 5) combined with IR.Scale bar: 200 μm. Magnification: × 100.

Background We screened resected tumor tissues from patients with lung cancer for EGFR mutations, ALK rearrangements, and rearranged during transfection (RET) gene variants (including RET rearrangements and the Kinesin Family Member 5B (KIF5B)-RET fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome. Method We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using RET break-apart and KIF5B-RET SY translocation probes) and a commercial RET transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining. Results Among the 581 specimens, 51 (8.8%) specimens harbored RET rearrangements, including 12 cases (2.1%) carrying a KIF5B-RET fusion gene. Surprisingly, RET expression was lower in KIF5B-RET fusion gene-positive than in RET wild-type specimens. We detected activating EGFR mutations in 11 (21.6%) of the 51 RET variant-positive specimens. Among the KIF5B-RET fusion gene-positive specimens, p-ERK expression was significantly lower in the EGFR mutation subgroup showing RET expression than in the EGFR mutation subgroup that did not express RET. Similarly, the RET rearrangement group showed significant variation in the expression level of p-AKT (P = 0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the RET rearrangement group regardless of RET expression. Conclusion This result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring RET variants.

Background: To evaluate the anti-tumor efficacy of CKD-516 in combined with irradiation (IR) and examine tumor necrosis, delayed tumor growth, and expression of molecules involved in hypoxia and angiogenesis. Methods : A xenograft mouse model of lung cancer was established. The tumor was exposed to irradiation (IR) for 5 days per week. CKD-516 was administered with ​​two treatment schedules (day 1 or days 1 and 5) at one hour after IR. After the administration, tumor tissues were stained with hematoxylin and eosin and pimonidazole. HIF1 , Glut-1, VEGF, CD31 and Ki-67 expression were evaluated by Immunohistochemical staining. Results: With short-term administration, IR and CKD-516+IR (d1) significantly reduced tumor size ( p = 0.0062 and p = 0.0051, respectively). CKD-516+IR groups were remarkably reduced blood vessels ( p < 0.005). In particular, CKD-516+IR (d1) resulted in the most extensive tumor necrosis, which was significantly increased with large hypoxic area ( p = 0.02) and decreased HIF1 , Glut-1, VEGF, and Ki-67 expressions. Long-term administration of CKD-516+IR reduced tumor size and delayed tumor growth. This combination also greatly reduced the number of blood vessels ( p = 0.0006) and significantly enhanced tumor necrosis ( p = 0.004). CKD-516+IR notably increased HIF1 expression ( p = 0.0047), but significantly diminished VEGF expression ( p = 0.0046). Conclusion: Taken together, our results demonstrate that CKD-516 in combination with IR can significantly enhance the anti-tumor efficacy compared to CKD-516 or IR alone in lung cancer xenograft mice. Keywords: Irradiation, Tumor necrosis, Hypoxia, Squamous cell carcinoma of lung, Xenograft mice

Simple Summary In this study, Sel-CapTM, a next-generation sequencing (NGS)-based genotyping platform, showed high sensitivity for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma samples collected from 185 patients with non-small cell lung cancer (NSCLC). In the early-stage NSCLC, Sel-Cap liquid biopsy was able to detect more than half the EGFR mutations, which were detected in tumor tissue (sensitivity: 50% and 78% for Ex19del and L858R respectively, with tumor results as the references), while the conventional NGS could not detect any. Sel-Cap liquid biopsy was particularly sensitive for resistant mutation T790M (sensitivity: 88%). In addition, we conducted a retrospective study to monitor T790M using Sel-Cap in 34 patients who progressed on first-line tyrosine kinase inhibitors (EGFR-TKIs). The study suggested that the first appearance of T790M in plasma, ranging from at treatment baseline to over three years post-EGFR-TKI initiation, may be useful for prediction of disease progression (around 5 months in advance). Abstract Sel-CapTM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R, and T790M, and compared results to those of PNAclampTM tumor biopsy (reference method, a peptide nucleic acid-mediated polymerase chain reaction clamping) and two other NGS liquid biopsies. Over two-thirds of activating mutations (Ex19del and L858R), previously confirmed by PNAclamp, were detected by Sel-Cap, which is 4–5 times more sensitive than NGS liquid biopsy. Sel-Cap showed particularly high sensitivity for T790M (88%) and for early-stage plasma samples. The relationship between initial T790M detection in plasma and progression-free survival (PFS) following first-line EGFR-TKIs was evaluated in 34 patients. Patients with T790M detected at treatment initiation (±3 months) had significantly shorter PFS than patients where T790M was first detected >3 months post treatment initiation (median PFS: 5.9 vs. 26.5 months; p < 0.0001). However, time from T790M detection to disease progression was not significantly different between the two groups (median around 5 months). In conclusion, Sel-Cap is a highly sensitive platform for EGFR mutations in plasma, and the timing of the first appearance of T790M in plasma, determined via highly sensitive liquid biopsies, may be useful for prediction of disease progression of NSCLC, around 5 months in advance.

Background: c-MET is co-expressed with EGFR in approximately 70% of EGFR mutant-positive tumors. In addition, c-Met amplification was found in 10-20% of lung cancer patients with acquired tolerance to EGFR TKI, and c-Met overexpression was found in 14-69% of patients receiving EGFR TKI. CKD-702, a double antibody, is designed to simultaneously block both EGFR and c-MET expression. Purpose: We evaluated the anti-tumor efficacy of CKD-702 with PDX model derived from lung adenocarcinoma patients. Methods: We constructed the PDX model by transplanting surgical tissue from lung adenocarcinoma patient into NOD/SCID mice after the approval of research protocol in Seoul St. Mary's Hospital IRB. We confirmed the co-existence of activating EGFR mutation and c-MET amplification with PNAclamp and FISH methods. We also checked overexpression of EGFR and c-MET protein by immunohistochemistry. Results: The characteristics of tumor tissue transplanted for PDX model were: 1) Korean, lung cancer with EGFR TKI chemotherapy failure confirmed c-MET amplification (> 5 copies) and overexpression (IHC score 3+), 19del Positive EGFR mutation, 2) European lung adenocarcinoma, c-MET amplification (> 2.5 copy) and overexpression (IHC score 3+) were confirmed, and L858R EGFR mutation positive tumor tumor confirmed. The anti-cancer efficacy of CKD-702 was reduced by more than 80% compared to the tumor size of the control group regardless of the concentration. In the CKD-702 20mg/kg group, there was no significant difference in the change in the overall tumor size until the time of disappearing or ending during drug administration. The %TGI was 93.6 and 97.5 according to the concentration of CKD-702, and the % TGD was 118.5 and 311.8, respectively. CKD-702 has a significantly higher tumor growth inhibitory effect regardless of the concentration, but the tumor growth retardation effect is 2.6 times different depending on the concentration. % TGI of CKD-702 20mg / kg group was similar to Hu8c4 group or Hu8c4 + Vectibix group. However, % TGD was 1.4 times higher than Hu8c4 group efficacy and 3.1 times higher than Hu8c4 + Vectibix group% TGD. Even after the completion of drug administration, tumor still remained at 47 days and 67 days in the CKD-702 10 mg/kg and 20 mg/kg groups, respectively. Although c-MET protein expression decreased during the administration of CKD-702, it was confirmed that c-MET protein expression increased again in tumor tissue at the time of tumor growth. Although the tumor size was rather large (605.6 ~ 816.2mm3), CKD-702 showed twice the tumor growth inhibition effect (% TGI: 32.8 vs. 68.1) and the tumor growth delay effect was 4.4 times higher depending on the drug concentration (% TGI: 2.8 vs 12.3). Conclusion: Our data showed that CKD-702 have effective anti-tumor activity in EGFR TKI-resistant PDX model harboring both activating EGFR mutation and c-MET overexpression. Citation Format: Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Jeong-Oh Kim, Eun-Ju Jeon, Ji Hye Choi, Soon-Ki Hong, Kyu-Jin Park, Jin Hyoung Kang. Anti-tumor efficacy of CKD-702 in EGFR TKI-resistant patient-derived xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3046.

Purpose Dose-limiting toxicities of docetaxel are widely considered to be neutropenia, anemia, skin toxicity, and nausea. One of the factors that limit the use of docetaxel is its unpredictability of inter-individual variation in toxicity. Materials and methods In order to identify the genetic factors that affect the risk of docetaxel-induced toxicities, we recruited patients who received docetaxel chemotherapy. We genotyped 92 patients with single-nucleotide polymorphisms (SNPs) in 5 genes: CYP3A4 (CYP3A4(*)1B, CYP3A4(*)18, and CYP3A4(*)3), CYP3A5 (CYP3A5(*)2 and CYP3A5(*)3), ABCB1 (C1236T, G2677G/T, and C3435T), SLCO1B3 (rs11045585), and ABCC2 (rs12762549). Results Out of 92 patients, 70 had grade 3 or 4 neutropenia; 4 had grade 1 or 2; and 18 had no toxicity (76.1%, 4.3%, and 19.6%, respectively). The findings of the SNP analysis showed that patients with TT genotype of ABCB1 3435C>T polymorphism showed significantly higher risk of neutropenia and anemia (p=0.029 and p=0.044, respectively). There were significant associations between docetaxel-induced leucopenia and 2677G/T of ABCB1 and rs12762549 of ABCC2 (p=0.025 and p=0.028, respectively). In a multivariate analysis, we observed that patients carrying 2677G>T in ABCB1might be associated with higher risk of chemo-resistance when treated with docetaxel (odds ratio [OR], 6.48; confidence interval, 1.92 to 21.94; p=0.003). In a subgroup analysis of non-small cell lung cancer patients, a significant association of tumor response with G2677T/A (OR, 4.54) in ABCB1 and SLCO1B3 (OR, 9.44) was observed. Conclusion Our data suggest that ABCB1 (2677G/T) and SLCO1B3 (rs11055585) might be major genetic predictors of docetaxel-related toxicities in patients receiving docetaxel chemotherapy.

The KRAS gain-of-function mutation confers intrinsic resistance to targeted anti-cancer drugs and cytotoxic chemotherapeutic agents, ultimately leading to treatment failure. KRAS mutation frequency in lung adenocarcinoma is ~15-30%. Novel therapeutic strategies should be developed to improve clinical outcomes in these cases. Deregulation of the p16/cyclin-dependent kinase (CDK) 4/retinoblastoma (Rb) pathway is frequently observed in various cancers and it represents an attractive therapeutic target. We compared the anti-tumor efficacy of genetically knocked-down CDK4 and a pharmacological inhibitor of CDK4/6, CINK4, in KRAS mutation-positive lung adenocarcinoma cells. We also investigated changes in anti-proliferative activity and downstream molecules with these treatments in combination with paclitaxel. CDK4 short interfering RNA (siRNA) significantly increased paclitaxel sensitivity in KRAS mutation-positive H23 cells. CINK4 demonstrated concentration- and time-dependent anti-proliferative activity in 5 adenocarcinoma lines. CINK4 induced G 1 arrest by downregulating the p16/cyclin D1/Rb pathway, resulting in apoptotic induction via increased expression of cleaved caspase3, cleaved PARP and Bax. Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells. These data suggest CDK4 is a promising target for development of anti-cancer drugs and CINK4 combined with paclitaxel may be an effective therapeutic strategy for enhancing anti-tumor efficacy in KRAS mutation-positive lung adenocarcinoma.

Patients with HPV-positive oropharyngeal cancer show better tumor response to radiation or chemotherapy than patients with HPV-negative cancer. HPV oncoprotein E6 binds and degrades a typically wild-type p53 protein product. However, HPV16 infection and p53 mutation infrequently coexist in a subset of HNSCCs. The purpose of this study was to investigate the mechanisms through which tumor biology and molecular genetic mechanisms change when two HPV-negative, p53-mutated oropharyngeal cell lines (YD8, non-disruptive p53 mutation; YD10B, disruptive p53 mutation) derived from patients with a history of heavy smoking are transfected with HPV E6 and E7 oncogenes in vitro. Transfection with HPV E6 and E7 oncogenes in YD8, reduced the abundance of proteins encoded by tumor suppressor genes, such as p-p53 and p-Rb. Cell proliferative activity was increased in the cells transfected with E6E7 compared to cells transfected with vector alone (P=0.09), whereas the invasiveness of E6E7-transfected cells was significantly reduced (P=0.02). cDNA microarray of the transfected cells with E6E7 showed significant changes in mRNA expression in several signaling pathways, including focal adhesion, JAK-STAT signaling pathway, cell cycle and p53 signaling pathway. Regarding the qPCR array for the p53 signaling pathway, the mRNA expression of STAT1 was remarkably upregulated by 6.47-fold (P

All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Epilepsy is the most prevalent chronic neurological disorder and can be controlled by antiepileptic drugs (AEDs) in up to 70% of patients. We performed an association study between adverse drug reactions and the genetic polymorphisms of CYP2C9, CYP2C19, ABCB1, and SCN1A. The clinical data of 83 epilepsy patients who had received AEDs containing carbamazepine (CBZ) were collected. We extracted genomic DNA from peripheral blood and then genotyped CYP2C9 (CYP2C9*2, CYP2C9*3), CYP2C19 (CYP2C19*2, CYP2C19*3), ABCB1 (C3435T), and SCN1A (IVS5N+5 G>A) using direct sequencing. The allele frequencies of CYP2C9*3, CYP2C19*2, CYP2C19*3, ABCB1 (3435C>T), and SCN1A (IVS5N+5 G>A) were 0.93, 0.72, 0.91, 0.61, and 0.55, respectively. Statistically significant differences were indicated from the data obtained. Patients with SCN1A genotype CC or CT were compared with patients with SCN1A genotype TT while using more than 500mg of carbamazepine. We have associated functional polymorphisms with the dose used in regular clinical practice for Korean epilepsy patients who had received antiepileptic drugs (AEDs) containing carbamazepine. For AEDs, we found that one of the SCN1A genotypes is associated with a 500 mg dose. There was no association found with CNS ADR caused by AEDs.

Pemetrexed has demonstrated clinical activity in non-small cell lung cancer (NSCLC) as well as other solid tumors. It transports into the cells via reduced folate carrier (RFC) and is polyglutamated by folypolyglutamate synthetase (FPGS). Pemetrexed directly inhibits several folate-dependent enzymes such as thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). We investigated the effects of genetic variations and the expression of RFC, FPGS, TS and DHFR enzymes on drug sensitivity to pemetrexed in NSCLC cells. Polymorphisms in RFC, FPGS, and DHFR were genotyped in four NSCLC cells - A549, PC14, HCC-1588, and H226. Real-time RT-PCR and Western blot was performed to evaluate mRNA transcripts and protein of these genes. The cytotoxicity of pemetrexed was measured by SRB assay. In PC14 and H226 cells, increased mRNA expressions of RFC and FPGS were associated with higher cytotoxicity to pemetrexed. 2R/2R genotype of TS and its increased mRNA expression were associated with drug resistance to pemetrexed in A549 cells, whereas 3R/3R genotype in TS with decreased mRNA expression was associated with higher sensitivity in H226 cells. After pemetrexed treatment, an inverse change of DHFR mRNA and protein expression was found. The strongest linkage disequilibrium (LD) was discovered between-1726C＞T and -1188A＞C SNP of DHFR gene. Our findings suggest the cytotoxic effect of pemetrexed may be associated with genetic polymorphisms and the expression level of genes involved in pemetrexed metabolisms in NSCLC cells.

Background: The KIF5B-RET rearrangement is detected with the frequency of 1 similar to 2 % in 'triple marker'-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational changes are known to be mutually exclusive, but the co-existence of ALK rearrangement with activating mutations of EGFR is rarely found. Methods: We examined the KIF5B-RET fusion gene in frozen tissues from 154 surgically resected lung tumors using RT-PCR with direct sequencing and the mutation status of EGFR and KRAS genes using PNA clamping. We tested KIF5B-RET translocation in Formalin Fixed Paraffin Embedded using fluorescence in situ hybridization. We also measured RET mRNA and protein expression by RT-PCR and immunohistochemistry, respectively. Results: The existence of KIF5B-RET fusion gene was identified in 9 patients. The mean age was 67.2 and M: F ratio 4: 5. Of 9 patients, 3 patients harbored wild type of EGFR and KRAS gene. However, KIF5B-RET fusion gene coincided with EGFR or KRAS mutation in 6 patients. These six pts were also positive for both RET break-apart probes (23.9 %) and KIF5B-RET fusion (44.4 %). However, there were no correlations between RET mRNA and protein expression in the KIF5B-RET-positive patients. The median disease free survival and overall survival were 23.9 months and 29.5 months, respectively. Conclusions: Taken together, our data suggest one-step screening platform for KIF5B-RET as well as EGFR, K-RAS, ALK oncogenic mutations be necessary for lung adenocarcinoma patients because EGFR or KRAS mutation are not infrequently found in KIF5B-RET-positive patients.

e20610 Background: RET rearrangements have been identified in 1-2% of lung adenocarcinomas. The most common fusion is the KIF5B-RET, the function and roles of the RET fusion oncogene, and its downstream signaling molecules remain unclear. Methods: We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from lung adenocarcinoma patients and investigated them using FISH with RET break-apart and KIF5B-RET SY translocation probes. NanoString’s nCounter technology was used to assay RETtranscripts. We evaluated the protein expressions of RET and RET-related signaling molecules, including p-AKT and p-ERK, using TMA-based IHC staining. Results: Using FISH, we identified 51 cases (8.8%) of RET variants and 10 cases (1.7%) of KIF5B-RET fusion genes among the 581 cases. RET protein expression was lower in the group harboring KIF5B-RET fusion gene than that in the group harboring a wild type RET gene. We found the activating EGFR mutations in 11 (21.6%) cases of 51 RET variants. For the group with KIF5B-RET fusion gene, the expression of p-ERK was significantly lower in EGFR mutation subgroup with presence of RET protein compared to EGFR mutation subgroup with absence of RET protein. For the group with RET rearrangement, there were significant differences in the expression level of p-AKT (P = 0.028) and, p-ERK protein expression was remarkably increased, especially in cases with no RET protein expression. Conclusions: Taken together, the expression of p-ERK protein was meaningfully increased in the RET variants group regardless of RET protein expression. This result suggests that RET inhibitors combined with ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring the RET variants.

Background: The mechanism of primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small-cell lung cancer (NSCLC) has not been clearly understood. Patients and methods: Eleven patients exhibiting primary resistance (disease progression

Background & Purpose LY2835219(LY), a novel CDK4/6 inhibitor, inhibits phosphorylation of RB and E2F activation, thereby arresting the cell cycle in the G1 phase and suppressing the cell proliferation and cell division. Docetaxel(DTX) is a cytotoxic anti-cancer drug, which inhibits mitotic cell division, thus induces G2/M arrest and apoptosis of various cancer cells. In present study, we evaluated the activities of CDK4/6 inhibitor alone or combined with docetaxel on the anti-proliferation, cell cycle and apoptosis in lung cancer cell lines harboring KRAS mutations. Methods We measured the anti-proliferative activities of LY or DTX single and their combinations (DTX+LY 72h and DTX 24h->LY 48h) on cell proliferation in A549(G12S) and H727(G12V) cells using CCK-8 asssy. We evaluated the expression of CDK2, CDK4, cPARP and caspase-3 by Western blot. The cell cycle distribution and apoptosis in subG1 phase were analyzed by flow cytometry. Results The IC50 values of the LY and DTX alone were 0.4 ± 0.2 uM and 0.9 ± 0.2 nM in A549 cells and 2.0 ± 0.7 uM and 3.1 ± 0.3 nM in H727 cells, respectively. The CI (Combination index) of the DTX->LY sequential and the DTX+LY simultaneous treatments were 0.8 and 0.9 (CILY showed decreased cell viability by 60% at very low concentration of DTX (3.0e−5 nM), but DTX+LY showed significantly decreased cell viability by 40% at higher concentration of DTX (IC50 values can not be measured). In A549 cells, after DTX treatment for 72h, the cell population in G2/M phase increased by 54.5% relative to control (24.6%). With LY alone, the fraction of A549 cells in G0/G1 phase increased compared to control (78.4% vs. 49.5%). In case of DTX+LY combination, the G2/M fraction of A549 cells significantly increased by 33% vs 24.6% in a dose-dependent manner. Meanwhile, in DTX->LY treatment, cell fractions in G2/M were increased (44.5% vs 24.6%), especially those in G0/G1 phases were remarkably reduced (14.7% vs 49.5%). In addition, the subG1 fraction was accumulated in response to individual drug treatments. In case of DTX single treatment, the subG1 fractions of both A549 and H727 cells increased in a dose-dependent manner (0.7% vs. 17.4% and 2.5% vs. 28.1%, respectively). We were not able to observe the change of subG1 fraction in DTX+LY combination treatment in A549 cells. However, in DTX->LY sequential treatment, the subG1 fraction increased in a dose-dependent manner by relative to control (23.3% vs 10.9%). The expression of c-PARP and caspase-3 was increased by the DTX alone and two different combination schedule and CDK2 and CDK4 expressions were decreased by LY alone and DTX->LY sequential treatment. Conclusions Taken together, our results suggest that DTX->LY sequential treatment has enhanced antitumor efficacy with synergistic interaction in lung cancer cell lines harboring KRAS mutations. Citation Format: Kyoung Hwa Son, Jung-Young Shin, Jeong-Oh Kim, JinHyoung Kang. Synergistic effect of combined cdk4/6 inhibitor with docetaxel in lung cancer cell lines harboring KRAS mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2832.

Background & Purpose In recent, KIF5B-RET gene rearrangement has been discovered as a driver oncogene in non-small cell lung cancers. This fusion gene was demonstrated to be able to induce tumorigenesis in vivo mouse model. Hereby, we investigated how KIF5B-RET fusion gene affect tumor differentiation and which transcription factors and signaling pathway are activated. Methods We analyzed cell proliferative activity in transformed HEK293T cells with KIF5B-RET fusion gene (K4) and empty vector (V5) using CCK8 assay. We used light microscopy and H&E staining to observe morphological changes of transformed cells. We evaluated protein and mRNA expressions of four EMT markers (E-cadherin, N-cadherin, Snail, and Twist) and two transcription factors (FOXA2, p-STAT5A) using transcription factor PCR array, Western blot, RT-PCR, immunofluorescence staining. Results K4 cells showed decreased cell proliferative activity compared with V5 cells. V5 cells had spreading pattern alike mesenchymal phenotype when their density was lower. However, their morphology changed to epithelial phenotype as they were overgrown with higher density. On the other hands, K4 cells exhibited the island growing pattern similar to epithelial phenotype in lower density of tumor cells, but changed to mesenchymal phenotype as the density of tumor cells gradually increased. On H&E staining, the cytoplasm of K4 cells was much smaller than that of V5 cells. We observed that E-cadherin mRNA was down-regulated, but N-cadherin and Twist were up-regulated in K4 cells. Based on the results of repeated screening assay, we finally selected two transcription factors, FOXA2 and STAT5A, which were significantly overexpressed in K4 cells. In K4 cells, phosphorylated-STAT5A was increased and FOXA2 was decreased compared to V5 cells. In addition, the expression of E-cadherin was reduced, but p-GSK3β, p-AKT, and p-ERK were markedly increased in K4 cells. The pattern of immunofluorescence staining of V5 and K4 cells were similar to the protein expressions of E-cadherin and p-STAT5A in Western blot analysis. Conclusion Taken together, FOXA2 and E-cadherin are down-regulated and p-STAT5A is up-regulated in HEK-293T cells stably expressing the KIF5B-RET. Our data suggest that KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. Citation Format: Min-Young Kim, Jung-Young Shin, Jeong-Oh Kim, Kyoung-Hwa Son, Jin Hyoung Kang. KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1687.

e20017Background: Activating or resistant somatic mutations (19 Del, L858R, T790M) occurring in the specific sites of EGFR gene are the powerful predictive biomarker for EGFR tyrosine kinase inhibi...

e20014Background: KIF5B-RET gene rearrangement occurs in about 1% of lung adenocarcinomas. In recent, the targeted agents to inhibit RET phosphorylation have been examined in several clinical studies. However, little was known about how this fusion gene drive lung cancer. Methods: We established transformed HEK293T cells with KIF5B-RET fusion gene using Lenti virus system. In transformed HEK293T cells, we identified transcription factors which are activated using qRT-PCR array and then, checked mRNA, protein, expressions of FOXA2. We performed immunohistochemistry to evaluate the expressions of FOXA2 protein in tumor tissues obtained from 38 lung adenocarcinoma patients who were screened by FISH, RT-PCR, and Nanostring analysis. Results: In the qRT-PCR array, the transcription factors of which the relative expression increased more than 1.5 in KIF5B-RET transformed HEK293T cells were MYF5, STAT5A, ESR1 and FOXA2. We confirmed the increased expressions of FOXA2 mRNA and protein in transformed HEK293T cells...

Studies on non-small cell lung cancer (NSCLC) patients with KRAS or BRAF mutations are urgently needed to improve clinical outcomes. We evaluated the cytotoxicities of paclitaxel and sorafenib alone and in combination in NSCLC cell lines with KRAS or BRAF mutations and investigated the mechanism of the interaction between the drugs. We found synergistic antitumor efficacy with paclitaxel followed by sorafenib in in vitro and in vivo models of NSCLC. And, we determined that downregulation of the phosphorylated ERK and Rb, and Mcl-1 plays a critical role in the synergistic activity of the drugs. Further clinical trials are needed to verify the antitumor efficacy of this combination.

Warfarin is a commonly prescribed anticoagulant drug for the prevention of thromboembolic disorders. We investigated the contribution of genetic variations of four genes and clinical factors to warfarin dose requirement and provided a warfarin-dosing algorithm based on genetic and clinical variables in Korean patients. We recruited 564 Korean patients on stable anticoagulation. Single nucleotide polymorphisms (SNPs) for the VKORC1, CYP2C9, CYP4F2 and GGCX were analyzed. Using multiple regression analysis, we developed a model to predict the warfarin requirement. The SNPs of VKORC1, CYP2C9, CYP4F2 and GGCX showed significant correlation with warfarin dose. Patients with the 3730AA genotype received significantly higher doses of warfarin than those with the 3730GG (P=0.0001). For CYP2C9, the highest maintenance dose was observed in the patients with wild-type genotype compared with the variant allele carriers (P

The KIF5B-RET gene is a novel fusion gene resulting from pericentric inversion between the kinesin family member 5B (KIF5B) and the rearranged during transfection (RET) gene, which recently isolated as an oncogene in non-small cell lung cancers. In the present study, we investigated the mechanisms associated with tumorigenic activity of KIF5B-RET fusion gene. We used a HEK-293T cell line stably expressing the KIF5B-RET fusion gene in pLOC lenti-viral vector. We investigated the tumor formation in a xenograft mouse models after subcutaneous injection and confirmed the expression of KIF5B-RET by PCR and western blot analysis. We also explored the mechanism of KIF5B-RET fusion gene in stable cell line. The KIF5B-RET stable cells showed upregulation of the cell growth, migration and colony formation. Furthermore, the KIF5B-RET stable cells induced the activation of BRAF, AKT and ERK compared with the parental cells. These results suggest that the KIF5B-RET fusion gene promotes the cell proliferation and tumorigenic activity though BRAF, AKT and ERK activation. These findings may also provide the possible that KIF5B-RET fusion gene as a ‘driver’ in some subgroups of lung cancers. Citation Format: Jung-Young Shin, Min-Young Kim, Kyoung-Hwa Son, Jeong-Oh Kim, Jin-Hyoung Kang. Tumorigenic activity of a novel KIF5B-RET fusion gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2710. doi:10.1158/1538-7445.AM2015-2710

e18528 Background: KIF5B-RET fusion genes are formed when part of the RET gene fuses with part of the KIF5B gene. This fusion gene induces the proliferation and differentiation of tumor cells by tr...

7537 Background: The KIF5B-RET rearrangement is detected with the frequency of 1~2% in ‘triple marker’-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational chang...

1540 Background: Activating EGFR mutations are strongly predictive of EGFR tyrosine kinase inhibitor (TKI) activity in NSCLCs, however, some patients having wild type EGFRdo not infrequently show clinically favorable outcome to EGFR TKIs. The key proapoptotic proteins have been reported to be involved in TKI-induced cell death. The up-regulation of BIM, a pro-apoptotic protein of BCL2 family, is required for TKIs to induce apoptosis in cancers. Methods: For 150 advanced or metastatic NSCLC pts. receiving gefitinib or erlotinib, we assayed mutation status of EGFR in paraffin embedded tumor tissue using PNA clamping (PANAGENE, Korea) and direct sequencing. Six different genetic variants of EGFR (-191C>A, -216 G>T, CA repeat number, R497K, 2607G>A, and D994D) and BIMdeletion in genomic DNA extracted from peripheral blood were analyzed. Results: M:F ratio was 72:78 and median age was 63 (35~82). Histological subtypes are as follows: 131 adenocarcinoma (87.3%), 16 squamous cell ca. (10.7%) and 3 large cell ca. (1.3%). Objective tumor response (CR+PR) was observed in 60 pts. (40.0%) and SD in 44 pts. (29.3%). Median PFS and OS were 4.3 (0.4~67.4) and 18.6 (0.9~78.1) months, respectively. Of 129 pts. in whom EGFR mutation could be analyzed, 41 pts. (31.8%) had activating EGFR mutation. In univariate analysis, only gender was associated with objective response to EGFR TKI in activating EGFR mutant group (P=0.048). In wild type EGFR group (n=88), R497K (P=0.04) and BIM (P=0.04) as well as gender (P=0.05) were significantly correlated with objective response. In addition, multivariate analysis demonstrated that gender, R497K and BIM were associated with objective response to EGFR TKIs in wild type EGFR group with statistical significance (P=0.023, 0.027 and 0.018, repectively). Neither six EGFR polymorphisms nor BIM was correlated with PFS and OS in activating EGFR mutant group. Meanwhile, in wild type EGFR group, R497K was significantly associated with OS (P=0.028). Conclusions: Taken together, our data suggest that R497K of EGFR and BIM deletion polymorphisms are useful pharmacogenetic biomarkers to predict objective tumor response for EGFR TKIs in advanced NSCLC pts. harboing wild type EGFR.

e18050 Background: The EGFR mutation on particular exons has been known as a strong predictive biomarker to EGFR TKIs (tyrosine kinase inhibitors) in NSCLC pts. However, some pts having wild type EGFR do not infrequently show clinically favorable outcome to EGFR TKIs. Accordingly, we hypothesized that clinical outcome and skin toxicity to EGFR TKIs might be related to specific single nucleotide polymorphisms regulating the expression of EGFR gene as well as EGFR sensitive mutation. Methods: In 211 advanced or metastatic NSCLC pts receiving gefitinib or erlotinib, we assayed mutation status of EGFR in paraffin embedded tumor tissue using PNA clamping method and direct sequencing. Six different SNPs in genomic DNA extracted from peripheral blood were analyzed; promoter 191C>A, 216 G>T, CA repeat number, exon 13 R497K, exon 20 2607G>A, and exon 25 D994D. Results: M:F ratio was 106:105 and mean age was 63.2 (35.0-82.0). Histological subtypes are as follows: 175 adenocarcinoma, 32 squamous cell ca. and 3 large cell ca. Objective response (CR+PR) was observed in 78 pts (36.9%) and SD in 69 pts (32.7%). Median PFS and OS were 8.7 ± 11.3 and 15.9 ± 14.8 months, respectively. Of 167 pts in whom EGFR mutation was analyzed, 68 pts (40.7%) had EGFR harboring sensitive mutation. In R497K, wild type (RR) was 35 (21.0%) and K alleles (RK + KK) were 132 (79.0%). In D994D, wild type (GG) was 76 (45.5%) and heterozygote and homozygote variants (GA+AA) were 91(54.5%). Statistically significant differences of PFS and OS were observed between wild and hetero-/homozygote variants of R497K (11.9 m vs. 36.2 m, p=0.037 and 23.7 m vs. 39.1 m, p=0.054, respectively) in the pts harboring mutant type EGFR. Meanwhile, in the pts harboring wild type EGFR, GA+ AA genotype of D994D demonstrated much longer PFS and OS compared with GG genotype (PFS: 18.5 vs. 3.7 m, p=0.013 and 37.6 vs. 15.2 m, p=0.035, respectively). In addition, skin rash showed statistically significant association with R497K polymorphism (P=0.031). Conclusions: Our data suggest that two germline genetic variations of EGFR gene, R497K and D994D, be useful pharmacogenetic biomarker to predict longer PFS and OS for EGFR TKIs in advanced NSCLC pts harboring mutant type and wild type EGFR, respectively.

Background High-risk type of human papillomavirus (HPV) has recently been found to be etiologically associated with 20 to 25% of SCCHN, particularly common in or pharyngeal tumors. We investigated the protein expression of PTEN/PI3K/AKT/mTOR signal pathway on HPV 16 +/− tonsillar squamous cell carcinomas. And, we analyzed microRNA expression profile in tongue cancer cell line transfected with HPV 16 E6/E7 oncogene. Method Tonsil cancer patients who had been treated at Seoul St. Mary's Hospital between 2000 and 2009 were reviewed. Tissue microarrays were prepared from formalin-fixed, paraffin-embedded tumor tissues of 65 surgical specimens, and stained with antibodies against PTEN, PIK3CA, phosphorylated AktSer473, and phosphorylated mTORSer2448. High-risk HPV infection status was determined by in situ hybridization. The protein expression of EGFR, PIK3CA, pAkt, mTOR, and PTEN was assessed by immunohistochemistry. MicroRNA expression was studied using microarrays (PANArray™ miRNA expression profiling kit) in two tongue cancer cell lines (YD8 and YD10B) transfected with HPV 16 E6/E7 oncogene. Results Of the 65 pts, 26 (40%) pts were HPV positive. Overall survival and relapse-free survival were significantly different between two groups (P = 0.05 and P = 0.036, respectively). PTEN expression was significantly higher in HPV-positive- than in HPV-negative patients (P = 0.02). PIK3CA, phosphorylated AktSer473, and phosphorylated mTORSer2448 expression was negatively correlated with HPV status, yet not significantly. HPV status (P = 0.03) and nodal stage (P = 0.05) were found to significantly affect overall survival, as determined by multivariate analysis. The expression profile of miRNAs was different in HPV 16 E6/E7 transformed cells compared with vector alone cells. In the final round, 8 of 158 miRNAs were selected to be most differently expressed miRNAs; let-7b, miR-1, miR-107, miR-122, miR-127-5p, miR-95, miR-23b and miR-370 (>1.2-fold). Conclusion We observed the higher expression of PTEN in HPV 16 positive tonsil tumor than HPV 16 negative tumor. Our data suggest that some microRNAs induced by E6/E7 oncogene might directly affect PTEN expression level independent of p53. We are underway on the experiments confirming that PTEN is regulated by which among these miRNAs. 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 185. doi:1538-7445.AM2012-185

Backgraound The eIF4B, a key molecule for initiation of protein translation, play an important role in cell growth, proliferation and survival. We investigated that eIF4B is associated with the molecular mechanism of de novo resistance against EGFR TKIs and its role on cell proliferation and survival in NSCLC cells. Method Among five NSCLC cell lines harboring EGFR and K-RAS wild type, PC14 cells showing the highest IC50, were selected for our experiment. In eIF4B silenced PC14 cells by shRNA, we evaluated cell proliferation by direct cell counts and SRB assay and cell cycle distribution using flow cytometry. The expression of signal molecules, transcription factors and apoptosis associated proteins was measured by Western blot. In vivo tumor model established by subcutaneous injection of eIF4B silenced PC14 cells, the retardation of tumor growth was measured. Results In PC14 cells transformed with eIF4B shRNA, we observed the protein expression of eIF4B was reduced (>70% at day 5). The proliferation of eIF4B silenced cells was greatly inhibited both in vitro and in vivo tumor model. Between eIF4B silenced cells and control cells, there was no significant difference in anti-proliferative activity of several target inhibitors (enzastaurin, gefitinib, LY294002, vandetanib) against EGFR-PI3K-AKT signaling molecules. The expression of proteins involved in cell survival (pEGFR, pAKT and mTOR), transcription factors (pNFkB and TS) and anti-apoptotic protein (bcl-2), was decreased and apoptosis associated proteins (phospho-p53 and active caspase-3) were increased. The G0/G1 cell fraction in eIF4B silenced cells was reduced (47.3% vs. 55.1% in control cells) and accumulation of apoptotic cells was observed (12.7% vs. 1.5% in control cells; shown as sub G1 population). Conclusion Our results demonstrated that eIF4B silencing resulted in a marked decrease of several anti-apoptotic and pro-proliferative proteins and eIF4B was required for cell proliferation and survival by regulating cell cycle and cell signaling molecules. 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 1291. doi:1538-7445.AM2012-1291

Background: Deregulation of the p16INK4A-cyclin D-cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is frequently observed in various cancers and represents one of the attractive therapeutic targets. The gain of function mutation in KRAS gene confers intrinsic resistance to targeted anti-cancer drugs as well as cytotoxic chemotherapeutic agents leading to treatment failure. More effective treatment for adenocarcinomas harboring KRAS mutations should be developed to improve clinical outcomes. Purpose: CINK4, a chemical inhibitor of cyclin-dependent kinase 4, has demonstrated potent anti-proliferative activity in the presence of pRb. Herein, when combined with paclitaxel, we investigated that CINK4 may increase its anti-proliferative activity in the NSCLC cells harboring KRAS mutant. Methods: We measured the anti-proliferative activities of CINK4 in A549 (KRAS mutant), H358 (KRAS mutant), SK-LU-1 (KRAS mutant), H23 (KRAS mutant), PC14 (KRAS wild), and Calu-3 (KRAS wild) cell lines using SRB assay, cell cycle distribution quantified by flow cytometric analysis, and the expression of cell cycle regulators and apoptosis-related proteins was evaluated by Western blotting. Multiple drug effect was analyzed to study an interaction between two drugs. The nature of the interaction between two agents was calculated for the combination index (CI). Results: Concentration- and time- dependent anti-proliferative effects of CINK4 (0.1∼ 40 µM concentration range) and paclitaxel (0.1∼300 nM) alone were observed in six NSCLC cell lines, and were not significantly different between each cell line (48 h IC50: 10.4 ± 0.2 µM and 5.5 ± 0.9 nM, 10.1± 1.9 µM and 5.2 ± 0.9 nM, 6.6 ± 0.8 µM and 3.6 ± 1.0 nM, 8.6 ± 0.8 µM and >10 nM, 8.9 ± 0.5 µM and 3.5 ± 0.2 nM, and 8.4 ± 0.2 µM and > 10 nM, in A549, H358, PC14, and Calu-3, SKLU-1, and H23 cells, respectively). After 48 h CINK4 treatment alone, the G0/G1phase cell population increased with increment of subG1 faction in concentration-dependent manner. CINK4 reduced cyclinD1 and Rb phosphorylation and increase the expression of cleaved PARP in concentration-dependent manner in A549, H358, and PC14 cells. The CINK4 combined with paclitaxel in four KRAS mutant cell lines enhanced anti-proliferative activity of paclitaxel. The CI values of CINK4 and palitaxel in A549, H358, SKLU-1 and H23 were 0.51 ± 0.16, 0.63± 0.12, 0.60 ± 0.15, and 0.55 ± 0.15, respectively. Conclusions: Taken together, our data suggest that CINK4 alone has promising anti-tumor activity in NSCLC cells with or without KRAS mutation. In addition, CDK4/6 inhibitor in combined with paclitaxel demonstrated more enhanced anti-tumor activity in NSCLC cells harboring mutant KRAS. 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 4896. doi:1538-7445.AM2012-4896

64 Background: Metastasis of cancer cells is associated with numerous activating molecules, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α. Excess VEGF production induces hematogenous metastasis in gastric cancer (GC) cells. To understand the anti-metastatic effect of bevacizumab, an anti-VEGF monoclonal antibody developed for selective inhibition of tumor angiogenesis, we investigated VEGF-induced expression of key adhesion molecules in human umbilical vein endothelial cells (HUVECs) and the inhibitory effect of bevacizumab on the adhesion of GC cells expressing high levels of VEGF. Methods: We measured the soluble VEGF (sVEGF) produced by 7 GC cells by ELISA. We evaluated intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin expression in HUVECs pretreated with SNU-1-conditioned medium (SNU-1 CM) or recombinant VEGF (rhVEGF) at different time points by western blotting. Results: We identified that SNU-1 cells secreted the highest sVEGF concentration in 7 GC cells. After rhVEGF pretreatment, ICAM-1 and E-selectin expression were highest at 4–6 h and 2 h, respectively, but VCAM-1 expression was unchanged. Bevacizumab cotreatment markedly decreased VCAM-1 and E-selectin expression to basal levels, whereas ICAM-1 expression was unaffected. The SNU-1 adherent cell percentage decreased following bevacizumab cotreatment of SNU-1 CM- or rhVEGF-pretreated HUVECs. Investigation of SNU-1-cell invasiveness through activated HUVECs revealed less than 10 invasive adherent cells, whereas no cells were found after bevacizumab cotreatment. Conclusions: Our preclinical data suggests that bevacizumab, might contribute to anti-metastasis by inhibiting SNU-1 cell adhesion to HUVECs by reducing E-selectin and VCAM-1 expression.

Background: Deregulation of the p16INK4A-cyclin D-cyclin-dependent kinases (CDK) 4/6-retinoblastoma (RB) pathway is a common paradigm in various solid tumors and represents one of the attractive targets in cancer treatment. KRAS mutation confers intrinsic resistance to cytotoxic chemotherapeutic agents, and to EGFR TKIs leading to treatment failure. An effective systemic treatment for adenocarcinomas harboring KRAS mutations are urgently needed to improve clinical outcomes. Purpose: CINK4, a chemical inhibitor of cyclin-dependent kinase 4, has demonstrated effective anti-proliferative activity in vivo in the presence of pRb. Herein, we investigated that CINK4 may increase its anti-proliferative activity in the NSCLC cells harboring KRAS mutation Methods: We measured the anti-proliferative activities of CINK4 in A549 (KRASG12S), H358 (KRASG12C), PC14 (KRASwild), and Calu-3 (KRASwild) cell lines using SRB assay, cell cycle distribution quantified by flow cytometry, and the expression of cell cycle regulators and apoptosis-related proteins was evaluated by Western blotting. Multiple drug effect was analyzed to study an interaction between two drugs. The nature of the interaction between two agents was calculated for the combination index (CI). Results: Concentration- and time-dependent anti-proliferative effects of CINK4 (0.1∼40 μM concentration range) and pacitaxel (0.1∼300 nM) alone were seen in four NSCLC cell lines, and were not significantly different between each cell line (48 h IC50: 10.4 ± 0.2 μM and 5.5 ± 0.9, 10.1± 1.9 μM and 5.2 ± 0.9, 6.6 ± 0.8 μM and 3.6 ± 1.0, and 8.4 ± 0.2 μM and > 10 nM, in A549, H358, PC14, and Calu-3, respectively). After 48 h CINK4 treatment alone, the G0/G1phase cell population increased with increment of subG1 faction in concentration-dependent manner. CINK4 reduced cyclinD1 and Rb phosphorylation and increase the expression of cleaved PARP in concentration-dependent manner in A549, H358, and PC14 cell lines. And CINK4 combined with paclitaxel in two KRAS mutated cell lines showed enhanced anti-proliferative activity of paclitaxel. The CI values of CINK4 and palitaxel combination in A549 and H358 were 0.51 ± 0.16 and 0.63± 0.12, respectively. Conclusions: CINK4 showed promising anti-tumor activity in NSCLC cell lines. NSCLC harboring KRAS mutation may have clinical benefit from CDK4/6 inhibitor in combined with chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A66.

7592 Background: It has been well known that EGFR mutation on particular exons is a strong predictive biomarker to EGFR TKIs (tyrosine kinase inhibitors) in NSCLC pts. However, some pts having wild...

Background As EBV-associated gastric cancer has unique features that are different from EBV (-) gastric cancer, EBV is considered to have a key role in gastric carcinogenesis. It has been reported that viral latent membrane protein 2A (LMP2A) in EBV-transformed tumor cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which provides a survival signal and chemo-resistance to cytotoxic anti-cancer drugs. This study was to evaluate anti-proliferative effect and cell cycle change when 5-FU and LY294002 (LY), a selective inhibitor of PI3K, were treated separately or combined with different schedules in EBV positive gastric cancer cell line, SNU-719. Methods After single treatment and sequential combination of 5-FU and LY, cytotoxic activity was measured by MTS assay. When 5-FU and LY were treated in single and sequential combinations, the expression of p-AKT, p-NFkB, p-p53 and bcl-2 was observed on different concentrations by Western blot analysis. We also investigated the effect on apoptosis and cell cycle distribution using flow cytometry. The LMP2A siRNA inhibition was done to confirm the reversal of decreased 5-FU activity and p-AKT. Results When 5-FU was sequentially combined with LY, the combination index (CI) value indicated synergistic anti-proliferative effect. The expression of p-AKT and p-NFκB was upregulated by 5-FU alone but sequential treatment of 5-FU and LY decreased the expression of both p-AKT and p-NFκB. When 5-FU was combined with LY, G0/G1 and sub G1 cell population (%) increased. When 5-FU was added to the cells transfected with LMP2A siRNA, its anti-proliferative effect increased and the expression of p-AKT decreased. In sequential combination of 5-FU and LY, the expression of p-p53 was increased and bcl-2 expression was diminished compared to 5-FU alone. Conclusion These data suggest that sequential combination of 5-FU and LY induce synergistic cytotoxicity and overcome intrinsic and acquired resistance of 5-FU via downregulation of activated p-AKT and mitochondria-dependent apoptosis in EBV gastric cancer cell line, SNU-719.

Sorafenib (BAY 43-9006), a multi-kinase inhibitor, targets a serine-threonine kinase, BRAF as well as several tyrosine kinases. Paclitaxel has been known to exhibit potent anti-tumor activity in non-small cell lung cancer (NSCLC). Our aim of this study was to examine anti-proliferation, anti-angiogenesis and apoptosis induction when Sorafenib combined with Paclitaxel in vitro and in vivo NSCLC models. We measured the inhibitory activities of Sorafenib or Paclitaxel single and their sequential combinations(PS: Paclitaxel 24h → Sorafenib 48h and SP: Sorafenib 48h → Paclitaxel 24h) on cell proliferation in A549 (KRAS mutation), H1666 (BRAF mutation), and Calu-3 (p53 mutation) by SRB assay. We evaluated phosphorylation of ERK, AKT and BRAF proteins using Western bloting and analyzed the cell cycle distribution in A549 cell line. Xenograft tumor model was established with A549 tumor cells implanted in the Balb/c nu/nu mice. Together with single treatments, two different combination schedules of Sorafenib with Paclitaxel were examined. PPS, 20mg/kg of paclitaxel IP at 2 times in 1st wk followed by 40mg/kg of Sorafenib PO during 5 consecutive days in 2nd wk, PSS, 20mg/kg of Paclitaxel IP one time at 1st day followed by 40mg/kg of Sorafenib PO during 5 consecutive days in 1st week. Intratumoral molecular changes were studied on paraffin-embedded tumor tissue obtained from xenograft mice using immunohistochemistry and the apoptotic induction was evaluated with TUNEL assay. Single Sorafenib or Paclitaxel showed active cytotoxicities with dose-dependent manner in three cell lines. Their IC50s at 72hrs were as follows: A549, 5.7 ± 1.3 µM and 1.6 ± 0.1 nM; H1666, 3.7 ± 0.2 µM and 2.2 ± 0.6 nM; Calu-3, 8.8 ± 1.0 µM and 1.4 ± 1.3 nM, respectively, and the combination effect of PS was superior to SP in the three cell lines. A549 cells were treated with Paclitaxel (24h/48h), Sorafenib (24h/48h), PS and SP. Sorafenib significantly reduced the expressions of pBRAF, pERK and pAKT. PS showed greater reduction of pBRAF, pERK and pAKT compared to Sorafenib. However, downregulation of pERK expression was not detected in SP. Sorafenib induced G0/G1 arrest of cell cycle (54.5% at baseline vs. 61.9% at 48hr). PS demonstrated greater increase of G0/G1 arrest than SP (66.8% vs. 50.8%). In xenograft tumor model, Sorafenib combined with Paclitaxel (PPS or PSS) more effectively suppressed tumor growth compared to their single treatments. In immunohistochemistry study, greater inhibition of Ki-67 as well as pAKT, pERK, and CD31 were observed in two sequential combinations compared to single treatment. Additionally, significant increase of apoptotic change was also found in these two combinations. Taken together, sequential combination of Sorafenib with Paclitaxel demonstrated synergistic interaction and potent anti-tumor efficacy compared to their single treatment in vitro and in vivo NSCLC models. 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 3618.

It has been known that Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of Rho family GTPase. We identified a RhoGDI2 protein through the protein expression profile and then investigated its functional role on EGFR downstream signal in NSCLC (non-small cell lung cancer). Three adenocarcinoma cell lines were selected according to the EGFR and K-RAS mutation status; PC9 (E476-A750 del in exon 19, wild type K-RAS), H1975 (T790M in exon 20 and L858R in exon 21, wild type K-RAS), A549 (wild type EGFR, K-RAS mutation in codon 12). Among 342 protein spots detected on the 2D gel, 134 spots were initially selected as differentially expressed proteins using proteome analysis. It was noteworthy that the ubiquitination-related proteins were highly expressed in EGFR TKIs-sensitive PC9 cells. On the other hand, the EGFR TKIs-resistant H1975 cells harboring both T790M and L858R mutations expressed the signal transduction-related proteins such as Zinc finger protein, G protein-coupled receptor and Ras oncogene family protein. We observed that p-EGFR (Y1047), p-AKT, and p-ERK were highly activated in PC9 cells in contrast to no or weak activation of these signaling proteins in A549 and H1975 cells. In PC9 cell, the expression of RhoGDI2 was significantly reduced after gefitinib treatment. On the contrary, its expression was enhanced by gefitinib in H1975 cells. Accordingly, RhoGDI2 of the final six protein spots was selected for further study. In six NSCLC cell lines, RhoGDI2 mRNA and protein expressions were evaluated using RT-PCR and Western blot analysis, respectively. The highest expression level was observed in PC-9 cells. When RhoGDI2 siRNA was transfected in PC9 cells, it was shown that RhoGDI2 protein expression was markedly reduced. Additionally, in this transfected PC-9 cells, no changes in the expression of p-ERK and truncated caspase-3 were detected, but total EGFR and p-AKT expression were increased. When gefitinib was added in the PC9 cells transfected with RhoGDI siRNA, the expression level of total EGFR was less decreased compared to that of PC-9 cells transfected with GAPDH (positive control). The RhoGDI2 siRNA transfected PC-9 cells treated with gefitinib showed more increased expression level of actin compared to that of positive control. We observed that the metastatic potential of the RhoGDI2 siRNA transfected PC-9 cells with lack of its expression was significantly increased in transwell migration assay. Taken together, it is suggested that RhoGDI2 protein is an important down-stream molecule of EGFR signaling pathway in PC9 cells harboring EGFR sensitive mutation, and plays a functional role in cell migration and metastasis. 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 1582.

Purpose The aim of this study is to investigate the effect of genetic variations and the expression of the reduced folate carrier (RFC) and dihydrofolate reductase (DHFR) on the drug sensitivity to methotrexate (MTX) in different cancer cell lines. Materials and methods We examined the six human cancer cell lines (MCF-7, AGS, A549, NCI-H23, HCT-116 and Saos-2). The cytotoxicity of MTX was measured by sulforhodamine B (SRB) assay. The expressions of the DHFR and RFC were evaluated by real-time PCR and western blotting. Four single nucleotide polymorphisms (SNPs) of the DHFR and two SNPs of the RFC were genotyped. Results The IC₅₀s of MTX was in an extensively broad range from 6.05±0.81 nM to>1,000 nM in the cell lines. The Saos-2 (>1,000 nM) and MCF-7 (114.31±5.34 nM) cells were most resistant to MTX; in contrast, the AGS and HCT-116 cells were highly sensitive to MTX with an IC(50) of 6.05±0.81 nM and 13.56±3.76 nM, respectively. A reciprocal change of the RFC and DHFR mRNA expression was found between the MTX-sensitive AGS and MTX-resistant Saos-2 cells. There was no significant difference in the expression levels of RFC protein in both the AGS and Saos-2 cells, whereas DHFR protein was more increased in the MTX-resistant Saos-2 cells treated with MTX. The genotype of the MTX-sensitive AGS cells were mutant variants of the DHFR; in contrast, the Saos-2 cells had the wild-type of the DHFR. Conclusion In conclusion, this study showed that inverse change of the RFC and DHFR mRNA and protein expression was associated with RFC and DHFR polymorphisms and it is postulated that this phenomenon might play an important role in sensitivity of certain cancers to MTX.

Background: Sorafenib (BAY 43-9006) is a multi-kinase inhibitor that targets a serine-threonine kinase, B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, several potential mechanisms of its inhibitory activity on tumor cell proliferation, angiogenesis and anti-apoptosis have been proposed. Paclitaxel exhibits unique and potent anti-cancer activity in the treatment of non-small cell lung cancer (NSCLC). Our aim of this study, is to examine the anti-proliferative, anti-angiogenesis effect and apoptosis induction when sorafenib combined with paclitaxel in NSCLC tumor model. Methods: We investigated sorafenib's effect on cell proliferation, cell cycle distribution, ERK phosphorylation in A549 NSCLC cell line using SRB assay, flow cytometry, and Western blot analysis, respectively. In vivo, A549 tumor cells were implanted in the Balb/c nu/nu mice. Five different combination schedules of sorafenib and paclitaxel were applied with their single treatment. PPS and PSS combination therapy. Tumor volume and body weight was measured at two times per week. Intratumoral molecular changes were studied on paraffin-embedded tumor tissue block from xenograft mice using immunohistochemistry and the apoptotic change was measured with TUNEL assay. Result: In vitro study, single treatment of sorafenib or paclitaxel inhibited cell proliferation in A549 cells with dose-dependent manner. Sorafenib effectively inhibited the phosphorylation of ERK and induced G0/G1 arrest of cell cycle. When these two agents were combined with different schedules, schedule-dependent cytotoxicity and cell cycle arrest were observed. In A549 xenograft model, combination of sorafenib and paclitaxel more significantly suppressed tumor growth than their single treatment. However, PSS combination caused profound body weight loss compared with other treatment schedules. In immunohistochemistry study, greater inhibition of Ki-67 as well as pAKT and pERK, and CD31 were observed in PPS combination compared with single treatment. In TUNEL assay, significant apoptosis was observed in combination treatment. Conclusion: Taken together, sorafenib, paclitaxel, and their combination effectivity inhibited tumor growth and angiogenesis via down regulation of pAKT and pERK signaling and apoptosis induction in NSCLC tumor model. The PPS and PSS combination schedule demonstrated more potent antitumor activity including anti-proliferation, anti-angiogenesis, and apoptosis compared to other combination schedules. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A81.

e22166 Background: Several techniques are in use for HPV DNA detection: Hybrid Capture IIR(HCIIR) assay, PCR-based DNA microarray and Luminex-based assay. This study was aimed to examine the performances of three validated HPV assays. We compared the concordance between the three methods for detecting HPV in cervical specimen. Methods: Cervical samples of 189 Korean women (21∼77 ages, average 41.2 age) were tested for HPV detection in this study. We examined the performances of three validated HPV assays according to each method's instructions : 1) Hybrid Capture IIR (HCIIR) assay (HCIIR; Digene Corporation, USA) was used to test for detection of 18 types of HPV (high-risk types: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and low-risk types types: HPV 6, 11, 42, 43, 44). 2) HPV DNA Chip, PCR-based DNA microarray system (MyGene Co., Seoul, South Korea) was used to test for genotyping of 24 types of HPV: 15 types of high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68) and 9 types of low-risk types (HPV6, 11, 34, 40, 42, 43, 44, 54, 70). 3) Luminex-based HPV test (YBT, Seoul, Korea) was performed for genotyping 26 types of HPV including 15 high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68) and 11 low-risk types (HPV-6, 11, 34, 40, 42, 43, 44, 54, 55, 67, 70). Results: We compared HPV detection results obtained by the HC II assay with those from the Luminex assay. 51 cases were discordant: 29 samples were HC II negative/Luminex positive and 22 samples were HCII positive/Luminex negative. We also compared HPV detection results obtained by the DNA chip assay with those from the Luminex assay. 37 cases were discordant: 5 samples were DNA chip negative/Luminex positive and 32 samples were DNA chip positive/Luminex negative. Conclusions: The distribution of the HPV detection results obtained for 91 samples using three different methods suggested that the results of DNA chip assay are more often discordant (17.9% instead of 5.1% for Luminex assay and 5.1% for the HC II assay). No significant financial relationships to disclose.

17014 Background: FMTC is defined as the presence of MTC in kindred with four or more affected members and with objective evidence of the absence of adrenal and parathyroid gland involvement. An id...