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Saito K.,Aichi Cancer Center Research Institute | Takigawa N.,Kawasaki Medical School | Ohtani N.,Cancer Institute of Japanese Foundation for Cancer Research JFCR | Iioka H.,Aichi Cancer Center Research Institute | And 10 more authors.
Molecular Cancer Therapeutics | Year: 2013

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 p14ARF expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14ARF triggered themost 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.

Takeuchi S.,Cancer Institute of Japanese Foundation for Cancer Research JFCR | Takeuchi S.,Kanazawa University | Takeuchi S.,Tokushima University | Takahashi A.,Cancer Institute of Japanese Foundation for Cancer Research JFCR | And 12 more authors.
Cancer Research | Year: 2010

Although the p16INK4a and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16INK4a and p21Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16INK4a and p21Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16 INK4a and p21Waf1/Cip1 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras-induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either p16INK4a or p21 Waf1/Cip1, indicating that p16INK4a and p21 Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz (a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16INK4a loss. Our findings establish an intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo. ©2010 AACR.

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