Gefitinib-resistant Non-small Cell Lung Cancer Research Articles

Restricting extracellular Ca2+ on gefitinib-resistant non-small cell lung cancer cells reverses altered epidermal growth factor-mediated Ca2+response, which consequently enhances gefitinib sensitivity.

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.

Abstract 1874: Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway

Abstract Mesenchymal epithelial transition factor (MET) activation has been considered to mediate primary and acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC), but the mechanisms are not wholly clear. Meanwhile, the effective therapeutic strategy for these patients remains controversial. We previously presented a lung adenocarcinoma patient with dual EGFR-MET alterations benefited from bevacizumab + erlotinib as first-line treatment, achieving 13 months partial response (PR). PFS of another phase reached to 9 months with the combination of osimertinib + crizotinib + bevacizumab when disease proceeded due to increased MET copy number. Persistent PFS benefit was confirmed when the patient was administrated with different TKIs treatment in the presence of bevacizumab. However, the underlying mechanism remains elusive. Thus, we assumed that VEGF might be involved in the regulation of the MET signaling pathway and the addition of anti-VEGF therapy might be promising. In this study, we firstly confirmed whether MET overexpression alone might causally responsible for resistance to gefitinib treatment. Thus, we conducted in vitro forward genetic experiments, we took advantage of the EGFR-mutant lung cancer cell line PC-9, which had been shown to be sensitive to gefitinib. Ectopic overexpression of MET in PC-9 parental cells (PC-9/MET) was able to confer resistance to gefitinib by activating AKT and ERK signaling and sustaining proliferation.Meanwhile we got a gefitinib resistant EGFR mutant NSCLC cell line, HCC827 (HCC827-GR), which showing acquired MET amplification. Interestingly, we found that VEGF promoted cell survival and elicited adaptive resistance to gefitinib and crizotinib alone or in combination in MET transduced PC9-MET cells. This phenomenon also existed in HCC827-GR which harboring MET amplification. Importantly, addition of the VEGF inhibitor bevacizumab was able to restore partial sensitivity to gefitinib in both PC-9/MET and HCC827-GR cells while triple inhibition of EGFR, MET, and VEGF could suppresses the cell proliferation more remarkable. Moreover, we observed that VEGF could facilitate MET phosphorylation and activate several intracellular signaling pathways, including ERK, AKT, and Stat3. Then we pretreated cells with several specific kinase inhibitors, followed by treatment with VEGF. We found that VEGF-mediated up-regulation of MET depended on the ERK pathway and was independent of AKT and Stat3 pathways. These findings were further confirmed in human NSCLC Xenograft models. Addition of bevacizumab to TKIs resulted in stronger inhibition of tumor growth than TKI alone or TKIs combination treatment, by blocking both EGFR and MET signal transduction. In conclusion, bevacizumab + TKIs enhanced antitumor activity in NSCLC with EGFR/MET co-alteration through inhibition of the VEGF/ ERK/MET signaling axis. Citation Format: Qian Chu, Shanshan Huang, Yuan Gao, Jie Lin, Yuan Chen, Bo Zhu, Kongming Wu. Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1874.

FGL1 regulates acquired resistance to Gefitinib by inhibiting apoptosis in non-small cell lung cancer

BackgroundThis study investigated the role of fibrinogen-like protein 1 (FGL1) in regulating gefitinib resistance of PC9/GR non-small cell lung cancer (NSCLC).MethodsThe effect of different concentrations of gefitinib on cell proliferation were evaluated using the CCK-8 assay. FGL1 expression in the normal human bronchial epithelial cell line Beas-2B, as well as four lung tumor cell lines, H1975, A549, PC9, and PC9/GR, was investigated by using western blotting and qRT-PCR. FGL1 was knocked down using small interfering RNA to evaluate the effects of FGL1 on PC9 and PC9/GR. The correlation between FGL1 expression and gefitinib resistance was determined in vitro via CCK-8 and colony formation assays, and flow cytometry and in vivo via flow cytometry and immunohistochemistry.ResultsFGL1 expression was significantly upregulated in non-small cell lung cancer cells with EGFR mutation and higher in the gefitinib-resistant NSCLC cell line PC9/GR than in the gefitinib-sensitive NSCLC cell line PC9. Further, FGL1 expression in PC9 and PC9/GR cells increased in response to gefitinib treatment in a dose-dependent manner. Knockdown of FGL1 suppressed cell viability, reduced the gefitinib IC50 value, and enhanced apoptosis in PC9 and PC9/GR cells upon gefitinib treatment. Mouse xenograft experiments showed that FGL1 knockdown in PC9/GR tumor cells enhanced the inhibitory and apoptosis-inducing actions of gefitinib. The potential mechanism of gefitinib in inducing apoptosis of PC9/GR cells involves inhibition of PARP1 and caspase 3 expression via suppression of FGL1.ConclusionsFGL1 confers gefitinib resistance in the NSCLC cell line PC9/GR by regulating the PARP1/caspase 3 pathway. Hence, FGL1 is a potential therapeutic target to improve the treatment response of NSCLC patients with acquired resistance to gefitinib.

CircSETD3 Contributes to Acquired Resistance to Gefitinib in Non-Small-Cell Lung Cancer by Targeting the miR-520h/ABCG2 Pathway

Gefitinib is a first-line treatment for patients with non-small-cell lung cancer (NSCLC), but acquired resistance is a major obstacle to its therapeutic efficacy, and the underlying mechanisms are not fully elucidated. Recent studies have indicated that circular RNAs play a crucial role in chemoresistance, but their expression and function in NSCLC cells with acquired resistance to gefitinib are largely unknown. In this study, we determined that circSETD3 was significantly upregulated in gefitinib-resistant NSCLC cell lines and the plasma of gefitinib-resistant NSCLC patients. circSETD3 markedly decreased the gefitinib sensitivity of NSCLC cells both in vitro and in nude mice xenografts. It could directly bind to miR-520h and lead to the upregulation of ATP-binding cassette subfamily G member 2 (ABCG2), an efflux transporter of gefitinib, resulting in a reduced intracellular gefitinib concentration. Moreover, we reported that the downregulation of serine/arginine splicing factor 1 (SRSF1) contributed to, at least in part, the increased expression of circSETD3 in NSCLC cells with acquired resistance to gefitinib. Taken together, our findings indicated that circSETD3 may serve as a prognostic biomarker and a potential therapeutic target for acquired resistance to gefitinib in NSCLC.

MiR-365a-5p suppresses gefitinib resistance in non-small-cell lung cancer through targeting PELI3.

Aim: Demonstrate the function of dysregulated miR-365a-5p-PELI3 signaling axis in the generation of gefitinib resistance during treatment for non-small-cell lung cancer (NSCLC). Patients & methods: All the NSCLC patients who participated in this research were recruited from the Second Hospital of Hebei Medical University. PC9 cells and PC9GR cells were cultured for in vitro experiments. Results: Patients who were primary resistant to EGFR-tyrosine kinase inhibitorhad lower miR-365a-5p levels. MiR-365a-5p directly targeted PELI3 mRNA. MiR-365a-5p overexpression enhanced the function of gefitinib in inhibiting cell viability. Tumor growth was suppressed through miR-365a-5p in nude mice. Conclusion: Dysregulated miR-365a-5p-PELI3 signaling axis triggered the generation of gefitinib resistance in NSCLC.

Chelidonine selectively inhibits the growth of gefitinib-resistant non-small cell lung cancer cells through the EGFR-AMPK pathway

Tyrosine kinase inhibitors (TKIs) have been widely used for the clinical treatment of patients with non-small cell lung cancer (NSCLC) harboring mutations in the EGFR. Unfortunately, due to the secondary mutation in EGFR, eventual drug-resistance is inevitable. Therefore, to overcome the resistance, new agent is urgently required. Chelidonine, extracted from the roots of Chelidonium majus, was proved to effectively suppress the growth of NSCLC cells with EGFR double mutation. Proteomics analysis indicated that mitochondrial respiratory chain was significantly inhibited by chelidonine, and inhibitor of AMPK effectively blocked the apoptosis induced by chelidonine. Molecular dynamics simulations indicated that chelidonine could directly bind to EGFR and showed a much higher binding affinity to EGFRL858R/T790M than EGFRWT, which demonstrated that chelidonine could selectively inhibit the phosphorylation of EGFR in cells with EGFR double-mutation. In vivo study revealed that chelidonine has a similar inhibitory effect like second generation TKI Afatinib. In conclusion, targeting EGFR and inhibition of mitochondrial function is a promising anti-cancer therapeutic strategy for inhibiting NSCLC with EGFR mutation and TKI resistance.

Genome-wide identification and characterization of long non-coding RNAs involved in acquired resistance to gefitinib in non-small-cell lung cancer

Acquired resistance is a major obstacle to the therapeutic efficacy of gefitinib in non-small-cell lung cancer (NSCLC). Current knowledge about the role of long non-coding RNAs (lncRNAs) in this phenomenon is insufficient. In this study, we searched RNA sequencing data for lncRNAs associated with acquired resistance to gefitinib in NSCLC, and constructed a functional lncRNA-mRNA co-expression network and protein-protein interaction (PPI) network to analyze their putative target genes and biological functions. The expression levels of 14 outstanding dysregulated lncRNAs and mRNA were verified using real-time PCR. Changes in the expression levels of 39 lncRNAs and 121 mRNAs showed common patterns in our two pairs of gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. The co-expression network included 1235 connections among these common differentially expressed lncRNAs and mRNAs. The significantly enriched signaling pathways based on dysregulated mRNAs were mainly involved in the Hippo signaling pathway; proteoglycans in cancer; and valine, leucine, and isoleucine biosynthesis. The results show that LncRNAs play an important part in acquired gefitinib resistance in NSCLC by regulating mRNA expression and function, and may represent potential new molecular biomarkers and therapeutic targets for gefitinib-resistant NSCLC.

Inhibition of Wnt/β-catenin pathway reverses multi-drug resistance and EMT in Oct4+/Nanog+ NSCLC cells

Cancer drug resistance and epithelial-mesenchymal transition (EMT), a critical process of cancer invasion and metastasis, have recently been associated with the existence of cancer stem cells (CSCs). However, there are no appropriate CSC-markers of non-small cell lung cancer (NSCLC)-associated drug resistance and EMT. It is unknown if and how the drug-resistant and EMT phenotypes in NSCLC cells link to specific stemness-related pathways. Here, we found a significant elevated expression of both Oct4 and Nanog in gefitinib-resistant NSCLC cells, which displayed multi-drug resistance (MDR) properties and exhibited EMT phenotype. Ectopic co-expression of Oct4/Nanog empowered NSCLC cells with cancer stem cell properties, including self-renewal, drug resistance, EMT and high tumorigenic capacity. Following molecular mechanism investigation indicated Oct4/Nanog-regulated drug resistance and EMT change through Wnt/β-catenin signaling activation. Moreover, silencing β-catenin abrogated Oct4/Nanog-mediated MDR and EMT process in NSCLC cells. Our findings propose Wnt/β-catenin pathway as a promising therapeutic target for the treatment of progression and metastasis of NSCLC with CSC-like signatures and epithelial-mesenchymal transition phenotype.

Cancer cell‑specific anticancer effects of Coptischinensis on gefitinib‑resistant lung cancer cells are mediated through the suppression of Mcl‑1 and Bcl‑2.

The epidermal growth factor receptor (EGFR)‑tyrosine kinase inhibitor (TKI), gefitinib, is an effective therapeutic drug used in the treatment of non‑small cell lung cancers (NSCLCs) harboring EGFR mutations. However, acquired resistance significantly limits the efficacy of EGFR‑TKIs and consequently, the current chemotherapeutic strategies for NSCLCs. It is, therefore, necessary to overcome this resistance. In the present study, the anticancer potential of natural extracts of Coptischinensis (ECC) against gefitinib‑resistant (GR) NSCLC cells were investigated invitro and invivo. ECC inhibited the viability, migration and invasion, and effectively induced the apoptosis of GR cells. These effects were associated with the suppression of EGFR/AKT signaling and the expression of anti‑apoptotic proteins, Mcl‑1 and Bcl‑2, which were overexpressed in GR NSCLC cells. Combination treatment with ECC and gefitinib enhanced the sensitivity of GR cells to gefitinib invitro, but not invivo. However, ECC increased the survival of individual zebrafish without affecting the anticancer effect to cancer cells invivo, which indicated a specific cytotoxic effect of ECC on cancer cells, but not on normal cells; this is an important property for the development of novel anticancer drugs. On the whole, the findings of the present study indicate the potential of ECC for use in the treatment of NSCLC, particularly in combination with EGFR‑TKI therapy, in EGFR‑TKI‑resistant cancers.

LncRNA CCAT1 Acts as a MicroRNA-218 Sponge to Increase Gefitinib Resistance in NSCLC by Targeting HOXA1

Long non-coding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) has been reported to play important roles in the development and progression of multiple human malignancies. However, the functional role and molecular mechanism of CCAT1 on gefitinib resistance in non-small cell lung cancer (NSCLC) are largely unclear. The aim of this study is to explore the roles of CCAT1 on gefitinib resistance in NSCLC and to explore the underlying mechanisms. The quantitative real-time PCR (qRT-PCR) analysis was to investigate the expression pattern of CCAT1 in gefitinib-resistant NSCLC patient tissues and cell lines, and then the effects of CCAT1 on gefitinib resistance of NSCLC in vitro and in vivo. Furthermore, bioinformatics online program predictions and luciferase reporter assay were used to validate the association of CCAT1 and miR-218 in NSCLC cells. In this study, CCAT1 was observed to be upregulated in gefitinib-resistant patient tissues and cell lines. In vitro and in vivo experiments demonstrated that CCAT1 knockdown impaired cell proliferation and promoted the gefitinib-induced cell apoptosis. Furthermore, we demonstrated that CCAT1 acts as a sponge for miR-218, and verified that HOXA1 is a novel target of miR-218. These results suggest that CCAT1 may serve as a promising therapeutic target for the treatment of epidermal growth factor receptor (EGFR) plus NSCLC patients.

Inhibition of Alternative Cancer Cell Metabolism of EGFR Mutated Non-Small Cell Lung Cancer Serves as a Potential Therapeutic Strategy.

Targeted therapy is an efficient treatment for patients with epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Therapeutic resistance invariably occurs in NSCLC patients. Many studies have focused on drug resistance mechanisms, but only a few have addressed the metabolic flexibility in drug-resistant NSCLC. In the present study, we found that during the developing resistance to tyrosine kinase inhibitor (TKI), TKI-resistant NSCLC cells acquired metabolic flexibility in that they switched from dependence on glycolysis to oxidative phosphorylation by substantially increasing the activity of the mitochondria. Concurrently, we found the predominant expression of monocarboxylate transporter 1 (MCT-1) in the TKI-resistant NSCLC cells was strongly increased in those cells that oxidized lactate. Thus, we hypothesized that inhibiting MCT-1 could represent a novel treatment strategy. We treated cells with the MCT-1 inhibitor AZD3965. We found a significant decrease in cell proliferation and cell motility in TKI-sensitive and TKI-resistant cells. Taken together, these results demonstrated that gefitinib-resistant NSCLC cells harbored higher mitochondrial bioenergetics and MCT-1 expression. These results implied that targeting mitochondrial oxidative phosphorylation proteins or MCT-1 could serve as potential treatments for both TKI-sensitive and –resistant non-small cell lung cancer.

Screening Circular RNAs Related to Acquired Gefitinib Resistance in Non-small Cell Lung Cancer Cell Lines

Background: Gefitinib is a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR) used to treat EGFR mutation-positive patients with non-small cell lung cancer (NSCLC). However, the efficacy of gefitinib is limited by the development of acquired resistance. Studies have shown that circular RNAs (circRNAs) are involved in the acquired resistance to many anticancer agents. However, the expression profiles and functions of circRNAs in gefitinib resistance in NSCLC are poorly understood so far.Methods: In this study, circRNA expression profiling was explored in two gefitinib-resistant NSCLC cell lines (HCC827/GR and PC9/GR) and their parental sensitive cells (HCC827 and PC9) using high-throughput RNA sequencing. Quantitative real-time PCR (qRT-PCR) was used to confirm the expression of selected differentially expressed circRNAs. Bioinformatic tools including gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), network analysis, and Kaplan-Meier plotter database were used to predict the functions and pathways of these differentially expressed circRNAs.Results: We identified 46 and 56 differentially expressed circRNAs in HCC827/GR and PC9/GR cell lines, respectively, compared with those in their parental cell lines. Gene ontology and KEGG pathway analysis identified that the host linear transcripts of these differentially expressed circRNAs were involved in many critical biological pathways and molecular functions. We found that hsa_circ_0000567 was consistently up-regulated, and hsa_circ_0006867 was consistently down-regulated in two resistant cell lines. We further used hsa_circ_0000567 and hsa_circ_0006867 as key circRNAs to construct circRNA-miRNA-mRNA networks. Several target mRNAs of these two circRNAs had been shown to significantly associate with the overall survival of patients with lung cancer.Conclusions: In this study, we generated the comprehensive expression and functional profiles of the differentially expressed circRNAs between gefitinib-resistant and -sensitive NSCLC cells, and showed that dysregulation of circRNAs might play an important role in the development of acquired resistance to gefitinib in NSCLC.

Cyclooxygenase-2 mediates gefitinib resistance in non-small cell lung cancer through the EGFR/PI3K/AKT axis.

Gefitinib is a potent inhibitor of EGFR and represents the front-line treatment for non-small cell lung cancer (NSCLC) therapeutics. However, NSCLC patients are prone to develop acquired resistance through as yet, undefined mechanisms of resistance. Here, we investigated the role of COX-2 during gefitinib resistance in NSCLC cells and revealed its underlying mechanism(s) of action. We report the upregulation of COX-2 in gefitinib-resistant NSCLC tissues and cells, which is associated with poor prognosis. In vitro assays in NSCLC cells (PC9/GR) showed that COX-2 facilitates gefitinib resistance in NSCLC cells through its effects on P-gp, MRP1, and BCRP, and cancer cell migration and invasion. In vivo, COX-2 silencing could repress tumor growth. We found that the overexpression of COX-2 enhances the transcription of MMP-2, MMP-7, and MMP-9 which mediates PI3K-AKT activation. In summary, we demonstrate that COX-2 mediates the gefitinib resistance of NSCLC cells through its interaction with EGFR and the PI3K-AKT axis. This highlights COX-2 as a novel molecular target for NSCLC.

Combined treatment with N-acetylcysteine and gefitinib overcomes drug resistance to gefitinib in NSCLC cell line.

We aimed to explore the molecular substrate underlying EGFR‐TKI resistance and investigate the effects of N‐acetylcysteine (NAC) on reversing EGFR‐TKI resistance. In the current research, the effects of NAC in combination with gefitinib on reversing gefitinib resistance were examined using CCK‐8 assay, combination index (CI) method, matrigel invasion assay, wound‐healing assay, flow cytometry, western blot, and quantitative real‐time PCR (qRT‐PCR). CCK8 assay showed that NAC plus gefitinib combination overcame EGFR‐TKI resistance in non‐small cell lung cancer (NSCLC) cells by lowering the value of half maximal inhibitory concentration (IC50). CI calculations demonstrated a synergistic effect between the two drugs (CI < 1). Matrigel invasion assay and wound healing assay demonstrated a decrease in migration and invasion ability of PC‐9/GR cells after NAC and gefitinib treatment. Flow cytometry displayed enhanced apoptosis in the combination group. Western blot and qRT‐PCR revealed that increased E‐cadherin and decreased vimentin in the combination group. When PP2 was administered with gefitinib, the same effects were seen. Our findings suggest that NAC could restore the sensitivity of gefitinib‐resistant NSCLC cells to gefitinib via suppressing Src activation and reversing epithelial‐mesenchymal transition.

Overcoming the Intrinsic Gefitinib-resistance via Downregulation of AXL in Non-small Cell Lung Cancer.

BackgroundResistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is a limited factor in the treatment of non-small-cell lung cancer (NSCLC) patients. Therefore, ongoing studies are trying to identify EGFR-TKIs-resistant mechanisms and to discover novel therapeutic strategies and targets for NSCLC treatment.MethodsIn the present study, the possibility of overcoming intrinsic gefitinib-resistance was examined by regulating the expression of AXL. A natural product-derived antitumor agent, yuanhuadine (YD) was employed to modulate the expression of AXL in the cells.ResultsTreatment with YD effectively downregulated AXL expression in AXL-overexpressed gefitinib-resistant H1299 cells. The combination of gefitinib and YD exhibited a synergistic grwoth-inhibitory activity in H1299 cells by downregulation of AXL expression.ConclusionsBased on these findings, AXL was found to be a promising therapeutic target to overcome the intrinsic resistance to gefitinib in NSCLC. Furthermore, YD is able to effectively regulate the expression of AXL and thus it may be applicable as a potential lead compound for the treatment of gefitinib-resistant NSCLC.

MiR-762 activation confers acquired resistance to gefitinib in non-small cell lung cancer

BackgroundEpidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) (e.g. gefitinib) currently remain the first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC) with activating EGFR mutation. However, acquired resistance to gefitinib, which occurs frequently through unidentified mechanisms, significantly attenuate therapeutic effectiveness. Previous miRNA microarray analysis reveals that expression levels of a conserved oncomiR miR-762 are significantly upregulated in gefitinib-resistant NSCLC cells. We therefore aim to elucidate the role and underlying mechanisms of miR-762 during the pathogenesis of gefitinib resistance.MethodsmiR-762 expression in gefitinib-resistant NSCLC tissues and cells was evaluated using RT-qPCR. The potential regulation of miR-762 expression by IL-6 was studied using pharmacological and biochemical approaches. Effects of miR-762 manipulation on sensitivity to gefitinib was assessed using MTT, apoptotic ELISA and xenograft model. Finally, the posttranscriptional regulation of active BCR related protein (ABR) by miR-762 was determined using luciferase assay and site-directed mutagenesis.ResultsmiR-762 expression was upregulated in gefitinib-resistant NSCLC tissues and cells, and this upregulation predicted a poor post-chemotherapy prognosis in NSCLC patients. miR-762 upregulation, induced by IL-6 signaling, significantly enhanced cell survival and rendered NSCLC cells unresponsiveness to gefitinib-elicited cell death. We finally provided the evidence that the oncogenic effect of miR-762 was mediated mainly through posttranscriptional repression of ABR in gefitinib-resistant NSCLC cells.ConclusionsOur findings provide a rationale for future efforts testing miR-762 inhibition and ABR restoration co-treatment in patients with recurrent EGFR mutant NSCLC to therapeutically combat the heterogeneity of EGFR-TKIs resistance mechanisms.

RETRACTED: lncRNA UCA1 Promotes Gefitinib Resistance as a ceRNA to Target FOSL2 by Sponging miR-143 in Non-small Cell Lung Cancer

Exosomes could mediate cell-cell crosstalk in cancer progression by transferring long noncoding RNAs (lncRNAs). The aim of this study is to explore the roles of the exosomal lncRNA urothelial carcinoma-associated 1 (UCA1) on gefitinib resistance in non-small cell lung cancer (NSCLC). First, we detected the expression of UCA1 in gefitinib-resistant and gefitinib-sensitive NSCLC by quantitative real-time PCR; the expression occurred in tissues, cell lines, and exosomes. Cell phenotypes and animal experiments were performed to determine the effects of UCA1 and exosomal UCA1. Furthermore, bioinformatics online programs and luciferase reporter assay were used to validate the association of UCA1 and miR-143 in NSCLC cells. We observed that UCA1 was increased in both gefitinib-resistant NSCLC cells and their secreted exosomes. In vitro and in vivo experiments demonstrated that UCA1 knockdown impaired cell proliferation and promoted the gefitinib-induced cell apoptosis. Then we demonstrated that repressed UCA1 promoted the miR-143 expression, and miR-143 could bind to the predicted binding site of UCA1. We then dissected the effect of miR-143 on gefitinib resistance in NSCLC and proved the suppressive role of miR-143. Furthermore, we found that miR-143 displayed its role via modulating the FOSL2 expression. In summary, our findings indicate that exosomal UCA1 may serve as a promising therapeutic target for the treatment of epidermal growth factor receptor-positive (EGFR+) NSCLC patients.

Long Noncoding RNA LINC01116 Contributes to Gefitinib Resistance in Non-small Cell Lung Cancer through Regulating IFI44

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, have been established as first-line treatments for non-small cell lung cancer (NSCLC) patients and have exhibited notable clinical efficacy. However, resistance to TKIs has become one of the major obstacles in improving the therapeutic efficacy of patients with NSCLC. This study aims to investigate the role of the long non-coding RNA (lncRNA) LINC01116 in gefitinib resistance of NSCLC and explore its underlying mechanism. In this study, we found that LINC01116 is upregulated in the gefitinib-resistant NSCLC cells and tissues. Loss- and gain-of-function assays uncovered that LINC01116 downregulation sensitized gefitinib resistance, whereas the overexpression of LINC01116 conferred PC9/R cells to gefitinib treatment. Moreover, LINC01116 silencing increased IFI44 expression. Overexpression of IFI44 reversed the resistance to gefitinib in PC9/R cells, and rescue experiments confirmed that LINC01116 affects the gefitinib resistance of PC9/R cells partly dependent on regulating IFI44 expression. Moreover, downregulation of LINC01116 increased the sensitivity of PC9/R cells to gefitinib in vivo. Our study demonstrates that LINC01116 plays a critical role in gefitinib resistance of NSCLC cells by affecting IFI44 expression, providing a novel therapeutic target to overcome TKI resistance in NSCLC.

Tanshinone IIA Reverses Gefitinib-Resistance In Human Non-Small-Cell Lung Cancer Via Regulation Of VEGFR/Akt Pathway.

BackgroundGefitinib-resistance is a primary obstacle for the treatment of non-small-cell lung cancer (NSCLC). It has been shown that tanshinone IIA (Tan IIA) could induce apoptosis of NSCLC cells. However, the role of combination of gefitinib with Tan IIA on gefitinib-resistance NSCLC cells remains unclear. Thus, this study aimed to investigate the role of combination on the proliferation, apoptosis and invasion of gefitinib-resistance NSCLC cells.MethodsCCK-8, flow cytometric and transwell assays were applied to detect proliferation, apoptosis and invasion in gefitinib-resistance NSCLC cells, respectively. In addition, Western blotting assay was used to detect the expressions of p-EGFR, p-VEGFR2, and p-Akt in HCC827/gefitinib cells.ResultsIn this study, Tan IIA enhanced the cytotoxic effect of gefitinib in gefitinib-resistance NSCLC cells. In addition, the inhibitory effects of gefitinib on the proliferation, migration and invasion of gefitinib-resistance NSCLC cells were enhanced in the presence of Tan IIA. Moreover, Tan IIA enhanced the pro-apoptotic effect of gefitinib in gefitinib-resistance NSCLC cells via increasing the level of cleaved caspase 3. Meanwhile, Tan IIA enhanced the sensitivity of HCC827/gefitinib cells to gefitinib via downregulation of the VEGFR2/Akt pathway. In vivo experiments further confirmed that combination of gefitinib with Tan IIA inhibited tumor growth in mouse xenograft model of HCC827/gefitinib.ConclusionWe found that Tan IIA could enhance gefitinib sensitivity in gefitinib-resistance NSCLC cells. Therefore, combination of gefitinib with Tan IIA might be considered as a therapeutic approach for the treatment of gefitinib-resistant NSCLC.

FGFR1 Induces Acquired Resistance Against Gefitinib By Activating AKT/mTOR Pathway In NSCLC.

ObjectiveAs an epidermal growth factor, receptor-tyrosine kinase inhibitor (EGFR-TKI), gefitinib demonstrates a good therapeutic effect in patients with EGFR-mutant non-small-cell lung cancer (NSCLC). However, an overwhelming majority of these patients inevitably develop resistance against gefitinib. Unfortunately, the mechanism underlying this phenomenon is still not fully understood. Here we aim to reveal the mechanism of gefitinib resistance in NSCLC induced by FGFR1.Materials and methodsWe used high-throughput sequencing to compare the mRNA expression profiles of PC9 and PC9-GR (gefitinib-resistant) cells. The clinical significance of fibroblast growth factor receptor 1 (FGFR1) in NSCLC was also investigated using immunohistochemistry and Kaplan-Meier survival analysis. Finally, the in vitro molecular mechanisms were analyzed using confocal laser microscopy, Western blotting, transwell assay, colony formation assay, CCK-8 assay, and apoptosis assay.ResultsWe observed that FGFR1 was highly expressed in NSCLC tissues and was closely associated with poor prognosis. Cytological experiments showed that FGFR1 promoted the proliferation and migration of PC9-GR cells and mediated their resistance to gefitinib. Furthermore, studies aimed at unraveling this mechanism revealed that FGFR1 activated the AKT/mTOR signaling pathway. These findings show that the FGFR1/AKT/mTOR signaling pathway plays a vital role in acquired resistance against gefitinib in NSCLC.ConclusionThis work provides new evidence that FGFR1 functions as a key regulator of gefitinib resistance, thereby demonstrating its potential as a novel biomarker and therapeutic target for NSCLC.