Cheol Hyeon Kim

Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line

Epithelial to mesenchymal transition (EMT) has been reported to be related with reduced sensitivity to EGFR tyrosine kinase (EGFR-TK) inhibitors. We performed this study to investigate whether this phenomenon would play a role in acquired resistance to gefitinib. In this study, we established a gefitinib-resistant subline (A549/GR), which was derived from the parental A549 cell line by chronic, repeated exposure to gefitinib. Compared with the A549 cells, the A549/GR cells were approximately 7.7-fold more resistant to gefitinib and they showed the cross-resistance against other EGFR-TK inhibitors, including CL-387,758, erlotinib and ZD6478. Phenotypic changes such as a spindle-cell shape and increased pseudopodia formation suggesting EMT was present in the A549/GR cells. These changes were accompanied by a decrease of E-cadherin and an increase of vimentin, which is a mesenchymal marker. In addition, the ability of invasion and migration was increased in the A549/GR cells. TGF-beta1 treatment for 72 h also induced EMT in the A549 cells and this transition led to resistance to gefitinib. Conversely, this was reversed through the removal of TGF-beta1. In conclusion, induction of EMT may contribute to the decreased efficacy of therapy in primary and acquired resistance to gefitinib.

Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs.

BACKGROUND Epithelial-to-mesenchymal transition (EMT), which was related with an acquired resistance to gefitinib, was found in the A549 lung cancer cell line. However, the clinical feasibility of this finding is still questionable. Here, we investigated whether EMT could be detected in a more clinically suitable situation using patients tumor and cells with deletion mutation on exon 19 of EGFR gene. METHODS HCC827 cell line was used to establish the subline resistant to EGFR-TKIs. The induction of EMT was analyzed by immunostainings and Western blots in resistant cells and biopsied tissue from a patient with acquired resistance to erlotinib. Migration and invasion assay was performed to characterize the resistant cells. EMT-related genes expression was evaluated by cDNA microarray. Phospho-receptor tyrosine kinase array analysis was carried out to find bypass activating signals such as MET. RESULTS We found that EMT developed in a lung cancer patient who had an acquired resistance to erlotinib while there were no known resistant mechanisms such as T790M and MET amplification. CL-387,785-resistant cells (HCC827/CLR) were obtained by long-term exposure to increasing concentrations of CL-387,785 (an irreversible EGFR-TKI). The morphological and molecular maker changes compatible with EMT were also found in HCC827/CLR cells. However, there were also no secondary T790M mutation and MET amplification. Furthermore, the activity of most of tested RTKs including receptor HER family was decreased suggesting that there was no bypass activating signal leading to resistance. These cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold). CONCLUSION EMT should be considered as one of possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells.

EGFR Mutation and Brain Metastasis in Pulmonary Adenocarcinomas

Background: This study aimed to explore the potential association of mutation in the epidermal growth factor receptor (EGFR) with brain metastases in patients with pulmonary adenocarcinoma. Methods: We analyzed clinical data on 314 patients who were tested for EGFR mutation and underwent brain magnetic resonance imaging at diagnosis. The relationship between EGFR mutation status and brain metastases at the initial presentation was analyzed. In addition, prognostic significance of EGFR mutational status on the risk of brain metastasis was evaluated in subgroups of surgically treated patients. Results: Of the 314 patients, 138 patients (43.9%) had EGFR mutations. The frequency of EGFR mutation was statistically higher for patients with brain metastases (64.7%, brain metastases; 39.8%, no metastases; 40.2%, extracranial metastases; p = 0.005). A strong association between EGFR mutation status and brain metastasis was observed (adjusted odds ratio = 3.83, p = 0.001), whereas no association was observed between EGFR mutation status and extracranial metastases (adjusted odds ratio = 1.73, p = 0.079). In addition, the number of brain metastases was significantly correlated with the EGFR mutation status (p = 0.029). Further analysis of 133 patients treated with surgical resection showed that EGFR mutation status was a poor prognostic factor for the risk of brain metastasis (hazard ratio = 4.49, p = 0.026) after adjustment for pathologic N stage. Conclusions: We found a significant association between EGFR mutation and risk of brain metastases at the time of diagnosis and follow-up after curative resection for pulmonary adenocarcinoma. This result indicates the distinct clinical features of EGFR-mutated tumors in terms of brain metastases.

p53 Enhances Gefitinib-Induced Growth Inhibition and Apoptosis by Regulation of Fas in Non–Small Cell Lung Cancer

Treatment with gefitinib, a specific inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), has resulted in dramatic responses in some patients with non-small cell lung cancer (NSCLC). Most patients who respond to gefitinib have EGFR-TK mutations; however, >10% of patients with EGFR-TK mutations do not respond. Similarly, some patients without EGFR-TK mutations respond to this drug, suggesting that other factors determine sensitivity to gefitinib. Aberrations of the tumor suppressor gene p53 are frequently associated with drug resistance. In this study, we investigated the role of p53 in growth-inhibitory and apoptotic effects of gefitinib in the human NSCLC cell lines NCI-H1299 and A549, which have no EGFR-TK mutations. NCI-H1299 cells, which had a p53-null genotype, were more resistant to gefitinib compared with A549 cells, which were wild-type p53 (IC(50), 40 micromol/L in NCI-H1299 and 5 micromol/L in A549). Treatment of A549 with gefitinib resulted in the translocation of p53 from cytosol to nucleus and the up-regulation of Fas, which was localized to the plasma membrane. In the stable H1299 cell line with tetracycline-inducible p53 expression, induced p53 enhanced growth inhibition and apoptosis by gefitinib through the up-regulation of Fas and restoration of caspase activation. A caspase inhibitor, Z-VAD-fmk, reduced these effects. Conversely, inhibition of p53 using antisense oligonucleotide in A549 caused a significant decrease in apoptosis by gefitinib and down-regulation of Fas under the same conditions. In conclusion, p53 may play a role in determining gefitinib sensitivity by regulating Fas expression in NSCLC.

The Role of MET Activation in Determining the Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) seems almost inevitable, even in patients with lung cancer that initially respond well to EGFR-TKIs. MET amplification was recently found to be a mechanism of escape from the anticancer effect of EGFR inhibitors. In the present study, we investigated the means whereby MET affects sensitivity to EGFR-TKIs in PC-9 cells. Gefitinib- or erlotinib-resistant sublines were established by exposing the parental PC-9 cell line to chronic, repeated treatments with these drugs. These resistant sublines showed more than 100-fold more resistance to gefitinib and erlotinib and acquired cross-resistance to other EGFR-TKIs. The T790M EGFR mutation was found by pyrosequencing, and this seemed to be the cause of drug resistance. Resistant cells also showed MET activation, although gene amplification was not detected. Furthermore, the induction of MET activity was not found to be associated with sensitivity to EGFR-TKIs. Interestingly, increased passage number without exposure to gefitinib or erlotinib caused MET activation, but this did not affect sensitivity to EGFR-TKIs. In addition, hepatocyte growth factor was found to block the ability of EGFR-TKIs to inhibit MET activation. However, sustained MET activation by hepatocyte growth factor did not modulate the cellular effects of gefitinib or erlotinib. Rather, activated MET enhanced migration and invasion abilities. Summarizing, MET activation may be acquired during cancer cell proliferation and enhances migratory and invasive abilities without affecting cellular sensitivity to EGFR-TKIs. Accordingly, the present study suggests that MET activation caused by factors other than MET gene amplification is not a suitable surrogate marker of resistance to EGFR-TKIs. (Mol Cancer Res 2009;7(10):1736–43)

Use of Complementary and Alternative Medicine among Korean Cancer Patients

Background : Complementary and alternative medicine (CAM) is now being increasingly used among cancer patients. The objectives of our study were to assess the prevalence, types, cost, subjective effects, and side effects of CAM use, reasons for CAM use, characteristics of CAM users compared to those of nonusers, and patients’ expectations of doctors regarding their CAM use among Korean cancer patients at a single cancer center. Methods : From April to August, 2003, we interviewed 186 cancer patients hospitalized in the Korea Cancer Center Hospital using a structured questionnaire, and analyzed the data. Results : 78.5% of experimental subjects (146 patients) had been treated with at least one type of CAM, in addition to conventional Western treatment, with a mean monthly cost of 1,380,000 Won/person (approximately, 1,100 U.S. dollars on July, 2004). The most prevalent types of CAM used by these patients included medicinal mushrooms (67.1%), herbs (54.1%), vegetable diets (50.6%), and ginseng (46.5%). The main reported reasons for the use of CAM in addition to conventional medicine were nutritional support (19.1%) and physical strengthening (17.8%). 5% of CAM users experienced side effects. The younger and more educated the patients were, the more likely they were to employ CAM. 66% of CAM users wanted to discuss CAM techniques with their doctors. Conclusion : More than two-thirds of cancer patients used various kinds of CAM, incurring considerable costs. Therefore, in order to help patients make informed decisions, medical society should be open to communication with patients. Not only the scientific aspects, but also the economic aspects of CAM usage should be examined more thoroughly, in order to ensure proper distribution of medical resources.

18F-FDG uptake and EGFR mutations in patients with non-small cell lung cancer: A single-institution retrospective analysis

This retrospective study was performed to evaluate a possible association between the presence of epidermal growth factor receptor (EGFR) mutations and the standardized uptake value (SUV) of (18)F-fluoro-2-deoxy-glucose ((18)F-FDG) uptake in patients with non-small cell lung cancer (NSCLC). We included 100 patients who were tested for EGFR mutations by direct sequencing of resected tissues and who underwent preoperative positron emission tomography/computed tomography at the time of diagnosis. The maximum SUV by the primary tumor was chosen for further analysis. EGFR mutations in exons 19 and 21 were detected in 21 NSCLC patients (21%). EGFR mutations were more frequent in never-smokers than ever-smokers (35% versus 11%; P=0.003), in adenocarcinomas than non-adenocarcinomas (34% versus 6%; P=0.001), and in females than males (41% versus 12%; P=0.001). The SUV ranged from 1.3 to 33.0 (median 10.6). Area under receiver operating characteristic curve for SUVs in respect to the presence of EGFR mutations was 0.74 (95% CI: 0.62-0.85). When a cut off value was used, patients with low SUVs were more likely to have EGFR mutations than those with high SUVs (40% versus 11%; P=0.001). On multivariate analysis, a low SUV remained a significant predictors for EGFR mutations (P=0.025). (18)F-FDG uptake was associated with the presence of EGFR mutation. These results extrapolate that (18)F-FDG uptake might be helpful to discriminate patients who harbor EGFR mutations, especially when a genetic test is not feasible.

Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors

PurposeH1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer.MethodsThree human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots.ResultsH1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors α-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR.ConclusionCombined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.

18F-Fluoro-2-Deoxy-Glucose Uptake Predicts Clinical Outcome in Patients with Gefitinib-Treated Non–Small Cell Lung Cancer

Purpose: To evaluate response and survival according to 18F-fluoro-2-deoxy-glucose uptake at presentation in patients with gefitinib-treated non–small cell lung cancer. Experimental Design: We retrospectively analyzed 84 positron emission tomography/computed tomography findings. Patient characteristics, response rates, and survivals were evaluated according to the maximum standardized uptake value (SUV) of primary tumor. The cutoff value of SUVs was obtained from receiver operating characteristic analysis. Results: The response rate (RR) was higher for never-smokers (41%) than ever-smokers (9%; P = 0.001). Patients with adenocarcinoma showed higher RR than those with other tumor histopathology (35% versus 9%; P = 0.009). The SUV was significantly lower in patients who were never-smokers (P = 0.005), patients with adenocarcinoma (P < 0.001), and female patients (P = 0.017). Patients with a low SUV showed higher RR compared with those with a high SUV (53% versus 18%; P = 0.003). Prolonged progression-free survival was observed in patients with low SUVs compared with those with high SUVs (median, 33.1 weeks versus 8.6 weeks; P = 0.003). While controlling for performance status, smoking history, and pathology, the high SUV conferred unfavorable outcome (hazard ratio, 2.3; P = 0.012). In terms of overall survival, a low SUV was associated with favorable outcome in univariate analysis (P = 0.011). Patients with a low SUV showed prolonged survival in multivariate analysis (P = 0.043). Conclusions: These results suggest that low SUVs at presentation can predict favorable response and survival in gefitinib-treated non–small cell lung cancer patients.

Combined treatment with silibinin and epidermal growth factor receptor tyrosine kinase inhibitors overcomes drug resistance caused by T790M mutation.

Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) produce an initially dramatic response in lung cancer patients harboring a mutation in the EGFR gene, development of acquired resistance is almost inevitable. A secondary mutation of threonine 790 (T790M) is associated with approximately half of the cases of acquired resistance. This study investigated whether the addition of silibinin to therapy with gefitinib or erlotinib could overcome T790M-mediated drug resistance considering that silibinin has various antitumor effects, including EGFR modulation. Silibinin selectively reduced the activity of the EGFR family (EGFR, ErbB2, and ErbB3) through the inhibition of receptor dimerization in lung cancer cells with EGFR mutations, but not in those harboring the wild type. In primary and acquired resistant cells with T790M, addition of silibinin enhanced the ability of EGFR-TKIs to downregulate EGFR signals and to inhibit cell growth. Similarly, the combination of silibinin and erlotinib effectively suppressed tumor growth in erlotinib resistance-bearing PC-9 xenografts. The results indicate that the addition of silibinin to EGFR-TKIs is a promising strategy to overcome T790M-mediated drug resistance. Mol Cancer Ther; 9(12); 3233–43. ©2010 AACR.