Kallikrein-related peptidase 6 overexpression promotes non-small cell lung cancer cell proliferation and is associated with poor patient outcome

Abstract

Introduction The human kallikrein-related peptidases (KLK) are a family of serine proteases that are often aberrantly expressed in common human malignancies and contribute to neoplastic progression through multifaceted roles. Methods We evaluated KLK6 expression in the tumoral and normal adjacent lung tissue of 56 patients with Non-Small Cell Lung Cancer (NSCLC) by real-time RT-PCR and immunohistochemistry. To determine the impact of KLK6 overexpression on the growth of lung cancer cells, we integrated the cDNA encoding the complete sequence of KLK6, through homolog recombination, in a NSCLC line (A549 Flp-In) and determined the growth rate of two independent clones. Progression of the KLK6- and parental cells inside the cell cycle was assessed by flow cytometry following synchronization of cells at the end of the G1 phase with starvation and hydroxyurea treatments. Key regulator proteins of the cell cycle were analyzed by Western blot in synchronized and unsynchronized cells. Results We found KLK6 transcript up-regulation in tumor tissues from patients with NSCLC and association of KLK6 status with low patient survival. KLK6 immunoreactivity was restricted to epithelial cells of normal bronchi and detected in most of cancer samples, in which KLK6 signal intensity correlated with well differentiated tumors. Ectopic expression of KLK6 dramatically enhanced NSCLC cell growth. Analysis of cell cycle progression revealed that promotion of cell growth caused by KLK6 results from an acceleration of cell cycle progression through G1/S transition, which was accompanied with a marked increase of cyclin E and repression of p21. In addition, expression of KLK6 in NSCLC cells was associated with an increase of c-Myc that is well-know to promote cell-cycle progression via regulation of cyclin D/E activation and down-regulation of p21. Conclusion ectopic expression of KLK6 facilitates cell cycle progression, certainly through alteration of c-Myc and downstream key regulators, and thus promotes cell proliferation. Moreover, KLK6 is overexpressed in NSCLC and associated with poor prognosis. Altogether, those findings suggest that KLK6 might play a central role in NSCLC development and progression.