Methylation induced gene silencing of HtrA3 in smoking-related lung cancer.
OBJECTIVE
Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer.


METHODS
We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment.


RESULTS
We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2'-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history.


CONCLUSIONS
Collectively, these results suggest that cigarette smoke-induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer.
Beleford
Daniah
D
Department of Experimental Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Liu
Zhixue
Z
Rattan
Ramandeep
R
Quagliuolo
Lucio
L
Boccellino
Mariarosaria
M
Baldi
Alfonso
A
Maguire
Jacie
J
Staub
Julie
J
Molina
Julian
J
Shridhar
Viji
V
D000230:Adenocarcinoma / Q000209:etiology / Q000235:genetics; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D002283:Carcinoma, Bronchogenic / Q000188:drug therapy / Q000209:etiology* / Q000235:genetics; D018287:Carcinoma, Large Cell / Q000209:etiology / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000209:etiology / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002945:Cisplatin / Q000008:administration & dosage; D019175:DNA Methylation / Q000502:physiology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005047:Etoposide / Q000008:administration & dosage; D020868:Gene Silencing / Q000502:physiology; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000209:etiology* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009602:Nitrosamines / Q000009:adverse effects; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D012907:Smoking / Q000009:adverse effects*
D009602:Nitrosamines; D005047:Etoposide; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; C474013:HTRA3 protein, human; D012697:Serine Endopeptidases; D002945:Cisplatin