Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity
Jeremy Chien, … , Scott H. Kaufmann, Viji Shridhar
Jeremy Chien, … , Scott H. Kaufmann, Viji Shridhar
Published July 3, 2006
Citation Information: J Clin Invest. 2006;116(7):1994-2004. https://doi.org/10.1172/JCI27698.
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Research Article Oncology

Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity

Abstract

Resistance to chemotherapy presents a serious challenge in the successful treatment of various cancers and is mainly responsible for mortality associated with disseminated cancers. Here we show that expression of HtrA1, which is frequently downregulated in ovarian cancer, influences tumor response to chemotherapy by modulating chemotherapy-induced cytotoxicity. Downregulation of HtrA1 attenuated cisplatin- and paclitaxel-induced cytotoxicity, while forced expression of HtrA1 enhanced cisplatin- and paclitaxel-induced cytotoxicity. HtrA1 expression was upregulated by both cisplatin and paclitaxel treatment. This upregulation resulted in limited autoproteolysis and activation of HtrA1. Active HtrA1 induces cell death in a serine protease–dependent manner. The potential role of HtrA1 as a predictive factor of clinical response to chemotherapy was assessed in both ovarian and gastric cancer patients receiving cisplatin-based regimens. Patients with ovarian or gastric tumors expressing higher levels of HtrA1 showed a higher response rate compared with those with lower levels of HtrA1 expression. These findings uncover what we believe to be a novel pathway by which serine protease HtrA1 mediates paclitaxel- and cisplatin-induced cytotoxicity and suggest that loss of HtrA1 in ovarian and gastric cancers may contribute to in vivo chemoresistance.

Authors

Jeremy Chien, Giovanni Aletti, Alfonso Baldi, Vincenzo Catalano, Pietro Muretto, Gary L. Keeney, Kimberly R. Kalli, Julie Staub, Michael Ehrmann, William A. Cliby, Yean Kit Lee, Keith C. Bible, Lynn C. Hartmann, Scott H. Kaufmann, Viji Shridhar

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Figure 3

HtrA1 is upregulated and activated during chemotherapeutic drug treatment.

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HtrA1-induced cell death is dependent on serine protease activity. OV202...
(A and B) SKOV3 cells were treated with cisplatin or paclitaxel, and lysates were taken at various time points. Immunoblot analysis of HtrA1 expression in these lysates (30 μg/lane) indicated upregulation of HtrA1 by cisplatin and paclitaxel. β-Actin immunoblots in the lower panels represent loading controls. (C) Immunoblot analysis of cells treated with paclitaxel for 24 hours indicated upregulation of HtrA1 (indicated by the dashed oval) and proteolysis of HtrA1 (35-kDa product in lane 2 indicated by an asterisk) that is dependent on HtrA1 protease activity, since protease mutant SA transfectants did not produce the smaller fragment in the presence of paclitaxel (lane 4). Forced expression of WT HtrA1 also produced a similar 35-kDa product (lane 5). (D) Pretreatment with 10 μM Z-VAD-FMK or cotransfection with dnCasp9 did not prevent proteolytic processing of HtrA1 (lanes 2 and 3). However, catalytic inactivation of HtrA1 (S328A) inhibited proteolysis of HtrA1 (lanes 4–6). Autocatalytic products are indicated by an asterisk. (E) To determine the domain composition of the 35-kDa product, it was immunoprecipitated (IP) with anti-HtrA1. Immunoblot and silver stain analyses detected 35-kDa and 50-kDa bands (indicated by asterisks and arrowheads, respectively). (F) LC-MS/MS analysis of the 35-kDa band showed peptide coverage missing in the Mac25 domain (underlined) but present in the 50-kDa band (red). (G) To compare the activities of 35-kDa and 50-kDa HtrA1, plasmid constructs (full-length and ΔMac25) were transfected into OV202 cells, and apoptotic activity was analyzed by annexin V labeling. Transfection of WTΔMac induced higher cell death compared with full-length HtrA1 (WT HtrA1).