PDF file - 1361K, Supplementary Figure 1: Cell cycle regulation and gene expression by gefitinib in the NSCLC. Supplementary Figure 2: p16INK4a and p14ARF expressions in response to gefitinib. Supplementary Figure 3: Regulation of p14ARF by chemotherapetic drugs. Supplementary Figure 4: Mutations of p16INK4a, p14ARF and p53 in the NSCLC cells. Supplementary Figure 5: Subcellular localization of the truncated GFP-p14ARF fusion proteins. Supplementary Figure 6: Growth suppression of the tumor cells by various anti-tumor peptides. Supplementary Figure 7: Immunohistochemistry of p14ARF and p53 in the NSCLC tissues. cells.
ARTICLE ABSTRACT
Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates the proliferation of tumors. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as a molecular-targeting agent for the inactivation of EGFR signaling and show considerable therapeutic effect in non–small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib-resistant mutations in EGFR, which is a critical issue for current therapeutics. We aimed to characterize the distinct molecular response to gefitinib between the drug-resistant and drug-sensitive lung adenocarcinoma cells in order to learn about therapeutics based on the molecular information. From the quantitative PCR analysis, we found a specific increase in p14ARF expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14ARF triggered the most augmented apoptosis in both clones. We identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14ARF (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of the gefitinib-resistant clones without affecting nonneoplastic cells. Notably, transduction of the minimized dose of the p14 38-65 peptide restored the response to gefitinib like that in the sensitive clones. These findings suggest that the region of p14ARF 38-65 a.a. is critical in the pharmacologic action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers. Mol Cancer Ther; 12(8); 1616–28. ©2013 AACR.
