Masashi Maeda1, Yuichi Murakami2, Kosuke Watari1, Michihiko Kuwano3, Hiroto Izumi4, Mayumi Ono5. 1. Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. 2. 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. 3. Laboratory of Molecular Cancer Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. 4. Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan. 5. Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: mono@phar.kyushu-u.ac.jp.
Abstract
OBJECTIVES: 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. MATERIAL AND 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. CONCLUSION: 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.
OBJECTIVES: We have previously reported that decreased expression of PTEN in lung cancerPC9 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. MATERIAL AND 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. CONCLUSION: 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: Christine Aaserød Pedersen; Maria Dung Cao; Thomas Fleischer; Morten B Rye; Stian Knappskog; Hans Petter Eikesdal; Per Eystein Lønning; Jörg Tost; Vessela N Kristensen; May-Britt Tessem; Guro F Giskeødegård; Tone F Bathen Journal: Breast Cancer Res Date: 2022-06-24 Impact factor: 8.408