Paclitaxel Resistance and Multicellular Spheroid Formation Are Induced by Kallikrein-Related Peptidase 4 in Serous Ovarian Cancer Cells in an Ascites Mimicking Microenvironment

Ying Dong,Joakim E Swedberg,Peter G Parsons,Jonathan M Harris,Carson Stephens,Michael A Mcguckin,Glen M Boyle,Judith A Clements,Carina Walpole,Georgia Sotiropoulou

doi:10.1371/journal.pone.0057056

Abstract

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Highlights

Serous epithelial ovarian carcinoma (EOC) accounts for .50% of ovarian cancer [1] which is the leading cause of death from gynecological malignancies [2]
Western blot analysis confirmed that the V5-tagged over-expressed KLK4 (33 kDa), an extracellular serine protease, is secreted in the conditioned media (CM) of both stable and transient KLK4 transfectants but not vector-only, mock-transfected or native SKOV-3 cells (Fig. 1A, top panel)
KLK4 Induced urokinase plasminogen activator (uPA) Expression in SKOV-3 Cells Given that aprotinin can inhibit the action of other serine proteases, such as uPA that was activated by KLK4 in previous biochemical studies [17,18], we examined the expression of uPA in KLK4 transfected SKOV-3 cells

Summary

Introduction

Serous epithelial ovarian carcinoma (EOC) accounts for .50% of ovarian cancer [1] which is the leading cause of death from gynecological malignancies [2]. The serine protease, urokinase plasminogen activator (uPA) and its receptor uPAR in EOC cells are induced by ascites [9] and the expression of uPA is associated with chemoresistance, progression and poor prognosis in women with this cancer [10,11]. These studies indicated that the tumor microenvironment clearly influences EOC progression [12,13], but the effect of suspension per se, mimicking the ascites microenvironment, on survival of EOC cells and chemosensitivity is not clear. The involvement of other serine proteases remains largely unknown

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