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Blocking Heat Shock Protein-90 Inhibits the Invasive Properties and Hepatic Growth of Human Colon Cancer Cells and Improves

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Blocking Heat Shock Protein-90 Inhibits the Invasive Properties and Hepatic Growth of Human Colon Cancer Cells and Improves 2868 Blocking heat shock protein-90 inhibits the invasive properties and hepatic growth of human colon cancer cells and improves the efficacy of oxaliplatin in p53-deficient colon cancer tumors in vivo Christian Moser,1 Sven A. Lang,1 Silvia Kainz,2 significantly reduced tumor growth and vascularization. Andreas Gaumann,3 Stefan Fichtner-Feigl,1,2 Furthermore, blocking Hsp90 reduced hepatic tumor Gudrun E. Koehl,2 Hans J. Schlitt,1 burden and metastatic nodules in an experimental model Edward K. Geissler,2 and Oliver Stoeltzing1,2 of hepatic colon cancer growth. Importantly, combining oxaliplatin with 17-DMAG in vivo significantly improved 1Departments of Surgical Oncology and 2Experimental growth inhibitory and proapoptotic effects on p53- Surgery, 3Institute of Pathology, University of Regensburg deficient cells, compared with either substance alone. In Medical Center, Regensburg, Germany conclusion, inhibition of Hsp90 abrogates the invasive properties of colon cancer cells and modulates the Abstract expression of the antimetastatic factor activating tran- scription factor-3. Hence, targeting Hsp90 could prove We recently showed that inhibition of heat shock protein valuable for treatment of advanced colorectal cancer 90 (Hsp90) decreases tumor growth and angiogenesis in by effectively inhibiting colon cancer growth and hepa- gastric cancer through interference with oncogenic sig- tic metastasis and improving the efficacy of oxaliplatin. naling pathways. However, controversy still exists about [Mol Cancer Ther 2007;6(11):2868–78] the antimetastatic potential of Hsp90 inhibitors. More- over, in vitro studies suggested that blocking Hsp90 could overcome p53-mediated resistance of cancer cells Introduction to oxaliplatin. We therefore hypothesized that blocking Invasion into adjacent tissues and metastasis to distant sites oncogenic signaling with a Hsp90 inhibitor would impair are major features of malignant cancer cells, which are metastatic behavior of colon cancer cells and also improve complex processes that require coordinated actions of a the efficacy of oxaliplatin in vivo. Human colon cancer large assortment of growth factors and their receptors, as cells (HCT116, HT29, and SW620) and the Hsp90 well as downstream signaling intermediates (1). With inhibitor 17-(dimethylaminoethylamino)-17-demethoxy- respect to colorectal cancer metastasis, the epidermal geldanamycin (17-DMAG) were used for experiments. growth factor (EGF) receptor (EGFR) system and c-Met, In vitro, 17-DMAG substantially inhibited phosphorylation the primary receptor for hepatocyte growth factor/scatter of epidermal growth factor receptor, c-Met, and focal factor (HGF), have been shown to play an important role adhesion kinase, overall resulting in a significant decrease (2–5). Both receptor systems are overexpressed in colorec- in cancer cell invasiveness. Importantly, 17-DMAG led to tal cancer and colorectal cancer liver metastases. In an up-regulation of the transcription factor activating addition, both kinase systems are involved in carcinogenic transcription factor-3, a tumor suppressor and antimeta- processes such as cell proliferation, motility, survival, static factor, on mRNA and protein levels. In a cell death apoptosis, and tumor angiogenesis (3, 6–8). Hence, EGFR ELISA, 17-DMAG markedly induced apoptosis in both and c-Met systems represent interesting molecular targets p53-wt and p53-deficient cells. In vivo, 17-DMAG for reducing metastasis and angiogenesis of colorectal cancer (4, 9). However, development of c-Met inhibitors has been challenging with studies currently ongoing. Interestingly, inhibition of heat shock protein 90 (Hsp90) may be a novel approach to achieve effective interference Received 6/18/07; revised 9/12/07; accepted 10/1/07. with the function of both receptor systems and respective Grant support: AACR-Littlefield Grant for Metastatic Colon Cancer Research (O. Stoeltzing) and the German Cancer Society (Deutsche downstream signaling intermediates. Hsp90 is a molecular Krebshilfe, Max-Eder Program, Bonn, Germany; O. Stoeltzing). chaperone that is ubiquitously and abundantly expressed. The costs of publication of this article were defrayed in part by the There are numerous proteins involved in the control of payment of page charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to physiologic and pathophysiologic processes that require indicate this fact. Hsp90 for their biogenesis, regulation, and function (10, 11). Requests for reprints: Oliver Stoeltzing, Departments of Surgery and In cancer, the expression of Hsp90 is increased at 2- to 10- Surgical Oncology, University of Regensburg Medical Center, Franz-Josef- fold higher levels when compared with that of normal Strauss-Allee 11, 93042 Regensburg, Germany. Phone: 49-941-944- 6801; Fax: 49-941-944-6802. E-mail: oliver.stoeltzing@ tissues (12). Indeed, compounds that inhibit Hsp90 action, klinik.uni-regensburg.de such as geldanamycin or its derivates 17-allylamino-17- Copyright C 2007 American Association for Cancer Research. demethoxygeldanamycin (17-AAG) and 17-dimethylami- doi:10.1158/1535-7163.MCT-07-0410 noethylamino-17-demethoxygeldanamycin (17-DMAG), Mol Cancer Ther 2007;6(11). November 2007 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 2869 have been shown to effectively inhibit tumor growth in chased from Invivogen (Cayla-Invivogen). Antibodies several preclinical xenograft models (13–16), and the against MAPK/Erk kinase 1/2, phospho-Ser217/221 MAPK/ efficacy of 17-AAG is currently being investigated in cancer Erk kinase 1/2, Akt, phospho-Thr308 Akt, p44/42 MAPK, patients in clinical phase I/II trials (17–20). phospho-Tyr202/204 p44/42 MAPK, signal transducer Importantly, a number of cancer-associated proteins have and activator of transcription-3, phospho-Tyr705 signal been identified as Hsp90 clients, including EGFR family transducer and activator of transcription 3, focal adhesion members, c-MET, AKT, mutant p53, mitogen-activated kinase (FAK), phospho-Tyr925 FAK, phospho-Tyr1349 c-Met, protein kinase (MAPK)/extracellular signal–regulated ki- and EGFR were purchased from Cell Signaling Technolo- nase (Erk), and the transcription factor hypoxia-inducible gies. Anti–h-actin and anti–activating transcription factor-3 factor-1a (8, 21–23). Because these are important partic- (ATF3) antibodies were purchased from Santa Cruz Bio- h ipants in pathways driving tumor cell survival, prolifera- technology. -Actin serves as a loading control in Western tion, and progression, Hsp90 seems to be an excellent target blot analyses. Recombinant human EGFand HGFwere in cancer therapy (22, 24, 25). In a previous study, we obtained from R&D Systems. showed that blocking Hsp90 significantly interferes with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazo- EGFR signaling in gastric cancer, which, in part, is lium Bromide Analyses mediated through down-regulation of EGFR itself (26). To evaluate cytotoxic effects of 17-DMAG on tumor cells, Moreover, we also showed that targeting multiple path- HCT116, HT29, and SW620 cells were seeded into 96-well  3 ways does not require therapy with Hsp90 inhibitors at plates (1 10 per well) and exposed to various j maximum tolerated doses to achieve potent inhibition of concentrations of 17-DMAG for 24 or 48 h at 37 C. We tumor growth and angiogenesis. Nevertheless, controver- used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- sies still exist about the antimetastatic potential of Hsp90 lium bromide assay to assess cell viability, as previously inhibitors. In one report, Price et al. (27) have shown an described (30). enhanced incidence of bone metastasis and osteolytic Immunoblot Analysis of Signaling Intermediates lesions with 17-AAG treatment in a human breast cancer To determine the effects of Hsp90 inhibition on signaling mouse model. This crucial aspect has not been investigated intermediates, cancer cells were incubated for 16 h with for colon cancer and associated hepatic metastases. In favor FCS-RPMI containing 17-DMAG (250 nmol/L) before sti- of a Hsp90-based approach is evidence that Hsp90 mulation with either recombinant human EGF(100 ng/mL, inhibition may overcome resistance of p53-deficient colon 10 and 30 min) or HGF(50 ng/mL, 10 and 30 min) under cancer cells to oxaliplatin through prevention of S-phase serum-reduced conditions (1% FCS-RPMI). Pretreatment of cells for a minimum of 16 h is required to down-regulate/ accumulation, thus leading to additive or synergistic inhibit Hsp90 client proteins, as recently reported (8, 16, 26). apoptotic effects in vitro (28, 29). However, this hypothesis Whole-cell lysates were prepared, as described elsewhere has not yet been validated in in vivo models. (30). Protein samples (75 Ag) were subjected to Western Because these are very important aspects to highlight blotting on a denaturating 10% SDS-PAGE. before initiating clinical trials with Hsp90 inhibitors for ELISA for Vascular Endothelial Growth Factor-A therapy of colorectal cancer (metastatic to liver), we Secretion particularly focused in this study on determining the To determine changes in vascular endothelial growth potential of the Hsp90 inhibitor 17-DMAG to inhibit the factor (VEGF)-A secretion by cancer cells, we used an migratory and invasive properties of p53-wt and p53- ELISA kit specific for human VEGF-A (BioSource Europe), deficient colon cancer cells, and
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