[CANCER RESEARCH 61, 4003–4009, May 15, 2001] Inhibition of Signal Transduction by the Hsp90 Inhibitor 17-Allylamino-17- demethoxygeldanamycin Results in Cytostasis and Apoptosis1 Isabelle Hostein, David Robertson, Francesca DiStefano, Paul Workman,2 and Paul Andrew Clarke Cancer Research Campaign Centre for Cancer Therapeutics [I. H., F. D., P. W., P. A. C.] and Confocal Microscope Facility [D. R.], Institute of Cancer Research, Sutton, Surrey SM2 5NG, United Kingdom ABSTRACT apoptosis regulators such as mutant p53 (5, 6). Unfortunately, geldanamycin has limited clinical potential because of its excessive 17-Allylamino-17-demethoxygeldanamycin (17AAG) is a first-in-class liver toxicity (5). However, subsequent derivatization of geldanamy- heat shock protein 90 (Hsp90) molecular chaperone inhibitor to enter cin has yielded 17AAG, an analogue with reduced liver toxicity that clinical trials. The downstream molecular and cellular consequences of Hsp90 inhibition are not well defined. 17AAG has shown activity against retains the potent antitumor activity of the parent compound (3, 9, 10). human colon cancer in cell culture and xenograft models. In this study, we This derivative has very recently entered Phase I clinical trial as the demonstrated that in addition to depleting c-Raf-1 and inhibiting ERK-1/2 first-in-class Hsp90 inhibitor at our center and in the US, under the phosphorylation in human colon adenocarcinoma cells, 17AAG also de- auspices of the United States NCI and the United Kingdom CRC. pleted N-ras, Ki-ras, and c-Akt and inhibited phosphorylation of c-Akt. A Mutation and activation of Ki-ras occurs in ϳ30% of all colon consequence of these events was the induction of cell line-dependent cancers, and certain mutations are associated with a poorer outcome cytostasis and apoptosis, although the latter did not result from dephos- (11). In addition, constitutive activation of ERK-1/2 is common in phorylation of proapoptotic Bad. One cell line, KM12, did not exhibit colon cancers, and inhibitors of the Ras/Raf/MEK/ERK signaling apoptosis and in contrast to the other cell lines overexpressed Bag-1, but pathway effectively inhibit the growth of colon cancer cells in vitro did not express Bax. Taken together with other determinants of 17AAG and in vivo (12, 13). Given that 17AAG depletes c-Raf-1 and inhibits sensitivity, these results should contribute to a more complete understand- ing of the molecular pharmacology of 17AAG, which in turn should aid ERK-1/2 phosphorylation, we hypothesized that 17AAG could be an the future rational clinical development and use of the drug in colon and effective anticancer agent against colon cancer, among other tumor other tumor types. types. Indeed, we have recently demonstrated that 17AAG inhibits the growth of human ovarian and human colon carcinomas in vitro and INTRODUCTION delayed the growth of two colon cancer xenograft lines in vivo (3). Inhibition of signal transduction by agents such as 17AAG could One promising therapeutic strategy for treating cancer is to specif- potentially result in cytostasis or cell death, and the balance of these ically target signal transduction pathways that have a key role in biological fates could influence patient treatment strategy and clinical oncogenic transformation and malignant progression (1, 2). The ben- outcome. For example, a cytostatic response would require a chronic zoquinone ansamycin antibiotics, such as geldanamycin and herbimy- administration schedule, as is used for various signal transduction cin A, have been identified as agents that revert transformation by inhibitors (e.g., Ref. 13), whereas a cell-killing effect might allow a v-Src and exhibit potent antitumor activity (3–6). Subsequent studies more intermittent dosing regimen. Moreover, understanding the mo- have revealed that inhibition of c-Src catalytic activity, the expected lecular basis of these responses to 17AAG might provide pharmaco- mechanism of action, was not responsible for antitumor activity (6, 7). dynamic end points and the identification of factors that could lead to Affinity binding experiments identified the chaperone Hsp903 as one a selection of patients most likely to be responsive to the drug. of the primary targets of geldanamycin (6). Hsp90 has an important Although we have previously shown that 17AAG is active against role in maintaining the correct conformation and stability of a number colon cancer in vitro and in vivo, the signaling pathways affected by of client proteins, but it is less promiscuous than other chaperones in 17AAG in colon cancer are not well understood and the ability of this that the number of its client proteins is limited (8). The interaction of agent to induce apoptosis has not been determined in any tumor geldanamycin with Hsp90 results in competition for ATP binding to model. This present study is the first to show that in human colon Hsp90 and inhibition of its chaperone function, a consequence being adenocarcinoma cells, 17AAG depletes c-Raf-1 and inhibits ERK-1/2 the ubiquitination and degradation of client proteins by the protea- phosphorylation, as has been described for other tumor types (3, 6, some pathway (6). The antitumor activity of geldanamycin has been 10). Moreover, we also demonstrate that treatment of human colon attributed to destabilization of key client proteins, including receptor adenocarcinoma cells with 17AAG results in depletion of N-ras, and nonreceptor kinases (Erb-B2, epidermal growth factor receptor, Ki-ras, and c-Akt together with inhibition of c-Akt phosphorylation. and Src family kinases), serine/threonine kinases (c-Raf-1 and Cdk4), Furthermore, we show that 17AAG treatment results in both cytostasis steroid hormone receptors (androgen and estrogen), and cell cycle and and apoptosis to an extent depending on the particular colon cancer cell line, and we identify factors that may influence and regulate these Received 9/19/00; accepted 3/15/01. responses. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with MATERIALS AND METHODS 18 U.S.C. Section 1734 solely to indicate this fact. 1 Cancer Research Campaign funding is gratefully acknowledged by P. C. and P. W. (Grant SP2330); P. W. is a Cancer Research Campaign Life Fellow. I. H. is supported by Tissue Culture. Human colon adenocarcinoma cell lines (HCT15, Ligue Nationale Contre Le Cancer and the Haddow Fund of the Institute of Cancer HCT116, HT29, and KM12) were cultured in DMEM (Life Technologies, Inc., Research. Paisley, United Kingdom) supplemented with 10% FCS (PAA Laboratories, 2 To whom requests for reprints should be addressed, at E Block, CRC Centre for Kingston, United Kingdom), 1ϫ nonessential amino acids (Life Technolo- Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom. Phone: 44 208 722 4301; Fax: 44 208 642 1140; E-mail: gies), and 2 mML-glutamine in a 5% CO2 atmosphere. Cells in mid-log phase [email protected]. of growth were exposed to 17AAG (kindly supplied via Dr. E. Sausville, NCI, 3 The abbreviations used are: Hsp, heat shock protein; 17AAG, 17-allylamino-17- Bethesda, MD) for 24 h. The cells were then washed and supplemented with demethoxygeldanamycin; NCI, National Cancer Institute; CRC, Cancer Research Cam- fresh drug-free medium and harvested for molecular analysis either immedi- paign; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; TBST, Tris- buffered saline-Tween; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, ately or at 48 and 72 h. The viability of adherent cells was measured at 72 h poly(ADP) ribose polymerase; PI3 kinase, phosphatidylinositol-3Ј-kinase. by trypan blue exclusion. 4003 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. INDUCTION OF APOPTOSIS BY AN Hsp90 INHIBITOR Analysis of Protein. Cells were resuspended in lysis buffer [150 mM NaCl, morphism that disables DT-diaphorase are particularly insensitive to 50 mM Tris-HCl (pH 8.0), 0.2% (w/v) SDS, 0.2% (v/v) NP40, 1% (v/v) 17AAG (3). HT29, HCT116, KM12, and HCT15 do not have the glycerol, 1 mM EDTA, 0.5 mM sodium orthovanadate, 10 mM sodium PPi, 100 polymorphism and express relatively similar, high levels of DT- mM NaF, and 1ϫ protease inhibitors (Complete Mini; Roche Diagnostics, diaphorase (14, 15). Therefore, DT-diaphorase is unlikely to contrib- Indianapolis, IN)] and an equal volume of 80 mM Tris-HCl containing 6 mM ute in a major way to any difference seen between the four colon MgCl . An aliquot was removed for protein estimation by Lowry assay 2 cancer cell lines. (Bio-Rad, Hercules, CA). Equal amounts of protein and Rainbow molecular weight markers (Amersham Pharmacia Biotech, Amersham, United Kingdom) The cell lines were treated with a range of 17AAG concentrations were separated by electrophoresis through polyacrylamide gels and electro- for 24 h, followed by incubation in the absence of drug. This allowed transferred to Hybond-C nitrocellulose (Amersham Pharmacia Biotech). Im- analysis of molecular response and recovery to the drug. Fig. 1 shows munoblots were blocked with 5% nonfat milk in TBST1 [10 mM Tris-HCl (pH dose-response data for the four colon cancer cell lines. The IC50 7.6), 142 mM NaCl, 0.1% Tween-20] and then incubated with 0.4 ␮g/ml each values for adherent cell viability at 72 h, calculated from three of anti-c-Raf-1 rabbit polyclonal antibody, anti-Bag-1 rabbit polyclonal anti- independent experiments, were 0.2 ␮M (HT29), 0.8 ␮M (HCT116), 0.9 body (Santa Cruz Biotechnology Biotech, Santa Cruz Biotechnology, CA), ␮M (KM12), and 46 ␮M (HCT15). There was evidence that geldana- anti-N-ras mouse monoclonal antibody, and anti-Ki-ras mouse monoclonal mycin and 17AAG are substrates for efflux by P-glycoprotein. This ␮ antibody (Oncogene Research, Cambridge, MA); 1 g/ml each of anti-ERK- may potentially be a contributing factor to the high IC value ob- 1/2 rabbit polyclonal antibody, anti-phospho-ERK-1/2 rabbit polyclonal anti- 50 served for HCT15 cells because these have a MDR phenotype (3).