Methylation Induced Gene Silencing of HtrA3 in Smoking-Related Lung Cancer

Abstract

Purpose: Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer.Experimental Design: 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. Clin Cancer Res; 16(2); 398-409. ©2010 AACR.

Structural insights into the activation mechanisms of human HtrA serine proteases

“…A reduced expression of HtrA3 in lung tumors from smokers was correlated with an increased frequency of methylation within the first exon of the gene and also corresponded with a poorer response to chemotherapy. These observations supplemented with in vitro studies led to a conclusion that cigarette smoke-induced methylation and silencing the HtrA3 gene could contribute to the etiology of chemoresistant disease in smoking-related lung cancer [75]. On the other hand, HtrA3 was up-regulated in some other cancers, including seminoma and several hematologic malignancies (reviewed by Ref.…”

HTRA3 is Reduced in Ovarian Cancers Regardless of Stage

“…Resistance of cancer cells to chemotherapy, such as cisplatin and etoposide mediated cytotoxicity, has been suggested to be a consequence of disturbances in HtrA3 pro-apoptotic activity (16). Taken together, these studies suggest HtrA3 may function as a pro-apoptotic protein and as a tumor suppressor (15,16). The pathogenesis and behavior of each class or subtype of ovarian cancer is different.…”

“…Disturbances in HtrA expression and function may contribute to the development of cancers and neurodegenerative disorders (9)(10)(11). HtrA3, initially identified as a pregnancy-related protease (12)(13)(14), was reported to be down-regulated in endometrial, ovarian and lung cancers (15)(16)(17)(18)(19)(20) and up-regulated in thyroid tumors (21), suggesting that the expression of HtrA3 depends on cancer type. The variable expression of HtrA3 is manifested in hematologic malignancies and depends on specific molecular alterations.…”

Maternal HtrA3 optimizes placental development to influence offspring birth weight and subsequent white fat gain in adulthood

“…To date, four mammalian HtrAs (HtrA1-4) have been identified [1][2][3][4][5][6][7] , and their dysregulation is associated with a number of diseases, including cancer, arthritis, neurodegenerative disorders, age-related macular degeneration, and pregnancy diseases [8][9][10][11][12][13][14][15][16][17] . In particular, HtrA1 and HtrA3 have been suggested as tumor suppressors, because they are down-regulated in a number of cancers and this reduction is suggested to promote tumorigenesis [18][19][20][21][22] .…”

“…In particular, HtrA1 and HtrA3 have been suggested as tumor suppressors, because they are down-regulated in a number of cancers and this reduction is suggested to promote tumorigenesis [18][19][20][21][22] . HtrA3 down-regulation in lung cancer is believed to occur because cigarette smoking induces methylation of the HtrA3 gene 8 . The reduced HtrA3 expression is further linked to diminished effectiveness of anti-cancer treatment of lung cancer 23 , and increased risk of postoperative recurrence of the tumor 24 .…”