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Lnc-TMEM132D-AS1 induces acquired resistance to osimertinib in NSCLC by the miR-766-5p/ENTPD1 axis.

Gefitinib is the first-line treatment for non-small cell lung cancer (NSCLC) harboring EGFR sensitive mutation. However, acquired resistance significantly limits its therapeutic efficacy. CircSETD3 has been reported to promote gefitinib resistance in NSCLC cells, however, its underlying mechanisms have not been fully clarified.The expression of circSETD3 were detected in NSCLC patients who received gefitinib as first-line treatment, including 20 gefitinib-sensitive patients and 20 acquired gefitinib-resistant patients. Cell viability were examined by CCK8 assay. The mRNA and protein levels were detected by qRT-PCR and western blot. Using RNA pull-down assay followed by mass spectrometry to identified proteins that interact with circSETD3. The interaction between circSETD3 and fragile X-related protein-1 (FXR1) were further validated by RNA immunoprecipitation (RIP) and pull-down analysis. Fuorescence in situ hybridization (FISH) and immunofluorescence (IF) assays was used for the identification of sub-location of circSETD3 and FXR1 in cells. The effect of circSETD3 overexpression and knockdown on NSCLC tumor growth to gefitinib sensitivity was detected using the mouse xenograft model.CircSETD3 was significantly upregulated in gefitinib-resistant NSCLC cells, and decreased the gefitinib sensitivity in vitro and in vivo. Mechanically, circSETD3 facilitated FXR1 binding to its downstream mRNA target, epithelial cell-transforming sequence 2 (ECT2), promoting ECT2 mRNA decay, which further inhibited cellular apoptosis.CircSETD3/FXR1/ECT2 axis plays a critical role in the acquired resistance to gefitinib in NSCLC. Our results highlight the potential of circSETD3 as a biomarker and therapeutic target for NSCLC patients with acquired gefitinib resistance.

Objective: Acquired resistance is a major obstacle to the therapeutic efficacy of osimertinib in non-small-cell lung cancer (NSCLC). Current knowledge about the role of long non-coding RNAs (lncRNAs) in this phenomenon is insufficient. Here, we aim to identify candidate lncRNAs associated with acqured resistance to osimertinib in NSCLC, and explore the role and underlying mechanism of one of these lncRNAs, lnc-TMEM132D-AS1. Methods and Results: RNA Sequencing (RNA-Seq) and quantitative real-time PCR (qPCR) were used to screen and validate the differentially expressed lncRNAs between osimertinib-sensitive and -resistant NSCLC cell lines. Lnc-TMEM132D-AS1 was screened out, and was found to be upregulated in the osimertinib-resistant NSCLC cell lines as well as the plasma of NSCLC patients. Results from CCK-8, colony formation, flow cytometry assays showed that lnc-TMEM132D-AS1 knockdown significantly increased the sensitivity of osimertinib-resistant NSCLC cells to osimertinib. After identifying the cytoplasmic localization of lnc-TMEM132D-AS1, a functional lnc-TMEM132D-AS1-miRNA-mRNA interaction network and protein-protein interaction (PPI) network were constructed to analyze its putative target genes and biological functions. Using qPCR and TISIDB database analysis, ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) was confirmed to be a target mRNA of lnc-TMEM132D-AS1, and was associated with tumor infiltration of immunosuppressive cells and poor prognosis in patients with NSCLC.Conclusion: In summary, lnc-TMEM132D-AS1 plays a crucial role in osimertinib resistance. It may serve as a prognostic biomarker and a potential therapeutic target for acquired resistance to osimertinib in NSCLC.

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., Graphical Abstract, Wu and colleagues identified circSETD3, which could regulate acquired resistance to gefitinib in NSCLC. They characterized the SRSF1/circSETD3/miR-520h/ABCG2 axis, which decreased intracellular concentration of gefitinib after drug resistance. They also demonstrated the effect of circSETD3 on acquired resistance to gefitinib in vivo.

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.

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.

Background: Gefitinib is approved for first-line treatment of advanced 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 still unknown. Methods: CircSETD3 expression was anaylzed in gefitinib-sensitive and -resistant NSCLC cell lines and in the plasma of gefitinib-sensitive and -resistant NSCLC patients. The influence of circSETD3 on gefitinib sensitivity was elucidated in vitro and in nude mice xenografts. The regulatory actions of circSETD3 on the miR-520h and ATP-binding cassette subfamily G member 2 (ABCG2) were assessed. Furthermore, the molecular mechanism of circSETD3 upregulation was also investigated. Findings: circSETD3 was significantly upregulated in gefitinib-resistant NSCLC cell lines and in the plasma of gefitinib-resistant NSCLC patients, and it decreased the gefitinib sensitivity both in vitro and in nude mice xenografts. CircSETD3 could directly bind to miR-520h and lead to the upregulation of ABCG2, an efflux transporter of gefitinib, resulting in reduced intracellular gefitinib concentration. The downregulation of serine/arginine splicing factor 1 (SRSF1) contributed to the aberrantly expression of circSETD3 in NSCLC cells with acquired resistance to gefitinib. Interpretation: circSETD3 decreases the gefitinib sensitivity through targeting the miR-520h/ABCG2 pathway, and may serve as a prognostic biomarker and a potential therapeutic target for acquired resistance to gefitinib in NSCLC. Trial Registration: Registered with the Chinese Clinical Trial Registry (No. ChiCTR1800014660). Funding Statement: The National Scientific Foundation of China (No. 81473284, 81603201). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee Board of Chongqing Medical University (No. 2017009). All animal experiments were approved by the Animal Ethics and Experimental Committee of the Chongqing Medical University, and performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), is used clinically as first-line therapy in patients with advanced non-small cell lung cancer (NSCLC) with EGFR activating mutations, but the inevitable development of acquired resistance limits its efficacy. In up to 30–40% of NSCLC cases, the mechanism underlying acquired resistance remains unknown. ATP-binding cassette (ABC) transporters are a family of membrane proteins that can significantly influence the bioavailability of numerous drugs, and have confirmed to play an essential role in multidrug resistance (MDR) in cancer chemotherapy. However, their role in acquired resistance to gefitnib in NSCLC has not been well studied. Here, through RNA sequencing (RNA-Seq) technology we assessed the differentially expressed ABC transporters in gefitinib-sensitive (PC9 and H292) and gefitinib-resistant (PC9/GR and H292/GR) NSCLC cells, with ABCC10 identified as a transporter of interest. Both ABCC10 mRNA and protein were significantly increased in acquired gefitinib-resistant NSCLC cells, independent of EGFR mutation status. In vitro transport assay showed that ABCC10 could actively efflux gefitinib, with an efflux ratio (ER) of 7.8. Further results from in vitro cell line models and in vivo xenograft models showed that overexpression of ABCC10 led to a reduction in gefitinib sensitivity through decreasing the intracellular gefitinib accumulation. Our data suggest that ABCC10 has an important role in acquired resistance to gefitinib in NSCLC, which can serve as a novel predictive marker and a potential therapeutic target in gefitinib treatment.