Chang-Min Choi

MET and AXL Inhibitor NPS-1034 Exerts Efficacy against Lung Cancer Cells Resistant to EGFR Kinase Inhibitors Because of MET or AXL Activation

In non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations, acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKI) can occur through a generation of bypass signals such as MET or AXL activation. In this study, we investigated the antitumor activity of NPS-1034, a newly developed drug that targets both MET and AXL, in NSCLC cells with acquired resistance to gefitinib or erlotinib (HCC827/GR and HCC827/ER, respectively). Characterization of H820 cells and evaluation of NPS-1034 efficacy in these cells were also performed. The resistance of HCC827/GR was mediated by MET activation, whereas AXL activation led to resistance in HCC827/ER. The combination of gefitinib or erlotinib with NPS-1034 synergistically inhibited cell proliferation and induced cell death in both resistant cell lines. Accordingly, suppression of Akt was noted only in the presence of treatment with both drugs. NPS-1034 was also effective in xenograft mouse models of HCC827/GR. Although the H820 cell line was reported previously to have T790M and MET amplification, we discovered that AXL was also activated in this cell line. There were no antitumor effects of siRNA or inhibitors specific for EGFR or MET, whereas combined treatment with AXL siRNA or NPS-1034 and EGFR-TKIs controlled H820 cells, suggesting that AXL is the main signal responsible for resistance. In addition, NPS-1034 inhibited cell proliferation as well as ROS1 activity in HCC78 cells with ROS1 rearrangement. Our results establish the efficacy of NPS-1034 in NSCLC cells rendered resistant to EGFR-TKIs because of MET or AXL activation or ROS1 rearrangement.

Transglutaminase 2 as an independent prognostic marker for survival of patients with non-adenocarcinoma subtype of non-small cell lung cancer

BackgroundExpression of transglutaminase 2 (TGase 2) is related to invasion and resistance to chemotherapeutic agents in several cancer cells. However, there has been only limited clinical validation of TGase 2 as an independent prognostic marker in cancer.MethodsThe significance of TGase 2 expression as an invasive/migratory factor was addressed by in vitro assays employing down-regulation of TGase 2. TGase 2 expression as a prognostic indicator was assessed in 429 Korean patients with early-stage non-small cell lung cancer (NSCLC) by immunohistochemical staining.ResultsTGase 2 expression increased the invasive and migratory properties of NSCLC cells in vitro, which might be related to the induction of MMP-9. In the analysis of the immunohistochemical staining, TGase 2 expression in tumors was significantly correlated with recurrence in NSCLC (p = 0.005) or in the non-adenocarcinoma subtype (p = 0.031). Additionally, a multivariate analysis also showed a significant correlation between strong TGase 2 expression and shorter disease-free survival (DFS) in NSCLC (p = 0.029 and HR = 1.554) and in the non-adenocarcinoma subtype (p = 0.030 and HR = 2.184). However, the correlation in the adenocarcinoma subtype was not significant.ConclusionsTGase 2 expression was significantly correlated with recurrence and shorter DFS in NSCLC, especially in the non-adenocarcinoma subtype including squamous cell carcinoma.

Multiple resistant factors in lung cancer with primary resistance to EGFR-TK inhibitors confer poor survival.

OBJECTIVESnEGFR activating mutations have been recognized as the most important predictor of response to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, 20-30% of patients harboring EGFR activating mutations show a poor response requiring investigation for underlying mechanisms.nnnMATERIALS AND METHODSnCharacteristics of 541 patients with lung cancer harboring EGFR activating mutations were analyzed to determine contributing factors that could differentiate responders and non-responders. In addition, previously suggested moleculo-pathologic factors of resistance such as IκB, IGF1R, PTEN, MET, AXL and BIM were evaluated in patients exhibiting primary resistance who had sufficient biopsied tissues available for analyses.nnnRESULTSnResponders to EGFR-TKIs had a higher incidence of deletion mutations and more frequent presence of EGFR amplifications than non-responders. The median OS was 21 months (95% CI 26.1-30.4) in responders compared to 8 months (95% CI 8.7-15.8) in non-responders (p<0.001). In analyses of patients with primary resistance, we found that 27.3% (6/22) of them exhibited decreased expression of IκB, and 9.1% (2/22) of patients showed increased expression of IGF1R. Loss of PTEN was noted in 54.5%, and BIM polymorphism was found in 19% of patients. No patients had MET amplification, while expression of AXL was detected in 5 patients. Two patients had simultaneous T790M EGFR or PIK3CA mutation alongside EGFR activating mutation. Most of patients exhibited multiple abnormalities of these factors. The overall survival was worse in the group with multiple resistant factors.nnnCONCLUSIONnOur study suggests that mechanisms of primary resistance may be more complex than those underlying acquired resistance, with several factors concomitantly contributing to primary resistance.

Role of IGF-Binding Protein 3 in the Resistance of EGFR Mutant Lung Cancer Cells to EGFR-Tyrosine Kinase Inhibitors

Most patients treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) eventually develop acquired resistance. Loss of expression of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been suggested as a possible mechanism of resistance to EGFR-TKIs in the A431 and HN11 cell lines. Here, we investigated IGFBP-3 expression in two EGFR mutant lung cancer cell lines with resistance to EGFR-TKIs and examined the value of serum IGFBP-3 level as a marker of resistance. The effect of the induction or suppression of IGFBP-3 expression on resistance was also evaluated. HCC827 sublines with resistance to gefitinib (HCC827/GR) and erlotinib (HCC827/ER) were established. Loss of IGFBP-3 expression was detected by Western blotting in both cell lines without changes in transcriptional activity, and ELISA showed significantly lower amounts of secreted IGFBP-3 in the culture media of the mutant cell lines than in that of the parental line. Despite the loss of IGFBP-3 expression, IGFR signalling activity remained unchanged. Forced expression of IGFBP-3 by adenovirus-mediated transfection or recombinant IGFBP-3 slightly increased the growth-inhibitory and apoptotic effects of EGFR-TKIs, whereas suppression of IGFBP-3 did not affect sensitivity to EGFR-TKI. Serum IGFBP-3 levels measured by ELISA before and after the development of EGFR-TKI resistance in 20 patients showed no significant changes (1815.3±94.6 ng/mL before treatment vs. 1778.9±87.8 ng/mL after EGFR-TKI resistance). In summary, although IGFBP-3 downregulation is associated with the acquisition of resistance to EGFR-TKIs regardless of the mechanism, its effect on resistance was not significant, indicating that IGFBP-3 may not play an important role in resistance to EGFR-TKIs and serum IGFBP-3 level is not a reliable indicator of resistance.

Epidermal growth factor receptor mutations and brain metastasis in patients with nonadenocarcinoma of the lung

OBJECTIVEnThis study explored the potential association between epidermal growth.factor receptor. (EGFR) mutation status and brain metastasis in patients with nonadenocarcinoma nonsmall cell lung cancer. (NSCLC).nnnPATIENTS AND METHODSnWe analyzed clinical data from 286 patients with nonadenocarcinoma NSCLC, who were tested for EGFR mutations and underwent brain magnetic resonance imaging at diagnosis. We examined the relationship between EGFR mutation and brain metastasis at initial presentation.nnnRESULTSnOf the 286 patients, 20 patients (7.0%) had EGFR mutations. EGFR mutations were more frequent in younger patients (11.1% in patients =64 years vs. 3.3% in patients >64 years: P = 0.01), females (21.4% vs. 3.5% in males: P <0.001), never-smokers (25.0% vs. 3.4% in smokers: P < 0.001), and tumors with nonsquamous histology (25.0% vs. 4.1% in squamous histology: P < 0.001). At diagnosis, the frequency of EGFR mutations was significantly different in patients with metastasis to different sites (4.0% [no metastases] vs. 10.4% [extracranial metastases] vs. 40.0% [brain metastases], P < 0.001). The strong association between EGFR mutation and brain metastasis remained significant in multivariate analysis (adjusted odds ratio [OR] = 9.68, 95% confidence interval [CI] =2.32-40.45; P = 0.002). Associations were also found for EGFR mutation status with nonsquamous histology (adjusted OR = 4.46, 95% CI = 1.46-13.56; P = 0.008).nnnCONCLUSIONnThis study indicates that the likelihood of nonadenocarcinoma patients having EGFR mutant tumors may differ according to brain metastasis and squamous cell histology.

AXL and MET receptor tyrosine kinases are essential for lung cancer metastasis

The AXL and MET receptors regulate key processes in tumor growth, metastasis, and drug resistance; thus, they have recently been implicated as promising therapeutic targets in various tumors. We investigated the metastatic potential and crosstalk between these receptors in non‑small cell lung cancer (NSCLC). We found that the treatment of NSCLC cells with hepatocyte growth factor (HGF) and growth arrest-specific 6 (Gas6), as ligands for MET and AXL, respectively, promoted their migration and invasion ability. However, treatment with inhibitors of each of these receptors significantly reduced the migratory and invasiveness of the cells, although their inhibitory rates varied according to the inhibition of each receptor. In addition, the suppression of each receptor by shRNA resulted in reduced migration and invasiveness. Notably, the suppression of AXL was more effective than the suppression of MET in the inhibition of migration and invasion. In accordance with in vitro results, when the cells were transferred via tail vein injection, AXL inhibition was more efficient in attenuating metastasis than MET inhibition. Clinically, AXL or MET expression is associated with a poor prognosis in primary tumors of NSCLC. In summary, AXL and MET can regulate tumor metastasis, but AXL was shown to be more potent than MET in lung metastasis. Thus, we conclude that AXL might be a suitable therapeutic target for the inhibition of lung metastasis.

Association between thyroid cancer and epidermal growth factor receptor mutation in female with nonsmall cell lung cancer

BACKGROUND: The aim of this study was to investigate the association between epidermal growth factor receptor (EGFR) mutation and thyroid cancer in female patients with nonsmall-cell lung cancer (NSCLC). METHODS: In a retrospective study, we examined 835 female patients who were diagnosed with NSCLC and underwent an EGFR mutation test between June 2003 and August 2013. The associations of EGFR mutation with thyroid cancer and a family history of thyroid cancer were evaluated using logistic regression models. RESULTS: EGFR mutation was found in 378 of 835 patients. In addition to adenocarcinoma (P < 0.001), EGFR mutations were positively associated with a personal history of thyroid cancer (5.8% versus 2.6%; P = 0.020), while showing a trend toward inverse association with a personal history of nonthyroid cancer (5.8% vs. 9.0%; P = 0.086). Likewise, the incidence of EGFR mutations was associated with a family history of thyroid cancer (2.9% vs. 0.9%; P = 0.028), while showing a trend toward inverse association with a family history of nonthyroid cancer (27.8% vs. 33.7%; P = 0.066). Multivariate logistic regression showed that the incidence of EGFR mutations was different in women with thyroid or nonthyroid cancer (P = 0.035) and in women with a family history of thyroid or nonthyroid cancer (P = 0.023). CONCLUSIONS: Our data suggest that thyroid cancer and a family history of thyroid cancer are associated with EGFR-mutated NSCLC in female patients. The differences in the incidence of thyroid cancer and a family history of thyroid cancer by EGFR mutational status provide new insight into pathogenesis of this genetic change.

Abstract 55: Glucose supports EGFR-mutated lung adenocarcinoma survival by sustaining EGFR stability

Tumor cells are genetically unstable, driven by genetic mutations that stimulate cancer cell proliferation and survival. These genetic mutations are generally linked to particular types of cancer. Oncogenic epidermal growth factor receptor (EGFR) is frequently mutated in non-small cell lung cancer (NSCLC), which is essential for NSCLC development and growth. However, the precise roles of EGFR in lung cancer metabolism are still unclear. Here we show that glucose (Glc) metabolism is critical for EGFR stability required for tumor growth and survival. EGFR mutation leads to a robust increase in Glc uptake, lactate production and glycolytic flux. Moreover, EGFR-mutated NSCLC cell lines are much more sensitive than EGFR WT to inhibition of Glc metabolism through EGFR degradation, suggesting that EGFR mutant NSCLCs rely more heavily than EFGR WT on aerobic glycolysis. Knockdown of EGFR in EGFR-mutated NSCLCs results in a decrease in expression level of genes that encode glycolysis enzymes, indicating that EGFR mutation leads to an increase in Glc metabolism and Glc dependence for growth and survival. Furthermore, inhibition of Glc metabolism shows a significant decrease in intracellular ATP, oxygen consumption and level of TCA cycle intermediates. Indeed, the addition of pyruvate dramatically rescues apoptotic cell death caused by EGFR degradation upon inhibition of Glc metabolism, suggesting that Glc to fuel the TCA cycle is essential for tumor survival. In addition, the inhibition of Glc metabolism overcomes T790M-mediated resistance to EGFR-TKIs. Together, our data suggest that the EGFR mutation mediates the reprogramming of Glc metabolism and this metabolic alteration represents an attractive target for new therapy of EGFR-mutated NSCLC. Citation Format: Jin Kyung Rho, Yun Jung Choi, Chang-Min Choi, Jae Cheol Lee. Glucose supports EGFR-mutated lung adenocarcinoma survival by sustaining EGFR stability. [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 55.

Abstract 1837: A HSP90 inhibitor, AUY922, is effective to overcome the MET- and AXL-mediated resistance to EGFR-TKI in lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnActivation of bypass signals such as MET and AXL has been identified as one of possible resistant mechanisms to EGFR-TKI. Since various oncoproteins are dependent on HSP90 for its maturation and stability, we investigated the effect of AUY922, a newly developed HSP90 inhibitor of non-geldanamycin class, in lung cancer cells with MET- and AXL-mediated resistance. We established resistant cell lines with HCC827 harboring the deletion mutation on exon 19 of EGFR gene by long-term exposure to increasing concentrations of gefitnib and erlotinib (designated as HCC827/GR and HCC827/ER, respectively). The resistance of HCC827/GR was mediated by MET activation, whereas AXL activation caused resistance in HCC827/ER. AUY-922 treatment effectively suppressed the proliferation and induced cell death in both resistant cell lines. Accordingly, down-regulation of MET and AXL leading to decreased activation of Akt was noted. The inhibitory effect of AUY-922 on AXL and MET was also confirmed in AXL or MET-transfected LK-2 cells. In addition, AUY-922 led to reduced capability of invasion and migration of resistant cells. AUY-922 was effective to control the tumor growth in xenograft mouse models of HCC827/GR and HCC827/ER. Our study showed AUY-922 is one of promising therapeutic options for the MET- and AXL-mediated resistance to EGFR-TKI in lung cancer.nnCitation Format: Jin Kyung Rho, Yun Jung Choi, Seon Ye Kime, Gwang Sup So, Se Hoon Choi, Chang-Min Choi, Jae Cheol Lee. A HSP90 inhibitor, AUY922, is effective to overcome the MET- and AXL-mediated resistance to EGFR-TKI in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1837. doi:10.1158/1538-7445.AM2014-1837