The role of peroxiredoxin 1 in lung cancer: Tumor development and radiotherapy resistance
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Peroxiredoxin 1 (Prx1), a major member of peroxiredoxin family, has been observed in various human cancers including lung cancer and its elevation appears to increase the stress resistance of cancer cells. Most studies have been restricted to observations of the elevated expression of Prx1 in various cultured cell systems and some human tissues. However, the effects of Prx1 expression in human tumors, its influence on cancer therapy, and the regulatory basis for its expression in cancers are not understood. Overall hypotheses of this study are that environmental redox change leads to the abnormal up-regulation of Prx1 in lung cancer and that elevated Prx1 may contribute to the lung cancer progression and radiotherapy resistance by controlling critical signaling molecules. The studies outlined in this thesis are directed towards (a) determining the molecular mechanisms of Prx1 up-regulation in lung cancer; (b) investigating the role of Prx1 in pro-growth effect during lung carcinogenesis and further malignant progression, and (c) testing the physiological significance of Prx1 in radiotherapy resistance of lung cancer. The human prx1 promoter was cloned and NF-E2-related factor 2 (Nrf2) was identified as a key transcription factor. Hypoxia/reoxygenation, an in vitro condition suited to mimic changes of oxygenation, increased Nrf2 nuclear localization and its binding to the electrophile responsive elements (EpRE) located at the proximal (-536 to -528) and distal (-1429 to -1421) regions of the prx1 promoter. A significant reduction of both steady-state and hypoxia/reoxygenation-mediated prx1 gene expression was demonstrated in Nrf2 knockout cells. The results indicated that decreased Kelch-like ECH-associated protein, Keap1, might be an important mechanism for the increased nuclear translocation and activation of Nrf2 in response to hypoxia/reoxygenation. A constitutive elevation of prx1 mRNA and protein was observed in Keap1 knockout cells. Previous studies have shown expression of Prx1 was increased during proliferation with serum stimulation, whereas its expression decreased when cells underwent differentiation. These results suggest a role of Prx1 in cell proliferation-enhancing function, however, the effect of Prx1 on promoting tumor growth and progression is not clear. In order to test the effect of Prx1 in tumor progression in vivo , we established stable transfectants of A549 human lung carcinoma cell lines with Prx1 shRNA. Down-regulation of Prx1 significantly inhibited tumor growth ( P < 0.05) and reduced the incidence of spontaneous pulmonary metastasis. Reduction of tumor growth rate and metastasis potential was accompanied by the decreased pAkt levels in Prx knock-down tumors. The cell survival enhancing function of Prx1 is traditionally attributed to its ROS-removing capacity, the physiological significance of its peroxidase activity in irradiated cells is unclear because the catalytic Cys 52 is easily inactivated by ROS due to its over-oxidation to sulfinic or sulfonic acid. The role of Prx1 in radiation sensitivity of human lung cancer cells was investigated with special emphasis on the redox status of the catalytic Cys 52 . Overexpression of Prx1 enhances the clonogenic survival of irradiated cells, and suppresses IR-induced JNK activation and apoptosis. The peroxidase activity of Prx1, however, is not essential for inhibiting JNK activation. The latter effect is mediated through its association with the GSTpi-JNK complex, thereby preventing JNK release from the complex. Reduced JNK activation is observed when the peroxidase activity of Prx1 is compromised by Cys 52 over-oxidation or in the presence of the Cys 52 to Ser 52 mutant (Prx1C52S) lacking peroxidase activity. Both Prx1 and Prx1C52S interact with the GSTpi-JNK complex, and suppress the release of JNK from the complex. Studies from this dissertation suggest that Prx1 maybe an useful target for early detection of lung cancer and inhibiting Prx1 may provide an approach for simultaneous targeting of important cellular signaling proteins in intervening tumorigenesis of lung epithelial cells. This study provides new insight into the anti-apoptotic function of Prx1 in modulating radio-sensitivity, and provides the impetus to monitor the influence of Prx1 levels in the management of lung cancer.