Gefitinib In Non-small Cell Lung Cancer Cells Research Articles

Cytochalasin H enhances sensitivity to gefitinib in non-small-cell lung cancer cells through inhibiting EGFR activation and PD-L1 expression

In our previous study, we have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove plant and found that CyH inhibited the proliferation of non-small cell lung cancer (NSCLC) cells. Recently, epidermal growth factor receptor (EGFR) activation and programmed cell death 1 ligand (PD-L1) expression have been demonstrated to mediate NSCLC resistance to gefitinib, first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI). Here, we further investigated the effect of CyH on EGFR activation, PD-L1 expression, and gefitinib sensitivity in NSCLC cell lines, A549 (wild-type EGFR), HCC827 (EGFR mutation), and NCI-H1975 (dual EGFR mutations and acquired gefitinib resistance) and animal model. Our results showed that CyH significantly inhibited EGFR activation and PD-L1 expression in NSCLC cells. Additionally, CyH dramatically promoted the inhibitory effect of gefitinib on the proliferation of A549 and HCC827 cells, and enhanced the sensitivity to gefitinib in NCI-H1975 cells. Moreover, CyH increased the inhibitory effect of gefitinib on EGFR activation and PD-L1 expression in HCC827 and NCI-H1975 cells. Animal experiments further demonstrated that CyH significantly promoted the inhibitory effect of gefitinib on the growth of NSCLC and the expression of Ki-67, p-EGFR, and PD-L1 in NCI-H1975 NSCLC xenograft tumors of nude mice. Furthermore, CyH inhibited the activation of JAK3/STAT signaling pathway. Taken together, our findings suggest that CyH promotes the sensitivity to gefitinib in NSCLC cells through the inhibition of EGFR activation and PD-L1 expression.

Single-cell mass spectrometry studies of drug metabolism heterogeneity and primary resistance to gefitinib in non-small cell lung cancer cells

Patients with epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) often show primary resistance to gefitinib therapy. It is thus necessary to study the metabolism of gefitinib in NSCLC cells to comprehensively reveal the reasons for the primary resistance of tumors. Herein, we develop a platform for studying drug metabolism heterogeneity based on single-cell mass spectrometry (sDMH-scMS) by integrating living-cell electrolaunching ionization MS (ILCEI-MS) and high-performance liquid chromatography-MS (HPLC-MS) analysis, and the primary resistance of NSCLC cells to gefitinib was studied using this platform. The ILCEI-MS analysis showed that approximately 11.9% of NSCLC single cells contained the gefitinib metabolite M11; HPLC-MS detection diluted the intensity of M11 in subpopulations and concealed the heterogeneity of drug metabolism in tumor single cells. The intensity of gefitinib in EGFR wild-type A549 cells was markedly lower than in mutant PC9 cells, and the intensity of gefitinib metabolites was significantly higher than in PC9 cells, suggesting that the primary resistance of NSCLC cells is related to gefitinib metabolism. Moreover, the combination of gefitinib and the drug-metabolizing enzyme inhibitor α-naphthoflavone was shown to overcome the primary resistance of the NSCLC cells. Overall, the results of this study are expected to be applicable for clinical drug resistance diagnosis and treatment at the single-cell level.

TRIP13 overexpression promotes gefitinib resistance in non‑small cell lung cancer via regulating autophagy and phosphorylation of the EGFR signaling pathway.

Non‑small cell lung cancer (NSCLC) accounts for the majority of lung cancers and remains the most common cause of cancer‑related death. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR‑TKIs) have been used as first‑line treatment for patients with NSCLC showing EGFR mutations. Unfortunately, drug resistance is a crucial barrier affecting the treatment of patients with NSCLC. Thyroid hormone receptor interactor 13 (TRIP13) is an ATPase that is overexpressed in numerous tumors and is involved in drug resistance. However, whether TRIP13 plays a role in regulating sensitivity to EGFR‑TKIs in NSCLC remains unknown. TRIP13 expression was evaluated in gefitinib‑sensitive (HCC827) and ‑resistant (HCC827GR and H1975) cell lines. The effect of TRIP13 on gefitinib sensitivity was assessed using the MTS assay. The expression of TRIP13 was upregulated or knocked down to determine its effect on cell growth, colony formation, apoptosis and autophagy. Additionally, the regulatory mechanism of TRIP13 on EGFR and its downstream pathways in NSCLC cells were examined using western blotting, immunofluorescence and co‑immunoprecipitation assays. The expression levels of TRIP13 were significantly higher in gefitinib‑resistant than in gefitinib‑sensitive NSCLC cells. TRIP13 upregulation enhanced cell proliferation and colony formation while reducing the apoptosis of gefitinib‑resistant NSCLC cells, suggesting that TRIP13 may facilitate gefitinib resistance in NSCLC cells. In addition, TRIP13 improved autophagy to desensitize gefitinib in NSCLC cells. Furthermore, TRIP13 interacted with EGFR and induced its phosphorylation and downstream pathways in NSCLC cells. The present study demonstrated that TRIP13 overexpression promotes gefitinib resistance in NSCLC by regulating autophagy and activating the EGFR signaling pathway. Thus, TRIP13 could be used as a biomarker and therapeutic target for gefitinib resistance in NSCLC.

Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition.

Introduction: G-protein coupled receptor 30 (GPR30) is associated with cell metastasis and drug resistance in many different cancer cells. The present study aimed to reveal the sensitivity of GPR30 to gefitinib in non-small cell lung cancer (NSCLC) cells.Methods: Cell viability and proliferation were detected using cell counting kit 8 and 5-ethynyl-2'-deoxyuridine assays, respectively. Western blotting and quantitative real-time reverse transcription PCR were used to detect GPR30 or epithelial-mesenchyme transition (EMT)-related mRNA and protein expression.Results: The results showed that GPR30 expression is associated with gefitinib sensitivity. G15, as a GPR30 antagonist, reduced GPR30 expression. We chose the maximum concentration of G15 with minimal cytotoxicity to detect cell viability after combined treatment with gefitinib in NSCLC cells, which indicated that G15 could increase sensitivity to gefitinib. However, the effect of G15 on gefitinib sensitivity disappeared after treatment with a small interfering RNA targeting GPR30. Further research showed that G15 or GPR30 siRNA treatment could upregulate E-cadherin and downregulate vimentin levels.Conclusion: Taken together, these data suggested that G15 could enhance NSCLC sensitivity to gefitinib by inhibition of GPR30 and EMT.

Inhibition of autophagy by YC-1 promotes gefitinib induced apoptosis by targeting FOXO1 in gefitinib-resistant NSCLC cells

Non-small cell lung cancer (NSCLC) is the most common cancer in the world. Gefitinib, an inhibitor of EGFR tyrosine kinase, is highly effective in treating NSCLC patients with activating EGFR mutations (L858R or Ex19del). However, despite excellent disease control with gefitinib therapy, innate resistance and inevitable acquired resistance represent immense challenges in NSCLC therapy. Gefitinib potently induces cytoprotective autophagy, which has been implied to contribute to both innate and acquired resistance to gefitinib in NSCLC cells. Currently, abrogation of autophagy is considered a promising strategy for NSCLC therapy. In the present study, YC-1, an inhibitor of HIF-1α, was first found to significantly inhibit the autophagy induced by gefitinib by disrupting the fusion of autophagosomes and lysosomes and thereby enhancing the proapoptotic effect of gefitinib in gefitinib-resistant NSCLC cells. Furthermore, the combinational anti-autophagic and pro-apoptotic effect of gefitinib and YC-1 was demonstrated to be associated with an enhanced of forkhead box protein O1 (FOXO1) transcriptional activity which resulted from an increase in the p-FOXO1 protein level in gefitinib-resistant NSCLC cells. Our data suggest that inhibition of autophagy by targeting FOXO1 may be a feasible therapeutic strategy to overcome both innate and acquired resistance to EGFR-TKIs.

Novel Hsp90 Inhibitor C086 Potently Inhibits Non-Small Cell Lung Cancer Cells As A Single Agent Or In Combination With Gefitinib.

PurposeInhibition of heat shock protein 90 (Hsp90) can lead to degradation of multiple client proteins, which are involved in tumor progression. Elevated Hsp90 expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). Discovery of effective drug is a promising strategy to improve patient survival. This study aims to investigate the synergistic antitumor mechanism of C086 combined with gefitinib in NSCLC cells in vitro.MethodsThe binding of C086, gefitinib, and the combinations to Hsp90 was characterized by fluorescence quenching experiments. The inhibition of A549 or NCI-H1975 cell proliferation and apoptosis by C086 and gefitinib as a single agent or in combinations were performed using CFSE staining assays, AnnexinV–APC/PI and Western blot.ResultsC086 alone or with gefitinib reduces proliferation and increases proapoptotic caspase activation of both wild-type and mutation NSCLC, with NCI-H1975 cells showing much greater sensitivity to C086 and the combinations than A549 cells. The combination of C086 and gefitinib showed synergistic reduction of EGFR expression and the downstream PI3K/Akt and Ras-Raf-Erk pathways enhanced suppression of Erk signaling.ConclusionC086 combined gefitinib has a good synergistic antitumor effect in vitro. Therefore, the combination of C086 and gefitinib may provide a new theoretical basis and ideas for the treatment of NSCLC patients.

The Effects and Mechanisms by which Saikosaponin-D Enhances the Sensitivity of Human Non-small Cell Lung Cancer Cells to Gefitinib.

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR)-sensitive mutations benefit from epidermal growth factor receptor tyrosine kinase inhibitors (EGFR- TKIs). However, drug resistance is a major cause of therapeutic failure. This study examined whether saikosaponin-d (SSD) enhances the anti-tumor effect of gefitinib in NSCLC cells. Cell Counting Kit-8 (CCK-8) was used to determine cell viability. Cell apoptosis was examined by flow cytometry. Signal transducer and activator of transcription (STAT3), phosphor-STAT3 (P-STAT3), and B-cell lymphoma 2 (Bcl-2) were detected by Western blot. An HCC827/GR tumor model was established to observe the effect of combination therapy in vivo. The combination of SSD with gefitinib had an enhanced inhibitory effect by reducing cell viability and inducing cells apoptosis in NSCLC cells. Furthermore, SSD decreased and increased the expression of P-STAT3 and Bcl-2, respectively. Down-regulated STAT3 promoted the sensitivity of lung cancer cells to gefitinib. The results of animal experiments also showed that SSD enhanced the anti-tumor effect of gefitinib. These results indicated that the combination of SSD with gefitinib had an increased antitumor effect in NSCLC cells and that the molecular mechanisms were associated with the inhibition of STAT3/Bcl-2 signaling pathway. Our findings suggest a promising approach for the treatment of NSCLC patients with EGFR-TKI resistance.

Ginsenoside Rg3 promotes the antitumor activity of gefitinib in lung cancer cell lines.

Lung cancer is one of the most common types of cancer with one of the highest incidence and mortality rates. Gefitinib is widely used for the treatment of non-small cell lung cancer (NSCLC). However, issues regarding drug resistance, toxicity and limited applicability have been associated with gefitinib. The aim of the present study was to investigate whether ginsenoside Rg3 enhances the anticancer activity of gefitinib in NSCLC cells. MTT assay demonstrated that ginsenoside Rg3 increased the cytotoxic effect of gefitinib in NSCLC cell lines in a dose- and time-dependent manner. In addition, flow cytometric analysis revealed that the combined treatment with gefitinib and ginsenoside Rg3 significantly increased apoptosis in NSCLC cell lines. Transwell migration assays demonstrated that the combined treatment with gefitinib and ginsenoside Rg3 significantly decreased NSCLC cell migration compared with gefitinib or ginsenoside Rg3 treatment alone. Furthermore, western blot analysis revealed that in NSCLC cell lines, the combined treatment with gefitinib and ginsenoside Rg3 increased protein expression levels of pro-apoptotic proteins Bax and cleaved-caspase-3, whilst the expression level of anti-apoptotic protein Bcl-2 decreased. In addition, western blot analysis revealed that, in NSCLC cell lines, the combined treatment with gefitinib and ginsenoside Rg3 decreased the protein expression levels of pro-migration factors SNAIL and SLUG, whilst the expression level of anti-migration protein E-cadherin increased. In conclusion, ginsenoside Rg3 may be able to enhance the anticancer activity of gefitinib, making NSCLC cells more sensitive to gefitinib.

Highlights of This Issue

Obg-like ATPase 1 (OLA1) was identified as a protein that functions in centrosome regulation together with BRCA1. Yoshino and colleagues identified five missense mutants of OLA1 that are deficient in the regulation of centrosome number. Three of them did not bind to BARD1, a heterodimer partner of BRCA1. Two phosphomimetic mutations restored the binding to BARD1. A BARD1 mutant, reported in cancer, failed to bind to OLA1 and rescue the BARD1 knockdown-induced centrosome amplification. These findings demonstrate that the OLA1-BARD1 interaction is critical for the regulation of centrosome number. Thus, OLA1 is important for the genome integrity to prevent tumor development.Resistance to radiation therapy is a hallmark of glioblastoma multiforme (GBM). Radiation can induce ER stress and downstream signaling associated with the endoplasmic reticulum stress response (ERSR). Protein kinase R-like endoplasmic reticulum kinase (PERK) is one of the regulators of ERSR. The current Rapid Impact, investigates the significance of PERK in GBM after ionizing radiation (IR). It was determined that radiation enhanced PERK-eIF2α-ATF4 pathway in GBM, thereby influencing survival and death processes. The dual function of PERK as a mediator of survival and death provides multiple approaches to enhance the efficacy of radiation therapy.Development of resistance to small molecule EGFR inhibitors in non-small cell lung cancer (NSCLC) patients with activating EGFR mutations has limited the efficacy of such treatment. Though recent studies have deepened the understanding of the molecular mechanisms, fully understanding and overcoming the resistance is still a challenge. This study, by Lu and colleagues, demonstrates that hypoxia promotes resistance to gefitinib in NSCLC cells in a pathway linked to features of epithelial-to-mesenchymal transition (EMT) and dependent on the function of the histone lysine demethylases, LSD1 and PLU-1. The results provide the rationale for combining EGFR inhibitors with LSD1 inhibitors as a therapeutic strategy for NSCLC.Cholangiocarcinoma (CCA) is a rare tumor type with very limited treatment options. To identify new molecular susceptibilities, this study interrogated the Hippo signaling pathway and its effector protein YAP. In the models tested, YAP regulation was independent of the Hippo pathway and was driven by SRC family kinase-mediated tyrosine phosphorylation. Tyrosine phosphorylation lead to nuclear retention of YAP, a so-called nuclear retention signal. Subsequently, LCK was identified as the most potent mediator of YAP phosphorylation, a previously undescribed function for LCK. Finally, LCK-mediated YAP phosphorylation was found to be targetable in vivo, utilizing the pan-SFK inhibitor dasatinib.

Functional cooperation between HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR

ObjectiveHypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are important transcription factors regulating expression of genes involved in cell survival. HIF-1α and c-Jun are key components of HIF-1 and AP-1, respectively, and are regulated by epidermal growth factor receptor (EGFR)-mediated cell signaling and tumor microenvironmental cues. The roles of HIF-1α and c-Jun in development of resistance to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) with activating mutation of EGFR have not been explored. In this study, we investigated the roles of HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR. Materials and methodsChanges in HIF-1α protein and in total and phosphorylated c-Jun levels in relation to changes in total and phosphorylated EGFR levels before and after gefitinib treatment were measured using Western blot analysis in NSCLC cells sensitive or resistant to gefitinib. The impact of overexpression of a constitutively expressed HIF-1α (HIF-1α/ΔODD) or a constitutively active c-Jun upstream regulator (SEK1 S220E/T224D mutant) on cell response to gefitinib was also examined. The effect of pharmacological inhibition of SEK1-JNK-c-Jun pathway on cell response to gefitinib was evaluated. ResultsDownregulation of HIF-1α and total and phosphorylated c-Jun levels correlated with cell inhibitory response to gefitinib better than decrease in phosphorylated EGFR did in NSCLC cells with intrinsic or acquired resistance to gefitinib. Overexpression of HIF-1α/ΔODD or SEK1 S220E/T224D mutant conferred resistance to gefitinib. There exists a positive feed-forward regulation loop between HIF-1 and c-Jun. The JNK inhibitor SP600125 sensitized gefitinib-resistant NSCLC cells to gefitinib. ConclusionsHIF-1α and c-Jun functionally cooperate in development of resistance to gefitinib in NSCLC cells. The translational value of inhibiting HIF-1α/c-Jun cooperation in overcoming resistance to EGFR TKI treatment of NSCLC cells with activating mutation of EGFR deserves further investigation.

P3.02b-123 Lysimachia Capillipes Capilliposide Inhibits AKT Activation and Restores Sensitivity to Gefitinib in NSCLC with Acquired Gefitinib Resistance: Topic: EGFR RES

Most non-small cell lung cancer (NSCLC) patients responding to gefitinib will eventually develop the resistance. Lysimachia capillipes (LC) capilliposide extracts from LC hemsl shows both in vitro and in vivo anti-cancer effects. We investigated whether LC capilliposide combined with gefitinib could overcome the resistance of NSCLC cells to gefitinib, and to identify the involved molecular signaling. NSCLC cell lines with different sensitivities to gefitinib were studied. Cell proliferation was assessed with MTT assay. Cell apoptosis and cell cycle distribution were measured using cytometry. EGFR-related signaling proteins and Human Phospho-Kinase were analyzed using Western blotting and protein array, respectively. Tumor growth inhibition were evaluated in PC-9-GR xenograft. CC3, Ki67 and pEGFR were assessed by IHC on tumor tissues. LC capilliposide inhibited cell growth in gefitinib-sensitive and -resistant cells. In gefitinib resistant cell PC-9-GR with T790M mutation, the LC capilliposide combined with gefitinib was potent in cell growth inhibition and apoptosis induction, but no obvious effect on gefitinib-induced G0/G1 arrest. LC capilliposide remarkable blocks the phosphorylation of EGFR downstream signaling molecule AKT, on which LC capilliposide and gefitinib alone had no obvious effect. The Human Phospho-Kinase array further confirmed the enhanced inhibitory effect on the AKT signaling. LC capilliposide treatment also enhanced tumor growth inhibition when combined with gefitinib in PC-9-GR xenografts. LC capilliposide restored the sensitivity to gefitinib in NSCLC cells with acquired gefitinib resistance, suggesting that combination of LC and gefitinib may be a promising therapeutic strategy to overcome gefitinib resistance in NSCLCs with T790M mutation.

Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells

Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14–17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 × 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC.

Mechanism of TRIM24 to Regulate Resistance of Gefitinib in NSCLC cells

背景与目的表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TKI)的原发和继发性耐药已成为其在临床肺癌治疗中的拦路虎，在肺癌组织中研究发现TRIM24呈高表达状态，在肺癌细胞中能调控细胞增殖、周期和细胞凋亡，本研究旨在进一步探讨TRIM24调控肺癌细胞吉非替尼耐药的分子机制。方法应用MTT方法和流式细胞仪检测干扰TRIM24及干扰TRIM24后加入吉非替尼对肺癌细胞系增殖能力及凋亡率的抑制率的变化，同时应用Western blot方法检测凋亡相关基因的变化及AKT信号通路中蛋白的表达。结果在A549细胞中使用干扰TRIM24可以提高吉非替尼对肿瘤增殖能力的抑制率，增加吉非替尼诱导的凋亡水平，同时干扰TRIM24及干扰TRIM24加入吉非替尼后促凋亡相关基因p-BAD、Bcl-2和AKT及AKT信号通路相关蛋白PIK3CA均出现降低。结论TRIM24能够调控肺癌细胞吉非替尼耐药，并且通过AKT信号通路引起肺癌细胞EGFRTKI耐药。

Overexpression of Sphk2 is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer is the major cause of cancer-related death worldwide, and 80% of them are non-small cell lung cancer (NSCLC) cases. Recent studies have shown that sphingosine kinase 2 (SphK2) could promote tumor progression; however, whether SphK2 could affect the chemoresistance of NSCLC to chemotherapy remains unclear. To determine whether SphK2 serves as a potential therapeutic target of NSCLC, we utilized small interference RNA (siRNA) to knock down SphK2 expression in human NSCLC cells and analyzed their phenotypic changes. The data demonstrated that knockdown of SphK2 led to decreased proliferation and enhanced chemosensitivity and apoptosis to gefitinib in NSCLC cells. In this study, we describe the findings that overexpression of SphK2 promotes chemoresistance in NSCLC cells. Inhibition of SphK2 might be considered as a strategy in NSCLC treatment with gefitinib.

Short-hairpin RNA library: identification of therapeutic partners for gefitinib-resistant non-small cell lung cancer.

Somatic mutations of the epidermal growth factor receptor often cause resistance to therapy with tyrosine kinase inhibitor in non-small cell lung cancer (NSCLC). In this study, we aimed to identify partner drugs and pathways that can induce cell death in combination with gefitinib in NSCLC cells. We undertook a genome-wide RNAi screen to identify synthetic lethality with gefitinib in tyrosine kinase inhibitor resistant cells. The screening data were utilized in different approaches. Firstly, we identified PRKCSH as a candidate gene, silencing of which induces apoptosis of NSCLC cells treated with gefitinib. Next, in an in silico gene signature pathway analysis of shRNA library data, a strong correlation of genes involved in the CD27 signaling cascade was observed. We showed that the combination of dasatinib (NF-κB pathway inhibitor) with gefitinib synergistically inhibited the growth of NSCLC cells. Lastly, utilizing the Connectivity Map, thioridazine was identified as a top pharmaceutical perturbagen. In our experiments, it synergized with gefitinib to reduce p-Akt levels and to induce apoptosis in NSCLC cells. Taken together, a pooled short-hairpin library screen identified several potential pathways and drugs that can be therapeutic targets for gefitinib resistant NSCLC.

Suppression of Dicer increases sensitivity to gefitinib in human lung cancer cells.

Accumulating evidence is revealing an important role of microRNA (miRNA) in tumor progression and chemotherapeutic resistance. Dicer is a cytoplasmic endoribonuclease type III crucial for production of mature miRNAs. The aberrant expression of Dicer has also been reportedly associated with clinical aggressiveness, prognosis, and patient survival in various cancer types. However, the molecular mechanisms of Dicer in acquired gefitinib resistance are still not clear. In this study, we analyzed the protein level of Dicer between gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/GR) non-small-cell lung cancer (NSCLC) cell lines by Western blot analysis. Silence and overexpression of the Dicer were performed to investigate the effects on gefitinib sensitivity, as assessed by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and sub-G1 assay of flow cytometry. To further explore the mechanism of chemoresistance, we examined whether Dicer knockdown led to modulating specific miRNAs and its miRNA target genes. Dicer expression was significantly increased in PC9/GR compared with PC9 cells. Knockdown of Dicer restores gefitinib sensitivity in resistant cells, and overexpression of Dicer enhances resistance to gefitinib in sensitive cells. Silencing of Dicer induces sensitivity to gefitinib in NSCLC cells through the downregulation of miR-30b/c and miR-221/222 to increase the protein level of caspase-3, resulting in an increase in gefitinib-induced apoptosis. Dicer contributes to the resistance to gefitinib in lung cancer. These results indicate that Dicer may be a target for diagnosis and therapy of patients with resistance to gefitinib.

Abstract B276: A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells.

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumours initially respond well to EGFR targeted monotherapy (e.g. gefitinib), but the benefit is usually limited due to the emergence of drug resistance. A major challenge in the field is to identify the most promising combination approaches that inhibit or prevent this resistance and then to prioritise these options for clinical testing in order to produce more durable patient benefits. Our aim in this study was to identify compounds that selectively and rapidly induced cell death but only when combined with gefitinib in EGFR M+ cells. Methods: We developed a high-throughput 96-well live-cell phenotypic assay (based on an acute apoptosis end point) to assess the combination potential of probe inhibitors targeting kinases (n=402), as well as compounds that are approved or in clinical development for the treatment of cancer (n=326). Screens were carried out using EGFR M+ (PC-9, HCC-827, HCC-2935, H1975) and EGFR WT (A549, H1993) NSCLC cells, and a non-tumor immortalized lung epithelial cell line (BEAS2B). Cells were treated with gefitinib (0-1 µM) and test compounds (0-10 µM) for 18 h. Hoechst 33258 (25 µM) was added and 30 min later cells with condensed nuclei and a high nuclear intensity were scored as apoptotic, and the percentage of apoptotic cells was calculated. SYTOX green DNA staining confirmed that apoptotic cells were scored positive using our simple Hoechst staining method. Results: The screening assay was robust and reproducible with Pearson correlation coefficients ranging from 0.66-0.92 when comparing inter-assay apoptosis scores. Using the compound library we identified 21 positive hits (Z-score≥10) that selectively induced apoptosis in the presence of gefitinib in EGFR M+ cells, but not in EGFR WT cells. Positive hits included compounds in four major target categories; BCL inhibitors (n=3), anti-mitotic agents (n=11), HDAC inhibitors (n=4), and AKT/PI3K inhibitors (n=5). Several of these targets have been suggested in the literature using different methodologies to our own, thereby validating our overall screening approach. Importantly, we have also identified several previously unreported combinations that induced marked apoptosis within 18 h of gefitinib treatment, and which we are currently evaluating in in vivo studies. In addition, we observed that combinations of EGFR TKIs with gefitinib were antagonistic in EGFR M+ cells. Finally, in the probe inhibitor library set, we have identified 20 diverse chemical structures that are strongly positive in our combination assay, suggesting new targets for selective killing of EGFR M+ NSCLC cells. Unexpectedly, screening the chemical probe library also identified 7 compounds that selectively induced cell death in combination with gefitinib in EGFR wild-type KRAS mutant A549 cells. Conclusion: We have developed a robust and reproducible high-throughput live cell apoptosis assay to screen compound libraries in combination with gefitinib in NSCLC cells. We have identified several novel combinations that are highly selective for EGFR M+ cells in the presence of gefitinib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B276. Citation Format: Sivan M. Bokobza, Aoife M. Devery, Anika M. Weber, Anderson J. Ryan. A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B276.

Blockade of DNA methylation enhances the therapeutic effect of gefitinib in non-small cell lung cancer cells

The sensitivity of lung cancer to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has been found to be associated with mutations in the tyrosine kinase domain of EGFR. However, not all mutations are sensitive to gefitinib. While CpG island methylation in the promoter region of the EGFR gene and transcriptional silencing are common in solid tumors, the role of the EGFR gene promoter methylation in affecting resistance to TKIs in non-small cell lung cancer (NSCLC) remains unknown. In this study, we examined the correlation between EGFR gene promoter methylation and the therapeutic effect of gefitinib in NSCLC cells. Three NSCLC cell lines with different EGFR mutation statuses and levels of sensitivity to EGFR-TKIs were used in this study: H1650 (del E746-A750), H1299 (wild-type EGFR) and PC-9 (del E746-A750). Cells were treated with gefitinib or 5-aza-2'-deoxy cytidine (5-aza-CdR), a methylation inhibitor, alone or in combination. Subsequently, the methylation status of the EGFR gene promoter was examined by methylation-specific PCR (MSP). Cell survival and apoptosis assays were performed using the Cell Counting Kit-8 (CCK-8) and flow cytometry. In addition, western blot analysis and quantitative real-time PCR were used to examine the expression levels of EGFR protein and mRNA. Our study showed that the promoter region of the EGFR gene in PC-9 cells was unmethylated, and that the cells were sensitive to gefitinib. By contrast, the promoter region of the EGFR gene in the H1650 and H1299 cells was methylated, and the cells were resistant to gefitinib. Of note, the combination treatment with 5-aza-CdR and gefitinib further enhanced the growth inhibitory effects and led to the induction of apoptosis, while a significant reduction in the expression of EGFR protein and mRNA was observed in the H1650 and H1299 cells. These results suggest that blockade of DNA methylation may enhance the antitumor effects of EGFR-TKIs and gefitinib in NSCLC cells. Thus, EGFR gene promoter methylation may be a potential mechanism for acquired resistance to gefitinib.

The role of celecoxib in Rad51 expression and cell survival affected by gefitinib in human non-small cell lung cancer cells

Celecoxib (Celebrex R) is a cyclooxygenase-2 (COX-2) selective inhibitor and gefitinib (Iressa R, ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). The addition of celecoxib to gefitinib to prolong the survival of patients with NSCLC still remains controversial and needs to be investigated. The Rad51 protein is essential for homologous recombination repair, and is overexpressed in chemo- or radioresistant carcinomas. In this study, we characterize the role of celecoxib in the cytotoxicity, ERK1/2 activation and Rad51 expression affected by gefitinib in NSCLC cells. We show that celecoxib can enhance the cytotoxicity induced by gefitinib in NSCLC cells. Treatment with celecoxib alone has no effect on the ERK1/2 activation, Rad51 mRNA and protein levels, however, combined treatment with gefitinib results in a significant reduction of phospho-ERK1/2 and Rad51 protein levels, and triggers the degradation of Rad51 via a 26S proteasome-dependent pathway. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescues the decreased ERK1/2 activity, and restores Rad51 protein levels and cell survival under co-treatment with gefitinib and celecoxib. Furthermore, blocking ERK1/2 activation by U0126 (MKK1/2 inhibitor) and knocking down Rad51 expression by transfection with small interfering RNA of Rad51 can enhance the cytotoxicity of celecoxib.

Synergistic antitumor effect of S-1 and the epidermal growth factor receptor inhibitor gefitinib in non-small cell lung cancer cell lines: role of gefitinib-induced down-regulation of thymidylate synthase

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the therapeutic response to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced non-small cell lung cancer (NSCLC). The response rate to these drugs remains low, however, in NSCLC patients with wild-type EGFR alleles. Combination therapies with EGFR-TKIs and cytotoxic agents are considered a therapeutic option for patients with NSCLC expressing wild-type EGFR. We investigated the antiproliferative effect of the combination of the oral fluorouracil S-1 and the EGFR-TKI gefitinib in NSCLC cells of differing EGFR status. The combination of 5-fluorouracil and gefitinib showed a synergistic antiproliferative effect in vitro in all NSCLC cell lines tested. Combination chemotherapy with S-1 and gefitinib in vivo also had a synergistic antitumor effect on NSCLC xenografts regardless of the absence or presence of EGFR mutations. Gefitinib inhibited the expression of the transcription factor E2F-1, resulting in the down-regulation of thymidylate synthase at the mRNA and protein levels. These observations suggest that gefitinib-induced down-regulation of thymidylate synthase is responsible, at least in part, for the synergistic antitumor effect of combined treatment with S-1 and gefitinib and provide a basis for clinical evaluation of combination chemotherapy with S-1 and EGFR-TKIs in patients with solid tumors.