Gefitinib-resistant Non-small Cell Lung Cancer Cell Lines Research Articles

MiRNA-153-3p promotes gefitinib-sensitivity in non-small cell lung cancer by inhibiting ATG5 expression and autophagy.

To clarify the function of miRNA-153-3p in gefitinib-sensitive non-small cell lung cancer (NSCLC) and the underlying mechanism. The expressions of miRNA-153-3p, LC3B and ATG5 in gefitinib-resistant and gefitinib-sensitive NSCLC tissues were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between miRNA-153-3p to LC3B or ATG5 was analyzed. We evaluated autophagy level in gefitinib-resistant NSCLC cells by calculating the percentage of PC-9/GR and HCC827/GR cells with positive GFP-LC3, as well as determining autophagy-related gene levels. The potential binding between ATG5 and miRNA-153-3p were verified by Dual-Luciferase reporter gene assay. The regulatory effects of miRNA-153-3p/ATG5 on gefitinib-sensitivity and apoptosis were finally examined by cytotoxicity assay and Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) staining, respectively. MiRNA-153-3p was lowly expressed in gefitinib-resistant NSCLC relative to the gefitinib-sensitive ones. MiRNA-153-3p was negatively correlated with autophagy activity marker LC3B in gefitinib-resistant NSCLC patients. Compared with parental cells, gefitinib-resistant NSCLC cell lines PC-9/GR and HCC827/GR presented a lower level of miRNA-153-3p and a higher level of autophagy. The overexpression of miRNA-153-3p greatly inhibited autophagy level. ATG5 could directly bind to miRNA-153-3p, and ATG5 was highly expressed in gefitinib-resistant NSCLC. The correlation analysis found a negative correlation between ATG5 and miRNA-153-3p and a positive correlation between ATG5 and LC3B in gefitinib-resistant NSCLC. More importantly, ATG5 reversed the regulatory effects of miRNA-153-3p on autophagy, gefitinib-sensitivity and apoptosis of PC-9/GR and HCC827/GR cells. MiRNA-153-3p is lowly expressed in gefitinib-resistant NSCLC patients. The overexpression of miRNA-153-3p enhances gefitinib-sensitivity in NSCLC by inhibiting autophagy via downregulating ATG5.

Abstract 1674: Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism

Abstract Lung cancer is the leading cause of cancer mortality. While non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer and dominates almost 85% of all lung cancer cases. Conventional therapy, such as surgery, chemotherapy, or radiotherapy, scarify normal cells during the curing progress are becoming unfavorable. To avoid harmful side-effect on normal cell, target-based therapy is the mainstream of modern cancer therapy. For example, gefitinib is a clinically-marketed drug which acts as tyrosine kinase inhibitor to inhibit epidermal growth factor receptor (EGFR) activating signaling pathway. For the NSCLC patients who harbor activating substitution from leucine to arginine at amino acid 858 (L858R) point mutation and in-frame exon 19 deletion on EGFR, response well to gefitinib and these two common mutation are used a biomarkers for gefitinib prescription. However, gefitinib-resistance commonly happens due to further substitution mutation from threonine to methionine at amino acid position 790 (T790M) occurred after one year or less gefitinib treatment. Therefore, developing new treatment strategy to tackle gefitinib-resistance is urgently. Abnormal energy metabolism is accepted as new cancer hallmark. Recently, a metabolism rate-limiting enzyme 5'-adenosine menophosphate-activated protein kinase (AMPK) has become a promising anti-cancer target. In this study, we have applied computational docking to perform in silico virtual screening, and we have identified a novel direct AMPK agonist, D561-0775 from a compound library. Using enzyme activation assay, we demonstrated that D561-0775 activates directly on AMPK α subunit. In addition, D561-0775 exhibited significant inhibitory effect on gefitinib-resistant NSCLC cell lines but is less cytotoxicity on normal lung fibroblast cells. D561-0775 also activates AMPK/mTOR signaling pathway, resulting in induction of apoptosis, cell cycle arrest, suppressing glycolysis and cholesterol synthesis in gefitinib-resistant H1975 lung cancer cells. Taken together, D561-0775 has provided clue on a new chemical structure that could be developed as cancer drug for gefitinib-resistant NSCLC patients through inhibition lipid metabolism by directly targeting at AMPK directly. Acknowledgments: This work was supported by Macau Science and Technology Development Fund (Project No: 021/2013/A1, 074/2011/A3 & 082/2013/A3) Citation Format: Xi Chen, Chun Xie, Xing Xing Fan, Ze Bo Jiang, Vincent Wong, Jia Hui Xu, Xiao Jun Yao, Liang Liu, Elaine L. Leung. Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1674.

Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Molecular mechanism of action and potential biomarkers of growth inhibition of synergistic combination of afatinib and dasatinib against gefitinib-resistant non-small cell lung cancer cells.

Epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI) is the first choice of treatment for advanced non-small cell lung cancer (NSCLC) patients harbouring activating EGFR mutations. However, single agent usually has limited efficacy due to heterogeneous resistant mechanisms of cancer cells. Thus drug combination therapy would offer more benefits by synergistic interactions and avoidance of resistance emergence. In this study, we selected 8 NSCLC cell lines with different genetic characteristics as research models to investigate the efficacy of 4 agents (gefitinib, cetuximab, afatinib and dasatinib) and their combinations. As a single agent, both afatinib and dasatinib showed more inhibition against cell proliferation than gefitinib and cetuximab. Afatinib combined with dasatinib demonstrated significantly high efficacy against 7 gefitinib-resistant NSCLC cell lines. Moreover, it reversed the resistance to the 4 studied single agents in PTEN mutated NSCLC cells. By studying the activity of EGFR, Src and their downstream signalling pathways including PI3K/PTEN/Akt, Ras/Raf/MEK/ERK, Src/FAK and JAK/Stat, we demonstrated the synergistic interaction between afatinib and dasatinib was not only due to their blockage of different signalling pathways but also the complemental inhibition of the related signalling molecules such as Stat3. We also found that the level of Src, Stat3, and MAPK may be useful biomarkers predicating synergism between afatinib and dasatinib for the treatment of gefitinib-resistant NSCLC cells.

Sensitivity to chemotherapeutics of NSCLC cells with acquired resistance to EGFR-TKIs is mediated by T790M mutation or epithelial-mesenchymal transition.

Chemotherapy is one of the methods to treat patients with non-small cell lung cancer (NSCLC) developing resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib. Previous studies revealed that the sensitivity to chemotherapy may depend on different cellular mechanisms of acquired EGFR-TKIs resistance. Thus, the present study aimed to investigate the sensitivity of distinct gefitinib-resistant NSCLC cell lines to chemotherapy in order to help select effective treatment regimens for patients with EGFR-TKI resistance. In the present study, we established two gefitinib-resistant cell lines (PC-9/ZD and PC-9/GR) with the human lung adenocarcinoma cell line PC-9 (carrying the delE746-A750 mutation in the EGFR gene). PC-9/ZD cell line expressed the T790M mutation, while PC-9/GR presented the phenotypes of epithelial to mesenchymal transition (EMT). PC-9/ZD cells were more sensitive to paclitaxel and docetaxel than PC-9 cells and knockdown of T790M decreased this sensitivity. In addition, PC-9/GR cells were less sensitive to chemotherapeutic drugs tested, including cisplatin, gemcitabine, pemetrexed, paclitaxel and docetaxel, compared to PC-9 and PC-9/ZD cells. CDH1 transfection reversed the EMT and restored the sensitivity to chemotherapy in PC-9/GR cells. Furthermore, PC-9 cells became resistant to chemotherapy after TGF-β1-induced EMT. The EMT in NSCLC cells significantly increased cancer stem cell (CSC) properties and tumorgenicity. Collectively, the present study revealed that gefitinib-resistant NSCLC cells carrying the T790M mutation were sensitive to taxane chemotherapy, indicating that T790M is a useful biomarker for the selection of chemotherapy. EMT in NSCLC cells confers resistance to chemotherapy, which may be associated with enhanced CSC properties.

ERK inhibition represses gefitinib resistance in non-small cell lung cancer cells.

Gefitinib, an EGFR tyrosine kinase inhibitor, is used to treat non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, the resistance to gefitinib eventually emerges in most of the patients. To understand its mechanism, we generated two acquired gefitinib-resistant NSCLC cell lines. The resistant cells have slower growth rates, but are more resistant to apoptosis in the presence of gefitinib, compared with their sensitive counterparts. In addition, our genome-wide transcriptome analysis reveals unexpected pathways, particularly autophagy, are dysregulated in the gefitinib-resistant cells. Autophagy is significantly enhanced in resistant cells. Importantly, inhibition of autophagy reduces gefitinib resistance. Furthermore, the phosphorylation of ERK, the extracellular signal-regulated kinase, is activated in resistant cells. Inhibition of ERK phosphorylation abrogates gefitinib resistance by suppressing autophagy both in vitro and in vivo. These findings establish a link between ERK and autophagy in gefitinib resistance, and suggest that the ERK signaling may serve as the potentially therapeutic target for treating gefitinib resistance in NSCLC patients.

Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism.

Drug resistance is becoming an obstacle in anti-cancer therapies. For target-based therapy of lung cancer, gefitinib, as the first generation of tyrosine kinase inhibitors (TKIs), demonstrated good initial response to the non-small cell lung cancer (NSCLC) patients whom harbors epidermal growth factor receptor (EGFR) mutation. However, within one year, additional EGFR mutation occurred, leading to eventual gefitinib-resistance. Therefore, it is urgently to discover novel effective small molecule inhibitors for those patients. Abnormal energy metabolism is accepted as new cancer hallmark. Recently, a metabolism rate-limiting enzyme 5’-adenosine menophosphate-activated protein kinase (AMPK) has become a promising anti-cancer target. In this study, we have identified a novel direct AMPK agonist, D561-0775 from a compound library by using molecular docking screening technique. We demonstrated that D561-0775 exhibited significant inhibitory effect on gefitinib-resistant NSCLC cell lines but less cytotoxicity on normal cells. Furthermore, D561-0775 demonstrated a remarkable in vitro AMPK enzyme activation effect. Taken together, D561-0775 showed potential anti-cancer activity via inducing apoptosis, cell cycle arrest, suppressing glycolysis and cholesterol synthesis after activation of AMPK in gefitinib-resistant H1975 cells. D561-0775 has provided a new chemical structure that could be developed as cancer drug for gefitinib-resistant NSCLC patients through inhibition lipid metabolism by directly targeting at AMPK directly.

MiR-19a contributes to gefitinib resistance and epithelial mesenchymal transition in non-small cell lung cancer cells by targeting c-Met

Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). However, most NSCLC patients inevitably develop gefitinib resistance, and the mechanisms underlying this resistance are not fully understood. In this study, we show that miR-19a is significantly down-regulated in gefitinib-resistant NSCLC cell lines compared with gefitinib-sensitive cell lines. In addition, the down-regulation of miR-19a suppressed the expression of epithelial markers but induced the expression levels of mesenchymal markers. A mechanistic analysis revealed that miR-19a regulated c-Met expression by directly targeting the c-Met 3′UTR. Overexpression of miR-19a decreased c-Met expression and re-sensitized gefitinib-resistant NSCLC cells in vitro and in vivo. Consistent with the in vitro findings, the miR-19a serum level was significantly decreased in NSCLC patients with acquired gefitinib resistance compared with the level observed prior to the acquisition of resistance in each patient, indicating that miR-19a expression may be a valuable biomarker for the prediction of acquired gefitinib resistance in a clinical setting. Our data demonstrate that the miR-19a/c-Met pathway plays a critical role in acquired resistance to gefitinib and that the manipulation of miR-19a might provide a therapeutic strategy for overcoming acquired gefitinib resistance.

Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC).

Gefitinib, erlotinib or afatinib are the current treatment for non-small-cell lung cancer (NSCLC) harboring an activating mutation of the epidermal growth factor receptor (EGFR), but less than 5% of patients achieve a complete response and the median progression-free survival is no longer than 12 months. Early adaptive resistance can occur as soon as two hours after starting treatment by activating signal transducer and activation of transcription 3 (STAT3) signaling. We investigated the activation of STAT3 in a panel of gefitinib-sensitive EGFR mutant cell lines, and gefitinib-resistant PC9 cell lines developed in our laboratory. Afatinib has great activity in gefitinib-sensitive as well as in gefitinib-resistant EGFR mutant NSCLC cell lines. However, afatinib therapy causes phosphorylation of STAT3 tyrosine 705 (pSTAT3Tyr705) and elevation of STAT3 and RANTES mRNA levels. The combination of afatinib with TPCA-1 (a STAT3 inhibitor) ablated pSTAT3Tyr705 and down-regulated STAT3 and RANTES mRNA levels with significant growth inhibitory effect in both gefitinib-sensitive and gefitinib-resistant EGFR mutant NSCLC cell lines. Aldehyde dehydrogenase positive (ALDH+) cells were still observed with the combination at the time that Hairy and Enhancer of Split 1 (HES1) mRNA expression was elevated following therapy. Although the combination of afatinib with STAT3 inhibition cannot eliminate the potential problem of a remnant cancer stem cell population, it represents a substantial advantage and opportunity to further prolong progression free survival and probably could increase the response rate in comparison to the current standard of single therapy.

Chronic treatment of non-small-cell lung cancer cells with gefitinib leads to an epigenetic loss of epithelial properties associated with reductions in microRNA-155 and -200c.

BackgroundThe EGFR tyrosine kinase inhibitor gefitinib is used in therapy for non-small-cell lung cancer (NSCLC). However, its application is limited by resistance-accelerated disease progression, which is accompanied by the epithelial-to-mesenchymal transition (EMT). In the present study, we performed multiple expression analyses of microRNAs (miRNAs) and quantified the expression of several related EMT players in gefitinib-resistant NSCLC cells.Methods and resultsTo establish gefitinib-resistant NSCLC cells, gefitinib-sensitive HCC827 cells, which exhibit an in-frame deletion [E746-A750] in EGFR exon 19, were exposed to gefitinib for at least 1.5 months. Next, to profile “gefitinib-resistant HCC827 (HCC827GR)” cells, which have a secondary T790M mutation in EGFR exon 20, a miRNA array analysis was performed in HCC827 and HCC827GR cells. The greatest differences were seen in the levels of miR-155 and miR-200c, which essentially disappeared in HCC827GR cells. In addition to these reductions, the levels of smad2 and zeb1, which are both key players in EMT and targets for miR-155 and miR-200c, respectively, were dramatically increased in HCC827GR cells. In HCC827GR cells, the expression of epithelial-cadherin (E-cadherin) was greatly reduced with repressive histone modifications, whereas vimentin, which is expressed in mesenchymal cells, was dramatically increased with active histone modifications. In another gefitinib-resistant NSCLC cell line (H1975 cells), similar to the findings in HCC827GR cells, both miR-155 and miR-200c were absent, and the EMT was induced along with epigenetic modifications. Interestingly, the inhibition of both miR-155 and miR-200c in HCC827 cells without gefitinib induced significant increases in smad2 and zeb1 along with a dramatic decrease in E-cadherin and a slight increase in vimentin. Furthermore, although the inhibition of these miRNAs in HCC827 cells decreased gefitinib sensitivity, this dual-inhibition in HCC827 cells without gefitinib did not produce a secondary T790M mutation in EGFR exon 20.Conclusion and implicationsThese results suggest that chronic treatment of NSCLC cells with gefitinib changes the expression of miRNAs, including dramatic reductions in miR-155 and miR-200c along with an EGFR mutation. Furthermore, this depletion of miR-155 and miR-200c may be associated with the EMT along with histone modifications, and may contribute to the decrease in the sensitivity to gefitinib independent of a secondary EGFR mutation.

MiR-124 modulates gefitinib resistance through SNAI2 and STAT3 in non-small cell lung cancer.

Gefitinib is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). Unfortunately, most NSCLC patients inevitably develop gefitinib resistance during treatment. In addition to EGFR mutation status, the mechanisms involved are largely unknown. In this study, we showed that miR-124, a tumor suppressor, was significantly down-regulated in gefitinib-resistant NSCLC patients and cell lines compared with gefitinib-sensitive patients and cell lines. In addition, the miR-124 depletion induced gefitinib resistance, and miR-124 overexpression sensitized gefitinib-resistant cells to gefitinib. Mechanistic analysis revealed that miR-124 decreased SNAI2 and STAT3 expression by directly targeting their 3'UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by miR-124 depletion. Our data demonstrate that the miR-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of miR-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.

MiR-30a-5p Overexpression May Overcome EGFR-Inhibitor Resistance through Regulating PI3K/AKT Signaling Pathway in Non-small Cell Lung Cancer Cell Lines.

Lung cancer is one of the most common deadly diseases worldwide, most of which is non-small cell lung cancer (NSCLC). The epidermal growth factor receptor (EGFR) mutant NSCLCs frequently respond to the EGFR tyrosine kinase inhibitors (EGFR-TKIs) treatment, such as Gefitinib and Erlotinib, but the development of acquired resistance limits the utility. Multiple resistance mechanisms have been explored, e.g., the activation of alternative tyrosine kinase receptors (TKRs) sharing similar downstream pathways to EGFR. MicroRNAs (miRNAs) are short, endogenous and non-coding RNA molecules, regulating the target gene expression. In this study, we explored the potential of miR-30a-5p in targeting the EGFR and insulin-like growth factor receptor-1 (IGF-1R) signaling pathways to overcome the drug resistance. IGF-1R is one of the tyrosine kinase receptors that share the same EGFR downstream molecules, including phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT). In this work, an in vitro study was designed using EGFR inhibitor (Gefitinib), IGF-1R inhibitor (NVP-AEW541), and miRNA mimics in two Gefitinib-resistant NSCLC cell lines, H460 and H1975. We found that the combination of EGFR and IGF-1R inhibitors significantly decreased the phosphorylated AKT (p-AKT) expression levels compared to the control group in these two cell lines. Knockdown of phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) had the same effect with the dual inhibition of EGFR and IGF-1R to reduce the expression of p-AKT in the signaling pathway. Overexpression of miR-30a-5p significantly reduced the expression of the PI3K regulatory subunit (PIK3R2) to further induce cell apoptosis, and inhibit cell invasion and migration properties. Hence, miR-30a-5p may play vital roles in overcoming the acquired resistance to EGFR-TKIs, and provide useful information for establishing novel cancer treatment.

Abstract 3034: Overcoming gefitinib resistance in non-small-cell lung cancer by inducing epidermal growth factor receptor (EGFR) degradation via methionine 790 oxidation

Abstract Personalized therapy is becoming a dominant cancer therapeutic strategy, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to treat non-small-cell lung cancer (NSCLC) patients with EGFR mutation but they only inhibit EGFR phosphorylation, acquired-resistance still remains unavoidable without EGFRT790M removal. Here, we report a new treatment strategy to overcome such drug resistance by selectively inducing EGFRT790M degradation via specific stimulation of methionine 790 (M790) oxidation. The basal reactive oxygen species (ROS) levels in EGFRT790M containing gefitinib-resistant NSCLC cell lines were substantially high and 63 clinical lung tumors showed higher NADPH oxidase 2 (NOX2) expression than 61 normal lung tissues which may contribute to high basal ROS in cancer. Interestingly, only NOX3 was upregulated by sanguinarine, a pharmacological agent to elevate ROS, resulted in EGFR over-oxidation and degradation as well as apoptosis. By contrast, such responses were lacking in EGFRWild-type (WT) cells, suggesting a new promising drug development strategy with wide therapeutic window between EGFRT790M and EGFRWT. Selective EGFRT790M degradation was manipulated by REDOX imbalance between NOX3 and methionine reductase (MsrA). When NOX3 was activated, NADPH, the key substrate of MsrA, was depleted to generate excessive ROS, resulting in inhibition of MsrA physiological reduction function on oxidized-methionine, thus localized methionine 790 oxidation of EGFRT790M occurred. Furthermore, the in vivo tumor suppression effect of sanguinarine, NOX3 upregulation and EGFR degradation were confirmed. These findings indicate that specific inducing EGFRT790M degradation via manipulating REDOX imbalance between NOX3 and MsrA would be promising strategy for treating gefitinib-resistant NSCLC. Citation Format: Elaine Lai-Han Leung, Xing-Xing Fan, Maria Pik Wong, Zhi-Hong Jiang, Zhong-Qiu Liu, Xiao-Jun Yao, Lin-Lin Lu, Yan-Ling Zhou, Li-Fong Yau, Vicky Pui-Chi Tin, Liang Liu. Overcoming gefitinib resistance in non-small-cell lung cancer by inducing epidermal growth factor receptor (EGFR) degradation via methionine 790 oxidation. [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 3034.

Targeting Adenine Nucleotide Translocase-2 (ANT2) to Overcome Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in Non-Small Cell Lung Cancer.

EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy has achieved favorable clinical outcomes in non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, patients eventually develop resistance to EGFR-TKIs by several mechanisms. Adenine nucleotide translocase-2 (ANT2) is an oncogenic mitochondrial membrane-associated protein. We investigated the therapeutic potential of ANT2 inhibition to EGFR-TKI resistance in NSCLC using gefitinib-sensitive (PC9 and HCC827) and gefitinib-resistant (H1975 and HCC827/GR) NSCLC cell lines. ANT2 was inhibited by transfecting cells with an ANT2-specific shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR cells compared with the PC9 and HCC827 cells. ANT2 upregulation in gefitinib-resistant cells was associated with increased SP1 binding to the ANT2 promoter. ANT2-specific shRNA decreased NSCLC cell viability. Moreover, ANT2-specific shRNA sensitized the H1975 and HCC827/GR cells to gefitinib, accompanied by HSP90 and EGFR downregulation. ANT2-specific shRNA also inactivated the PI3K/Akt signaling pathway in the H1975 and HCC827/GR cells, which was mediated by the suppression of miR-221/222 levels and by the subsequent restoration of PTEN. In EGFR-TKI-treated NSCLC patients, ANT2 expression was higher in patients exhibiting poor responses compared with patients showing excellent responses. Furthermore, ANT2 expression increased in tumor tissues biopsied after acquiring gefitinib resistance compared with tissues before gefitinib treatment. These findings suggest that ANT2 overexpression contributes to EGFR-TKI resistance in NSCLC and that ANT2 targeting may be considered a novel strategy for overcoming this resistance. Mol Cancer Ther; 15(6); 1387-96. ©2016 AACR.

Molecular Understanding of the Acquisition of Resistance to Anti-cancer Drugs Associated with the Exacerbation of Cancer.

Gefitinib and erlotinib target the ATP cleft in the tyrosine kinase EGFR, which is overexpressed in 40-80 percent of non-small-cell lung cancer (NSCLC) and many other epithelial cancers. However, the application of gefitinib is ultimately limited by the emergence of mutations and other molecular mechanisms conferring drug resistance. Furthermore, it has been considered that acquired resistance to gefitinib is associated with a clinically significant risk of accelerated disease progression. We previously established a new gefitinib-resistant NSCLC cell line, HCC827GR, which harbors the T790M mutation. Using HCC827GR, we found that the inhibition of adenosine A2a receptors of NSCLC regulated cancer proliferation and exacerbation, indicating that adenosine A2a receptors may be new targets for a novel strategy in NSCLC therapy. These findings suggest that multilayered crosstalk between G-protein coupled receptors (GPCRs) and EGFR may play an important role in regulating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.

Abstract 5563: Tripterygium induces apoptosis in NSCLC via mitochondria dysfunction

Abstract Lung cancer remains the leading cause of cancer deaths globally. Patients with non-small cell lung cancer (NSCLC) account for 80% of all lung cancer cases. Active mutation of EGFR was identified as one of the key anti-cancer target for NSCLC patients. Recently, tyrosine kinase inhibitor, like gefitinib which can specifically inhibit the activation of EGFR, emerges as a new potential anticancer drug. However, drug resistance remains an unsolved problem. Most of patient acquired resistance to this inhibitor within 12 months. Therefore, it is necessary and urgent to discover more effective potential agents as candidate drugs for treating gefitinib resistant NSCLC. Tripterygium, a triterpene extracted from the Chinese herb “Thunder of God Vine”, has been shown to have multiple bioactivities. Its anti-cancer effect has been proved in many kinds of cancers. However, the effect of Tripterygium on gefitinib-resistant NSCLC cells is still unknown. In this study, we examined the potency of Tripterygium in induction of apoptosis in a gefitinib-resistant NSCLC cell line and explored its underlying anti-cancer mechanism. Our results demonstrated that Tripterygium induced its apoptotic effect on NSCLC cells in a dose- and time-dependent manner. Moreover, the function of mitochondria was disrupted by the treatment of Tripterygium. The mitochondria membrane potential was gradually loss, and the ratio of Bax/Bcl-2 which is the indicator of mitochondria membrane integrity, was significantly up-regulated after the treatment of Tripterygium. Although the caspases were activated, the treatment of pan-caspase inhibitor could only partially inhibit the level of apoptosis. Moreover, the level of Hsp90 client proteins, EGFR and its downstream target AKT, were measured. Interestingly, both of them were remarkably decreased after Tripterygium treatment. Taken together, our data suggest that Tripterygium, which may be developed as a promising agent for gefitinib-resistant NSCLC patient, induced apoptosis through mitochondria dysfunction and EGFR degradation in gefitinib-resistant NSCLC cells. Citation Format: Xing-Xing Fan, Na Li, Jin-Lin Wu, Yan-Ling Zhou, Liang Liu, Jian-Xing He, Elaine L. Leung. Tripterygium induces apoptosis in NSCLC via mitochondria dysfunction. [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 5563. doi:10.1158/1538-7445.AM2015-5563

Homoharringtonine induces apoptosis and inhibits STAT3 via IL-6/JAK1/STAT3 signal pathway in Gefitinib-resistant lung cancer cells.

Tyrosine kinase inhibitors (TKIs) are mostly used in non-small cell lung cancer (NSCLC) treatment. Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic. Therefore, exploring novel compounds to overcome this resistance is urgently required. Here we investigated the antitumor effect of homoharringtonine (HHT), a natural compound extracted from Cephalotaxus harringtonia, on Gefitinib-resistant NSCLC cell lines in vitro and in vivo. NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR. HHT inhibited cells growth, cell viability and colony formation, as well as induced cell apoptosis through mitochondria pathway. Furthermore, we explored the mechanism of HHT inhibition on NSCLC cells. Higher level of interleukin-6 (IL-6) existed in lung cancer patients and mutant EGFR and TGFβ signal requires the upregulation of IL-6 through the gp130/JAK pathway to overactive STAT3, an oncogenic protein which has been considered as a potential target for cancer therapy. HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression. Gefitinib-resistant NSCLC xenograft tests also confirmed the antitumor effect of HHT in vivo. Consequently, HHT has the potential in Gefitinib-resistant NSCLC treatment.

Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT.

Gefitinib efficiency in non-small-cell lung cancer (NSCLC) therapy is limited due to development of drug resistance. The molecular mechanisms of gefitinib resistance remain still unclear. In this study, we first found that connexin 26 (Cx26) is the predominant Cx isoform expressed in various NSCLC cell lines. Then, two gefitinib-resistant (GR) NSCLC cell lines, HCC827 GR and PC9 GR, from their parental cells were established. In these GR cells, the results showed that gefitinib resistance correlated with changes in cellular EMT phenotypes and upregulation of Cx26. Cx26 was detected to be accumulated in the cytoplasm and failed to establish functional gap-junctional intercellular communication (GJIC) either in GR cells or their parental cells. Ectopic expression of GJIC-deficient chimeric Cx26 was sufficient to induce EMT and gefitinib insensitivity in HCC827 and PC9 cells, while knockdown of Cx26 reversed EMT and gefitinib resistance in their GR cells both in vitro and in vivo. Furthermore, Cx26 overexpression could activate PI3K/Akt signaling in these cells. Cx26-mediated EMT and gefitinib resistance were significantly blocked by inhibition of PI3K/Akt pathway. Specifically, inhibition of the constitutive activation of PI3K/Akt pathway substantially suppressed Cx26 expression, and Cx26 was confirmed to functionally interplay with PI3K/Akt signaling to promote EMT and gefitinib resistance in NSCLC cells. In conclusion, the reciprocal positive regulation between Cx26 and PI3K/Akt signaling contributes to acquired gefitinib resistance in NSCLC cells by promoting EMT via a GJIC-independent manner.

3,6,2′,4′,5′-Pentahydroxyflavone, an Orally Bioavailable Multiple Protein Kinase Inhibitor, Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is the most lethal cancer, causing more than 150,000 deaths in the United States in 2013. The receptor tyrosine kinase inhibitors such as gefitinib are not perfect clinical therapeutic agents for NSCLC treatment due to primary or acquired tyrosine kinase inhibitor resistance. Herein, 3,6,2',4',5'-pentahydroxyflavone (36245-PHF) was identified as a multiple kinase inhibitor for NSCLC treatment based on the computational screening of a natural products database. 36245-PHF was shown to inhibit PI3K and Aurora A and B kinases and overcome gefitinib-resistant NSCLC growth. Our data clearly showed that 36245-PHF markedly inhibited anchorage-independent growth of gefitinib-resistant NSCLC cell lines and exerted a substantial chemotherapeutic effect following oral administration in a gefitinib-resistant NSCLC xenograft model. The evidence from three different subsequent methodological approaches, in vitro, ex vivo, and in vivo, all confirmed that 36245-PHF as a multiple protein kinase inhibitor. Overall, we identified 36245-PHF as a multiple protein kinase inhibitor and as a novel therapeutic agent to overcome gefitinib-resistant NSCLC growth, which could provide a new option for clinical NSCLC oral treatment.

Paris saponin I induces apoptosis via increasing the Bax/Bcl-2 ratio and caspase-3 expression in gefitinib-resistant non-small cell lung cancer in vitro and in vivo

Polyphyllins, a major component of Rhizoma paridis, have been extensively used in non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate the effects of Paris saponin I (PSI) on a panel of gefitinib-resistant NSCLC cell lines and its inhibition of tumor growth in a nude mouse model. The MTT assay was used to assess growth inhibition. The cell cycle was analyzed using flow cytometry and apoptosis was assessed using Annexin V/propidium iodide staining. The morphology of the apoptotic cells was determined by transmission electron microscopy. The protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were detected using western blot analysis. In addition, the glucose metabolism in tumor-bearing mice was evaluated using 18F-fludeoxyglucose (FDG) micro-positron emission tomography imaging. The PSI-induced growth inhibition rate was observed to significantly increase in a time- and dose-dependent manner. Furthermore, PSI induced significant G2/M-phase arrest and apoptosis. The expression levels of Bcl-2 decreased, while those of Bax and caspase-3 increased following PSI treatment. 18F-FDG-uptake in the PSI treatment groups was significantly decreased compared with that in the control group in vivo. In conclusion, PSI is a potent antitumor agent that acts by inhibiting the proliferation of gefitinib-resistant cells, and has potential as a candidate for a natural drug for gefitinib-resistant therapy. PSI-induced apoptosis, which occurred via multiple pathways, including G2/M-phase arrest and upregulation of the Bax/Bcl-2 ratio and caspase-3 expression, ultimately led to cell death and tumor inhibition.