Background:Our previous study revealed that proline-rich tyrosine kinase 2 (Pyk2) is implicated in both anchorage-independent growth and anoikis resistance in lung cancer cells. This study aims to explore the expression and clinical significance of Pyk2 and its phosphorylated forms in non-small-cell lung cancer (NSCLC).Methods:The mRNA and protein levels of Pyk2 or cancer stem cell markers (ALDH1a1, ABCG2 and Bmi-1) were either examined by reverse transcription-PCR or western blotting. An immunohistochemistry (IHC) assay was conducted to analyse the expression of Pyk2 and its phosphorylated forms in 128 NSCLC cases.Results:The levels of Pyk2 mRNA, total protein, and its phosphorylated form pY881 were higher in lung cancer lesions than in the paired noncancerous tissues. The IHC analysis showed the levels of the Pyk2 and Pyk2[pY881] proteins were highly expressed in 70 (54.7%) and 77 (60.2%) cases, respectively. Both Pyk2 and Pyk2[pY881] were independent prognostic factors for NSCLC patients. The gain and loss study of Pyk2 function revealed that Pyk2 could upregulate the expression of ALDH1a1, ABCG2 and Bmi-1 and enhance the ability of colony formation in soft agar assay in A549 and H460 cells.Conclusion:Both Pyk2 and phosphorylated Pyk2[pY881] are potential prognostic factors and therapeutic targets for NSCLC.British Journal of Cancer advance online publication, 6 August 2013; doi:10.1038/bjc.2013.439 www.bjcancer.com.