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Sensitivity of non-small cell lung cancer to erlotinib is regulated by the Notch/miR-223/FBXW7 pathway.

ABSTRACT: Recent evidence supports a role for microRNA-223 (miR-223) in modulating tumor cell sensitivity to chemotherapeutic drugs; however, its role in cellular resistance to the effects of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) used in treatment of non-small cell lung cancer (NSCLC) remains to be elucidated. The levels of miR-223 in parental cell line (HCC827) and erlotinib resistant HCC827 cell line (HCC827/ER) were detected by qRT-PCR. HCC827/ER cells were treated with MK-2206 to block the Akt signaling pathway or RO4929097 to block the Notch signaling pathway, and then transfected with an miR-223 inhibitor or interference expression plasmid of F-Box/WD repeat-containing protein 7 (FBXW7) or insulin-like growth factor 1 receptor (IGF1R). HCC827 cells were transfected with miR-223 mimics. Next, CCK-8, colony formation, and flow cytometric apoptosis assays were used to assess cell resistance to erlotinib. When compared with its expression in HCC827 cells, miR-223 expression was significantly up-regulated in HCC827/ER cells. Blocking either the Akt or Notch signaling pathway and reducing miR-223 expression resulted in decreased resistance in HCC827/ER cells. Conversely, increasing miR-223 expression induced cell resistance to erlotinib in HCC827 cells. miR-223 enhanced resistance to erlotinib by down-regulating FBXW7 expression. Reducing FBXW7 expression lowered resistance to erlotinib in HCC827/ER cells, while interference with expression of IGF1R produced no significant effect. This study demonstrated that NSCLC cells can up-regulate their levels of miR-223 expression via the Akt and Notch signaling pathways. miR-223 may serve as an important regulator of erlotinib sensitivity in NSCLC cells by targeting FBXW7.

SUBMITTER: Zhang H

PROVIDER: S-EPMC5479025 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Sensitivity of non-small cell lung cancer to erlotinib is regulated by the Notch/miR-223/<i>FBXW7</i> pathway.

Zhang Haiwei H Chen Fanglin F He Yongpeng Y Yi Lin L Ge Chuang C Shi Xiaolong X Tang Chao C Wang Donglin D Wu Yongzhong Y Nian Weiqi W

Bioscience reports 20170621 3

Recent evidence supports a role for microRNA-223 (miR-223) in modulating tumor cell sensitivity to chemotherapeutic drugs; however, its role in cellular resistance to the effects of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) used in treatment of non-small cell lung cancer (NSCLC) remains to be elucidated. The levels of miR-223 in parental cell line (HCC827) and erlotinib resistant HCC827 cell line (HCC827/ER) were detected by qRT-PCR. HCC827/ER cells were treated wit ...[more]

PMID: 28507201

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Project description:BackgroundCisplatin is widely used as a first-line treatment for non-small cell lung cancer (NSCLC), but chemoresistance remains a major clinical obstacle for efficient use. As a microRNA, miR-223 was reported to promote the doxorubicin resistance of NSCLC. However, whether miR-223 is also involved in cisplatin resistance of NSCLC and the mechanism miR-223 involved in drug resistance is unclear. Accumulated evidence has shown that abnormal autophagy is associated with tumor chemoresistance. The study aimed to study the role of miR-223 on cisplatin sensitivity in NSCLC and uncover the potential mechanisms.MethodsNSCLC cells transfected with mimic or inhibitor for miR-223 was assayed for chemoresistance in vitro. MiR-223 expression was assessed by quantitative real-time PCR (qRT-PCR). Western blot were used to study the expression level of F-box/WD repeat-containing protein 7 (FBXW7) and autophagy-related protein. The effect of miR-223 on cisplatin sensitivity was examined by using CCK-8, EdU assays and Autophagic flux assay. Luciferase assays, EdU assays and small interfering RNA were performed to identify the targets of miR-223 and the mechanism by which it promotes treatment resistance. Xenograft models were established to investigate the effect of mir-223 on cisplatin sensitivity.ResultsIn the present study, we found that the level of miR-223 was significantly positively correlated with cisplatin resistance. MiR-223 overexpression made NSCLC cells resistant to cisplatin treatment. We further found that autophagy mediated miR-223-mediated cisplatin resistance in NSCLC cells. Further mechanistic research demonstrated that miR-223 directly targeted FBXW7. The overexpression of miR-223 could inhibit the level of FBXW7 protein expression, thus promoting autophagy and making NSCLC cells resistant to cisplatin. Finally, we confirmed the increased effect of cisplatin sensitivity by miR-223 Antagomir in xenograft models of NSCLC.ConclusionsOur results demonstrate that miR-223 could enhance autophagy by targeting FBXW7 in NSCLC cells. Inhibition of autophagy by miR-223 knockdown provides a novel treatment strategy to alleviate cisplatin resistance in NSCLC.

Project description:BackgroundCisplatin (DDP) is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and long-term DDP stimulation increased resistance of NSCLC cells to this drug by enriching cancer stem cells (CSCs), which contributed to recurrence and worse prognosis of NSCLC, but the molecular mechanisms are still not fully delineated.MethodsReal-Time qPCR and Western Blot analysis were conducted to examine gene expressions at mRNA and protein levels, respectively. Dual-luciferase reporter gene system was used to validate the targeting sites among circRNA CDR1as, miR-641 and HOXA9 mRNA. Cell growth was evaluated by CCK-8 assay, trypan blue staining assay and colony formation assay. The Annexin V-FITC/PI double staining method was employed to measure cell apoptosis ratio. Spheroid formation and flow cytometer assay was used to evaluate cell stemness. Xenograft mice models were established to measure tumorgenicity in vivo, and Ki67 expressions in mice tumor tissues were examined by immunohistochemistry (IHC).ResultsHere we identified a novel circRNA CDR1as/miR-641/Homeobox protein Hox-A9 (HOXA9) pathway regulated stemness and DDP chemoresistance in NSCLC. Mechanistically, circRNA CDR1as and HOXA9 were high-expressed, while miR-641 was low-expressed in DDP-resistant NSCLC cells, instead of their corresponding parental DDP-sensitive NSCLC cells. Additionally, we validated that circRNA CDR1as positively regulated HOXA9 in NSCLC cells by serving as an RNA sponge for miR-641, and knock-down of circRNA CDR1as increased the sensitivity of DDP-resistant NSCLC cells, which were reversed by downregulating miR-641 and upregulating HOXA9. Consistently, overexpression of circRNA CDR1as increased drug resistance of DDP-sensitive NSCLC cells by regulating miR-641/HOXA9 axis. In addition, the expression levels of stemness signatures (SOX2, OCT4 and Nanog) were higher in DDP-resistant NSCLC cells, which also tended to form spheres and enrich CD44+CD166+ population compared to their parental DDP-sensitive NSCLC cells, suggesting that CSCs were enriched in DDP-resistant NSCLC cells. Notably, knock-down of circRNA CDR1as inhibited stemness of DDP-resistant NSCLC cells by inhibiting HOXA9 through upregulating miR-641.ConclusionsTaken together, this study identified that circRNA CDR1as regulated stemness and DDP chemoresistance in NSCLC cells by targeting miR-641/HOXA9 axis.

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Sensitivity of non-small cell lung cancer to erlotinib is regulated by the Notch/miR-223/FBXW7 pathway.

Publications

Sensitivity of non-small cell lung cancer to erlotinib is regulated by the Notch/miR-223/<i>FBXW7</i> pathway.

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