Gefitinib Resistance Research Articles

Macrophage renewal modes affect acquired resistance to gefitinib in EGFR‑mutant lung cancer PC‑9 cells.

EGFR tyrosine kinase inhibitors (TKIs) have been successfully used in lung cancer treatment. Nevertheless, tumor cells may develop resistant phenotypes in the tumor microenvironment after a period of treatment with any generation of EGFR TKIs. Macrophages play a pivotal role in carcinogenesis and development. At the same time, macrophages renew continuously, and their polarized phenotype changes dynamically. Although macrophage polarization is generally considered to be involved in secondary resistance, it would be time consuming if renewed macrophages would only protect tumor cells from drug attacks after polarization. It is unclear how dynamic renewal of macrophage contributes to acquired resistance. By using two co‑culture approaches, the present study modeled two types of renewal of macrophages: Migrated macrophages (Mm) and locally self‑renewal resident macrophages (Mr). The EGFR‑mutant lung cancer cell line PC‑9 was induced to develop resistance to gefitinib in these co‑culture approaches and without macrophages co‑culture. PC‑9 cells induced in Mm co‑culture recovered fast from gefitinib attack, and formed the gefitinib resistant (GR) subline PC‑9/Mm/GR with the strongest resistant ability. PC‑9/Mm/GR cells exhibited epithelial characteristics, including stable expression of EGFR, phosphorylated EGFR, insulin‑like growth factor 1 receptor, TGF‑βII receptor and E‑cadherin. PC‑9 cells induced in Mr co‑culture and those without co‑culture developed similar weak resistance to gefitinib. TGF‑β secretion was inhibited when PC‑9 cells were in Mr co‑culture, but not in Mm co‑culture. Macrophages were not polarized in either co‑culture type. The present findings suggested that macrophages may affect resistance acquirement by different renewal modes. Mm could promote resistance acquirement by stabilizing tumor cell phenotypes, which would happen before polarization. It would be helpful to monitor the response to EGFR‑TKIs and design novel treatment strategies if macrophages from two renewal modes were distinguished effectively.

FBP1 induced by β-elemene enhances the sensitivity of gefitinib in lung cancer.

β-elemene is known to play a critical role in tumorigenesis as well as tyrosine kinase inhibitor (TKI) resistance in lung cancer. However, the biological function and molecular mechanism remain largely unknown. In this study, the common genes involved in gefitinib resistance and β-elemene were identified using bioinformatic analysis. The expression of FBP1 was examined by qRT-PCR and Western blot analysis. Cell proliferation, flow cytometry, clone formation and IC50 assays were performed to assess the effects of β-elemene and FBP1. Western blot analysis was used to evaluate apoptosis-related gene expression. Finally, in vivo experiments were conducted to assess the crucial role of FBP1 in gefitinib-resistant HCC827/GR cells in nude mice. Screening analysis demonstrated that fructose-1,6-bisphosphatase (FBP1) was induced by β-elemene and downregulated in gefitinib-resistant lung cells. Functionally, overexpression of FBP1 inhibited proliferation and gefitinib resistance and promoted apoptosis of PC9/GR and HCC827/GR cells in vitro. Mechanistically, FBP1 impeded the nuclear translocation of p-STAT3. The FBP1/STAT3 axis was required for FBP1-mediated apoptosis-related gene expression. In vivo experiments further confirmed the enhanced effects of FBP1 on lung cancer cell sensitivity to gefitinib. Our research indicated that β-elemene suppressed proliferation and enhanced sensitivity to gefitinib by inducing apoptosis through the FBP1/STAT3 axis in gefitinib-resistant lung cancer cells.

ANGPTL4 Regulates Lung Adenocarcinoma Pyroptosis and Apoptosis via NLRP3\ASC\Caspase 8 Signaling Pathway to Promote Resistance to Gefitinib.

Prior research has identified ANGPTL4 as a key player in the control of the body's lipid and glucose metabolism and a contributor to the onset of numerous cardiovascular conditions. Recently, it has been shown that ANGPTL4 also plays a critical role in tumor growth and progression. Nowadays, the number of EGFR-TKI resistant patients is increasing, and it is important to investigate the role of ANGPTL4 in regulating gefitinib resistance in PC9/GR non-small-cell lung cancer (NSCLC). The expression of ANGPTL4 in A549, PC9, H1975, BEAS-2B and PC9/GR cells was verified by Western blot and qRT-PCR assays, and the effect of gefitinib on the proliferative ability of each cell was probed by CCK-8 assay. By using shRNA to inhibit ANGPTL4 expression in cells, the effect of ANGPTL4 on cell migratory ability was examined and the effect of ANGPTL4 on cellular gefitinib sensitivity was confirmed using the CCK-8 assay and the edu proliferation test. Mouse transplantation tumors were constructed, and the effect of ANGPTL4 on cellular gefitinib sensitivity was investigated in vivo by flow cytometry, Tunel staining assay, immunohistochemical staining, and ROS fluorescence staining assay. ANGPTL4 expression in homoRNA overexpression cells was constructed, and the changes in the expression levels of ASC\NLRP3\Caspase 8 pathway and focal and apoptotic proteins were investigated in vitro, in vivo, afterknockdown and overexpression of ANGPTL4 expression by Westen blot assay. ANGPTL4 was highly expressed in PC9/GR cells. Interfering with ANGPTL4 expression resulted in decreased proliferation and migration ability, decreased resistance to gefitinib, and increased scorching and apoptosis in PC9/GR cells. Interfering with ANGPTL4 expression in PC9/GR cells was shown to promote sensitivity to gefitinib and to mediate the NLRP3/ASC/Caspase 8 pathway to induce cell scorching and apoptosis. ANGPTL4 promotes gefitinib resistance in PC9/GR cells by regulating the NLRP3/ASC/Caspase 8 pathway to inhibit scorch death. ANGPTL4 may be an effective new target for inhibiting EGFR-TKI resistance in lung adenocarcinoma cells.

Statement of Retraction: The lncRNA RHPN1-AS1 downregulation promotes gefitinib resistance by targeting miR-299-3p/TNFSF12 pathway in NSCLC

This article refers to:RETRACTED ARTICLE: The lncRNA RHPN1-AS1 downregulation promotes gefitinib resistance by targeting miR-299-3p/TNFSF12 pathway in NSCLC

The pseudogene DUXAP10 contributes to gefitinib resistance in NSCLC by repressing OAS2 expression.

Gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI),is the currently recommended first-line therapy for advanced EGFR-mutant lung cancer, and understanding the mechanism of resistance is the key to formulating therapeutic strategies for EGFR-TKIs. In this study, we evaluate the expression patterns and potential biological functions of the pseudogene DUXAP10 in gefitinib resistance. We find that pseudogene DUXAP10 expression is significantly upregulated in NSCLC gefitinib-resistant cells and tissues. Gain and loss of function assays reveal that knockdown of DUXAP10 by siRNA reverses gefitinib resistance both in vitro and in vivo. Furthermore, DUXAP10 interacts with the histone methyltransferase enhancer of zeste homolog 2 (EZH2) to repress the expression of 2',5'-oligoadenylate synthetase (OAS2). Overall, our study highlights the pivotal role of DUXAP10 in gefitinib resistance, and the DUXAP10/EZH2/OAS2 axis might be a promising therapeutic target to overcome acquired gefitinib resistance in NSCLC.

EGFR Mutant Lung Adenocarcinoma Showed Reduced Radiosensitivity after Acquired Resistance to EGFR TKIs

<h3>Purpose/Objective(s)</h3> Though earlier application of local radiotherapy for the primary lesion may improve the survival of patients, the timing of radiotherapy intervention for advanced EGFR mutant lung adenocarcinoma is still controversial. This study aimed to detect the difference of radiosensitivity before and after resistance and explore the related mechanism by constructing EGFR mutant lung adenocarcinoma cell lines with acquired resistance to gefitinib in vitro, so as to provide theoretical basis for determining the optimal intervention timing of radiotherapy. <h3>Materials/Methods</h3> Two lung adenocarcinoma cell lines harboring EGFR mutation-PC9 and HCC827, were used for establishment of acquired resistant cells to gefitinib. The radiosensitivity was detected using a cell counting kit. Flow cytometry was introduced to explore the cell cycle distribution and Western blot was applied to further test cell cycle and DNA repair related proteins. Soft agar assay and Western blot were performed to determine the cancer stem cell (CSC) characteristics. RT-PCR detected the expression of CSC markers. Finally, the relevant signal pathways were explored. <h3>Results</h3> The cell counting kit showed that the inhibitory activity of cells with irradiation after gefitinib resistance is weaker than that before resistance. Flow cytometry revealed that the G2/M phase proportion of PC9 and HCC827 were significantly higher than that of PC9/GR and HCC827/GR under the same irradiation dose, which is consistent with the results of western blot that the increased expression of G2/M regulatory protein, including p21, Cyclin B1, CyclinA2, pCDC2 and Myt1 were more significant increased in parental cells than resistant cells. The phosphorylated level of RAD17, ATM and CHK2 which were DNA damage repair protein were more upregulated in PC9GR and HCC827GR cells. Furthermore, GR had strong capability of sphere-forming and EMT phenotype. RT-PCR showed that the expression of many genes regulating CSC characteristics increased obviously after gefitinib resistance, including SOX2, OCT4, Nanog, Notch1, Notch3, ALDH1A1, ALDH1A3 and BMI. But the up-regulated genes in the two drug-resistant cells were not completely same. Western blot and RT-PCR showed that BMP/SMAD signal pathway was more activated in both resistant cells than that in parental cells. <h3>Conclusion</h3> Lung adenocarcinoma with EGFR mutations showed reduced radiosensitivity after acquired resistance to EGFR TKIs through obtaining CSC characteristics, which may be mediated by BMP/SMAD signal pathway.

E2F1-induced long non-coding RNA MCF2L-AS1 modulates Cyclin D1 mRNA stability through ELAVL1 to induce Gefitinib resistance in non-small cell lung cancer.

Gefitinib is a widely used therapeutic drug for non-small cell lung cancer (NSCLC), and its acquired resistance has become one of the barriers to the successful use of the drugs to treat NSCLC patients. Long non-coding RNA (lncRNA) has an essential role in developing cancer drug resistance. Hence, this study aimed to investigate the effect and modulatory mechanisms of lncRNA MCF2L-AS1 in Gefitinib resistance in NSCLC. IBEAS-2B and A549 cells and human NSCLC tissues were used. A549/GR cell line was constructed by continuous exposure to Gefitinib. Cell viability, apoptosis, migration, colony formation, and protein expression studies were done in transfected cells. Interactions of MCF2L-AS1, ELAVL1, and Cyclin D1 (CCND1 was also investigated. In patients with Gefitinib-resistant NSCLC, MCF2L-AS1 and CCND1 were both up-regulated. Knockdown of MCF2L-AS1 reduced Gefitinib-resistant NSCLC cell progression, indicating that inhibition of MCF2L-AS1 restrained Gefitinib-resistant NSCLC. Mechanically, MCF2L-AS1 enhanced CCND1 mRNA stability via combining with ELAVL1, thereby elevating the resistance of NSCLC cells to Gefitinib. Moreover, E2F1 could transcriptionally up-regulate MCF2L-AS1. The results manifest that lncRNA MCF2L-AS1, as an oncogene of NSCLC, controls CCDN1 via ELAVL1 to drive the growth of NSCLC cells and Gefitinib resistance.

Retraction Note: EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers.

Retraction Note: EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers.

Exploring the relationship between autophagy and Gefitinib resistance in NSCLC by silencing PDLIM5 using ultrasound-targeted microbubble destruction technology

BackgroundUltrasound-targeted microbubble destruction (UTMD) technology is a new drug and gene delivery strategy. This study investigates novel ultrasound (US) sensitive siRNA-loaded nanobubbles (siRNA-NBs) to explore the relationship between PDLIM5 mediated autophagy and drug resistance development using epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of non-small cell lung cancer (NSCLC).MethodsUS sensitive siRNA-NBs were designed to inhibit the expression of PDLIM5 in gefitinib-resistant human NSCLC PC9GR cells in vitro. The expression of autophagy-related proteins (P62 and LC3-II/I) and autophagosomes in PC9GR cells after PDLIM5 gene silencing were explored.ResultsUS-sensitive PDLIM5-targeted siRNA-NBs were effectively delivered into PC9GR cells, inhibiting PDLIM5 expression, increasing LC3-II/I and p62 expressions and increasing autophagosomes in PC9GR cells in vitro.ConclusionsUsing UTMD, US-sensitive siRNA-NBs have the potential as an ideal delivery vector to mediate highly effective RNA interference for NSCLC cells. Furthermore, PDLIM5 plays a role in the autophagy-mediated resistance in gefitinib-resistant PC9GR cells.

SH005S7 Overcomes Primary and Acquired Resistance of Non-Small Cell Lung Cancer by Combined MET/EGFR/HER3 Inhibition.

In this study, we have examined the anticancer effects of SH005S7 on MET-amplified and (HCC827GR) NSCLC cells and their primary HCC827 cells. In vitro, first of all, cell viability and colony formation assay confirmed the growth inhibitory effects of SH005S7 on both cells. Second, SH005S7 inactivated EGFR-related multiple cell signaling, which was associated with a marked decrease in the constitutive phosphorylation of EGFR, HER3, MET, AKT, and ERK. Third, SH005S7 attenuated the anchorage-independent cell growth. Fourth, SH005S7 blocked invasive and metastatic capability by downregulation of mesenchymal markers—vimentin, snail, and MMP-9. Fifth, BrdU assay confirmed the cell cycle arrest of SH005S7 on these cells. When administered orally to nude mice xenografically transplanted human NSCLC, SH005S7 inhibited the growth of tumor and did not cause hepatotoxicity and nephrotoxicity in animals. Immunohistochemical and Western blot analyses of tissue showed that the suppression of growth correlated with inhibition of proliferation (Ki-67, PCNA), invasiveness (vimentin, snail), and angiogenesis (CD31) marker and decrement in the constitutive and phosphorylation of EGFR, HER3, MET, AKT, and ERK. Additionally, SH005S7 had immune stimulatory effects by TNF-α cytokine release on macrophage, without cell cytotoxicity. Overall, our results suggest that SH005S7 can inhibit the growth of MET-amplified and gefitinib-resistant NSCLC cells through the suppression of EGFR-related multiple targets linked to overcome gefitinib resistance.

Identification of Gefitinib Resistance-Related lncRNA-miRNA-mRNA Regulatory Networks and Corresponding Prognostic Signature in Patients with Lung Adenocarcinoma.

PurposeTo identify and characterize gefitinib resistance-related (GefR-related) lncRNAs and construct a prediction model for lung adenocarcinoma (LUAD).MethodsDifferential expression analysis between PC9 and gefitinib-resistant PC9 (PC9GR) cell samples was performed to screen GefR-related lncRNAs and mRNAs based on the GSE34228 dataset. These lncRNAs, mRNAs, and their corresponding microRNAs (miRNAs) were used to construct the GefR-related network and PPI networks. Functional enrichment analyses were conducted using the STRING database. A prognostic signature was developed using the TCGA dataset. The reliability of the signature was tested using the Kaplan–Meier method and ROC curve. Lastly, the FZD4-associated ceRNA subnetwork was selected to confirm the in vitro expressions of the GefR-related lncRNAs using RT-qPCR assay.ResultsA GefR-related ceRNA network that consists of 35 miRNAs, 26 lncRNAs, and 179 mRNAs was constructed. Then, 20 hub genes were screened from the targeted mRNAs of the constructed PPI network, and enrichment analysis identified relevant enriched pathways. We also constructed a prognostic signature for LUAD based on nine mRNAs in the GefR-related ceRNA network. The 9-mRNA signature was an independent predictor of LUAD, the AUC produced by ROC analysis showed a good predictive power of the model, and Kaplan–Meier analysis showed poorer outcomes in the high-risk group, relative to the low-risk group. Lastly, MIR137HG and ZNF295-AS1 levels were found to be associated with gefitinib resistance and exerted their functions through the ceRNA mechanism.ConclusionWe established a prognostic signature and identified two lncRNAs (MIR137HG and ZNF295-AS1) with potential significant roles in gefitinib resistance.

BI6727, a polo-like kinase 1 inhibitor, synergizes with gefitinib to suppress hepatocellular carcinoma cells via a G2/M arrest mechanism.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death, which indicates that efficient intervention agents or strategies against HCC are urgently needed. In the present study, we firstly found that a combination of gefitinib (an ep i dermal growth factor receptor (EGFR) inhibitor) and B I 6727 (a pol o -like kinase 1 (PLK1) inhibitor) could significantly inhibit cell proliferation of HCC cells, which attenuated acquired resistance of gefitinib in HCC cells. Interestingly, our results showed that these anti-tumor effects of gefitinib in combination with BI6727 were associated with G2/M arrest. Moreover, further study revealed that BI6727 could downregulate the protein levels of cell division cycle 25C (Cdc25C) via ubiquitination-dependent pathway, which subsequently induced G2/M arrest. Furthermore, two critical checkpoints proteins ataxia telangiectasia-mutated (p-ATM)/ ATM and Rad-3 related(p-ATR) and another hallmark phosphorylated H2AX (γ-H2AX ) of DNA damage were positively regulated in HCC cells exposed to gefitinib in combination with BI6727. These results indicated that co-treatment induced G2/M arrest was closely related to DNA damage. In summary, the present study discovered that gefitinib synergizing with BI6727 could significantly facilitate DNA damage and overcome acquired resistance of HCC cells to gefitinib. Our study provides a promising approach for the combination of EGFR inhibitors and PLK1 inhibitors in the clinical treatment for HCC.

TRPML3 enhances drug resistance in non-small cell lung cancer cells by promoting Ca2+-mediated lysosomal trafficking

Lysosomes are emerging as versatile signaling hubs that mediate numerous cellular processes, including the development of drug resistance in cancer cells. Transient receptor potential mucolipin 3 (TRPML3), an endolysosomal Ca2+-permeable channel, is implicated in regulating lysosomal trafficking during endocytosis and autophagy. However, the role of TRPML3 in cancer progression remains unclear. In this study, we focused on identifying key molecules that modulate exosomal release triggered by lysosomal exocytosis during the development of gefitinib resistance in non-small cell lung cancer (NSCLC). We found that the basal release of exosomes and lysosomal exocytosis is higher in the gefitinib-resistant NSCLC cell line HCC827/GR than in the gefitinib-sensitive NSCLC cell line HCC827. Notably, exosomal release and lysosomal exocytosis were associated with an increase in TRPML3 expression. Lysosomal Ca2+ release via TRPML3 was triggered by the gefitinib-mediated elevation of lysosomal pH. Furthermore, TRPML3 deficiency enhanced the gefitinib-mediated increase in sub-G0 cell population, reduction of cell proliferation, and poly (ADP-ribose) polymerase cleavage. These data demonstrated that TRPML3 is a promising modulator of drug resistance. By sensing the elevation of lysosomal pH, it mediates lysosomal Ca2+ release, lysosomal trafficking and exocytosis, and exosomal release. Taken together, our study is the first to report the autonomous defense mechanism developed in NSCLC cells against the small-molecule tyrosine kinase inhibitor gefitinib, leading to acquired drug resistance.

Activation of the HSP27-AKT axis contributes to gefitinib resistance in non-small cell lung cancer cells independent of EGFR mutations

PurposeAlthough epidermal growth factor receptor (EGFR)-activating mutations in non-small cell lung cancer (NSCLC) usually show sensitivity to first-generation EGFR-tyrosine kinase inhibitors (TKIs), most patients relapse because of drug resistance. Heat shock protein 27 (HSP27) has been reported to be involved in the resistance of EGFR-TKIs, although the underlying mechanism is unclear. Here, we explore the mechanisms of HSP27-mediated EGFR TKI resistance and propose novel therapeutic strategies.MethodsTo determine the mechanism of HSP27 associated gefitinib resistance, differences were assessed using gefitinib-sensitive and -resistant NSCLC cell lines. In vivo xenograft experiments were conducted to elucidate the combinatorial effects of J2, a small molecule HSP27 inhibitor, and gefitinib. Analyses of human NSCLC tissues and PDX tissues were also used for comparison of HSP27 and phosphorylated AKT expression.ResultsLarge-scale cohort analysis of NSCLC cases revealed that HSP27 expression correlated well with the incidence of EGFR mutations and affected patient survival. Increased pAKT and HSP27 was observed in gefitinib-resistant cells compared with gefitinib-sensitive cells. Moreover, increased phosphorylation of HSP27 by gefitinib augmented its protein stability and potentiated its binding activity with pAKT, which resulted in increased gefitinib resistance. However, in gefitinib-sensitive cells, stronger binding activity between EGFR and HSP27 was observed. Moreover, these phenomena occurred regardless of EGFR mutation including secondary mutations, such as T790M. AKT knockdown switched HSP27-pAKT binding to HSP27-EGFR, which promoted gefitinib sensitivity in gefitinib-resistant cells. Functional inhibition of HSP27 yielded sensitization to gefitinib in gefitinib-resistant cells by inhibiting the interaction between HSP27 and pAKT.ConclusionsOur results indicate that combination of EGFR-TKIs with HSP27 inhibitors may represent a good strategy to overcome resistance to EGFR-TKIs, especially in cancers exhibiting AKT pathway activation.

Targeted Co-Delivery of Gefitinib and Rapamycin by Aptamer-Modified Nanoparticles Overcomes EGFR-TKI Resistance in NSCLC via Promoting Autophagy

Acquired drug resistance decreases the efficacy of gefitinib after approximately 1 year of treatment in non-small-cell lung cancer (NSCLC). Autophagy is a process that could lead to cell death when it is prolonged. Thus, we investigated a drug combination therapy of gefitinib with rapamycin—a cell autophagy activator—in gefitinib-resistant NSCLC cell line H1975 to improve the therapeutic efficacy of gefitinib in advanced NSCLC cells through acute cell autophagy induction. Cell viability and tumor formation assays indicated that rapamycin is strongly synergistic with gefitinib inhibition, both in vitro and in vivo. Mechanistic studies demonstrated that EGFR expression and cell autophagy decreased under gefitinib treatment and were restored after the drug combination therapy, indicating a potential cell autophagy–EGFR positive feedback regulation. To further optimize the delivery efficiency of the combinational agents, we constructed an anti-EGFR aptamer-functionalized nanoparticle (NP-Apt) carrier system. The microscopic observation and cell proliferation assays suggested that NP-Apt achieved remarkably targeted delivery and cytotoxicity in the cancer cells. Taken together, our results suggest that combining rapamycin and gefitinib can be an efficacious therapy to overcome gefitinib resistance in NSCLC, and targeted delivery of the drugs using the aptamer-nanoparticle carrier system further enhances the therapeutic efficacy of gefitinib.

The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer.

Objective:The novel compound GCJ-490A has been discovered as a pan-histone deacetylase (HDAC) inhibitor that exerts potent inhibitory activity against HDAC1, HDAC3, and HDAC6. Because of the important roles of HDACs in lung cancer development and the high distribution of GCJ-490A in lung tissue, we explored the anti-tumor potency of GCJ-490A against non-small cell lung cancer (NSCLC) in vitro and in vivo in this study.Methods:The in vitro effects of GCJ-490A alone or combined with the EGFR inhibitor gefitinib against NSCLC were measured with proliferation, apoptosis, and colony formation assays. NSCLC xenograft models were used to investigate the efficacy of GCJ-490A combined with gefitinib for the treatment of NSCLC in vivo. Western blot assays, luciferase reporter assays, chromatin immunoprecipitation assays, quantitative real time-PCR, immunohistochemistry, and transcription factor activity assays were used to elucidate possible mechanisms.Results:GCJ-490A effectively inhibited NSCLC cell proliferation and induced apoptosis in vitro and in vivo. Interestingly, inhibition of HDAC1 and HDAC6 by GCJ-490A increased histone acetylation at the IKKα promoter and enhanced IKKα transcription, thus decreasing c-Met. Moreover, this c-Met downregulation was found to be essential for the synergistic anti-tumor activity of GCJ-490A and gefitinib.Conclusions:These findings highlight the promising potential of HDAC inhibitors in NSCLC treatment and provide a rational basis for the application of HDAC inhibitors in combination with EGFR inhibitors in clinical trials.

Circ-MAN1A2 Contributes to the Acquired Resistance of Gefitinib by Binding to miR-409-3p to Induce TWIST1 Expression in Non-small-cell Lung Cancer

Circ-MAN1A2 Contributes to the Acquired Resistance of Gefitinib by Binding to miR-409-3p to Induce TWIST1 Expression in Non-small-cell Lung Cancer

Depleting hsa_circ_0000567 suppresses acquired gefitinib resistance and proliferation of lung adenocarcinoma cells through regulating the miR-377-3p / ZFX axis: an in vitro and in vivo study.

Circular RNAs (circRNAs) expression profile has been reported in lung adenocarcinoma (LUAD) cells resistant to gefitinib, and hsa_circ_0000567 was abnormally upregulated. However, its precise role in gefitinib resistance remains unclarified. Levels of hsa_circ_0000567, microRNA (miR)-377-3p and zinc finger protein X-linked (ZFX) were detected by real-time quantitative PCR. Direct attachment was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Gefitinib resistance was measured by MTT assay, colony formation assay, flow cytometry, western blotting, and xenograft in mice. Expression of hsa_circ_0000567 was upreguated in human gefitinib-resistant human LUAD tissues and cells (HCC827/GR and PC9/GR). Clinically, higher hsa_circ_0000567 correlated with advanced tumor burden. Blockage of hsa_circ_0000567 suppressed cell viability, half maximal inhibitory concentration (IC₅₀) value, colony formation ability, and expression of Bcl-2 and proliferating cell nuclear antigen (PCNA) in HCC827/GR and PC9/GR cells under gefitinib treatment or not, accompanied with enhanced apoptosis rate and Bax expression. In vivo, tumor growth of PC9/GR cells untreated and treated with gefitinib was restrained by silencing hsa_circ_0000567. miR-377-3p was directly regulated by hsa_circ_0000567, and then targeted ZFX. Similar to hsa_circ_0000567 knockdown, overexpressing miR-377-3p inhibited chemoresistance in genitinib-resistant LUAD cells in vitro, whereas, depleting miR-377-3p was able to promote gefitinib resistance in spite of hsa_circ_0000567 knockdown. Moreover, restoring ZFX abrogated miR-377-3p-mediated chemosensitivity in genitinib-resistant LUAD cells in vitro. The hsa_circ_0000567/miR-377-3p/ZFX axis might contribute to acquired gefitinib resistance in LUAD cells both in vitro and in vivo, suggesting hsa_circ_0000567 as a novel therapeutic target in treatment of gefitinib-resistant LUAD.

M6A methyltransferase METTL3-induced lncRNA SNHG17 promotes lung adenocarcinoma gefitinib resistance by epigenetically repressing LATS2 expression

Gefitinib has been widely applied for the treatment of lung adenocarcinoma (LUAD). However, the long-term application of gefitinib usually leads to acquired drug resistance in tumour patients, resulting in clinical treatment failure. Small nucleolar host gene 17 (SNHG17) has been shown to play a regulatory role in LUAD progression. Nevertheless, the role of SNHG17 in LUAD gefitinib resistance remains elusive. The expression pattern of SNHG17 was examined in tissues and cell lines of gefitinib-sensitive and gefitinib-resistant LUAD, respectively. Gain- and loss-of-function experiments were employed to assess the biological functions of SNHG17 in cell proliferation and apoptosis, as well as aggressive phenotypes of LUAD cells. MeRIP-qPCR and colorimetric quantificational analysis were performed to detect m6A modifications and contents. Fluorescence in situ hybridisation (FISH) and subcellular fractionation analysis were used to reveal the distribution of SNHG17. RIP and ChIP assays were performed to further validate the SNHG17/EZH2/LATS2 regulatory axis. A xenograft tumour growth assay was conducted to evaluate the role of SNHG17 in LUAD gefitinib resistance in vivo. SNHG17 was upregulated in gefitinib-resistant LUAD tissues and cell lines. Functional assays showed that SNHG17 aggravated the malignant phenotypes of gefitinib-resistant LUAD cells. In addition, METTL3-mediated N6-methyladenosine modification could induce the upregulation of SNHG17by stabilising its RNA transcript. Mechanistically, SNHG17 epigenetically repressed the expression of LATS2 by recruiting EZH2 to the promoter region of LATS2. The regulatory role of the SNHG17/EZH2/LATS2 axis in LUAD gefitinib resistance was further supported in vivo. Collectively, our findings suggested that SNHG17 induced by METTL3 could promote LUAD gefitinib resistance by epigenetically repressing LATS2 expression.

Structural dynamics and kinase inhibitory activity of three generations of tyrosine kinase inhibitors against wild-type, L858R/T790M, and L858R/T790M/C797S forms of EGFR

Mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR), including L858R/T790M double and L858R/T790M/C797S triple mutations, are major causes of acquired resistance towards EGFR targeted drugs. In this work, a combination of comprehensive molecular modeling and in vitro kinase inhibition assay was used to unravel the mutational effects of EGFR on the susceptibility of three generations of EGFR tyrosine kinase inhibitors (erlotinib, gefitinib, afatinib, dacomitinib, and osimertinib) in comparison with the wild-type EGFR. The binding affinity of all studied inhibitors towards the double and triple EGFR mutations was in good agreement with the experimental data, ranked in the order of osimertinib > afatinib > dacomitinib > erlotinib > gefitinib. Three hot-spot residues at the hinge region (M790, M793, and C797) were involved in the binding of osimertinib and afatinib, enhancing their inhibitory activity towards mutated EGFRs. Both double and triple EGFR mutations causing erlotinib and gefitinib resistance are mainly caused by the low number of H-bond occupations, the low number of surrounding atoms, and the high number of water molecules accessible to the enzyme active site. According to principal component analysis, the molecular complexation of osimertinib against the two mutated EGFRs was in a closed conformation, whereas that against wild-type EGFR was in an open conformation, resulting in drug resistance. This work paves the way for further design of the novel EGFR inhibitors to overcome drug resistance mechanisms.