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[Cancer Research 66, 8192-8199, August 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Taxotere-Induced Inhibition of Human Endothelial Cell Migration Is a Result of Heat Shock Protein 90 Degradation

Janice Murtagh, Haiyan Lu and Edward L. Schwartz

Department of Oncology, Albert Einstein College of Medicine, Bronx, New York

Requests for reprints: Edward L. Schwartz, Department of Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467. Phone: 718-920-4015; Fax: 718-882-4464; E-mail: eschwart{at}aecom.yu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
In addition to effects on tumor cell proliferation and apoptosis, microtubule-binding agents are potent inhibitors of angiogenesis. The cancer chemotherapeutic drug Taxotere (docetaxel) inhibited vascular endothelial growth factor (VEGF)–induced human umbilical vein endothelial cell (HUVEC) migration in vitro at concentrations substantially lower than required to cause cell cycle arrest or apoptosis. Here, we show that Taxotere caused the ubiquitination and subsequent proteasomal degradation of heat shock protein 90 (Hsp90) in HUVEC. This prevented signaling from the focal adhesions and VEGF receptors and inhibited integrin activation. Taxotere prevented the VEGF-induced phosphorylation of focal adhesion kinase, Akt, and endothelial nitric oxide synthase (eNOS), all of which are Hsp90 client proteins. Taxotere completely blocked the VEGF-induced increase in eNOS activity, and the addition of a NO donor reversed the inhibitory effect of Taxotere on VEGF-induced migration. A similar reversal occurred with a proteasomal inhibitor of Hsp90 degradation. Furthermore, overexpression of Hsp90 rescued HUVEC from the inhibition of VEGF-induced migration by Taxotere. Previous studies have suggested that tubulin is also a client protein of Hsp90, and immunocytochemical analysis showed that Taxotere caused the dissociation of Hsp90 from tubulin. We suggest that uncomplexed Hsp90 is more susceptible to ubiquitination and subsequent proteasomal degradation than the bound form. Although inhibitors of Hsp90 are currently under clinical investigation as antitumor agents, this seems to be the first account of a drug that reduces Hsp90 function by enhancing its proteasomal degradation. Further, the loss of Hsp90 and the inactivation of Hsp90 client proteins are previously undescribed actions of Taxotere that may contribute to its antiangiogenic activity. (Cancer Res 2006; 66(16): 8192-9)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Endothelial cell migration can be induced by chemotactic factors, such as vascular endothelial growth factor (VEGF), and is a key component of the angiogenic process (1). At the molecular level, the processes driving cell migration consist of a series of overlapping and interconnecting signaling pathways. Binding of VEGF to the Flk-1/KDR VEGF receptor-2 (VEGFR-2), the receptor subtype associated with its migration-stimulating actions, induces receptor dimerization, activation of intrinsic receptor kinase activity, and tyrosine autophosphorylation (2). Downstream signaling molecules implicated in the cellular actions of VEGF include phospholipase C{gamma}, the Raf-Mek-Erk-mitogen-activated protein kinases, phosphatidylinositol 3-kinase (PI3K), Akt, p38 stress kinase, and endothelial nitric oxide synthase (eNOS; ref. 3). VEGF has also been shown to have multiple effects on the signaling components of the focal adhesions, most notably on focal adhesion kinase (FAK) and the integrins (4–9). Recent data suggest that the VEGF, epidermal growth factor (EGF), and platelet-derived growth factor receptor families can form complexes with the integrins and that the reciprocal cross-talk and cooperation of the growth factor and extracellular matrix (ECM) receptors enhance their respective cellular actions (5, 6, 10, 11).

Microtubules are necessary for directed migration of endothelial and other cells, and there are several possible mechanisms by which microtubule-disrupting compounds could block cell motility (12, 13). These include impairment of the repositioning of the microtubule organizing center, interference with microtubule interaction with developing focal adhesions, inhibition of Rho GTPase activation by the cycle of microtubule polymerization and depolymerization, blockade of the formation of lamellipodia and cell polarization as a consequence of the inhibition of intracellular protein trafficking and vesicle transport, and inhibition of microtubule-mediated integrin clustering and increased avidity (14–18). These processes vary in their sensitivity to inhibition by microtubule-targeting drugs. Thus, earlier studies, in which the high concentrations of these agents caused near complete microtubule breakdown, concluded that there was a loss in the ability of cells to polarize their actin activity; consequently, the cells had zones of cytoskeletal reorganization around their entire circumference rather than in a localized region as would be required for pseudopodia formation and directional motion (13, 19). These mechanisms are likely not relevant, however, to the actions of the microtubule-binding drugs at concentrations that occur clinically. It has been hypothesized that microtubules contribute to cell locomotion by their interaction with transient assemblies whose subsequent stabilization and/or maturation are required for motility, and this was suggested to be a target for microtubule-binding drugs at lower concentrations (13).

Microtubule-targeting agents have antiangiogenic properties (14, 20–23). These agents include the chemotherapeutic drugs Taxol (paclitaxel) and Taxotere (docetaxel), which inhibited endothelial cell migration in vitro at exceptionally low concentrations that did not affect microtubule gross morphology or inhibit cell proliferation, although they did produce more subtle effects on microtubule dynamics (14, 20–24). In contrast, the antiangiogenic effects of other agents, such as vinblastine and vinorelbine, were produced by direct cytotoxicity (25). The purpose of the present study was to determine the mechanism(s) by which low, pharmacologically relevant concentrations of Taxotere inhibited endothelial cell migration, which we hypothesized were occurring via previously undefined actions of the taxanes.

Heat shock protein 90 (Hsp90) is a highly conserved and essential cytoplasmic chaperone protein involved in the refolding of proteins in cells exposed to environmental stress and in the conformational maturation of key regulatory proteins (26). In addition to its cytoprotective role, Hsp90 is expressed under nonstress conditions, where it functions to maintain homeostasis by regulating protein folding quality control and the stability of numerous client proteins, including several key regulators of signal transduction (26). The natural product geldanamycin exerts its antitumor effects by binding to, and inhibiting, the intrinsic ATPase activity of Hsp90, resulting in degradation of Hsp90 client proteins via the ubiquitin proteasome pathway (27). A geldanamycin analogue, 17-alkylamino-geldanamycin, has shown promising antitumor activity in clinical trials. Taxol has been reported to directly bind to the murine homologue of Hsp90 in macrophage cell extracts; however, the consequence(s) of this interaction are not known (28). Recent reports have associated Hsp90 with the actions of VEGF in endothelial cells notably with the VEGF-induced phosphorylation of FAK, with the activation of the serine/threonine protein kinase Akt, and with an increase in eNOS activity and NO production (9, 29). Because Hsp90 functions to stabilize signaling molecules associated with VEGF-mediated endothelial cell migration, our studies examined its role in Taxotere-induced inhibition of endothelial cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture and materials. Human umbilical vein endothelial cells (HUVEC; GlycoTech, Gaithersburg, MD by arrangement with the Developmental Therapeutics Program Angiogenesis Resource Center, National Cancer Institute) were grown in MCDB131, 2% fetal bovine serum (FBS), 10 ng/mL EGF, 12 µg/mL endothelial cell growth supplement, 1 µg/mL hydrocortisone, 10 units/mL heparin, 2 mmol/L L-glutamine, penicillin G, and streptomycin sulfate. S-nitroso-N-acetylpenicillamine (SNAP) and N-benzyloxycarbonyl-L-isoleucyl-{gamma}-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) were from Calbiochem (La Jolla, CA). Geldanamycin was from the National Cancer Institute.

Endothelial cell migration assay. Assays used the CytoSelect 24-well cell migration assay kit (Cell Biolabs, San Diego, CA). Confluent HUVEC were cultured with non-growth factor-containing medium for 18 hours before harvesting with 0.05% trypsin/EDTA. Harvested cells were suspended at 106/mL in M199 with 1% serum, and 105 cells were seeded onto Transwell inserts (8 µm pore, Costar, Acton, MA) precoated with 10 µg/mL fibronectin. Inserts containing HUVEC were placed into a 24-well plate containing 700 µL M199 with 1% serum and incubated for 1 hour at 37°C. Migration was stimulated by addition of the VEGF (10 ng/mL) to the lower well of the chamber. Taxotere was added to the upper chamber, and in some experiments, its addition was delayed for various times after the addition of the VEGF. After 5 hours, inserts were swabbed to remove nonmigrated cells, placed in cell staining solution for 10 minutes, and then shaken with 200 µL extraction solution. HUVEC migration was quantitated by measuring the absorbance at 560 nm.

Modulation of Hsp90 expression. For knockdown of Hsp90 gene expression, HUVEC were transfected with 50 nmol/L human Hsp90{alpha} small interfering RNA (siRNA; a pool of four siRNA, each with a 19-bp duplex core; Dharmacon, Lafayette, CO) or siCONTROL nontargeting siRNA immediately before plating onto fibronectin-coated migration filters using siPORT NeoFX transfection reagent (Ambion, Austin, TX). For Hsp90 overexpression, HUVEC were transfected with expression vectors for Hsp90{alpha} (in pCMV-Sport6), Hsp90ß (in pOTB7), or both Hsp90{alpha} and Hsp90ß or with a control vector using SuperFect transfection reagent (Qiagen, Valencia, CA). Hsp90 expression vectors were obtained from American Type Culture Collection (Manassas, VA) through the Mammalian Gene Collection (NIH). After 24 hours, cells were used in a Boyden chamber migration assay.

Reverse transcription-PCR analysis. Total RNA was isolated from HUVEC using TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Aliquots of total RNA were treated with RNase-free DNase I (Invitrogen) and the RNA concentration was determined spectrophotometrically. RNA (2-10 µg) was transcribed with SuperScript II RNase H– reverse transcriptase (Invitrogen) in the presence of 250 ng random primers. First-strand cDNA synthesis reaction (2 µL) was used as template for PCR in a Taq-catalyzed amplification using the following primer pairs: Hsp90{alpha} forward 5'-GGCAGAGGCTGATAAGAACG-3' and reverse 5'-AGTCATCCCTCAGCCAGAGA-3', Hsp90ß forward 5'-ATGCTCCAGCAGAGCAAAAT-3' and reverse 5'-TCCCATCAAATTCCTTGAGC-3', and glyceraldehyde-3-phosphate dehydrogenase forward 5'-GTCAGTGGTGGACCTGACCT-3' and reverse 5'-AGGGGAGATTCAGTGTGGTG-3'. Each PCR cycle (30 total) consisted of a denaturing step (95°C for 30 seconds), an annealing step (55°C for 45 seconds), and primer elongation (72°C for 90 seconds).

Immunofluorescent staining. HUVEC were seeded on fibronectin-covered chamber slides. After 4 hours in serum-free M199, cells were treated with Taxotere for 1 hour and then stimulated with VEGF (10 ng/mL for 4 hours or 50 ng/mL for 10 minutes). For costaining analysis, cells were treated with 10 nmol/L Taxotere for the indicated times in the absence of VEGF stimulation. The cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 in PBS for 5 minutes at room temperature, and treated with blocking buffer (1% bovine serum albumin in PBS) for 1 hour at room temperature. Mouse anti-{alpha}-tubulin-Cy3 antibody (Sigma, St. Louis, MO), mouse anti-Hsp90 antibody (Chemicon, Temecula, CA), mouse anti-integrin {alpha}Vß3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and an engineered monovalent antibody that recognizes integrin {alpha}Vß3 in its high-affinity state [WOW-1 F(ab); ref. 30; kindly provided by Dr. S. Shattil, University of California San Diego, San Diego, CA] were used to assess the effects of VEGF and Taxotere on integrin activation.

Immunoprecipitation and Western blots. Confluent HUVEC, seeded on fibronectin-covered dishes and starved overnight in M199 with 1% FBS, were treated with Taxotere for 1 hour followed by VEGF. Monolayers were washed in PBS and treated with immunoprecipitation lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% (v/v) NP40, with phenylmethylsulfonyl fluoride, aprotinin, leupeptin]. Cell-free extracts were incubated on ice for 30 minutes and centrifuged. For immunoprecipitation, 300 µg total protein was incubated overnight at 4°C with protein G-agarose beads coated with saturating amounts of antibodies to Akt (Cell Signaling Technology, Danvers, MA) or free Hsp90 (both {alpha} and ß; Stressgen, Victoria, BC, Canada). The resulting immune complexes were recovered after centrifugation by boiling in SDS-PAGE loading buffer. For whole-cell ysates, cells were lysed in a modified radioimmunoprecipitation assay buffer (Biosource International, Carlsbad, CA).

For immunoblotting, aliquots of whole-cell lysates (30 µg) or isolated immunocomplexes were separated by SDS-PAGE under reducing conditions using 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes and immunoblotted using antibodies against VEGFR-2, FAK, ß-actin (Santa Cruz Biotechnology), phosphorylated VEGFR-2 Tyr951, phosphorylated FAK Tyr397, ubiquitin (Biosource International), phosphorylated Akt Ser473, phosphorylated eNOS Ser117, Akt, eNOS, total Hsp90 (Cell Signaling Technology), Hsp70, Hsp90{alpha}, and Hsp90ß (Stressgen).

eNOS activity. Homogenates were prepared by sonicating cells in buffer [1 mmol/L EDTA, 1 mmol/L EGTA, 25 mmol/L Tris-HCl (pH 7.4)]. After centrifugation, 50 µg were added to a reaction, which contained (in 50 µL) 25 mmol/L Tris-HCl (pH 7.4), 3 µmol/L tetrahydrobiopterin, 10 µg/mL calmodulin, 1 µmol/L flavin adenine dinucleotide, 1 µmol/L flavin adenine mononucleotide, 1 mmol/L NADPH, 0.6 mmol/L CaCl2, and purified [2,3,4,5-3H]L-arginine (0.8 µCi, 60 Ci/mmol; Amersham, Piscataway, NJ). Reactions were incubated for 60 minutes at 37°C. Parallel control reactions used boiled cell extracts to correct for background. The reaction was stopped by the addition of stop buffer [50 mmol/L HEPES (pH 5.5), 5 mmol/L EDTA] and the lysates in stop buffer were subjected to anion exchange chromatography. Enzyme activity was calculated based on the amount of [3H]L-citrulline generated as determined by liquid scintillation counting. Data represent three individual experiments.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Taxotere inhibited HUVEC migration in vitro at low concentrations that did not inhibit HUVEC proliferation and was inhibitory only when added during the early stages of migration. Taxotere is a widely used anticancer drug with significant clinical activity most notably in advanced ovarian, breast, lung, esophageal, and prostate cancers (31). It reversibly binds to the ß-subunit of tubulin in microtubules, enhances the formation of more stable microtubules, inhibits microtubule depolymerization, and causes cell cycle arrest via mitotic impairment (32, 33). In addition, Taxotere has been shown to have direct antiangiogenic actions, including inhibition of endothelial cell proliferation, motility, invasiveness, and tubule formation in vitro and the inhibition of neovascularization in corneal micropocket and s.c. Matrigel plug assays in vivo (14, 20–23). In the present studies, the effect of Taxotere on HUVEC migration was evaluated in a modified Boyden chamber assay, a chemotactic model of migration representative of tumor-induced endothelial cell migration. Stimulation by a directional gradient of VEGF resulted in migration of the HUVEC to the underside of the membrane, and this was inhibited by Taxotere in a concentration-dependent manner with an IC50 of 0.01 nmol/L. In contrast, the IC50 for the inhibition of HUVEC proliferation was ~1 nmol/L (34).

Migration is a complex multistep process, and there are several potential sites where Taxotere might be exerting its actions ranging from the inhibition of early signaling pathways to impeding the mechanical events directly responsible for cell motility. Although the migration process is rapidly initiated on the addition of VEGF, directional motility continues for several hours thereafter in the Boyden chamber assay used in this study. To determine if HUVEC were equally sensitive to the inhibitory actions of Taxotere over this entire period, the addition of Taxotere was delayed for varying intervals after the addition of the VEGF. The concentration of Taxotere used in this experiment (10 nmol/L) completely inhibited migration if added just before the VEGF (time 0 in Fig. 1A ). By 15 minutes after the addition of VEGF, however, Taxotere began to lose its efficacy and it no longer inhibited HUVEC migration if its addition was delayed >30 minutes after that of VEGF. Similar results were obtained when 0.1 nmol/L Taxotere was used (data not shown). Thus, the actions of Taxotere seem to be predominantly on the early events associated with VEGF-induced cell migration.


Figure 1
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  		Figure 1. Taxotere inhibited VEGF-induced HUVEC migration and integrin activation but not VEGFR-2 activation. A, HUVEC migration in Boyden chambers with Taxotere (10 nmol/L) added immediately before (time 0) or at the indicated times after the addition of VEGF (10 ng/mL). In all cases, migration was assessed 5 hours after the addition of VEGF. Points, mean of three determinations; bars, SE. B to G, immunocytochemistry for high-affinity state integrin {alpha}Vß3 [WOW-1 F(ab); B-D] and total integrin {alpha}Vß3 (E-G) on HUVEC treated with VEGF (C and F) or VEGF + 1 nmol/L Taxotere (D and G). No staining was observed in the absence of either primary antibody. H, Western blot analysis of VEGFR-2 activation. HUVEC were plated on fibronectin (10 µg/mL), starved overnight, and treated with the indicated concentrations of Taxotere for 1 hour. Cells were then stimulated with VEGF (10 ng/mL) for 5 minutes, and the extent of VEGFR-2 phosphorylated on Tyr951 was determined. Membranes were reprobed for total VEGFR-2.

 
Taxotere suppressed VEGF-induced integrin {alpha}Vß3 activation. In addition to mediating cell adhesion, integrins are intimately involved in inside-out and outside-in cell signaling, and all these properties play a critical role in cell migration (16). Integrin binding to ECM is regulated in part by conformational changes in the {alpha}ß heterodimers that increase integrin affinity for their ECM ligands and cause integrin activation (30). Previous work showed that VEGF stimulated the recruitment of activated, high affinity integrins to the leading edge of migrating endothelial cells (6, 14). To investigate whether Taxotere was capable of suppressing integrin activation, we used WOW-1, an engineered, monovalent, ligand-mimetic F(ab) fragment that reacts selectively with {alpha}Vß3 when it is in a high-affinity state (30). WOW-1 exhibited weak nuclear staining in control cells (Fig. 1B), reported previously to be due to nonspecific interactions (35). VEGF treatment resulted in {alpha}Vß3 activation (Fig. 1C), as judged by the more intense WOW-1 staining across the entire cell and this was blocked by 1 nmol/L Taxotere (Fig. 1D). In contrast, neither VEGF nor Taxotere had any effect on the levels or distribution of total {alpha}Vß3 integrins (Fig. 1E-G). In addition to blocking integrin-associated pathways, inhibition of integrin activation could also have an impact on the VEGFR-2 pathway, as the full induction of the effects of VEGF requires the direct interaction of VEGFRs with activated integrin cells (36, 37).

Taxotere decreased FAK, Akt, and eNOS phosphorylation and inhibited VEGF-induced eNOS activity. Addition of a NO donor reversed the inhibition of VEGF-induced HUVEC migration by Taxotere. The biological effects of VEGF are triggered by its binding to its receptors. Subsequent tyrosine phosphorylation of VEGFR-2 is one of the first events in the signaling cascade whereby VEGF induces cell migration (2). To determine if VEGFR-2 activation was affected by Taxotere, HUVEC were seeded on fibronectin, starved in serum-free medium overnight, treated with Taxotere for 1 hour, and stimulated with VEGF for 5 minutes. Using an antibody that was specific for Tyr951-phosphorylated VEGFR-2, we found that Taxotere had no effect on VEGF-induced VEGFR-2 phosphorylation when compared with cells stimulated with VEGF alone (Fig. 1H). Identical results were obtained when total VEGFR-2 was immunoprecipitated and then probed with an anti-phosphotyrosine antibody (data not shown).

Signaling through focal adhesions to trigger cell migration, initiated either by VEGFR-2 or by the integrins, is dependent on phosphorylation of FAK on multiple tyrosine residues (10). In agreement with previous reports, phosphorylation of FAK on Tyr397 (the autophosphorylation site) occurred rapidly on VEGF stimulation of HUVEC, reaching a maximum at 5 minutes and declining to basal level by 30 minutes (Fig. 2A ). Similar to VEGFR-2, previous studies have shown that tyrosine phosphorylation of FAK is tightly linked to its intrinsic kinase activity. Taxotere prevented FAK phosphorylation, with a reduction to near baseline levels at 0.1 nmol/L Taxotere (Fig. 2A). FAK plays a key role in the dynamic reorganization of the cytoskeletal network that precedes cell migration. Its phosphorylation provides sites for interaction with other focal adhesion-associated proteins, and we examined one of these, paxillin. Paxillin is phosphorylated in VEGF-treated cells on Tyr31, and this occurred maximally at 30 minutes after VEGF addition, indicating, as expected, that it was downstream and likely as a consequence of FAK phosphorylation. The phosphorylation of paxillin is dependent on FAK phosphorylation, and because the latter was inhibited by Taxotere, we expected that paxillin phosphorylation would likewise be inhibited. This can be seen in Fig. 2A, where the increase in paxillin phosphorylation was blocked by low concentrations of Taxotere.


Figure 2
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  		Figure 2. Effect of Taxotere on VEGFR-2 downstream signaling and reversal of the effect of Taxotere by a NO donor. A, HUVEC were treated with Taxotere and VEGF as in Fig. 1H, and extracts were prepared 5 minutes after the addition of VEGF for phosphorylated FAK analysis or after 30 minutes for phosphorylated paxillin, phosphorylated Akt, and phosphorylated eNOS analysis. Membranes were reprobed for total FAK, paxillin, Akt, and eNOS. B, eNOS activity was measured in extracts from untreated, VEGF-treated, and VEGF + Taxotere-treated cells. Columns, mean of three experiments; bars, SE. C, migration was determined in HUVEC treated with VEGF; VEGF + 10 nmol/L Taxotere (Tax); VEGF, Taxotere, and 10 µmol/L SNAP; SNAP alone; and VEGF + SNAP. *, P < 0.05, significantly different from VEGF alone; **, P < 0.05, significantly different from VEGF + Taxotere.

 
In addition to its effect on cell migration, VEGFR-2 mediates several other properties of the vasculature, including endothelial cell proliferation, survival, and vessel permeability. Some or all of these seem to be mediated by a VEGF-induced increase in the production of endothelial NO, which peaks at 5 to 20 minutes and is produced by the enzyme eNOS; NO then activates signaling cascades further downstream (38–40). The effects of VEGF on angiogenesis have been shown to be abrogated in endothelial cells from eNOS knockout mice (39). VEGF-induced activation of eNOS occurs via eNOS phosphorylation at Ser1177 by the protein kinase Akt, which itself is serine phosphorylated in response to VEGF and several stimuli in a PI3K-dependent manner; thus, inhibition of the VEGFR-2/FAK pathway by Taxotere would be anticipated to prevent the downstream activation of Akt and eNOS (41, 42). The serine phosphorylation of eNOS and to a lesser extent, Akt, was increased with VEGF treatment, and both were blocked in a concentration-dependent manner by Taxotere (Fig. 2A). In fact, the level of eNOS phosphorylation in particular was reduced below baseline levels by Taxotere. Consistent with this was the observation of a VEGF-induced increase and a Taxotere-mediated reduction to below basal levels in eNOS activity, measured in cell extracts from VEGF-treated and Taxotere-treated HUVEC (Fig. 2B).

Seeking to mechanistically link the inhibition of eNOS activation by Taxotere with cell migration, we examined the effect of a NO donor, SNAP. SNAP has been shown previously to increase the levels of NO and stimulate cell migration in HUVEC even in the presence of an eNOS inhibitor, L-NAME (43). SNAP (10 µmol/L) reversed the inhibitory effect of Taxotere on VEGF-induced migration by ~50% (Fig. 2C). This suggested that a substantial portion, but not all of the actions of Taxotere, was mediated by its effect on eNOS activity. When used alone, the same concentration of SNAP was sufficient to induce HUVEC migration equal to that of VEGF alone, indicating that this concentration of SNAP produced cellular levels of NO that were likely comparable with those induced by VEGF. Further, SNAP did not significantly further increase the extent of cell migration induced by VEGF, indicating that, at the concentration used, VEGF was producing an optimal amount of NO and confirming that the ultimate pathways activated by VEGF and SNAP substantially overlapped.

Taxotere caused the loss of Hsp90 protein, resulting in the breakdown of essential Hsp90 client protein interactions. The activation of integrins and the formation of VEGFR-2/FAK/integrin complexes at the focal adhesion do not occur spontaneously but rather require the participation of the cytoplasmic chaperone protein Hsp90, which serves as a bridging protein for the formation of heterocomplexes among integrins, VEGFR-2, and FAK (7). Similarly, Hsp90 is required for the phosphorylation of Akt and eNOS and the formation of an Akt/eNOS complex (7). We considered the possibility that the inhibition of the chaperone functions of Hsp90 could be the mechanism by which Taxotere was producing its antimigratory effects on HUVEC. Examination of Hsp90 expression levels by Western analysis led to the surprising observation that Taxotere nearly completed depleted endothelial cells of total Hsp90 protein. Taxotere potently abolished both Hsp90{alpha} and Hsp90ß expression at concentrations as low as 0.1 nmol/L (Fig. 3A ). This action was selective in that the expression of the related chaperone protein Hsp70 was not decreased, and in fact, a slight increase in Hsp70 expression was observed (Fig. 3A). Different cell lines vary in the extent to which depletion of Hsp90 leads to an increase in Hsp70 (44). Complete loss of Hsp90 expression seemed to occur ~2 hours after Taxotere treatment and the effect could still be seen after 48 hours (Fig. 3C). The Taxotere-induced loss of Hsp90 would be anticipated to prevent the formation of multiprotein complexes with its client proteins, and a representative example of this, the Akt/Hsp90 complex, is shown in Fig. 3B. The rapid and substantial loss of Hsp90 protein observed in the present study could have accounted for all the specific cellular actions of Taxotere found, including the inhibition of integrin, FAK, and eNOS activation.


Figure 3
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  		Figure 3. Effect of Taxotere on Hsp90 expression. A, Western blot analysis of total Hsp90, Hsp90{alpha}, Hsp90ß, and Hsp70 expression in HUVEC treated with Taxotere for 1 hour at the indicated concentrations followed by Taxotere + VEGF for an additional 30 minutes. Blots were reprobed for ß-actin. B, immunoprecipitation using an anti-Akt antibody followed by PAGE and immunoblotting for Hsp90. Protein loading was determined by reprobing for Akt. C, Western blot analysis of Hsp90 in HUVEC treated with Taxotere (10 nmol/L) for the indicated times. D, Western blot analysis of Hsp90 expression in HUVEC treated with laulimalide for 1 hour at the indicated concentrations followed by laulimalide + VEGF for an additional 30 minutes.

 
The effect of a second microtubule-binding agent, laulimalide, on Hsp90 was also examined. Although laulimalide stabilizes microtubules in a manner analogous to that of Taxotere, studies have shown that it binds to tubulin at a site distinct from that of the taxanes, and some of its effects on HUVEC were synergistic when combined with Taxotere (34). As illustrated in Fig. 3D, laulimalide caused a concentration-dependent depletion of Hsp90, with complete loss of protein expression at 1 nmol/L.

Knockdown of Hsp90 replicated the effect of Taxotere on migration, whereas overexpression of Hsp90 rescued HUVEC from the inhibition of VEGF migration by Taxotere. It has been suggested previously that the chaperoning function of Hsp90 is essential for the coordination of cell migration (7). In addition, pharmacologic inhibition of Hsp90 is antiangiogenic (7, 45). The Hsp90 inhibitor geldanamycin prevented VEGF-induced HUVEC migration (P < 0.05; Fig. 4A ), in agreement with a previous report (7). We also observed that knockdown of Hsp90 gene expression using siRNA significantly (P < 0.05) decreased VEGF-induced migration (Fig. 4A). Migration of HUVEC treated with a nonspecific scrambled siRNA was not significantly different from the VEGF control. Western analysis of extracts from cells transfected with Hsp90 siRNA showed a decrease in Hsp90 expression as well as a decrease of its client protein eNOS, suggesting that inhibition of migration may be either directly due to the loss of Hsp90 or a result of the loss of Hsp90 client proteins (Fig. 4B). To show that Hsp90 was in fact a specific target for Taxotere, we overexpressed Hsp90{alpha} or Hsp90ß in HUVEC 24 hours before analyzing migration (Fig. 4C). Cells overexpressing either isoform of Hsp90 were resistant to the inhibition of VEGF-induced migration by Taxotere (Fig. 4C), providing a direct link between the levels of Hsp90 in the cells and their sensitivity to inhibition by Taxotere. The Hsp90-transfected cells responded identically to VEGF (in the absence of Taxotere) as did the control vector-transfected cells (data not shown).


Figure 4
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  		Figure 4. Effect of other Hsp90 inhibitors on VEGF-induced cell migration; Hsp90 overexpression rescues HUVEC from the migration-inhibitory effect of Taxotere. A, HUVEC were treated with 0.1 nmol/L geldanamycin (GA) for 30 minutes before VEGF stimulation. HUVEC were also transfected with 50 nmol/L Hsp90 siRNA or a siRNA with a scrambled sequence (Scram) immediately before plating onto fibronectin-coated migration filters followed by the addition of VEGF. Data are expressed as a percentage of VEGF-induced migration. Columns, mean of three individual experiments; bars, SE. B, Western analysis confirms loss of Hsp90 and its client protein eNOS in response to Hsp90 siRNA transfection. C, HUVEC were transfected with expression vectors for Hsp90{alpha} ({alpha}), Hsp90ß (ß), and both Hsp90{alpha} and Hsp90ß ({alpha} + ß) or with a control vector (CV). After 24 hours, cells were used in a Boyden chamber migration assay. Cells were stimulated with VEGF (solid columns) in the presence and absence of Taxotere. Open column, control (C) without VEGF. Columns, mean of four individual experiments; bars, SE. D, Western analysis on extracts from transfected cells confirms the overexpression of Hsp90.

 
Taxotere increased the proteasomal degradation of Hsp90. We next examined possible mechanisms for the Taxotere-induced loss of Hsp90 in the HUVEC. mRNA levels of Hsp90 (both {alpha} and ß forms) were not reduced by Taxotere treatment (Fig. 5A ). Taxotere (10 nmol/L for 3 hours) also did not affect the synthesis of Hsp90 protein as judged by either pulse or continuous incorporation of [35S]methionine into immunoprecipitated Hsp90 protein (data not shown). Factors that might affect the stability of the Hsp90 protein were then examined, particularly its ubiquitination. Cell lysates from control, VEGF-treated, and Taxotere-treated cells were incubated with an antibody that preferentially immunoprecipitates free Hsp90 compared with Hsp90 complexed to either client proteins or cochaperone proteins. Western analysis of the immunoprecipitates with an anti-ubiquitin antibody showed increased Hsp90 ubiquitination in response to Taxotere (Fig. 5B). Although polyubiquitination of proteins often leads to the appearance of multiple bands or smears on Western blots, a previous report showed this not to always be the case for Hsp90 (44), in agreement with our observations.


Figure 5
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  		Figure 5. Possible mechanisms for the disruption of Hsp90 expression by Taxotere. A, Hsp90 (both {alpha} and ß) mRNA levels were measured by reverse transcription-PCR in cells treated with Taxotere. B, cell lysates from control, VEGF-treated, and Taxotere-treated cells were immunoprecipitated with an antibody which preferentially binds free Hsp90, relative to complexed Hsp90. Western blot analysis was then done using an anti-ubiquitin antibody and an anti-total Hsp90 antibody. C, HUVEC migration in response to VEGF (V), VEGF + 10 nmol/L Taxotere (V+T), or VEGF + Taxotere + 50 µmol/L PSI (V+T+P). *, P < 0.05, significantly different from VEGF alone. D, Western analysis of Hsp90 in cells treated with the indicated concentrations of Taxotere and with 50 µmol/L of the proteasomal inhibitor PSI.

 
Because ubiquitination typically leads to the subsequent degradation of the affected protein, we examined the expression of Hsp90 in cells pretreated with a proteasomal inhibitor before Taxotere treatment. The Taxotere-induced loss of Hsp90 protein could be prevented by the proteasomal inhibitor PSI (Fig. 5D). In addition, complete reversal of the inhibitory effect of Taxotere on endothelial cell migration was observed when cells were treated with the proteasomal inhibitor immediately before Taxotere treatment (Fig. 5C). This suggests that Taxotere induces the proteasomal degradation of Hsp90 via ubiquitination.

Evaluation of possible mechanisms for the disruption of Hsp90 by the taxanes. Although the proximal site of action by which Taxotere depletes Hsp90 is not known, two possibilities were considered. In addition to its many client proteins, Hsp90 also reversibly associates with microtubules in intact cells, where it binds to the COOH-terminal fragments of tubulin (46). Disruption of the microtubule network with nocodazole was found to disrupt Hsp90 binding to microfilaments (47). Although numerous studies have focused on the role Hsp90 plays in modulating the stability of its client proteins, little is known about the regulation of turnover of the Hsp90 protein itself. Thus, it was possible that by binding to tubulin, Taxotere could displace or otherwise disrupt the interaction of Hsp90 with microtubules, perhaps thereby enhancing its degradation. To test this possibility, immunofluorescence was used to determine the effect of Taxotere on the interaction of Hsp90 and tubulin in intact HUVEC. In untreated HUVEC and those treated with Taxotere for 5 minutes, Hsp90 and tubulin colocalize as judged by an overlap in staining (Fig. 6 ). However, longer treatment times (10 and 20 minutes) led to a dissociation of Hsp90 and tubulin as judged by independent Hsp90 (green) and tubulin (red) staining. By 60 minutes, little Hsp90 staining was observed, in agreement with the immunoblot data in Fig. 3A. By disrupting the interaction of Hsp90 with microtubules, Taxotere may have enhanced Hsp90 degradation.


Figure 6
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  		Figure 6. Taxotere disrupts the interaction of microtubules with Hsp90 in intact cells. Immunocytochemistry for Hsp90 (green) and tubulin (red) in untreated HUVEC (Control) and cells treated with 10 nmol/L Taxotere for the indicated times. Costaining in merged images appears in yellow.

 
Taxotere could also affect the turnover of Hsp90 by a direct interaction with the protein. A previous study, which used cell extracts, biotinylated Taxol, and affinity chromatography, identified Hsp90 as one of several potential taxane-binding polypeptides (28). The direct binding of Taxotere to Hsp90, therefore, would be an alternative mechanism by which the drug could disrupt the chaperone protein. We were able to confirm that [3H]Taxol binds directly to purified Hsp90 in a cell-free system (data not shown).

Although this is the first report of Taxotere inducing Hsp90 degradation in endothelial cells, the clinical use of other Hsp90 inhibitors as antiangiogenic and anticancer agents is currently being explored. Analogues of the Hsp90-binding antibiotic geldanamycin reduced angiogenesis by inhibiting endothelial cell migration, ECM invasion, and formation of capillary-like structures (45). In addition, Hsp90 may regulate angiogenesis via indirect mechanisms. Hsp90 regulates hypoxia-inducible factor-1{alpha}, which drives the transcription of many genes involved in tumor cell adaptation, including VEGF (48, 49). Therefore, Taxotere-associated inhibition of Hsp90 could inhibit angiogenesis at both the level of the endothelial cells, by inhibiting their migration toward the tumor, and within the tumor, by preventing the transcription of endothelial cell chemoattractants. Further, these observations could have broader implications for the anticancer actions of Taxotere, given the role of Hsp90 and chaperone proteins in maintaining the conformation of oncogenic signaling proteins, in guiding the disposition of key regulators of cell growth and differentiation, and in allowing tumor cells to tolerate mutations in signaling molecules that might otherwise be lethal.

We reported an additional novel action of Taxotere on VEGF-induced cell signaling molecular events: inhibition of the increase in integrin {alpha}Vß3 affinity. The Taxotere-induced loss of Hsp90 is likely directly responsible for this effect, because the level of Hsp90 found in complexes with {alpha}Vß3 integrin dramatically decreased following Taxotere treatment (data not shown). Direct associations between integrins and the VEGFR-2 and VEGFR-3 (Flt4) receptors have been reported previously in endothelial cells (36, 37), and as noted above, the full induction of the effects of VEGF requires the direct interaction of VEGFRs with activated integrins. A direct inhibitory effect of Taxotere at this level could lead to most or all of its other actions that we observed.

The effects of Taxotere on FAK Tyr397 phosphorylation and focal adhesion formation could have additional consequences for cell migration that were not explored in this study. Phosphorylated FAK and nascent focal adhesions are sites for the assembly of other signaling molecules, including actin-anchoring proteins, such as talin, vinculin, tensin, and {alpha}-actinin, which link the microfilament network to the adhesive molecule integrins at their sites of clustering (50). These proteins provide a structural link allowing the anchorage of stress fibers to the membrane and integrins, and the inhibition of their recruitment and assembly would likely prevent cell motility. Inhibition of the tyrosine phosphorylation of FAK would also prevent the association of FAK with the SH2 domains of Src family kinases and of the p85{alpha} subunit of PI3K and with the adapter protein Grb-2, thereby blocking their downstream signaling (51–53).

In summary, by showing that Taxotere decreased the expression of Hsp90, we provided insights into the mechanism by which low concentrations of the drug inhibited endothelial migration and tumor angiogenesis. The critical role that Hsp90 plays in a wide range of signal transduction pathways further suggests that these observations could have broader implications as to the mechanism of the antitumor actions of the taxanes. For example, Taxotere was also found to affect expression of Hsp90 client proteins in some, but not all, breast cancer cell lines tested.1 The concentrations at which these actions occurred are pharmacologically relevant. Typically administered clinically as a short (e.g., 1 hour) infusion once every week or once every 3 weeks, plasma concentrations of Taxotere required to substantially inhibit microtubule function, cause mitotic arrest, or induce apoptosis were achieved for ~24 hours after drug infusion. In contrast, concentrations that inhibit endothelial cell migration (1-10 nmol/L) were maintained in patients for the entire period (i.e., 7 or 21 days) between drug administrations (54). Thus, as currently used clinically, it is possible that Taxotere produces continuous and persistent inhibition of Hsp90 and endothelial cell function. This prolonged effect could be as important to the therapeutic efficacy of Taxotere as its direct effects on the tumor cells, which likely occur at plasma drug levels that are attained for a much shorter duration.


   Acknowledgments
 
Grant support: NIH grant CA98456.

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 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: J. Murtagh and H. Lu contributed equally to this work.

1 J. Murtagh and E.L. Schwartz, unpublished observations. Back

Received 2/27/06. Revised 4/24/06. Accepted 5/30/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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