Antitumor Impact ofp14ARFon Gefitinib-Resistant Non–Small Cell Lung Cancers

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 gefitinibresistant 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 p14 ARF expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14 ARF 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 p14 ARF (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 p14 ARF 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.

ARF Confers a Context-Dependent Response to Chemotherapy in Muscle-Invasive Bladder Cancer

“…However, in addition to its well-characterized tumor suppressive role, ARF has been shown to have tumor-promoting or anti-proliferative activities in some contexts (40–43). Moreover, ARF is upregulated following drug treatment in lung and ovarian cancer (44,45), and its nucleolar expression is associated with adverse outcome in lymphoma (46). Therefore, it has become increasingly apparent that the functions of ARF and its relationship to p53 status are relatively complex.…”

Peptide‐based tumor inhibitor encoding mitochondrial p14ARF is highly efficacious to diverse tumors

“…S1a). To evaluate the growth suppression by these four p14 peptides, the lung adenocarcinoma lines, both gefitinib‐sensitive PC‐9 and gefitinib‐resistant RPC‐9 clones, were employed for the treatment because they were already demonstrated to be very responsive to the original form, the r9‐p14 38‐65 peptide, in our previous study . In the MTT assay, the most potent inhibition was obtained with r9‐CatB‐p14 MIS, the shortest form (Fig.…”

“…MMNK‐1 cells (normal cholangiocytes), normal human dermal fibroblast (NHDF) cells and human cancer cell lines were prepared as previously described . Briefly, cells were maintained in RPMI 1640 containing 10% FBS with 100 unit/mL penicillin and 100 mg/mL streptomycin (Life Technologies, Tokyo, Japan) at 37°C with 5% CO 2 .…”

“…Antigen retrieval of paraffin‐embedded sections, immunohistochemistry and signal development were performed as described previously . The primary antibodies for p14ARF and Ki‐67 were diluted at 1:500 in CanGet signal solution (Toyobo, Tokyo, Japan), respectively.…”

“…lines were prepared as previously described. (14,15) Briefly, cells were maintained in RPMI 1640 containing 10% FBS with 100 unit/mL penicillin and 100 mg/mL streptomycin (Life Technologies, Tokyo, Japan) at 37°C with 5% CO 2 . NuLi-1 cells (normal human bronchus cells), TIME cells (human microvascular endothelium cells) and human pancreatic duct epithelial cell (HPNE) cells were purchased from the American Type Culture Collection (ATCC) and cultured following the instructions.…”