Gefitinib In Lung Cancer Cells Research Articles

LINC00969 inhibits proliferation with metastasis of breast cancer by regulating phosphorylation of PI3K/AKT and ILP2 expression through HOXD8.

Breast cancer (BC) is a malignancy that is inadequately treated and poses a significant global health threat to females. The aberrant expression of long noncoding RNAs (lncRNAs) acts as a complex with a precise regulatory role in BC progression. LINC00969 has been linked to pyroptotic cell death and resistance to gefitinib in lung cancer cells. However, the precise function and regulatory mechanisms of LINC00969 in BC remain largely unexplored. Cell proliferation, migration, and invasion of BC cells were evaluated using CCK-8 and Transwell assays. Western blotting was employed to analyze the protein expression levels of HOXD8, ILP2, PI3K, t-AKT, and p-AKT. LINC00969 was drastically reduced in BC tissues LINC00969 overexpression markedly suppressed proliferation, migration, and invasion, and blocked PI3K and p-AKT protein expression in MCF-7 cells. Activation of the PI3K/AKT pathway reversed the suppressive effect of LINC0096 overexpression on the proliferation, migration, and invasion of MCF-7 cells. Moreover, LINC00969 overexpression enhanced HOXD8 and blocked ILP2 protein expression in MCF-7 cells. In contrast, activating the PI3K/AKT pathway had no effect on HOXD8 and blocked ILP2 protein expression in MCF-7 cells overexpressing LINC00969. HOXD8 knockdown enhanced ILP2, PI3K, and p-AKT protein expression, and the proliferation, migration, and invasion of MCF-7 cells co-transfected with si-HOXD8 and ov-LINC00969. LINC00969 regulated HOXD8 via binding to miR-425-5p. LINC00969 inhibits the proliferation and metastasis of BC cells by regulating PI3K/AKT phosphorylation through HOXD8/ILP2.

MiR-142-5p reverses the resistance to gefitinib through targeting HOXD8 in lung cancer cells.

To investigate the role and potential mechanism of micro ribonucleic acid (miR)-142-5p in the acquired resistance to gefitinib in lung cancer cells. The drug resistance of PC9/G cells was detected via methyl thiazolyl tetrazolium (MTT) assay. Expression levels of miR-142-5p and HOXD8 in PC9 and PC9/G cells were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting. PC9/G cells were transfected with miR-142-5p mimic, while PC9 cells were transfected with miR-142-5p inhibitor. Subsequently, expression changes of HOXD8 were determined using qRT-PCR and Western blotting, cell sensitivity to gefitinib was detected through MTT assay, and the apoptosis was detected via flow cytometry. Moreover, Dual-Luciferase reporter assay was conducted to determine the relationship between HOXD8 and miR-142-5p. Finally, potential involvement of HOXD8 in miR-142-5p-regulated gefitinib sensitivity was confirmed via rescue tests. PC9/G cells were more significantly resistant to gefitinib compared with its parental PC9 cells. MiR-142-5p was down-regulated in PC9/G cells, while that of HOXD8 was up-regulated in PC9/G cells. Transfection of miR-142-5p mimic could inhibit the expression level of HOXD8 in PC9/G cells and reverse its resistance to gefitinib. Conversely, transfection of miR-142-5p inhibitor could upregulate HOXD8 in PC9 cells and promote its resistance to gefitinib. According to the Dual-Luciferase reporter assay, miR-142-5p could suppress the expression of HOXD8 through the targeted binding to the HOXD8 3'UTR. Moreover, miR-142-5p could regulate mitochondrial apoptosis pathway by targeting HOXD8. Finally, rescue tests confirmed that miR-142-5p regulated the sensitivity of PC9 cells to gefitinib by acting on the target gene HOXD8. Down-regulation of miR-142-5p induces the resistance of lung cancer PC9 cells to gefitinib by upregulating HOXD8.

Fucoidan increased the sensitivity to gefitinib in lung cancer cells correlates with reduction of TGFβ-mediated Slug expression

Gefitinib is a first tyrosine kinase inhibitor (TKI) designed with an EGFR tyrosine kinase for lung cancer targeted therapy. However, some lung cancer patients with wild-type EGFR (wtEGFR) or acquired secondary EGFR mutation showed lower response rate of gefitinib. In this study, we examined the efficacy of fucoidan on altering gefitinib-sensitivity on TKI-resistant lung cancer A549 and H1975 cells. We found that the simultaneous administration of fucoidan and gefitinib synergistically inhibited lung cancer cell viability via activating apoptotic response. Moreover, we found that fucoidan effectively downregulated expressions of mesenchymal-like molecules. Mechanistically, we demonstrated that fucoidan altered the gefitinib-inhibitory rate may result from induction of proteasome-dependent Slug degradation. Abolishment of TGFβ signaling enhanced gefitinib-inhibited cell viability and reduced N-cadherin, Twist and Slug levels. Moreover, knockdown of Slug contributed the increasing the gefitinib-sensitivity of H1975 cells. Our study is the first to find that fucoidan alters the gefitinib-sensitive of TKI-resistant cells by reduction of TGFβ receptor-mediated expressions of mesenchymal-like molecules and induction of Slug degradation. Together, our current results indicate that combination of fucoidan and gefitinib may be a potential and effective therapeutic strategy in gefitinib non-sensitive lung cancer.

Overcoming acquired resistance of gefitinib in lung cancer cells without T790M by AZD9291 or Twist1 knockdown in vitro and in vivo.

The T790M mutation is recognized as a typical mechanism of acquired resistance to first generation of epithermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib in non-small cell lung cancer (NSCLC) patients who are commonly treated by third generation of EGFR-TKI AZD9291 (osimertinib). However, the therapeutic strategy for overcoming acquired resistance to EGFR-TKIs in NSCLC patients without T790M remains to be definitively determined. In the present study, gefitinib-resistant H1650 (H1650GR) or AZD9291-resistant H1975 (H1975AR) was generated by exposing NSCLC cell line H1650 or H1975 to progressively increased concentrations of gefitinib or AZD9291 over 11months. The cytotoxic effects of gefitinib or AZD9291 in vitro were evaluated via the half maximal inhibitory concentrations (IC50s) determined by the MTT assay. IC50 of gefitinib in H1650GR (50.0 ± 3.0µM) significantly increased compared with H1650 (31.0 ± 1.0µM) (p < 0.05). Similarly, the IC50 of AZD9291 in H1975AR (10.3 ± 0.9µM) significantly increased compared with H1975 (5.5 ± 0.6µM) (p < 0.05). However, IC50 of AZD9291 on H1650GR (8.5 ± 0.5µM) did not increase compared with H1650 (9.7 ± 0.7µM). On the other hand, IC50 of AZD9291 on gefitinib-resistant A549 (A549GR established in our previous study) (12.7 ± 0.8µM) was significantly increased compared with A549 (7.0 ± 1.0µM) (p < 0.05). AZD9291 induced caspase 3/7 activation in A549, H1650, and H1650GR, but not in A549GR. Western blot analyses showed that p-Akt played a key role in determining the sensitivities of A549, A549GR, H1650, and H1650GR to gefitinib or AZD9291. Additionally, increased expression of Twist1 was observed in all cells with acquired EGFR-TKI resistance and knockdown of Twist1 by shRNA was found to significantly enhance the sensitivity of A549GR to gefitinib or AZD9291 via reversing epithelial-mesenchymal transition and downregulating p-Akt, but not of H1975AR to AZD9291. The enhanced cytotoxic effect of AZD9291 on A549GR by Twist1 knockdown in vitro was further validated by in vivo studies which showed that Twist1 knockdown could lead to significantly delayed tumor growth of A549GR xenograft with increased sensitivity to AZD9291 treatment in nude mice without any observed side toxic effects. In summary, our study demonstrated that the mechanisms of acquired resistance in different NSCLC cell lines treated by even the same EGFR-TKI might be quite different, which provide a rationale for adopting different therapeutic strategies for those NSCLC patients with acquired EGFR-TKI resistance based on different status of heterogeneous mutations.

Leptomycin B reduces primary and acquired resistance of gefitinib in lung cancer cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib has demonstrated dramatic clinical efficacy in non-small cell lung cancer (NSCLC) patients. However, its therapeutic efficacy is ultimately limited by the development of acquired drug resistance. The aim of this study was to explore the potential utility of chromosome region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) in combination with gefitinib to overcome primary and acquired gefitinib resistance in NSCLC cells. The combinative effects of gefitinib and LMB were evaluated by MTT and its underlining mechanism was assessed by flow cytometry and Western blot. LMB displayed a synergistic effect on gefitinib-induced cytotoxicity in A549 (IC50: 25.0±2.1μM of gefitinib+LMB vs. 32.0±2.5μM of gefitinib alone, p<0.05). Gefitinib+LMB caused a significantly different cell cycle distribution and signaling pathways involved in EGFR/survivin/p21 compared with gefitinib. A549 cells then were treated with progressively increased concentrations of gefitinib (A549GR) or in combination with LMB (A549GLR) over 10months to generate gefitinib resistance. IC50 of gefitinib in A549GLR (37.0±2.8μM) was significantly lower than that in A549GR (53.0±3.0μM, p<0.05), which indicates that LMB could reverse gefitinib-induced resistance in A549. Further mechanism investigation revealed that the expression patterns of EGFR pathway and epithelial-mesenchymal transition (EMT) markers in A549, A549GR, and A549GLR were significantly different. In conclusion, LMB at a very low concentration (0.5nM) combined with gefitinib showed synergistic therapeutic effects and ameliorated the development of gefitinib-induced resistance in lung cancer cells.

Abstract 3352: Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma

Abstract Lung cancer is the most lethal of all cancers world-wide and the majority of patients require chemotherapy or targeted therapy at presentation. Survival is compromised by drug resistance through multiple mechanisms such as by-pass mutations, micro-environment activation of survival programs and induction of tumor initiating cells (TIC). Induction of TIC phenotypes could be accompanied by upregulation of embryonic or developmental pathways but molecular mechanisms are incompletely understood. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) is a multifunctional serine/threonine kinase involved in growth and stress signals integration. A tumor-supportive role has been reported in leukaemia and thyroid cancer but reports on its involvement in TIC regulation are limited. We investigated the role and molecular mechanism of CaMK2A in lung cancer TIC regulation using in vitro and in vivo models. The results showed CaMK2A was overexpressed in lung cancer cell lines and human lung adenocarcinomas compared to normal lung. In cancer cells with stable CaMK2A-knockdown, in vitro reduction in tumor spheres formation, anchorage independent growth and increased reactive oxygen species, as well as in vivo reduction of xenograft tumorigenicity were observed. The opposite effects were observed in cancer cells with stable CaMK2A over-expression. Further, specific pharmacological inhibition of CaMK2A by KN93 led to significantly reduced IC50 for gefitinib in lung cancer cells harboring activating EGFR mutation (HCC827) and in HCC827 exogenously induced for gefitinib resistance. In contrast, IC50 of cisplatin treatment was increased in cancer cells with genetically-induced CaMK2A over-expression. Moreover, CaMK2A knockdown led to reduced mRNA expressions of pluripotency genes (SOX2, NANOG, OCT4) and TIC markers (ALDH, CD166). These results suggested CaMK2A is involved in lung tumorigenicity through TIC regulation. To further investigate the molecular mechanism, we showed tumorigenicity was restored in xenografts with CaMK2A knockdown rescued with SOX2 over-expression. In CaMK2A knockdown cells, reduced SOX2 expression was associated with increased H3K27me3 repressive histone mark, which has not been reported in the literature. Taken together, we have shown CaMK2A plays a role in lung adenocarcinoma and TIC maintenance through histone modification and regulation of SOX2 expression. Targeting TIC through CaMK2A modulation might be useful for overcoming drug resistance and improving long term lung cancer survival. Citation Format: Maria P. Wong, Jing Liu, Zhi-Jie Xiao, Si-Qi Wang, Vicky Pc Tin. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma. [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 3352.

Abstract 4458: EGFR endocytosis is associated with susceptibility to gefitinib in lung cancer cells with wild-type EGFR.

Abstract Although epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a rational targeted drug with dramatic survival benefit in lung cancer, susceptibility to the drug is restricted in lung cancer with activating mutations of EGFR. Nevertheless, the EGFR-TKI still remains an attractive anticancer drug in lung cancer with wild-type EGFR (wtEGFR). Therefore, in this study, we aimed to investigate unknown mechanism which can contribute to response of the EGFR targeted therapy using gefitinib, a well-known EGFR-TKI, in eight lung cancer cells with wtEGFR. We first examined differential effects of gefitinib on cell survival, morphology, motility, cell cycle and EGFR signaling between the TKI-sensitive and -resistant cells. As a result, although the TKI-resistant cells did not show any changes in cell phenotype by gefitinib treatment, phosphorylation of EGF induced EGFR and downstream effectors was inhibited by the drug treatment as if those in the TKI-sensitive cells. Additionally, we found difference in cellular distribution of EGF induced EGFR after gefitinib treatment between the TKI-sensitive and -resistant cells and it was associated with EGFR endocytosis. Based on our results, we next investigated that regulation of EGFR endocytosis could affect on the response of gefitinib in lung cancer cells with wtEGFR. Inhibition of EGFR endocytosis by combination treatment with gefitinib and two endocytosis inhibitors, dynasore and dynole 34-2, led to significantly decreased cell viability in the TKI-resistant cells compared with treatment of these drugs alone. Moreover, the reduced cell survival after the combination treatment was concerned with increased apoptotic cell death, accompanying by elevated cleavage of poly ADP ribose polymerase (PARP) and decreased myeloid cell leukemia-1 (Mcl-1), X-linked inhibitor of apoptosis protein (XIAP), Survivin and Livin among anti-apoptotic proteins. In addition, we found differentially expressed Rab25, a member of the RAS superfamily of small GTPases, between the TKI-sensitive and-resistant cells through genome-wide gene expression analysis. The expression status of Rab25 was partially associated with response of gefitinib in lung cancer cells with wtEGFR. Furthermore, the silencing of Rab25 in the TKI-sensitive cells with its normal expression reversed sensitivity to gefitinib treatment. In conclusion, our results provided molecular evidences that EGFR endocytosis might contribute to cell survival and response to the EGFR-TKI as a bypass survival mechanism in lung cancer with wtEGFR. Thus, targeting EGFR endocytosis could help us to overcome therapeutic limitation of EGFR-TKI in lung cancer with wtEGFR and Rab25 expression may be an important tool for predicting efficacy of the drug treatment. Citation Format: Ukhyun Jo, Young Mi Whang, Jae Sook Sung, Kyong Hwa Park, Seung Tae Kim, Yeul Hong Kim. EGFR endocytosis is associated with susceptibility to gefitinib in lung cancer cells with wild-type EGFR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4458. doi:10.1158/1538-7445.AM2013-4458

Abstract 1306: SR48692 inhibits EGF receptor transactivation and proliferation of lung cancer cells.

Abstract Neurotensin (NTS) is an autocrine growth factor for some lung cancer cells (Moody et al., 1985;Life Sci 36:1727). NTS binds with high affinity to the GPCR NTSR1 causing phosphatidyl inositol turnover and proliferation of lung cancer cells; SR48692 is a NTSR1 antagonist (Moody et al., 2001; Peptides 22:109). NTSR1 expression is associated with poor survival of non-small cell lung cancer (NSCLC) patients (Alfano et al., 2010; Clin Cancer Res 16:4401). NSCLC cells express the epidermal growth factor receptor (EGFR) whose tyrosine kinase activity is inhibited by gefitinib. Here the abillity of SR48692 to block EGFR transactivation caused by NTS was investigated. NTS and NTS8-13 but not NTS1-8 addition to NSCLC cell lines NCI-H1299 or A549 cells increased phosphorylation of Tyr1068 of the EGFR after 2 min which was blocked by 10 uM SR48692 or 10 ug/ml gefitinib. The ability of NTS to regulate EGF receptor transactivation was blocked by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant) and transforming growth factor TGF) α antibody. By ELISA, NTS significantly increased secretion of TGFα from lung cancer cells. NTS but not levocabastine (NTSR2 agonist) increased cytosolic Ca2+ within seconds after addition to NCI-H1299 cells which was antagonized by SR48692. NTS addition to NCI-H1299 cells increased tyrosine phosphorylation of beta-catenin, ERK, FAK, PYK-2 and Src which was antagonized by SR48692. SR48692 inhibited the proliferation of NCI-H1299 and A549 cells which have wild type EGFR. Also, SR48692 potentiated the ability of gefitinib to inhibit growth of NCI-H1299 and A549 cells. These results indicate that SR48692 is a NTSR1 antagonist which increases the potency of gefitinib in lung cancer cells. Citation Format: Terry W. Moody, Robert T. Jensen. SR48692 inhibits EGF receptor transactivation and proliferation of lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1306. doi:10.1158/1538-7445.AM2013-1306

Abstract 964: Gefitinib resistance due to STAT3-mediated Akt activation in lung cancer cells.

Abstract Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, are effective drugs for a subset of non-small cell lung cancer. Gefitinib binds to the tyrosine kinase domain of EGFR and hence inactivates its downstream prosurvival signaling pathways, including PI3k/Akt and MAPK pathways. However, most patients received gefitinib eventually developed resistance to this drug. In this study, we demonstrated that in gefitinib-treated A549 cells (human lung cancer cell line), Akt activation undergoes a time-dependent recovery following an initial inhibition by gefitinib. Since constant activation of Akt has been associated with treatment failure of gefitinib in lung cancer cells, exploring the mechanisms that lead to Akt restoration should provide new insights into the drug resistance and therapeutic efficacy. For that purpose, alterations in Akt activation and other signaling pathways, including MAPK and STAT3, involved in EGFR-mediated cellular responses were determined in A549 cells treated with gefitinib. Among them, we found that activation of STAT3 was inhibited in the cells without gefitinib treatment. Gefitinib treatment inhibited EGFR phosphorylation on tyrosine 1086 (Y1086), which leads to STAT3 activation. Further studies showed that interruption of the STAT3 signaling by either chemical inhibitor or siRNA against STAT3 prevented fast recovery of Akt activation and enhanced gefitinib-induced suppression of cell proliferation. Taken together, these data suggest that activation of STAT3 is an intrinsic mechanism of drug resistance in response to EGFR TKIs. Combinational targeting on both EGFR and STAT3 may enhance the efficacy of gefitinib or other EGFR TKIs on lung cancer. Citation Format: Kai Wu, Yongju Lu, Bailing Chen, Qingshan Chang, Ping Qiu, Miaomiao Yu. Gefitinib resistance due to STAT3-mediated Akt activation in lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2013-964

MicroRNAs reduce tumor growth and contribute to enhance cytotoxicity induced by gefitinib in non-small cell lung cancer

MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, involved in diverse physiological and pathological processes. An oncogenic or tumor-suppressive miRNA may have potential as a therapeutic target to control cancers. Gefitinib is a tyrosine kinase inhibitor that targets epidermal growth factor receptor (EGFR). H460 and A549 cells with EGFR receptor-independent over-activation of protein kinase B (Akt) or extracellular signal-regulated kinases (ERK) are significantly resistant to gefitinib. The first aim of this study was to confirm a role for three miRNAs (let-7a, hsa-miR-126, and hsa-miR-145) in the inhibition of proliferation in non-small cell lung cancer (NSCLC) cells. A second aim was to evaluate three miRNAs for their abilities to overcome cellular resistance and enhance the gefitinib cytotoxicity. The expression of miRNAs was estimated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was examined by sulforhodamine B assay and tumor xenografts were measured in SCID/beige mice. The activation of Akt and ERK was observed by Western blotting. Forced expression of individual miRNA suppressed the growth of two cell lines and xenografts. The effect varied among different miRNAs and cells. Restoration of hsa-miR-126 more obviously inhibited cell growth than did restoration of hsa-miR-145 in both cells, and the suppressive effect was more significant in H460 xenografts than in A549 xenografts. Western blotting revealed that the inhibition of cell proliferation resulted from the inhibition of the activation of Akt and ERK. Moreover, forced expression of miRNAs contributed to enhanced cytotoxicity induced by gefitinib in lung cancer cells; especially in hsa-miR-126, the highest value of half max inhibitory (IC50) was increased sixfold. These findings confirm that tumor-suppressive miRNAs can inhibit the growth of NSCLC cells and enhance the targeted agents cytotoxicity, suggesting novel potential approaches to an improvement in chemotherapy.