Expression of peroxiredoxin 1 and 4 promotes human lung cancer malignancy

Abstract

Members of the Peroxiredoxin (Prx) family are major cellular antioxidants that scavenge hydrogen peroxide and play essential roles in oxidative stress and cell signaling. 2-Cys Prxs, including Prx1, 2, 3 and 4, have been indicated in multiple oncogenic signaling pathways and thus may contribute to various processes of cancer development. The significance of 2-Cys Prxs in lung cancer development and their biological function in signal transduction have not been fully investigated. In this study we analyzed the expression of 2-Cys Prxs in lung cancer, and examined their levels of expression in a variety of cell lines established from human lung normal or cancer tissues. We found that 2-Cys Prxs, in particular, Prx1 and Prx4, were preferentially expressed in cell lines derived from human lung cancer. Through isoform specific knockdown of individual Prx, we demonstrated that Prx1 and Prx4 (but not Prx3) were required for human lung cancer A549 cells to form soft agar colony and to invade through matrigel in culture. Knockdown of Prx1 or Prx4 significantly reduced the activation of c-Jun and repressed the AP-1 mediated promoter activity. In mouse xenograft models, knockdown of Prx4 in A549 cells reduced subcutaneous tumor growth and blocked metastasis formation initiated through tail vein injection. Moreover, overexpression of Prx1 or Prx4 further enhanced the malignancy of A549 cells both in culture and in mouse xenografts in vivo. These data provide an in-depth understanding of the contribution of Prx1 and Prx4 to lung cancer development and are of importance for future development of therapeutic methods that targeting 2-Cys Prxs.

Keywords: Peroxiredoxin; cell signaling; lung cancer; tumor invasion and metastasis.

Figure 1

mRNA expression of 2-Cys Prxs in human lung normal and cancer tissues from published microarrays. Data were summarized using the Oncomine database (www.oncomine.com). A: Microarray results from Garber M.E., et al, PNAS USA 98:13784~9. Category and number of samples included: 0, normal lung (n = 6); 1, large cell lung carcinoma (n = 4); 2, lung adenocarcinoma (n = 42); 3, small cell lung carcinoma (n = 5); 4, squamous cell lung carcinoma (n = 16). B: Microarray results from Bhattacharjee A., et al, PNAS USA 98:13790~5. Category and number of samples included: 0, normal lung (n = 17); 1, lung adenocarcinoma (n = 139); 2, lung carcinoid tumor (n = 20); 3, small cell lung carcinoma (n = 6); 4, squamous cell lung carcinoma (n = 21).

Figure 2

Differential expression of 2-Cys Prxs in various cell lines and knockdown of individual Prx in human lung adenocarcinoma A549 cells. (A) Expression of 2-Cys Prxs in cell lines established from human lung normal epithelium or different types of lung cancer. Cell lines were originally derived from: ¹lung normal epithelium; ²small cell carcinoma; ³squamous cell carcinoma; ⁴adenocarcinoma; *cancer metastasis. For each cell line, cells were lysed in RIPA buffer at the concentration of 1× 10⁷ cell/ml and equal volume of lysates were loaded to the gel for Western blot. (B-D) Knockdown of endogenous Prx1 (B), Prx3 (C) or Prx4 (D) in A549 cells using isoform specific ShRNAs targeting the correspondent protein coding regions. Two sets of unique ShRNA (indicated by the number in the parenthesis) were used for each specified Prx target. The endogenous Prx levels were measured by Western blot from triplicate dishes of stable cells established by lentivrial infection and antibiotic selection. Note that knockdown of targeted Prx does not interfere with the expression of other isoforms of 2-Cys Prxs.

Figure 3

Knockdown of Prx1 or Prx4 in A549 cells abolishes their ability of to form anchorage independent colonies in soft agar and represses their capability of invading through matrigel. A, B: Anchorage independent colony formation in soft agar; C, D: Transwell cell invasion assay. Compared to wildtype parental A549 (Wt) or ShNT cells, *p<0.05 (n = 6, t test).

Figure 4

Overexpression of Prx1 or Prx4 in A549 cells enhances their ability to form anchorage independent colonies in soft agar and increase their capability of invading through matrigel. (A) The protein coding regions of human Prx1, Prx2, Prx3 or Prx4 gene in HEK293T cells were reverse transcribed and amplified by PCR, and then cloned into the pCDNA3.1-Myc vector for protein expression; (B) Myc tagged Prx1, 2, 3 or 4 expression in A549 stable cells; (C-F) Anchorage independent colony formation in soft agar (C, D) and transwell invasion assay (E, F) using cells with stably overexpression of individual Prx.

Figure 5

Knockdown of Prx4 represses, whereas overexpression of Prx4 activates c-Jun mediated AP-1 activation. (A, B) Proteome profiler human phosphokinase array (A) and quantification of phosphorylated c-Jun levels (B). -, no stimulation. +, serum stimulation. Compared to ShNT (+) cells, *p<0.05 (n = 4, t test). (C) Western blot of phosphorylated c-Jun after serum stimulation in A549 cells expressing ShNT, ShPrx4 or MycPrx4. (D) AP-1 luciferase reporter assay in A549 cells with or without expression of ShRNAs or MycPrxs. Compared to either ShNT or vector cells, *p<0.05 (n = 6, t test).

Figure 6

Knockdown of Prx4 represses, whereas overexpression of MycPrx4 enhances tumor xenograft growth and metastasis formation in mouse models in vivo. (A) Tumor growth curves of subcutaneously injected A549-ShNT, ShPrx4 or MycPrx4 cells into SCID mice; (B, C) Images (B) and average weight (C) of primary tumors extracted from injection sites 40 days post subcutaneous injection; (D) Lung tumor nodules found in SCID mice receiving tail vein injection of A549-ShNT, ShPrx4 or MycPrx4 cells. Arrow heads indicated tumor nodules. Bar graph shows the average number of tumor nodules found in each experimental group. Compared to mice receiving ShNT cells, *p<0.05 (n = 9, t test).