Arum Kim

Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells

BackgroundmTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.MethodsComponents of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.ResultsIGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.ConclusionsIn conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.

Notch1 destabilizes the adherens junction complex through upregulation of the Snail family of E-cadherin repressors in non-small cell lung cancer.

One of the critical steps driving cancer cell migration and metastasis is the repression of cell adhesion molecules resulting in loss of cell‑to-cell adhesion. Although interactions between Notch1 and components of the adherens junction complex have been suggested, little is known concerning the consequence of their interactions. In this study, we investigated the interaction between the Notch1 and the E‑cadherin/β‑catenin complex, its effect on the expression of adherens junction complex components and its influence on non-small cell lung cancer (NSCLC) cell proliferation. With progression of lung neoplastic lesions in LSL K-ras G12D mice, the expression of E‑cadherin was inhibited whereas that of Notch1 was increased with frequent nuclear localization, suggesting an inverse relationship between E‑cadherin and Notch1 expression with tumor progression. Transduction of the human Notch1 intracellular domain (N1ICD) into NSCLC cells inhibited expression of E‑cadherin and β‑catenin and induced changes in the localization of adherens junction molecules. The loss of E‑cadherin was mediated through upregulation of the Snail family of transcription factors, Snail and Slug. Experiments in which siRNA against E-cadherin was introduced into NSCLC cells revealed that N1ICD decreased the expression of β‑catenin in an E‑cadherin‑independent manner, leading to inhibition of markers of Wnt/β‑catenin signaling activation. Despite inhibition of Wnt/β‑catenin signaling in the N1ICD‑transduced cells, cells transduced with N1ICD showed no difference in cell cycle progression when compared with that of the control vector-transduced cells. In conclusion, Notch1 inhibited the expression of E‑cadherin through upregulation of the Snail family of transcriptional factors, resulting in inhibition of expression of β‑catenin and destabilization of adherens junctions.

Genetic heterogeneity of actionable genes between primary and metastatic tumor in lung adenocarcinoma

BackgroundBiopsy for lung cancer diagnosis is usually done at a single site. But it is unclear that genetic information at one biopsy site represents that of other lesions and is sufficient for therapeutic decision making.MethodsNon-synonymous mutations and insertions/deletions of 16 genes containing actionable mutations, and intron 2 deletion polymorphism of Bcl2-like11 were analyzed in 41 primary tumor and metastatic lymph node (L/N) matched, pStage IIA ~ IIIA non-small cell lung cancer (NSCLC) samples using a next generation sequencing based technique.ResultsA total of 249 mutations, including 213 non-synonymous mutations, 32 deletions, and four insertions were discovered. There was a higher chance of discovering non-synonymous mutations in the primary tumors than in the metastatic L/N (138 (64.8%) vs. 75 (35.2%)). In the primary tumors, 106 G > A:C > T transitions (76.8%) of 138 non-synonymous mutations were detected, whereas in the metastatic L/N, 44 (58.7%) of 75 were discovered. A total 24 (11.3%) out of 213 non-synonymous mutations were developed in the context of APOBEC signature. Of those, 21 (87.5%) was detected in the primary tumors and 4 (16.7%) was detected in the metastatic L/N. When the mutation profiles between primary tumor and metastatic L/N were compared, 13 (31.7%) of 41 cases showed discrepant mutation profile. There were no statistically significant differences in disease free survival and overall survival between groups showing identical mutation profiles and those with discrepancy between primary and metastatic L/N.ConclusionsGenetic heterogeneity between the primary and L/N metastatic lesions is not infrequent finding to consider when interpreting genomic data based on the result of one site inspection. A large prospective study may be needed to evaluate the impact of genetic heterogeneity on the clinical outcomes of NSCLC patients.

AZD6244 inhibits cisplatin-induced ERK1/2 activation and potentiates cisplatin-associated cytotoxicity in K-ras G12D preclinical models

Although cisplatin has been widely used as a component of standard treatments for advanced non-small cell lung cancers (NSCLC) with KRAS-activating mutations, clinical outcomes remain suboptimal. Among the resistance mechanisms to cisplatin, activation of the MAPK cascade, which plays an important role in cancer cell stress and death, offers a promising therapeutic target. Using KRAS-mutant NSCLC cells and a mouse model, we evaluated the efficacy of adding the MEK1/2 inhibitor AZD6244 as an addition for cisplatin-based chemotherapy. Cisplatin increased phosphorylation of MEK1/2 and ERK1/2 and reduced Bcl-2 like 11 (BIM) expression in NSCLC cells and the mouse model. BIM silencing in NSCLC cells using shRNA led to a blunted cytotoxic response to cisplatin, while prevention of BIM loss with the MEK1/2 inhibitor synergized cisplatin-mediated cell death. The combination of cisplatin and AZD6244 yielded a superior response to cisplatin alone in K-ras mice. In conclusion, an MEK1/2 inhibitor potentiated the anti-tumor effects of cisplatin in KRAS-dependent lung cancer cells and an animal model through inhibition of BIM degradation. These findings warrant further studies of clinical applications of MEK1/2 inhibitors in cisplatin-based chemotherapy for lung cancer.

KRAS oncogene substitutions in Korean NSCLC patients: Clinical implication and relationship with pAKT and RalGTPases expression

OBJECTIVES Since different conformation of each KRAS mutant leads to inherent downstream signaling, its distribution, influence on the clinical outcome, and effect on the signaling mediators were investigated in the Korean NSCLC patients whose tumor have KRAS mutation. MATERIALS AND METHODS Mutation at KRAS codons 12 and 13 was evaluated in 1420 Korean NSCLC by direct sequencing and expression of RalA, RalB, and pAKT-Ser473 was evaluated by immunohistochemistry in 30 cases whose KRAS mutant tumor tissues were available. RESULTS Eighty-two (5.8%) out of 1420 patients harbored a KRAS mutation either in codon 12 or 13. Gly12Asp was the most frequent (34.1%), followed by Gly12Cys (22.0%) and Gly12Val (13.4%). Transversion at codons 12 and 13, which includes Gly12Cys, Gly12Val, Gly12Ala, Gly13Cys, and Gly12Phe was detected in 45 cases (54.9%) and transition, including Gly12Asp, Gly12Ser, and Gly13Asp was detected in 37 cases (45.1%). Male and smoking history were associated with transversion (p=0.001 and 0.006, respectively; χ(2)-test), and multivariate analysis showed that gender was an independent influencing factor (p=0.026; Cochran-Mantel-Haenszel test). Multivariate analysis on survival revealed that KRAS mutation subtype did not influence overall survival of the patients with KRAS mutations after adjustment for age, gender, performance status, and stage. There were no differences in the nuclear and cytoplasmic expression of pAKT-Ser473 between transversion and transition mutants. Expression of Ral-GTPases, RalA and RalB, did not differ between transversion and transition mutants, however, strong expression of RalB in the tissue of patients with KRAS mutants was associated with advanced stages (P-value=0.020, χ(2)-test). CONCLUSIONS In this study population, not only the frequency of KRAS mutation but also the distribution of its subtypes differed from those of Western studies, with unique influencing factors. Clinical outcome and expression of pAKT-Ser473, RalA, and RalB did not differ among subtypes.

ABT-737 Synergizes with Cisplatin Bypassing Aberration of Apoptotic Pathway in Non-small Cell Lung Cancer

A subset of non-small cell lung cancer (NSCLC), which does not have a druggable driver mutation, is treated with platinum-based cytotoxic chemotherapy, but it develops resistance triggered by DNA damage responses. Here, we investigated the effect of activation of STAT3 by cisplatin on anti-apoptotic proteins and the effectiveness of a co-treatment with cisplatin and a BH3 mimetic, ABT-737. We analyzed the relationship between cisplatin and STAT3 pathway and effect of ABT-737, when combined with cisplatin in NSCLC cells and K-ras mutant mouse models. The synergism of this combination was evaluated by the Chou-Talalay Combination Index method. In vivo activity was evaluated by micro-CT. In NSCLC cells, there was a time and dose-dependent phosphorylation of SRC-JAK2-STAT3 by cisplatin, followed by increased expression of anti-apoptotic molecules. When the expression of the BCL-2 protein family members was evaluated in clinical samples, BCL-xL was most frequently overexpressed. Dominant negative STAT3 suppressed their expression, suggesting that STAT3 mediates cisplatin mediated overexpression of the anti-apoptotic molecules. ABT-737 displaced BCL-xL from mitochondria and induced oligomerization of BAK. ABT-737 itself showed cytotoxic effects and a combination of ABT-737 with cisplatin showed strong synergistic cytotoxicity. In a murine lung cancer model, co-treatment with ABT-737 and cisplatin induced significant tumor regression. These findings reveal a synergistic cytotoxic and anti-tumor activity of ABT-737 and cisplatin co-treatment in preclinical models, and suggest that clinical trials using this strategy may be beneficial in advanced NSCLC.

MYC expression correlates with PD-L1 expression in non-small cell lung cancer.

Objectives Programmed death-ligand 1 (PD-L1) is a widely used biomarker for predicting immune checkpoint inhibitors, but is of limited usefulness in the prediction of drug response. MYC, a transcription factor that is overexpressed in cancers, is involved in preventing immune cells from attacking tumor cells through inducing PD-L1 expression. This study evaluated the relationship between MYC and PD-L1 expression in 84 non-small cell lung cancer (NSCLC) patients who underwent curative surgical resection. Materials and Methods The relationship between MYC and PD-L1 was investigated by introducing pcDNA3-cMYC into A549 and H1299 cells with low PD-L1 expression and siRNA against MYC into H60 and H2009 cells with high PD-L1 expression. Expression of PD-L1 in NSCLC tissues was analyzed by immunostaining using a PD-L1 (22C3) PharmDx protocol using the Dako Automated Link 48 platform and expression of MYC was determined using anti-c-MYC (Y69) (ab320720). Results Of 84 patients, PD-L1 was expressed in 14 (16.7%) and MYC was overexpressed in 30 (35.7%). We investigated the relationship between PD-L1 and MYC expression. There were 49 (58.3%) double-negative patients and 9 (10.7%) double-positive patients. Significant positive correlation was observed between PD-L1 and MYC expression (γ=0.210, P=0.029). Double-negative patients showed better disease free (31.1 vs. 7.1 months, P=0.011) and overall survival (56.1 vs. 14.4 months, P=0.032) than double-positive patients. Conclusion Taken together, MYC expression significantly correlated with PD-L1 expression in NSCLC. The usefulness of MYC expression as a surrogate marker of treatment response assessment is worth evaluating for immune checkpoint inhibitor therapy and special interest are required for the subgroup of NSCLC patients, whose tumor expresses PD-L1 and MYC double positive.

BCL2-like 11 intron 2 deletion polymorphism is not associated with non-small cell lung cancer risk and prognosis

OBJECTIVES BCL2-Like 11(BIM), which encodes a BH3-only protein, is a major pro-apoptotic molecule that facilitates cell death. We hypothesized that a BIM intron 2 deletion polymorphism increases lung cancer risk and predicts poor prognosis in non-small lung cancer (NSCLC) patients. MATERIALS AND METHODS We prospectively recruited 450 lung cancer patients and 1:1 age, sex, and smoking status matched control subjects from February 2013 to April 2014 among patients treated at Severance, Gangnam Severance, and Chonnam Hwasoon Hospital. The presence of a 2903-bp genomic DNA deletion polymorphism of intron 2 of BIM was analyzed by PCR and validated by sequencing. Odds ratios were calculated by chi-square tests and survival analysis with Kaplan-Meier estimation. RESULTS AND CONCLUSION Sixty-nine out of 450 (15.3%) lung cancer patients carried the BIM deletion polymorphism, while 66 out of 450 (14.7%) control subjects carried the BIM deletion polymorphism, with an odds ratio of for lung cancer of 1.054 (95% CI; 0.731-1.519). We categorized 406 NSCLC patients according to the presence of the polymorphism and found that there were no statistically significant differences in age, sex, histologic type, or stage between subjects with and without the deletion polymorphism. The BIM deletion polymorphism did not influence overall survival (OS) or progression free survival (PFS) in our sample (OS; 37.6 vs 34.4 months (P=0.759), PFS; 49.6 vs 26.0 months (P=0.434)). These findings indicate that the BIM deletion polymorphism is common in Korean NSCLC patients but does not significantly affect the intrinsic biologic function of BH3-only protein. Furthermore, the BIM deletion polymorphism did not predict clinical outcomes in patients with NSCLC.

Keratinization of Lung Squamous Cell Carcinoma Is Associated with Poor Clinical Outcome

Background Although the World Health Organization (WHO) classification of lung squamous cell carcinoma (SCC) was revised in 2015, its clinical implications for lung SCC subsets remain unclear. We investigated whether the morphologic characteristics of lung SCC, including keratinization, were associated with clinical parameters and clinical outcome of patients. Methods A total of 81 patients who underwent curative surgical resection of diagnosed lung SCC, were enrolled in this study. Attributes such as keratinization, tumor budding, single cell invasion, and nuclear size within the tumor, as well as immunohistochemistry of Bcl-xL and pS6 expressions, were evaluated. Results The keratinizing and nonkeratinizing subtypes did not differ with respect to age, sex, TNM stage, and morphologic parameters such as nuclear diameter, tumor budding, and single cell invasion at the tumor edge. Most patients with the keratinizing subtype (98.0%) had a history of smoking, whereas the nonkeratinizing group had a relatively higher proportion of never-smokers relative to the keratinizing group (24.0% vs. 2.0%; p=0.008, chi-square test). Expression of pS6 (a surrogate marker of mammalian target of rapamycin complex 1 [mTORC1] signaling that regulates keratinocyte differentiation), and Bcl-xL (a key anti-apoptotic molecule that may inhibit keratinization), did not correlate significantly with the presence of keratinization. Patients with the keratinizing subtype had a significantly shorter overall survival (85.2 months vs. 135.7 months, p=0.010, log-rank test), and a multivariate analysis showed that keratinization was an independent, poor prognostic factor (hazard ratio, 2.389; 95% confidence interval, 1.090–5.233; p=0.030). Conclusion In lung SCC, keratinization is associated with a poor prognosis, and might be associated with smoking.

P2.13-12 The deNovo T790M Mutation Recovered by ddPCR is Associated with a Poor Response to 1st and 2nd Generation EGFR-TKI in Lung Adenocarcinoma.

clinical trials. Increased AXL expression is associated with innate immune suppression and the appearance of resistance to targeted therapies in models of NSCLC and pt samples. AXL inhibition via bemcentinib prevents the appearance of such resistance in vivo and has shown immunomodulatory effect in AML patients. Method: This study was designed to confirm the safety and tolerability of bemcentinib as a monotherapy and when administered in combination with erlotinib (arm A). Pts with activating EGFR mutation driven NSCLC who had progressed on an approved EGFR inhibitor (arm B) or were receiving erlotinib in the first line setting (arm C) were treated with bemcentinib at RP2D in combination with full dose erlotinib to evaluate the potential of bemcentinib to reverse or prevent resistance to EGFR targeted therapy, respectively. Plasma protein biomarker levels were measured using the DiscoveryMap v3.3 panel (Myriad RBM) at C1D1 and C2D1. Result: 2 out of 8 pts (25%) with stage IV disease who received bemcentinib monotherapy achieved SD for close to 1 year including evidence of tumour shrinkage of 19% in 1 pt. 1 pt who had progressed on previous erlotinib monotherapy (12.5%) achieved a PR receiving bemcentinib in combination with erlotinib and remains on treatment well beyond 2 years later (arm A). A further 3 pts had SD at 6 wks. 11 patients (4 female, median age 58; 38-67) were enrolled in arm B and had received a median of 2 (1 4) previous lines of cytotoxic chemotherapy and a median of 2 previous EGFR inhibitors. 2 of these 11 pts (18%) including 1 pt who was refractory to erlotinib therapy at the onset of combination therapy remain on treatment more than 6 months into therapy at the time of writing with best responses of PR and SD, respectively. 1 further pt had SD at 6 weeks. The most common treatment-related AEs have been gastrointestinal and rash.There was no evidence of any impact of bemcentinib on erlotinib pharmacokinetics. Protein biomarkers predictive of pt benefit following bemcentinib treatment were identified. Conclusion: Bemcentinib can be safely administered in combination with erlotinib to pts with NSCLC and achieves additional benefit in a proportion of patients who do not have T790M and have progressed on EGFR inhibition or are maintained on erlotinib alone. Clinical trial information: NCT02424617.