Regulation EGFR-TKI resistance by Long Noncoding RNA Clusterin in Non-Small Cell Lung Cancer

[Speaker] Hsiao-Hui Lin:1
[Co-author] Wen-Shu Chen:1, Jin-Yuan Shih:2, Kai-Chien Yang:1, Ching-Chow Chen:1
1:Pharmacology, National Taiwan University, Taiwan, 2:Internal Medicine, National Taiwan University Hospital, Taiwan

[Background] Non-small-cell lung cancer (NSCLC) is one of the leading cause of cancer death worldwide. The discovery of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) such as gefitinib or erlotinib leads to a great success against NSCLC with EGFR activating mutation. However, acquired resistance emerges after a period of treatment which is largely attributed to secondary EGFR T790M mutation. Although T790M-specific TKI AZD9291 could overcome acquired resistance, their effect is hindered by newly developed acquired resistance. Long noncoding RNAs (lncRNAs) have been implicated in carcinogenesis and cancer progression may also serve as novel potential biomarkers for cancer diagnosis and prognosis.

[Methods] High-throughput sequencing of RNA (RNA-Seq) revealed that a subset of lncRNAs such as Clusterin (CLU) was dysregulated in both HCC827/IR (Iressa resistant) and H1975/AR (AZD9291 resistant) cells, which showed Epithelial-mesenchymal transition (EMT) characteristics critical for EGFR-TKI resistance and stemness. Knockdown of CLU transcript variant 4 (CLU-v4) was performed by gapmers or short hairpin RNA (shRNA) to evaluater RNA and protein expression, migration assay, colony and sphere formation. In addition, the sensitivity of gefitinib or AZD9291 was examined by MTT assay, colony formation, and flow cytometry analysis in shRNA CLU-v4 downregulated cells.

 [Results] RNA-seq analysis revealed the upregulation of lncRNA CLU in both resistant cells. CLU-v4 were enriched in nucleus. Knockdown of CLU-v4 by gapmers or shRNA decreased the expression of vimentin, and the ability of cell migration, colony formation. In addition, the cancer stem cell (CSC) characteristics and the expression of stemness-related genes including Oct4 and ALDH were suppressed.

[Conclusion] Silencing of CLU-v4 in both EGFR-TKI resistant cells not only restored the sensitivity to TKI but also decreased cell proliferation, migration and CSC signature, suggesting the involvement of CLU-v4 in EMT to regulate EGFR-TKI resistance in lung cancer.
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