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1.  Acquired Resistance to EGFR Inhibitors Is Associated with a Manifestation of Stem cell–like Properties in Cancer Cells 
Cancer research  2013;73(10):3051-3061.
Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical problem in the treatment of lung cancer. Although several mechanisms have been shown to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR-mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported, alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial-to-mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited downregulation of miRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell–like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side-population, and self-renewal capability. Resistant sublines with stem cell–like properties were resistant to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was upregulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell–like property with EGFR-TKI treatment.
PMCID: PMC4506773  PMID: 23542356
2.  TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells 
PLoS ONE  2015;10(6):e0129838.
TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.
PMCID: PMC4474554  PMID: 26090892
3.  Anti-Cancer Effects of REIC/Dkk-3-encoding Adenoviral Vector for the Treatment of Non-small Cell Lung Cancer 
PLoS ONE  2014;9(2):e87900.
REIC/Dkk-3 is down-regulated in a broad range of human cancer cells and is considered to function as a tumor suppressor. We previously reported that REIC/Dkk-3-expressing adenovirus vector (Ad-REIC) induced endoplasmic reticulum (ER) stress and cancer-specific apoptosis in human prostate cancer. In this study, we examined the therapeutic impact of Ad-REIC on non-small cell lung cancer (NSCLC).
Materials and Methods
We examined the anti-tumor effect of Ad-REIC on 25 NSCLC cell lines in vitro and A549 cells in vivo. Two of these cell lines were artificially established as EGFR-tyrosine kinase inhibitor (TKI) resistant sublines.
Ad-REIC-treatment inhibited the cell viability by 40% or more in 13 (52%) of the 25 cell lines at multiplicity of infection (MOI) of 20 (20 MOI). These cell lines were regarded as being highly sensitive cells. The cell viability of a non-malignant immortalized cell line, OUMS-24, was not inhibited at 200 MOI of Ad-REIC. The effects of Ad-REIC on EGFR-TKI resistant sublines were equivalent to those in the parental cell lines. Here, we demonstrated that Ad-REIC treatment activated c-Jun N-terminal kinase (JNK) in NSCLC cell lines, indicating the induction of ER stress with GRP78/BiP (GRP78) up-regulation and resulting in apoptosis. A single intratumoral injection of Ad-REIC significantly inhibited the tumorigenic growth of A549 cells in vivo. As predictive factors of sensitivity for Ad-REIC treatment in NSCLC, we examined the expression status of GRP78 and coxsackievirus and adenovirus receptor (CAR). We found that the combination of the GRP78 and CAR expressional statuses may be used as a predictive factor for Ad-REIC sensitivity in NSCLC cells.
Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. Our study indicated that Ad-REIC has therapeutic potential against NSCLC and that the expression statuses of GRP78 and CAR may predict a potential therapeutic benefit of Ad-REIC.
PMCID: PMC3912155  PMID: 24498395
4.  Novel Germline Mutation in the Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas 
We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas.
PMCID: PMC3906987  PMID: 24317180
5.  Impact of aberrant methylation of microRNA-9 family members on non-small cell lung cancers 
Molecular and Clinical Oncology  2012;1(1):185-189.
MicroRNAs (miRs) contribute to cancer development and progression by acting as oncogenes and tumor suppressor genes. miR-9 family members (miR-9s), including miR-9-1, 9-2 and 9-3, have been shown to be oncogenically involved through the downregulation of E-cadherin expression, which promotes the epithelial-mesenchymal transition. Tumor suppressive roles of miR-9s have also been reported to silence miR-9 through methylation, which is associated with an shortened overall survival (OS) period in several types of cancer. In this study, the impact of miR-9s methylation on non-small cell lung cancers (NSCLC) was investigated. In total, 293 resected NSCLC samples were examined and the miR-9s methylation status was determined using a combined bisulfite restriction analysis. miR-9 expression was analyzed by in situ hybridization. Methylation of miR-9-1, 9-2 and 9-3 was present in 20 (7%), 33 (11%) and 34 (12%) of the cases, respectively. Methylation of any miR-9s (miR-9s methylation) was observed in 76 of the cases (26%), and miR-9 expression was silenced in cases with miR-9s methylation. Logistic regression analysis demonstrated that male gender [odds ratio (OR), 2.0; 95% confidence interval (95% CI), 1.1–3.6; P=0.01] and pathologically negative lymph node metastasis (OR, 4.8; 95% CI, 1.4–17.2; P=0.002) were independent relative factors for miR-9s methylation. Additionally, miR-9s methylation [hazard ratio (HR), 4.2; 95% CI, 1.2–27.0; P=0.026] and early pathological stage (HR, 8.3; 95% CI, 2.1–28.6; P=0.004) were found to be independent predictive factors for prolonged OS time by the Cox proportional hazard test. miR-9s methylation which induces expression silencing is common in NSCLC cases without lymph nodal metastasis, suggesting that miR-9s are oncogenically involved in NSCLC carcinogenesis through the promotion of tumor metastasis.
PMCID: PMC3956236  PMID: 24649145
lung cancer; non-small cell lung cancer; microRNA; miR-9; methylation; in situ hybridization
6.  Biological Sensors for Solar Ultraviolet Radiation 
Sensors (Basel, Switzerland)  2011;11(4):4277-4294.
Solar ultraviolet (UV) radiation is widely known as a genotoxic environmental agent that affects Earth ecosystems and the human population. As a primary consequence of the stratospheric ozone layer depletion observed over the last decades, the increasing UV incidence levels have heightened the concern regarding deleterious consequences affecting both the biosphere and humans, thereby leading to an increase in scientific efforts to understand the role of sunlight in the induction of DNA damage, mutagenesis, and cell death. In fact, the various UV-wavelengths evoke characteristic biological impacts that greatly depend on light absorption of biomolecules, especially DNA, in living organisms, thereby justifying the increasing importance of developing biological sensors for monitoring the harmful impact of solar UV radiation under various environmental conditions. In this review, several types of biosensors proposed for laboratory and field application, that measure the biological effects of the UV component of sunlight, are described. Basically, the applicability of sensors based on DNA, bacteria or even mammalian cells are presented and compared. Data are also presented showing that on using DNA-based sensors, the various types of damage produced differ when this molecule is exposed in either an aqueous buffer or a dry solution. Apart from the data thus generated, the development of novel biosensors could help in evaluating the biological effects of sunlight on the environment. They also emerge as alternative tools for using live animals in the search for protective sunscreen products.
PMCID: PMC3231322  PMID: 22163847
sunlight; UV radiation; biosensors; biological dosimetry; DNA damage
7.  The estrogen receptor influences microtubule-associated protein tau (MAPT) expression and the selective estrogen receptor inhibitor fulvestrant downregulates MAPT and increases the sensitivity to taxane in breast cancer cells 
Microtubule-associated protein tau (MAPT) inhibits the function of taxanes and high expression of MAPT decreases the sensitivity to taxanes. The relationship between estrogen receptor (ER) and MAPT in breast cancer is unclear. In this study, we examined the correlation of MAPT expression with the sensitivity of human breast cancer cells to taxanes, and the relationship between ER and MAPT.
The correlation between MAPT expression and sensitivity to taxanes was investigated in 12 human breast cancer cell lines. Alterations in cellular sensitivity to taxanes were evaluated after knockdown of MAPT expression. ER expression was knocked down or stimulated in MAPT- and ER-positive cell lines to examine the relationship between ER and MAPT. The cells were also treated with hormone drugs (tamoxifen and fulvestrant) and taxanes.
mRNA expression of MAPT did not correlate with sensitivity to taxanes. However, expression of MAPT protein isoforms of less than 70 kDa was correlated with a low sensitivity to taxanes. Downregulation of MAPT increased cellular sensitivity to taxanes. MAPT protein expression was increased by stimulation with 17-β-estradiol or tamoxifen, but decreased by ER downregulation and by fulvestrant, an ER inhibitor. The combination of fulvestrant with taxanes had a synergistic effect, whereas tamoxifen and taxanes had an antagonistic effect.
Expression of MAPT protein isoforms of less than 70 kDa is correlated with a low sensitivity to taxanes in breast cancer cells. ER influences MAPT expression and fulvestrant increases the sensitivity to taxanes in MAPT- and ER-positive breast cancer cells.
PMCID: PMC2917038  PMID: 20579400
8.  PIK3CA Mutations and Copy Number Gains in Human Lung Cancers 
Cancer research  2008;68(17):6913-6921.
We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.
PMCID: PMC2874836  PMID: 18757405
9.  Genetic predictors of MEK-dependence in non-small cell lung cancer 
Cancer research  2008;68(22):9375-9383.
Hyperactivated ERK signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS and upstream receptor tyrosine kinases. In order to characterize the MEK/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n= 87) and tumors (n=916) for BRAF mutations. We found that NSCLC cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition, compared to those harboring mutations in EGFR, KRAS, or ALK and ROS kinase fusions. Supporting its classification as a “driver” mutation in the cells in which it is expressed, MEK inhibition in V600EBRAF NSCLC cells led to substantial induction of apoptosis, comparable to that seen with EGFR kinase inhibition in EGFR-mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR-mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF-mutant cell lines were resistant to EGFR inhibition. These data, together with the non-overlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hot-spot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors, and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared to those whose tumors harbor kinase domain mutations in EGFR.
PMCID: PMC2649746  PMID: 19010912
BRAF; non-small cell lung cancer; MEK inhibitor; PD0325901; EGFR mutation; gefitinib; erlotinib
10.  MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors 
We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC.
PMCID: PMC2767331  PMID: 19117057
MET; amplification; EGFR; gefitinib; lung cancer
11.  Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells 
PLoS ONE  2009;4(10):e7464.
Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.
Methodology/Principal Findings
We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.
MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.
PMCID: PMC2757721  PMID: 19826477
12.  Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung 
We investigated EGFR and KRAS alterations among atypical adenomatous hyperplasia and small lung adenocarcinomas with bronchioloalveolar features to understand their role during multistage pathogenesis.
Sixty lesions measuring 2 cm or less were studied, including 38 noninvasive lesions (4 atypical adenomatous hyperplasias, 19 Noguchi type A and 15 type B) and 22 invasive lesions (type C) based on the World Health Organization classification and Noguchi’s criteria. EGFR and KRAS mutations were examined using PCR-based assays. EGFR copy number was evaluated using fluorescence in situ hybridization.
EGFR and KRAS mutations were found in 26 (43.3%) and 5 (8.3%) lesions, respectively. Increased EGFR copy number status was identified in 10 lesions (16.7%), both mutant and wild type. EGFR or KRAS mutations were present in 39.5% and 7.9% (respectively) of noninvasive lesions and 50% or 9.1% (respectively) of invasive lesions. EGFR copy number was increased in 7.9% and 31.8% of noninvasive and invasive lesions (P = 0.029). Multivariate analysis revealed that increased EGFR copy number was the only significant factor to associate with invasive lesions (P = 0.035).
EGFR and KRAS mutations occur early during the multistage pathogenesis of peripheral lung adenocarcinomas. By contrast, increased EGFR copy number is a late event during tumor development and plays a role in the progression of lung adenocarcinoma independent of the initiating molecular events.
PMCID: PMC2758162  PMID: 18379350
Multistage pathogenesis; EGFR; KRAS; Mutation; Amplification
13.  Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines 
PLoS ONE  2009;4(2):e4576.
Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.
Methodology/Principal Findings
We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results:
 1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance.
 2. mKRAS was associated with increased in vitro resistance to gefitinib.
Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.
PMCID: PMC2642732  PMID: 19238210

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