Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells

Kanmei Rai, Nagio Takigawa, Sachio Itou, Hiromi Kashihara, Eiki Ichihara, Tatsuji Yasuda, Kenji Shimizu, Mitsune Tanimoto, Katsuyuki Kiura

Research output: Contribution to journalArticle

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Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been strikingly effective in lung cancers harboring activating EGFR mutations. Unfortunately, the cancer cells eventually acquire resistance to EGFR-TKI. Approximately 50% of the acquired resistance involves a secondary T790M mutation. To overcome the resistance, we focused on EGFR suppression using microRNA-7 (miR-7), targeting multiple sites in the 3′-untranslated region of EGFR mRNA. Two EGFR-TKI-sensitive cell lines (PC-9 and H3255) and two EGFR-TKI-resistant cell lines harboring T790M (RPC-9 and H1975) were used. We constructed miR-7-2 containing miR-7-expressing plasmid. After transfection of the miR-7-expressing plasmid, using cationic liposomes, a quantitative PCR and dual luciferase assay were conducted to examine the efficacy. The antiproliferative effect was evaluated using a cell count assay and xenograft model. Protein expression was examined by Western blotting. The miR-7 expression level of the transfectants was approximately 30-fold higher, and the luciferase activity was ablated by 92%. miR-7 significantly inhibited cell growth not only in PC-9 and H3255 but also in RPC-9 and H1975. Expression of insulin receptor substrate-1 (IRS-1), RAF-1, and EGFR was suppressed in the four cell lines. Injection of the miR-7-expressing plasmid revealed marked tumor regression in a mouse xenograft model using RPC-9 and H1975. EGFR, RAF-1, and IRS-1 were suppressed in the residual tumors. These findings indicate promising therapeutic applications of miR-7-expressing plasmids against EGFR oncogene-addicted lung cancers including T790M resistance by liposomal delivery.

Original languageEnglish
Pages (from-to)1720-1727
Number of pages8
JournalMolecular Cancer Therapeutics
Volume10
Issue number9
DOIs
Publication statusPublished - Sep 2011

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MicroRNAs
Epidermal Growth Factor Receptor
Protein-Tyrosine Kinases
Lung Neoplasms
Plasmids
Insulin Receptor Substrate Proteins
Luciferases
Heterografts
Cell Line
Mutation
Residual Neoplasm
3' Untranslated Regions
Oncogenes
Liposomes
Transfection
Neoplasms
Cell Count
Western Blotting
Polymerase Chain Reaction
Messenger RNA

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells. / Rai, Kanmei; Takigawa, Nagio; Itou, Sachio; Kashihara, Hiromi; Ichihara, Eiki; Yasuda, Tatsuji; Shimizu, Kenji; Tanimoto, Mitsune; Kiura, Katsuyuki.

In: Molecular Cancer Therapeutics, Vol. 10, No. 9, 09.2011, p. 1720-1727.

Research output: Contribution to journalArticle

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abstract = "Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been strikingly effective in lung cancers harboring activating EGFR mutations. Unfortunately, the cancer cells eventually acquire resistance to EGFR-TKI. Approximately 50{\%} of the acquired resistance involves a secondary T790M mutation. To overcome the resistance, we focused on EGFR suppression using microRNA-7 (miR-7), targeting multiple sites in the 3′-untranslated region of EGFR mRNA. Two EGFR-TKI-sensitive cell lines (PC-9 and H3255) and two EGFR-TKI-resistant cell lines harboring T790M (RPC-9 and H1975) were used. We constructed miR-7-2 containing miR-7-expressing plasmid. After transfection of the miR-7-expressing plasmid, using cationic liposomes, a quantitative PCR and dual luciferase assay were conducted to examine the efficacy. The antiproliferative effect was evaluated using a cell count assay and xenograft model. Protein expression was examined by Western blotting. The miR-7 expression level of the transfectants was approximately 30-fold higher, and the luciferase activity was ablated by 92{\%}. miR-7 significantly inhibited cell growth not only in PC-9 and H3255 but also in RPC-9 and H1975. Expression of insulin receptor substrate-1 (IRS-1), RAF-1, and EGFR was suppressed in the four cell lines. Injection of the miR-7-expressing plasmid revealed marked tumor regression in a mouse xenograft model using RPC-9 and H1975. EGFR, RAF-1, and IRS-1 were suppressed in the residual tumors. These findings indicate promising therapeutic applications of miR-7-expressing plasmids against EGFR oncogene-addicted lung cancers including T790M resistance by liposomal delivery.",
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