Cancer Research Annual Meeting 2010 Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 9482, October 1, 2007. doi: 10.1158/0008-5472.CAN-07-0569
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Panner, A.
Right arrow Articles by Pieper, R. O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Panner, A.
Right arrow Articles by Pieper, R. O.

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Heat Shock Protein 90{alpha} Recruits FLIPS to the Death-Inducing Signaling Complex and Contributes to TRAIL Resistance in Human Glioma

Amith Panner1,2,3, Joseph C. Murray1, Mitchel S. Berger1,2,3 and Russell O. Pieper1,2,3

1 Department of Neurological Surgery, 2 The Brain Tumor Research Center, and 3 The UCSF Comprehensive Cancer Center, University of California-San Francisco, San Francisco, California

Requests for reprints: Russ Pieper, UCSF Cancer Center, 2340 Sutter Street, Room N219, San Francisco, CA 94115-0875. Phone: 415-502-7132; Fax: 415-502-6779; E-mail: rpieper{at}cc.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heat shock protein 90 (HSP90) is a molecular chaperone that contributes to the proper folding and stability of target proteins. Because HSP90 has been suggested to interact with FLIPS, the key regulator of tumor necrosis factor-{alpha}–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in glioma cells, we examined the role HSP90 played in controlling TRAIL response. HSP90{alpha} was found to associate with FLIPS in resting cells in a manner dependent on the ATP-binding NH2-terminal domain of HSP90{alpha}. Following TRAIL exposure, HSP90{alpha} and the client FLIPS protein were recruited to the death-inducing signaling complex (DISC). Short interfering RNA–mediated suppression of HSP90{alpha} did not alter the total cellular levels of FLIPS, but rather inhibited the recruitment of FLIPS and other antiapoptotic proteins such as RIP and FLIPL to the DISC, and sensitized otherwise resistant glioma cells to TRAIL-induced apoptosis. These results show that HSP90{alpha}, by localizing FLIPS to the DISC, plays a key role in the resistance of tumor cells to TRAIL, and perhaps other proapoptotic agents. The results also define a novel means of apoptotic control by a HSP90{alpha} that may in turn help explain the global antiapoptotic effects of this protein. [Cancer Res 2007;67(19):9482–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heat shock proteins (HSP) are a highly conserved group of intracellular proteins classified by molecular weight into groups of HSP110, HSP90, HSP70, HSP60, small molecular HSPs (<27 kDa), and ubiquitin (1–5). HSPs, as a group, are among the most abundant proteins in the cytosol and function in multicomponent complexes as molecular chaperones. HSP function is best understood under conditions of cellular stress (hypoxia, heat), during which levels of HSPs are dramatically increased (3, 6). Under these conditions, HSPs promote cell survival by preventing protein aggregation and promoting the refolding of damaged proteins (1–3, 7, 8).

In addition to having a key role in mitigating cellular stress, HSPs also play a critical role in unstressed cells. HSP70, and to a lesser extent HSP90, are critically involved in what are called "protein holding" functions (8–10). HSP70 binds to newly synthesized peptides and prevents premature protein misfolding, whereas HSP90 binds to proteins with unstable tertiary structures and helps to prevent protein degradation. HSP60 and HSP27, in contrast, are considered to function in protein folding (8–10). These HSPs form folding complexes that use ATP to help create the intramolecular interactions necessary for client protein folding (3, 11). The protein holding and folding functions of HSPs are critical in all normal cells, consistent with the embryonic lethal nature of HSP deletion (12).

The protein holding and protein folding functions of the HSPs are critical not only in normal unstressed cells, but also in transformed cells, in which the normal functions of HSPs are used to facilitate cell growth and cell cycle progression. As an example, HSP90 stabilizes Akt and oncogenic forms of mutant epidermal growth factor receptor, both of which contribute to the growth of a variety of cancers including gliomas (13–15). HSP70, in a complex with HSP90 and HSP40, also serves to regulate the function and turnover of the estrogen receptor, increasing its activation and driving estrogen-dependent cellular responses (16, 17). HSP70 and HSP90 are also critically involved in the DNA-binding properties and stability of p53, and by stabilizing mutant p53, contribute to its role in transformation (18, 19). These observations suggest that the normal protein folding functions of HSPs, and in particular HSP70 and HSP90, are subverted by tumors to stabilize proteins critical for the establishment and maintenance of the transformed phenotype.

Although HSPs play a key role in the regulation of cell growth and cell cycle progression, their antiapoptotic properties may be even more important to the persistence of malignant cells. The intrinsic apoptotic pathway is driven by mitochondrial release of cytochrome c to the cytosol, which in turn triggers the polymerization of Apaf-1. Apaf-1 recruits procaspase-9 and procaspase-3 into the apoptosome, where the caspases are activated (20, 21). HSP70 reportedly interacts with Apaf-1, thereby preventing the interaction of Apaf-1 with procaspase-9 (22–24). Overexpression of HSP27 also increases the resistance of cells to various apoptotic stimuli reportedly by directly binding to the cytosolic cytochrome c and sequestering it from Apaf-1 (25, 26). In addition, HSP20 has been reported to complex with the proapoptotic protein Bax, which prevents the translocation of Bax from the cytosol into the mitochondria during apoptotic insult (27). As a result, HSP20 may preserve the integrity of mitochondria, restrict the release of cytochrome c, and repress the activation of caspase-3. The antiapoptotic effects of HSPs seem to be of particular importance in the cancer setting because targeted disruption of HSP27 or HSP70 leads to the activation of programmed cell death pathways (28). These results suggest that the ability of HSPs to block programmed cell death may help sustain the transformed cells and perhaps also to contribute to resistance to proapoptotic therapeutic agents.

Although HSP20, HSP27, and HSP70 are recognized as controllers of programmed cell death, the role of HSP90 in this process is less clear. There are two forms of HSP90 in the cytosol, HSP90{alpha} and HSP90ß, which when combined, comprise ~1% to 2% of total cellular proteins (29, 30). Although these highly related isoforms differ in their inducibility (HSP90{alpha} is inducible, HSP90ß is constitutively expressed), the functional differences between the two proteins are not well defined. Both contain a highly conserved ATP-binding domain near their NH2 terminus, and the chaperoning activity of both requires the binding of ATP at this site (31, 32). The middle region of both HSP90{alpha} and HSP90ß has a key role in the binding of client proteins (33), whereas the COOH terminus domain facilitates the dimerization of HSP90 (34, 35). The COOH terminus domain of HSP90{alpha} also contains a conserved EEVD motif that recruits various co-chaperones, such as the immunophilins and HSP70/HSP90-organizing protein, which in turn, modify the specificity of the HSP90-containing complexes (34, 35). HSP90 has been reported to play a role in apoptosis induced by nicotinamide, and HSP90 also binds to a variety of clients, many of which play key roles in the control of apoptosis (14, 36–38). In particular, HSP90 has been reported to bind to FLIP, a protein that interacts with FADD and is a key suppressor of tumor necrosis factor-{alpha}–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in gliomas (37, 39). Although the exact isoforms of HSP90 and FLIP involved in this interaction have not been defined, our interest in TRAIL as a targeted chemotherapeutic agent, and in understanding the pathways that control TRAIL sensitivity/resistance in gliomas, led us to examine the possibility that HSP90{alpha}, via its potential ability to bind to and stabilize FLIPS, might also be a key regulator of TRAIL-induced apoptosis. We here show that HSP90{alpha} does regulate TRAIL-induced apoptosis, but not by altering FLIPS stability. Rather, HSP90{alpha} interacts with FLIPS and inhibits its ability to localize to its site of action in the death-inducing signal complex. Furthermore, the ability of HSP90{alpha} to localize antiapoptotic proteins to the death-inducing signaling complex (DISC) is not limited to FLIPS, but also applies to at least two other antiapoptotic proteins, RIP and FLIPL. These results identify a novel means of apoptotic control by a HSP, and also identify a novel mechanism by which HSP90{alpha} can control sensitivity to a variety of apoptotic stimuli including TRAIL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and drug treatment. Immortalized or Ras-transformed human astrocytes were generated and cultured as described previously (40). Human recombinant TRAIL was kindly provided by Avi Ashkenazi (Genentech, South San Francisco, CA). Cells were exposed to TRAIL (800 ng/mL) for 24 h prior to harvest.

Retroviral infection, transfection of plasmids, and short interfering RNA. The pBabe retroviral construct encoding constitutively active cdc42 (Q61L) was provided by David Stokoe (UCSF Cancer Center, San Francisco, CA). Pools of productively infected cells (9 µg/mL, obtained by selection with puromycin; Sigma) were used for further analysis. The pFLAG-CMV 6c mammalian expression vector encoding wild-type human HSP90{alpha} was kindly provided by Dario C. Altieri (University of Massachusetts Medical School, Worcester, MA). The pGEX vectors encoding various glutathione S-transferase–tagged NH2-terminal or COOH-terminal deletion mutants of the chicken HSP90{alpha} protein were kindly provided by Ahmed Chadli (Mayo Clinic, Rochester, MN). The pGEX vectors containing chicken HSP90{alpha} cDNA were digested with EcoRI and SalI, after which the cDNA was subcloned into EcoRI + SalI–digested mammalian expression vector pFLAG-CMV 6c (Sigma). Cells were then transiently transfected using Fugene 6 (Roche) according to the instructions of the manufacturer, after which the cells were harvested 48 h later. For short interfering RNA (siRNA) studies, 300 nmol/L of either HSP90{alpha}-targeted siRNA (Ambion) or scramble siRNA was transfected into cells using Fugene 6 (Roche) according to the manufacturer's instructions and protein levels were analyzed 24 to 72 h later by Western blot. In TRAIL plus siRNA studies (except where noted), siRNA was added to the cells for 48 h followed by incubation with TRAIL in the presence of siRNA for an additional 24 h prior to cell harvest.

Immunoprecipitation and analysis of DISC components. For the analysis of components of the assembled DISC, cells were incubated with TRAIL (0 or 800 ng/mL, 24 h) and lysed, after which, the DISC-related proteins were immunoprecipitated using an anti-FADD antibody. For immunoprecipitation of HSP90{alpha}, FLIPS, FLIPL, and RIP, cells were allowed to reach 70% confluency in DMEM. Following incubation with TRAIL (0 or 800 ng/mL) for 24 h, the cells were washed once with ice-cold PBS and harvested using 1x ice-cold cell lysis buffer supplemented with 1 mmol/L of phenylmethylsulfonyl fluoride and incubated on ice for 10 min. HSP90{alpha}, FLIPS, FLIPL, or RIP was selectively immunoprecipitated from 200 µg of protein (whole cell lysates) by combining the cell lysate with 20 µL of FADD antibody (Cell Signaling Technology) conjugated to agarose A/G beads (Santa Cruz Biotechnology) followed by gentle rotation for 4 h at 4°C. In some instances, an HSP90{alpha} antibody was used to immunoprecipitate FLIPS, FLIPL, or RIP following incubation with TRAIL. In order to immunoprecipitate the various FLAG-tagged NH2- or COOH-terminal HSP90{alpha} deletion mutants, the cell lysate of NH2- or COOH-terminal HSP90{alpha} deletion mutant–expressing cells was combined with an antibody targeting the FLAG epitope (Cell Signaling Technology). Samples were then centrifuged briefly (30 s, 2,000 x g) and pellets were washed twice with 1x lysis buffer. Immunocomplexes (pellets) were eluted in 3x SDS sample buffer. The levels of HSP90{alpha}, FLIPS, FLIPL, and RIP in the assembled DISC, as well as expression of the FLAG-tagged HSP90{alpha} NH2- or COOH-terminal deletion mutants, were assessed by Western blot analysis. Immunoprecipitations carried out using a nonspecific normal mouse IgG antibody were included as negative controls, as were the analyses of cells to which TRAIL (800 ng/mL) was added following lysis.

Immunoblot analysis. Cells were washed with ice-cold PBS, scraped from the culture dish, and incubated in tissue lysis buffer containing 10 mmol/L of KCl, 1 mmol/L of sucrose, 2 mmol/L of MgCl2, 0.5% Igepal CA-630, 1 mmol/L of EDTA, 1 mmol/L of DTT, 10 mmol/L of ß-glycerophosphate, 1 mmol/L of Na3VO4, 10 mmol/L of NaF, 100 µg/mL of phenylmethylsulfonyl fluoride, and 10 µg/mL of aprotinin (all reagents were purchased from Sigma) for 30 min on ice. The cell lysate was centrifuged, and the supernatant was stored at –80°C until use. The protein concentration of extracts was measured using Protein Assay reagent (Bio-Rad Laboratories). Protein (30 µg) was subjected to SDS-PAGE and electroblotted onto Immobilon-P membrane (Millipore). The membrane was blocked in 5% nonfat skim milk/TBST [20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Tween 20] at 4°C overnight and incubated with rabbit polyclonal antibody against HSP90{alpha}, FLAG, phospho-Akt, FLIPS, FADD, FLIPL, TANK, RIP, TRAF, or {alpha}-tubulin (all antibodies obtained from Cell Signaling Technologies). Bound antibody was detected with antirabbit IgG (Santa Cruz Biotechnology) using enhanced chemiluminescence Western blotting detection regents (Amersham Pharmacia Biotech, Inc.). Densitometric measurements of immunoreactive bands were acquired using an AlphaImager 2200 (Alpha Innotech Corporation). The expression of {alpha}-tubulin was used to verify equal loading.

Analysis of apoptosis by flow cytometry. For the analysis of apoptosis, cells were incubated with either TRAIL (800 ng/mL) or vehicle control, after which, cells were harvested and monitored for the percentage of cells with sub-G1 DNA content (apoptotic cells) using propidium iodide as previously described (41).

For cell cycle analysis, harvested cells were centrifuged and resuspended in PBS after which, the cells were fixed by the addition of ice-cold 70% ethanol and stored at –20°C for 1 h. Subsequently, cells were pelleted by centrifugation and resuspended in staining solution containing 1 mg/mL of propidium iodide (Sigma) and 50 mg/mL of RNase A (Sigma) in PBS. After 5 min of incubation at 37°C, the cell suspension was passed through a 40-µm pore size cell strainer (Becton Dickinson) and subjected to flow cytometry using a Becton Dickinson FACSort flow cytometer. The data was analyzed using CellQuest and ModFit software (Becton Dickinson). For the purpose of analysis, acquired events (20,000 cells) were gated to eliminate cell aggregates and debris.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because the protein stabilizing property of HSP90{alpha} has been linked to the suppression of apoptosis, we addressed the possible involvement of HSP90{alpha} in the control of TRAIL-induced apoptosis. We focused these studies on HSP90{alpha} because only HSP90{alpha}, and not HSP90ß, was up-regulated in glioma cells.4 To begin these studies, normal human astrocytes immortalized and/or transformed by the expression of one of four different forms of V12 H-Ras (E6/E7/hTERT/Ras cells expressing V12 H-Ras, G37 cells expressing V12 H-Ras E37G, S35 cells expressing V12 H-Ras T35S, or C40 cells expressing V12 H-Ras Y40C) were obtained. These cells, which exhibit different levels of FLIPS expression and TRAIL sensitivity (E6/E7/hTERT/Ras and G37 have low levels of FLIPS and >10-fold higher TRAIL sensitivity, S35 and C40 have 3-fold higher levels of FLIPS and low TRAIL sensitivity; ref. 42) were first incubated with siRNA targeting HSP90{alpha}. Levels of HSP90{alpha}, HSP90ß, and of two reported client proteins of HSP90{alpha}, pAkt (13, 37) and FLIPS (a key inhibitor of TRAIL-induced apoptosis in glioma cells; refs. 39, 43) were then monitored by Western blotting. As shown in Fig. 1A and B , incubation of cells with siRNA targeting HSP90{alpha} significantly decreased protein levels of HSP90{alpha} (relative to cells receiving a scrambled siRNA control) for up to 72 h following siRNA introduction without altering the levels of HSP90ß. Consistent with previous reports, suppression of HSP90{alpha} also resulted in a concomitant decrease in the protein levels of pAkt (Fig. 1C; refs. 14, 37). siRNA-mediated suppression of HSP90{alpha}, did not, however, alter the protein levels of FLIPS (Fig. 1D), which was surprising given that FLIPS has been reported to be both a client protein of HSP90{alpha} (43) and down-regulated in response to suppression of the pAkt-mTOR pathway (39).


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Effects of siRNA targeting HSP90{alpha} on HSP90{alpha}, HSP90ß, phospho-Akt, and FLIPS protein levels. Normal human astrocytes were serially infected with retroviruses encoding E6, E7, hTERT, and either a constitutively active form of mutant V12 H-Ras (E6/E7/hTERT/Ras), or a mutant V12 H-Ras that was additionally modified to selectively activate the Ral (G37), Raf (S35), or phosphoinositide-3-kinase (C40) pathway. Cells were then mock transfected (CTRL), or transiently transfected with siRNA targeting HSP90{alpha} or a nonspecific scrambled siRNA, and analyzed for levels of HSP90{alpha} (A), HSP90ß (B), phospho-Akt (C), and FLIPS (D) protein 24 to 72 h after siRNA addition. {alpha}-Tubulin was used as a loading control. FLIPS exposures in (A) are longer than those in (B) because of relatively low levels of FLIPS expression in the E6/E7/hTERT/Ras and G37 cells. The data are representative of one of three independent experiments for each group.

 
Having shown that siRNA targeting HSP90{alpha} could down-regulate HSP90{alpha} levels, the effects of HSP90{alpha} down-regulation on TRAIL sensitivity were examined. TRAIL-sensitive cells (E6/E7/HTERT/Ras and G37 cells) underwent significant apoptosis in response to TRAIL (800 ng/mL, 24 h; Fig. 2A, top, lanes 1 and 2 ), consistent with our previous observations that TRAIL induces a dose-dependent cleavage (activation) of caspases across a 0 to 1000 ng/mL range of exposure, with peak activation 24 h after exposure (42; data not shown). The levels of HSP90{alpha} and FLIPS in these cells were not altered by TRAIL exposure [Fig. 2A, bottom, lanes 1 and 2, FLIPS in (A) overexposed relative to the FLIPS in (B)]. Incubation of these cells with siRNA targeting HSP90{alpha} reduced levels of HSP90{alpha} (but not FLIPS) relative to cells receiving scrambled siRNA control (Fig. 2A, bottom, lanes 3 and 4), but did not significantly alter the high levels of TRAIL-induced apoptosis noted in these relatively FLIPS-deficient cells (Fig. 2A, top, columns 3 and 4). In TRAIL-resistant S35 and C40 cells (Fig. 2B, top, lanes 1 and 2) incubation with siRNA targeting HSP90{alpha} also reduced the levels of HSP90{alpha} (but not FLIPS) relative to cells receiving scrambled siRNA control (Fig. 2A, bottom, lanes 3 and 4), although in these TRAIL-resistant/FLIPS overexpressing cells, suppression of HSP90{alpha} levels significantly increased the extent of TRAIL-induced apoptosis (Fig. 2B, top, columns 3 and 4). These results show that HSP90{alpha} levels could control the extent of TRAIL-induced apoptosis, but that they do so in a manner apparently independent of FLIPS levels.


Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. siRNA targeting HSP90{alpha} sensitizes otherwise TRAIL-resistant S35 and C40 cells to TRAIL-induced apoptosis without altering total FLIPS levels. TRAIL-sensitive E6/E7/hTERT/Ras and G37 cells (A) and TRAIL-resistant S35 and C40 cells (B) were exposed to TRAIL (0 or 800 ng/mL, 24 h), or incubated with either a scramble siRNA control or siRNA targeting HSP90{alpha} for 48 h followed by the addition of TRAIL for 24 h, stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having a <2N DNA content (apoptotic cells; top). An aliquot of cells from each group described was lysed (top), after which, levels of HSP90{alpha} and FLIPS were monitored by Western blot (bottom). {alpha}-Tubulin was used as a loading control. Both 1x (30 s) and 5x FLIPS exposures are provided for the E6/E7/hTERT/Ras and G37 cells, which express low levels of FLIPS relative to the C40 and S35 cells (1x exposure only). Top, columns, means; bars, SEs (n = 3). Bottom, Western blots were representative of one of three independent experiments for each group.

 
Because suppressing levels of the chaperone protein HSP90{alpha} increased TRAIL sensitivity, but only in cells with higher levels of FLIPS, we considered the possibility that HSP90{alpha} might control TRAIL sensitivity not by altering FLIPS levels, but rather by altering FLIPS localization. To begin to address this possibility, TRAIL-sensitive cells (E6/E7/hTERT/Ras, G37) with high levels of HSP90{alpha} and low levels of FLIPS were first retrovirally infected with a construct encoding cdc42 (a protein shown to translationally up-regulate FLIPS expression; ref. 42), after which levels of FLIPS, HSP90{alpha}, and TRAIL-induced apoptosis were examined in TRAIL-treated cells. Introduction of the cdc42-encoding construct had no effect on the levels of HSP90{alpha} protein, but did increase the levels of FLIPS protein relative to cells receiving empty vector (Fig. 3, bottom, lanes 3 and 4 ), and to cells receiving the cdc42-encoding construct but not exposed to TRAIL (42). Consistent with previous reports (42, 44), the cdc42-mediated increase in FLIPS protein levels decreased TRAIL sensitivity relative to cells that received only empty vector (Fig. 3A, lanes 3 and 4). The cdc42-overexpressing, FLIPS up-regulated E6/E7/hTERT/Ras and G37 cells were then incubated with an siRNA targeting HSP90{alpha}, after which levels of HSP90{alpha} and FLIPS, and the extent of TRAIL-induced apoptosis were assessed. Incubation of cdc42-overexpresing cells with siRNA targeting HSP90{alpha} effectively reduced the levels of HSP90{alpha} protein whereas having no effect on the levels of FLIPS protein relative to cells receiving scramble siRNA control (Fig. 3C, lanes 5 and 6); however, as in cells with endogenously high levels of FLIPS (S35, C40; Fig. 2), siRNA targeting HSP90{alpha} sensitized the FLIPS overexpressing E6/E7/hTERT/Ras and G37 cells to TRAIL-induced apoptosis relative to cells that received a scrambled siRNA control (Fig. 3A, lanes 5 and 6).


Figure 3
View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. siRNA targeting HSP90{alpha} disrupts the recruitment of FLIPS to the DISC and sensitizes cells to TRAIL-induced apoptosis. TRAIL-sensitive E6/E7/hTERT/Ras and G37 cells were either left untransfected/uninfected, or were subjected to retroviral infection/selection with an empty or cdc42-encoding construct, and transfected with a scrambled siRNA or a siRNA targeting HSP90{alpha}. A, all cells were then exposed to vehicle (CTRL) or TRAIL (800 ng/mL, 24 h) 48 h after initiation of siRNA exposure (where siRNA was used). Cells were then stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having <2N DNA content (apoptotic cells). B, recruitment of HSP90{alpha} and FLIPS to the DISC was assessed from anti-FADD antibody DISC immunoprecipitates of groups described in (A). C, total levels of HSP90{alpha} and FLIPS proteins in the lysates of groups described in (A) was determined by Western blotting using an aliquot of lysate taken prior to immunoprecipitation. {alpha}-Tubulin was used as a loading control. A, columns, means; bars, SE (n = 3). B and C, Western blots representative of one of three independent experiments for each group.

 
Having shown that siRNA-mediated suppression of HSP90{alpha} confers TRAIL sensitivity in cells endogenously or exogenously overexpressing FLIPS, and that TRAIL-induced apoptosis was not associated with changes in absolute FLIPS levels, we directly examined the possibility that HSP90{alpha} enhances TRAIL-induced apoptosis by chaperoning FLIPS to its site of action in the DISC. To do so, lysates from TRAIL-incubated E6/E7/hTERT/Ras and G37 cells (low levels of FLIPS) or cdc42 overexpressing E6/E7/hTERT/Ras or G37 cells (high levels of FLIPS) were subjected to immunoprecipitation using an antibody targeting the DISC-related proteins FADD, after which protein levels of HSP90{alpha} and FLIPS in the immunoprecipitated DISC were monitored by Western blotting. Incubation with TRAIL resulted in the appearance of HSP90{alpha} in the FADD-immunoprecipitated DISC of TRAIL-sensitive E6/E7/hTERT/Ras and G37 cells (Fig. 3B, lane 1 versus lanes 2 and 3), and also in the same cells made TRAIL-resistant by virtue of cdc42-driven FLIPS overexpression relative to control cells (Fig. 3B, lane 1 versus lanes 4 and 5), or to cdc42-overexpressing cells not incubated with TRAIL (data not shown). Incubation with an siRNA targeting HSP90{alpha}, however, abolished the recruitment of both HSP90{alpha} and FLIPS to the DISC (relative to scramble controls; Fig. 3B, lane 5 versus lane 6). These results show that HSP90{alpha} serves to localize FLIPS to the DISC, and in doing so, allows FLIPS to interfere with the activation of the TRAIL-induced apoptotic signaling pathway.

The ability of HSP90{alpha} to localize FLIPS to the DISC following TRAIL exposure, as well as the suggestion that FLIPS is a client protein of HSP90{alpha}, suggests a physical interaction between the two proteins. To directly determine if, and under what circumstances, FLIPS and HSP90{alpha} interact, and which domains of the HSP90{alpha} protein were necessary for FLIPS binding and localization, cells with high levels of FLIPS (C40 and S35) were first transiently transfected with various constructs encoding avian FLAG-tagged deletion mutants of HSP90{alpha} (the avian HSP90{alpha} is 94% homologous to the human protein; ref. 2, forms a functional complex with HSP90{alpha} co-chaperones; ref. 34, and is widely used to study HSP90{alpha}-client protein interactions). Expression of the control FLAG-tagged NH2-terminal domain mutants, and the FLAG-tagged COOH-terminal domain deletion mutants were then verified by Western blot using an antibody to FLAG (Fig. 4A , S35; data not shown). Having established the expression of the FLAG-tagged HSP90{alpha} deletion mutants, the transfected C40 and S35 cells were lysed after which the lysate was subjected to immunoprecipitation using an antibody targeting FLAG. The levels of FLIPS protein were then assessed by Western blot following elution of the immunoprecipitated proteins and compared with total levels of FLIPS protein in the lysate prior to immunoprecipitation. Although total levels of FLIPS were similar in all groups (Fig. 4B), both FLAG-tagged NH2-terminal deletion mutants of HSP90{alpha} were less efficient at binding to FLIPS (lanes 4 and 6). Deletion of the COOH-terminal domain of the HSP90{alpha} protein, however, had no effect on the ability of HSP90{alpha} to bind to FLIPS (lanes 3, 5, and 7), and these deletion mutant HSP90{alpha} proteins bound FLIPS as well as the full-length HSP90{alpha} protein (lane 2). These results show that whereas the COOH-terminal domain of HSP90{alpha} is not necessary for interaction with FLIPS, the NH2-terminal domain, and specifically, the NH2-terminal 206 amino acid (which contains the ATP-binding domain) are necessary and sufficient to bind to FLIPS.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. The NH2-terminal ATP-binding domain of HSP90{alpha} is both necessary and sufficient to bind to FLIPS. TRAIL-resistant C40 and S35 cells were transiently transfected with an empty vector (pFLAG-CMV 6c), or a vector encoding various NH2-terminal or COOH-terminal deletion mutants of HSP90{alpha}. A, expression of the deletion mutants was verified by Western blot analysis using an anti-FLAG antibody 48 h following transfection (S35 cells not shown). B, the interaction between the HSP90{alpha} deletion mutants and FLIPS in C40 and S35 cells was assessed in protein eluted from anti-FLAG antibody immunoprecipitates using an antibody targeting FLIPS. To verify equal levels of FLIPS protein among the experimental groups, total levels of FLIPS protein in the lysates of the deletion mutant–expressing cells were determined by Western blot using an aliquot of lysate taken prior to immunoprecipitation. The data are representative of one of three independent experiments for each group.

 
Having shown that HSP90{alpha} regulates TRAIL-induced apoptosis by chaperoning FLIPS to its site of action in the DISC, we considered the possibility that HSP90{alpha} may serve as a chaperone for multiple DISC-related proteins, and in doing so, serve as a more global regulator of apoptosis. To address this possibility, TRAIL-resistant C40 cells were incubated with a scrambled siRNA or an siRNA targeting HSP90{alpha}, after which the cells were exposed to TRAIL. The levels of a variety of DISC-related proteins were then assessed in cell lysates and in DISC complexes immunoprecipitated using a FADD-directed antibody. siRNA-mediated suppression of HSP90{alpha} levels did not alter the total cellular levels of any of the DISC-related proteins relative to controls (TRAIL alone, scrambled siRNA plus TRAIL, and lysates exposed to TRAIL; Western blot; Fig. 5 ). It did, as expected, significantly decrease levels of FLIPS in the immunoprecipitated DISC (Fig. 5, lanes 5 versus 4). siRNA-mediated suppression of HSP90{alpha} levels, however, also significantly decreased the levels of FLIPL and RIP in the DISC, whereas not altering levels of FADD, TANK, or TRAF. These results show that HSP90{alpha} serves as a selective chaperone for a subfamily of DISC-related proteins, and that this action, rather than protein stabilization or alteration of protein levels, could allow HSP90{alpha} to globally regulate apoptosis initiated by a variety of stimuli.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. HSP90{alpha} serves as a chaperone for multiple DISC-related proteins. TRAIL-resistant C40 cells were either exposed to vehicle or TRAIL (800 ng/mL, 24 h), or were transfected with scrambled siRNA or siRNA targeting HSP90{alpha}, after which the transfected cells were exposed to TRAIL (800 ng/mL) during the last 24 h of the 72-h siRNA incubation. Cells were then lysed and the levels of total cellular DISC-related proteins (right, WB) or DISC-related proteins immunoprecipitated using a IgG antibody or an antibody targeting FADD (left) were determined by Western blot. Lane 2, total or DISC-immunoprecipitated proteins from lysate incubated with TRAIL (800 ng/mL, 24 h). {alpha}-Tubulin was used as a loading control. The data are representative of one of three independent experiments for each group.

 
Having established that HSP90{alpha} interacts with the FLIPS protein, we considered the possibility that HSP90{alpha} may interact with a complex of DISC-related proteins, maintaining the stability of these client proteins prior to recruitment to the DISC, as previously suggested by Lewis et al. (45). To do so, C40 cells with high levels of FLIPS and HSP90{alpha} were lysed, after which, the proteins in the lysate were immunoprecipitated using an antibody targeting HSP90{alpha}. Following elution of the immunoprecipitated proteins, levels of HSP90{alpha}, FLIPS, FLIPL, RIP, FADD, TANK, and TRAF proteins were then monitored by Western blot and compared with total levels of these proteins from an aliquot of lysate obtained prior to immunoprecipitation. Although the C40 cells expressed high levels of all DISC-related proteins as assessed by Western blot prior to immunoprecipitation (Fig. 6B ), only HSP90{alpha}, FLIPS, FLIPL, and RIP were present in the immunoprecipitated HSP90{alpha} protein complex (relative to proteins immunoprecipitated with an IgG antibody; Fig. 6A, lanes 1 and 2). Similar results were obtained using S35 cells (data not shown). These studies show that HSP90{alpha}, in a manner that is ATP-binding domain-dependent, interacts with a complex of DISC-related proteins consisting of FLIPS, FLIPL, and RIP in resting cells and allows the recruitment of these proteins to the DISC following apoptotic stimulation.


Figure 6
View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. HSP90{alpha} interacts with FLIPS, FLIPL, and RIP proteins in resting cells. C40 cells were lysed and the levels of HSP90{alpha}, FLIPS, FLIPL, RIP, FADD, TANK, and TRAF proteins were immunoprecipitated using either an IgG antibody or an antibody targeting HSP90{alpha} (A), or levels of total cellular HSP90{alpha}, FLIPS, FLIPL, RIP, FADD, TANK, and TRAF proteins (B) were determined by Western blot. {alpha}-Tubulin was used as a loading control. The data are representative of one of three independent experiments for each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of HSP90{alpha} to bind and stabilize potentially oncogenic proteins has long been recognized as critical to the tumorigenic process. Accumulating evidence, however, suggests that HSP90{alpha} also contributes to tumorigenesis in other ways. The present study provides evidence that in addition to binding and stabilizing potentially oncogenic proteins, HSP90{alpha} helps localize FLIPS and a subset of other antiapoptotic proteins to their site of action in the DISC. The immediate consequence of this action is that HSP90{alpha} regulates TRAIL-induced apoptosis and has the potential to contribute to TRAIL resistance. In a broader sense, however, the novel localization function of HSP90{alpha} may help explain the global antiapoptotic functions of HSP90{alpha} and its overexpression in a variety of cancers.

Although our observation that HSP90{alpha} down-regulation decreased pAkt levels was expected, the inability of HSP90{alpha} down-regulation to alter FLIPS levels was initially surprising. Interaction of client proteins with HSPs typically results in increased client protein stability and increased client protein levels. In the present study, however, FLIPS levels were not altered by HSP90{alpha} suppression or pAkt suppression. This was particularly surprising because decreased levels of pAkt (as seen following exposure to HSP90{alpha} siRNA) have been reported to decrease translation of FLIPS mRNA and lower FLIPS protein levels (39). Although HSP90{alpha} has the potential to regulate the Akt-mTOR-FLIPS pathway at multiple levels, the end result of suppression of HSP90{alpha} was clearly not decreased levels of FLIPS. FLIPS, however, as previously reported (39, 42, 46), did play a key role in controlling TRAIL sensitivity as cdc42 overexpression (which drives FLIPS overexpression independently of Akt levels; refs. 42, 44) increased both FLIPS levels and TRAIL resistance (Fig. 3). These results, as a whole, suggest that the although in some cases HSP90{alpha} serves to control the protein levels of client proteins, in other cases, it alters client protein function in more subtle ways.

The ability of HSP90{alpha} to interact with FLIPS, and guide it to the DISC, is both consistent with known HSP function and novel. HSPs typically interact with client proteins in a manner dependent on the NH2-terminal ATP-binding domain of the HSP. The interaction between HSP90{alpha} and FLIPS seems to follow this paradigm as the ATP-binding NH2-terminal domain was required for the HSP90{alpha}-FLIPS interaction. Although the interaction between HSPs and client proteins follows a standard route, the end result of HSP/client protein interactions can be variable. In some cases, the interaction could lead to stabilization and protection from degradation, whereas in other instances, intracellular targeting of the proteins occurs. As an example, HSP90 has been reported to stabilize RIP (ref. 45; although our studies with siRNA-targeting of HSP90{alpha} suggests that this action is dependent on geldanamycin-inhibitable forms of HSP90, but not HSP90{alpha}). HSP90{alpha} has also been reported to protect the mutant form of p53 from degradation in some tumors (18, 19), or localize the active form of the estrogen receptor from the cytosol to the nucleus where it interacts with the estrogen response element (47). In some instances, HSP90{alpha} seems to be involved in both stabilizing and localizing client proteins. As an example, Her-2 (p185erbB2), a receptor tyrosine kinase overexpressed in a significant proportion of malignancies, binds to HSP90{alpha} and the HSP90{alpha} endoplasmic reticulum homologue, Grp94 (48). Treatment with an HSP90 inhibitor leads to the disruption of these complexes, resulting in both protein instability as well as the inability of the nascent Her-2 peptide to localize to the endoplasmic reticulum (48). The present study represents the first example of an HSP-client interaction leading to localization of an antiapoptotic protein to its site of action in the DISC. Given the importance of HSP90{alpha} in the interaction, and that the FLIPS/HSP90{alpha} complex is recruited to the DISC only following proapoptotic stimuli, the results suggest that DISC assembly creates or reveals a binding site for the HSP90{alpha}-FLIPS complex through direct interaction with HSP90{alpha}. HSP90{alpha} displays a binding preference for the {alpha}C-ß4 loop region found in a variety of client proteins (49). One possibility, therefore, is that this HSP90{alpha}-binding motif is revealed following DISC assembly in response to apoptotic stimuli, although this possibility, and the site of HSP90{alpha} interaction with the DISC remains to be examined.

The ability of HSP to target FLIPS to the DISC in response to TRAIL seems to be critical for the key role HSP90{alpha} plays in TRAIL resistance in gliomas. Previous work from our lab showed that activation of the Akt-mTOR pathway translationally up-regulates FLIPS, and that high levels of FLIPS confer TRAIL resistance (39). The present data, although consistent with this idea, adds a new layer of complexity to FLIPS regulation. The present work shows that high levels of FLIPS in the cytosol is not sufficient to confer TRAIL resistance, and that in addition to high levels of FLIPS, cells also need HSP90{alpha} to localize FLIPS appropriately. Because many tumor cells (and all glioblastoma multiforme cells we have examined) have elevated levels of HSP90{alpha}, low levels of HSP90{alpha} do not seem to be a cause of TRAIL resistance. In work in preparation, it also seems that the same Akt-mTOR pathway which leads to a generalized translational up-regulation also leads to a specific translational up-regulation of HSP90{alpha}, suggesting a coordinate up-regulation of both client and chaperone protein by the same pathway. The present data does, however, take on increased significance in that they suggest that HSP90{alpha} inhibitors may be efficient TRAIL-sensitizing agents. In data not presented, the nonspecific HSP inhibitor geldanamycin does in fact sensitize glioma cells to TRAIL in an action which seems to be, at least in part, mediated through an HSP90{alpha}-dependent mechanism. HSP inhibitors therefore represent a second class of agents (in addition to mTOR inhibitors; ref. 50) that might be useful in sensitizing tumor cells to TRAIL.

In addition to playing a key role in FLIPS localization and TRAIL sensitivity, the present work suggests that HSP90{alpha} alters the DISC localization of several antiapoptotic proteins, and by doing so, may affect apoptosis in a more global manner than that previously suspected. The present work shows that HSP90{alpha} interacts with at least three antiapoptotic proteins (FLIPS, FLIPL, and RIP) in untreated cells, and targets these proteins to the DISC in response to TRAIL. Although FLIPL and RIP have been excluded from playing a role in controlling TRAIL sensitivity in glioma cells (39), both proteins have been reported to play key roles in controlling the sensitivity of various cell types to a variety of other proapoptotic agents (51, 52). The ability of HSP90{alpha} to localize all three proteins (and potentially other DISC components), suggests that HSP90{alpha} may facilitate global inhibition of apoptosis. Consistent with this idea, HSP90 has been shown to interact with and stabilize RIP, and by doing so, to suppress tumor necrosis factor–induced apoptosis (45). The up-regulation of HSP90{alpha} noted in many cancers may in fact be a prerequisite for tumor formation, serving as part of a global response to suppress apoptosis stimulated by undoubtedly harsh tumor microenvironmental conditions. Although this idea has not formally been tested, the ability of HSP inhibitors to induce apoptosis in a variety of tumor types suggests that an understanding of how HSP90{alpha} contributes to protein localization may contribute to a better understanding of cellular transformation, and ultimately to better cancer therapies.


   Acknowledgments
 
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
 
4 Unpublished data. Back

Received 2/14/07. Revised 5/14/07. Accepted 7/20/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lindquist S, Craig EA. The heat shock proteins. Annu Rev Genet 1988;22:631–7.[CrossRef][Medline]
  2. Georgopolis C, Welch WJ. Role of the major heat shock proteins as molecular chaperones. Ann Rev Cell 1993;9:601–34.
  3. Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded proteins. Science 2002;295:1852–8.[Abstract/Free Full Text]
  4. Morimoto RI, Kline MP, Bimston DN, et al. The heat shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem 1997;32:17–29.[Medline]
  5. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications of medicine and disease. Physiol Rev 1992;72:1063–81.[Free Full Text]
  6. Hickey E, Weber LA. Preferential translation of heat shock protein mRNAs in HeLa cells. In: Schlesinger M, Ashburner M, Tissieres A, editors. Heat shock; from bacteria to man. New York: Cold Spring Harbor Laboratory Press; 1982. p. 199–206.
  7. Hightower LE. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 1991;66:191–7.[CrossRef][Medline]
  8. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992;355:33–45.[CrossRef][Medline]
  9. Netzer WF, Hartl FU. Protein folding in the cytosol: chaperone-dependent and-independent mechanisms. Trends Biochem Sci 1998;22:68–74.
  10. Pratt WB, Toft DO. Regulation of signaling protein function and trafficking by the Hsp90/Hsp70-based chaperone machinery. Exp Biol Med 2003;228:111–33.[Abstract/Free Full Text]
  11. Fink AL. Chaperone-mediated protein folding. Physiol Rev 1999;79:425–49.[Abstract/Free Full Text]
  12. Voss A, Thomas T, Gruss P. Mice lacking HSP90ß fail to develop a placental labyrinth. Development 2000;127:1–11.[Abstract]
  13. Basso AD, Solit DB, Chiosis G, et al. Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J Biol Chem 2002;277:39858–66.[Abstract/Free Full Text]
  14. Sato S, Fujita N, Tsuruo T. Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci U S A 2000;97:10832–7.[Abstract/Free Full Text]
  15. Lavictoire SJ, Parolin DA, Klimowicz AC, et al. Interaction of Hsp90 with the nascent from of the mutant epidermal growth factor receptor EGFRvIII. J Biol Chem 2002;278:5292–9.[CrossRef][Medline]
  16. Pratt WB. The Hsp90 based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc Soc Exp Biol Med 1998;217:420–34.[CrossRef][Medline]
  17. Takahashi S, Mikami T, Watanabe Y, et al. Correlation of heat shock protein 70 expression with estrogen receptor levels in invasive human breast cancer. Am J Clin Pathol 1994;101:519–25.[Medline]
  18. Lehmann TA, Bennet WP, Metcalf RA. P53 mutations, ras mutations and p53-heat shock protein complexes in human lung carcinoma cell lines. Cancer Res 1991;51:4090–6.[Abstract/Free Full Text]
  19. Blagosklonny MV, Toretsky J, Bohen S, et al. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc Natl Acad Sci U S A 1996;93:8379–83.[Abstract/Free Full Text]
  20. Twiddy D, Cohen GM, MacFarlane M, et al. Caspase-7 is directly activated by the ~700-kDa apoptosome complex and is released as a stable XIAP-caspase-7 ~200-kDa complex. J Biol Chem 2006;281:3876–88.[Abstract/Free Full Text]
  21. Baliga B, Kumar S. Apaf-1/cytochrome c apoptosome: an essential initiator of caspase activity or just a sideshow? Cell Death Differ 2003;10:16–8.[CrossRef][Medline]
  22. Saleh A, Srinivasula SM, Balkir L, et al. Negative regulation of Apaf-1 apoptosome by Hsp70. Nat Cell Biol 2000;2:476–83.[CrossRef][Medline]
  23. Beere HM, Wolf BB, Cain K, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2000;2:469–75.[CrossRef][Medline]
  24. Beere HM, Green DR. Stress management—heat shock protein-70 and the regulation of apoptosis. Trends Cell Biol 2001;11:6–10.[CrossRef][Medline]
  25. Garrido C, Bruey JM, Fromentin A, et al. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J 1999;13:2061–70.[Abstract/Free Full Text]
  26. Bruey JM, Ducasse C, Bonniaud P, et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat Cell Biol 2000;2:645–52.[CrossRef][Medline]
  27. Fan GC, Ren X, Qian J, et al. Novel cardioprotective role of a small heat-shock protein, Hsp20, against ischemia/reperfusion injury. Circulation 2005;111:1792–9.[Abstract/Free Full Text]
  28. Gorman AM, Heavey B, Creagh E, et al. Antioxidant-mediated inhibition of the heat shock response leads to apoptosis. FEBS Lett 1999;445:98–102.[CrossRef][Medline]
  29. Csermley P, Schnaider T, Söti C, et al. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998;79:129–68.[CrossRef][Medline]
  30. Scheibel T, Buchner J. The Hsp90 family: an overview. In: Gething MJ, editor. Guidebook to molecular chaperones and protein catalyses. Oxford: Oxford University Press; 1997. p. 147–51.
  31. Prodromou C, Roe SM, O'Brien R, et al. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 1997;90:65–75.[CrossRef][Medline]
  32. Grenert JP, Sullivan WP, Fadden P, et al. The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J Biol Chem 1997;272:23843–50.[Abstract/Free Full Text]
  33. Hawle P, Siepmann M, Harst A, et al. The middle domain of Hsp90 acts as a discriminator between different types of client proteins. Mol Cell Biol 2006;26:8385–95.[Abstract/Free Full Text]
  34. Chadli A, Bouhouche I, Sullivan W, et al. Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90. Proc Natl Acad Sci U S A 2000;97:12524–9.[Abstract/Free Full Text]
  35. Carrigan PE, Nelson GM, Roberts PJ, et al. Multiple domains of the co-chaperone Hop are important for Hsp70 binding. J Biol Chem 2004;279:16185–93.[Abstract/Free Full Text]
  36. Song HY, Dunbar JD, Zhang YX, et al. Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor. J Biol Chem 1995;270:3574–81.[Abstract/Free Full Text]
  37. Georgakis GV, Li Y, Rassidakis GZ, et al. Inhibition of the heat shock protein 90 function by 17-allylamino-17-demethoxy-geldanamycin in Hodgkin's lymphoma cells down-regulates Akt kinase, dephosphorylates extracellular signal-regulated kinase, and induces cell cycle arrest and cell death. Clin Cancer Res 2006;12:584–90.[Abstract/Free Full Text]
  38. Wu YP, Kita K, Suzuki N. Involvement of human heat shock protein 90{alpha} in nicotine-induced apoptosis. Int J Cancer 2002;100:37–42.[CrossRef][Medline]
  39. Panner A, James CD, Berger MS, et al. mTOR controls FLIPS translation and TRAIL sensitivity in glioblastoma multiforme cells. Mol Cell Biol 2005;25:8809–23.[Abstract/Free Full Text]
  40. Sonoda Y, Ozawa T, Hirose Y, et al. Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma. Cancer Res 2001;61:4956–60.[Abstract/Free Full Text]
  41. Chang GH-F, Barbaro NM, Pieper RO. Phosphatidylserine-dependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, and glioma cells. Neuro-oncol 2000;2:174–83.[Abstract]
  42. Panner A, Nakamura JL, Parsa AT, et al. mTOR-independent translational control of the extrinsic cell death pathway by RalA. Mol Cell Biol 2006;26:7345–57.[Abstract/Free Full Text]
  43. Field N, Low W, Daniels M, et al. KSHV vFLIP binds IKK-{gamma} to activate IKK. J Cell Sci 2003;116:3721–8.[Abstract/Free Full Text]
  44. Fang Y, Park IH, Wu AL, et al. PLD1 regulates mTOR signaling and mediates cdc42 activation of S6K1. Curr Biol 2003;13:2037–44.[CrossRef][Medline]
  45. Lewis J, Devin A, Miller A, et al. Disruption of Hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-{kappa}B activation. J Biol Chem 2000;275:10519–26.[Abstract/Free Full Text]
  46. Panner A, Parsa AT, Pieper RO. Translational regulation of TRAIL sensitivity. Cell Cycle 2006;5:e1–4.
  47. Sabbah M, Radanyi C, Redeuilh G, et al. The 90-kDa heat-shock protein (hsp90) modulates the binding of the oestrogen receptor to its cognate DNA. Biochem J 1996;314:205–13.[Medline]
  48. Xu W, Mimnaugh EG, Kim J-S, et al. Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2. Cell Stress Chaperones 2002;7:91–6.[CrossRef][Medline]
  49. Citri A, Harari D, Shohat G, et al. Hsp90 recognizes a common surface on client kinases. J Biol Chem 2006;281:14361–9.[Abstract/Free Full Text]
  50. Panner A, Parsa AT, Pieper RO. Use of Apo2L/TRAIL with mTOR inhibitors in the treatment of glioblastoma multiforme. Expert Rev Anticancer Ther 2006;6:1313–22.[CrossRef][Medline]
  51. Meurette O, Rebillard A, Huc L, et al. TRAIL induces receptor-interacting protein 1-dependent and caspase-dependent necrosis-like cell death under acidic extracellular conditions. Cancer Res 2007;67:218–26.[Abstract/Free Full Text]
  52. Bajenova O, Tanq B, Pearse R, et al. RIP kinase is involved in arsenic-induced apoptosis in multiple myeloma cells. Apoptosis 2004;9:561–71.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Panner, A.
Right arrow Articles by Pieper, R. O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Panner, A.
Right arrow Articles by Pieper, R. O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online