title: CpG hypermethylation contributes to decreased expression of PTEN during acquired resistance to gefitinib in human lung cancer cell lines.
abstract: OBJECTIVE
  We have previously reported that decreased expression of PTEN in lung cancer PC9 cells harboring an EGFR-activating mutation (del E746-A750) results in acquisition of resistance to EGFR-TKIs, gefitinib and erlotinib, accompanied by enhanced phosphorylation of Akt and decreased nuclear translocation of a transcription factor EGR-1 [8]. In the present study, PTEN promoter methylation accounted for the decreased expression of PTEN in our gefitinib-resistant mutant.
  
  
  METHODS
  DNA methylation status of the PTEN promoter in PC9 and gefitinib-resistant cells were examined using methylation-specific PCR. The effect of DNA methylation on PTEN expression was evaluated by treatment of lung cancer cell lines with 5-aza-2'-deoxycytidine (5AZA-CdR).
  
  
  RESULTS
  We observed the characteristics of two gefitinib-resistant sublines, GEF1-1 and GEF2-1, derived from PC9 as follows. (1) PTEN overexpression suppressed AKT phosphorylation and restored the sensitivity to gefitinib and erlotinib in GEF1-1 cells. (2) EGR-1 siRNA mediated knockdown suppressed the expression of cyclin D1 and ICAM-1 genes but not of PTEN gene in PC9 cells. (3) Transfection of EGR-1 cDNA into a drug-resistant subline induced the expression of cyclin D1 and ICAM-1 but not of PTEN. (4) Treatment with 5AZA-CdR induced the expression of PTEN in resistant sublines but not in the parental line PC9. (5) A CpG site near the translational start point of the 5'-regulatory region was methylated in GEF1-1 and GEF2-1 but not in PC9.
  
  
  CONCLUSIONS
  Our results strongly suggest that CpG hypermethylation of the PTEN gene contributes to the decreased expression of PTEN during acquired resistance to gefitinib or erlotinib.
authors: Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors: Masashi
authors: M
authors: Maeda
authors: Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; St Mary's Institute of Health Sciences, St Mary's Hospital, Kurume 830-8543, Japan.
authors: Yuichi
authors: Y
authors: Murakami
authors: Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors: Kosuke
authors: K
authors: Watari
authors: Laboratory of Molecular Cancer Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors: Michihiko
authors: M
authors: Kuwano
authors: Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.
authors: Hiroto
authors: H
authors: Izumi
authors: Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: mono@phar.kyushu-u.ac.jp.
authors: Mayumi
authors: M
authors: Ono
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands; D019938:Cyclin D1 / Q000235:genetics; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D051766:Early Growth Response Protein 1 / Q000235:genetics; D000077156:Gefitinib; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics*; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D018799:Intercellular Adhesion Molecule-1; D019938:Cyclin D1; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; D000077156:Gefitinib
keywords: DNA methylation; EGFR-tyrosine kinase inhibitor; EGR-1; Gefitinib-resistance; Lung cancer; PTEN