Abstract
The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient.
Introduction
Targeting a specific molecule involved in regulating cellular radioresponse has received considerable attention as a potential strategy for enhancing the antitumor effectiveness of radiotherapy. In experimental models, numerous studies have reported that inhibiting a given radioresponse regulatory molecule could result in an increase in tumor cell radiosensitivity. However, there are also a number of examples in which targeting a selected radioresponse associated molecule (e.g., epidermal growth factor receptor, Chk1, and nuclear factor κB) affects the radiosensitivity of some tumor cell lines but not others (1–7). Such results indicate that whether a selected target has a major, minor, or no role in determining radioresponse is subject to at least some degree of cell type specificity. Clearly, the ability to predict those tumors in which the putative target plays a critical role in determining radiosensitivity would provide a significant advantage in the potential clinical application of a molecularly targeted radiosensitizer. In turn, this ability will require a thorough understanding of the genetic and/or epigenetic context that allows a given molecule to exert its regulatory action.
As a potential molecular target for tumor cell radiosensitization, a number of groups, including our own, have investigated the chaperone protein Hsp90 (8–12). In contrast to other molecular chaperones, Hsp90 has a relatively high selectivity for signaling proteins (13). Among the client proteins of Hsp90 are steroid hormone receptors, Src family kinases, cyclin-dependent kinases (Cdk4 and 6), and the HIF-1α transcription factor (13). In addition, Hsp90 acts to stabilize Raf-1, Akt, and ErbB2 (14, 15), each of which has been associated with protection against radiation-induced cell death; a reduction in their individual activities has been shown to result in radiosensitization in certain cell lines (16–19) Consequently, inhibition of Hsp90 results in the simultaneous loss of multiple molecules that could affect radiosensitivity. Thus, Hsp90 inhibitors provide a strategy for implementing a multitarget approach to radiosensitization.
Previous studies have shown that inhibition of Hsp90 activity with radicicol (11), geldanamycin (10), or the geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17AAG; refs. 9, 12) enhances the radiosensitivity of a variety of human tumor cell lines, consistent with Hsp90 being a molecular target for radiosensitization. Recently, we reported the effects of the 17AAG analogue 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) on tumor cell radiosensitivity in vitro and in vivo. 17DMAG, in contrast to 17AAG, is water-soluble, orally bioavailable, and does not seem to undergo extensive metabolism to toxic species (20, 21). In these studies, 17DMAG was found to enhance the in vitro radiosensitivity of three human cell lines originating from three different tumor histologies and to result in a greater than additive increase in radiation-induced tumor growth delay in a human tumor xenograft model (8). Whereas a number of radioresponse-associated proteins decreased after 17DMAG exposure, the radiosensitization appeared to correlate best with a decrease in the levels of ErbB2 (8). To further investigate the general relationship between Hsp90 inhibition and radiosensitization, we used 17 DMAG as a representative of clinically applicable Hsp90 inhibitors and have evaluated its effects on the radiosensitivity of a number of additional human tumor cell lines. During this evaluation, the AsPC1 pancreatic carcinoma cell line was found to be unique. Whereas exposure of AsPC1 cells to 17DMAG resulted in a loss of ErbB2, Raf, and Akt proteins, the Hsp90 inhibitor had no effect on cellular radiosensitivity. Its distinctive response suggested that further investigations of the AsPC1 cell line may provide mechanistic insight into the role of Hsp90 in regulating radiosensitivity. Specifically, we now report on the use of this cell line to identify a molecular determinant of cellular susceptibility to 17DMAG-induced radiosensitization. The data presented herein indicate that the expression of ErbB3 abrogates the increase in radiosensitivity induced by Hsp90 inhibition.
Materials and Methods
Cell lines and treatment. Six human tumor cell lines were evaluated: three pancreatic carcinomas (MiaPaCa, PSN1, and AsPC1), two breast carcinomas (T-47D and NCI/ADR-res) and a prostate carcinoma (DU145). MiaPaCa, AsPC1, and DU145 were obtained from the American Type Culture Collection (Gaithersburg, MD). The other cell lines were kindly supplied by the Screening Technologies Branch, In vitro Cancer Screening Program, National Cancer Institute-Frederick. AsPC1 was grown in RPMI 1640 (Life Technologies, Rockville, MD) containing 2 mmol/L l-glutamine, 2.5 g/L glucose, 10 mmol/L HEPES, and 1.0 mmol/L sodium pyruvate and 15% fetal bovine serum (FBS). T-47D, NCI/ADR-res were grown in RPMI 1640 containing 2 mmol/L l-glutamine and 10% FBS. The other cell lines were grown in RPMI 1640 containing 2 mmol/L l-glutamine and 5% FBS. They were maintained at 37°C in an atmosphere of 5% CO2 and 95% room air. 17DMAG, provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program of the National Cancer Institute, was dissolved in PBS to a stock concentration of 1 mmol/L and stored at −20°C. Cell lines were exposed to 17DMAG (50 nmol/L) for 16 hours. Cultures were irradiated using a Pantak (Solon, OH) X-ray source at a dose rate of 1.55 Gy/min.
Clonogenic assay. Cultures were trypsinized to generate a single cell suspension and a specified number of cells were seeded into each well of six-well tissue culture plates. After allowing cells time to attach (6 hours), 17DMAG or the vehicle control (PBS) was added at specified concentrations and the plates were irradiated 16 hours later. Immediately after irradiation, the growth media was aspirated and fresh media was added. Twelve to 14 days after seeding, colonies were stained with crystal violet, the number of colonies containing at least 50 cells was determined and the surviving fractions were calculated. Survival curves were generated after normalizing for cytotoxicity generated by 17DMAG alone. Data presented are the mean ± SE from at least three independent experiments.
Short interfering RNA treatment. The short interfering RNA (siRNA) SMARTpool containing four-pooled siRNA duplexes directed against ErbB2 (catalog number M-003126-01), ErbB3 (catalog number M-003127-02), and a nonspecific control pool (siRNA-negative control) as a nonspecific siRNA control (catalog number D-001206-13) were purchased from Dharmacon, Inc. (Chicago, IL). Transfection of siRNA was done on 30% to 50% confluent cells using OligofectAMINE (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Knockdown of the specified protein was optimized for siRNA concentration and time posttransfection.
Immunoprecipitation and immunoblot analyses. Cells were washed twice with ice-cold PBS and then lysed in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, 1 mmol/L NaF, 1 mmol/L NaO3V4, and protease inhibitor cocktail]. Protein concentrations were measured using a modification of the Bradford method or a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) according to the manufacturer's instructions. For immunoblot analysis, lysates (20 μg per sample) were resolved on 10% SDS gels. For immunoprecipitation, 1 mg of lysate was incubated with 10 μg of primary antibody overnight at 4°C. The next day, 40 μL of protein A agarose beads (Upstate Biotechnology, Lake Placid, NY) were added to the lysate and incubated for 1.5 hours at 4°C. Immunoprecipitates were washed thrice with cold coimmunoprecipitation lysis buffer and resuspended in 1× sample buffer, heated at 90°C for 5 minutes, and centrifuged. Supernatants were electrophoretically transferred for 2 hours to Immobilon-P membranes. Membranes were blocked with 5% nonfat dry milk or 3% bovine serum albumin (BSA) in 500 mmol/L NaCl, 20 mmol/L Tris (pH 7.5), and 0.1% Tween 20 (TBST) for 1 hour followed by overnight incubation with primary antibody, then washed thrice with TBST and incubated for 1 hour with horseradish peroxidase–conjugated secondary antibody (1:2,000 dilution in 3% nonfat dry milk/TBST or 3% BSA/TBST; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). This was followed by three 10-minute washes in TBST. Blots were developed using Western blotting luminol reagent (Santa Cruz) or Visualizer Western blot detection kit (Upstate Biotechnology) and the Luminescent Image Analyzer LAS-3000 (Fujifilm, Stamford, CT) according to the manufacturer's instructions. Antibodies against ErbB1 [sc-03 (IP); Santa Cruz and 610016 (Western); BD Biosciences, San Diego, CA]; ErbB-2 [OP15 (Western); EMD Biosciences, Inc., San Diego, CA]; ErbB-3 [sc-285 (IP), Santa Cruz; and 05-390 (Western), Upstate Biotechnology]; ErbB-4 [sc-283 (Western), Santa Cruz]; antiphosphotyrosine [05-321 (Western), Upstate Biotechnology]; c-Raf (sc-133, Santa Cruz) Akt (9272, Cell Signaling Technology, Beverly, MA) and actin (Chemicon, Temecula, CA) were purchased from commercial sources. Densitometric analyses were done using the General-Purpose Analysis Software Multi Gauge (ver 2.2) for Windows (Fujifilm).
ErbB1 kinase assay. Analysis of ErbB1 kinase activity was done using the Universal Tyrosine Kinase Kit purchased from Takara Mirus Bio (Madison, WI) according to the manufacturer's instructions. Briefly, immunoprecipitated ErbB1 protein was washed thrice with PBS, suspended in the kinase reacting solution including 2-mercaptoethanol. Bead suspensions (40 μL) were duplicately transferred into 96-well PTK substrate (poly Glu-Tyr) immobilized microplate, and the rest of beads were used for Western blotting to show that ErbB1 had been evenly immunoprecipitated. The kinase reaction was initiated by the addition of 10 μL of 40 mmol/L ATP-Na and allowed to proceed for 20 minutes at 37°C. Plates were washed thrice with PBS and 0.05% Tween 20, and phosphorylation was detected using antiphosphotyrosine–horseradish peroxidase antibodies (Upstate Biotechnology). After washing and enhancement according to the manufacturer's instructions, signal was detected using a Victor2 plate reader (wavelength 450 nm). ErbB1 tyrosine kinase activity was calculated on the basis of the standard curve with recombinant c-src.
Results
A number of different treatment protocols using Hsp90 inhibitors, including 17DMAG, have been reported to enhance the radiosensitivity of human tumor cell lines (8–12). The radioresponse of three cell lines (DU145 prostate carcinoma, PSN1 pancreatic carcinoma, and MiaPaCa pancreatic carcinoma) exposed to 50 nmol/L 17DMAG for 16 hours prior to irradiation are shown in Fig. 1. The 16 hours of 17DMAG treatment only resulted in surviving fractions of 0.3, 0.5, and 0.5 for DU145, MiaPaCa, and PSN1, respectively. The combination of 17DMAG and radiation resulted in the enhancement of radiosensitivity for each cell line with dose enhancement factors (at a surviving fraction of 0.1) for DU145, MiaPaCa, and SN1 were 1.7, 1.6, and 1.3, respectively. These results are consistent with dose enhancement factors reported in previous studies using Hsp90 inhibitors (8, 9). However, when the same 17DMAG treatment protocol was tested on the pancreatic carcinoma cell line AsPC1, there was no effect on radiosensitivity (Fig. 1D). Exposure to 50 nmol/L 17DMAG alone resulted in a surviving fraction of 0.8.
We previously reported that exposure of DU145 and MiaPaCa cells to 50 nmol/L 17DMAG for 16 hours decreased the levels of the radioresponse-associated proteins ErbB2, Raf, and Akt (8). These proteins were also reduced in the other radiosensitized cell line shown in Fig. 1 (PSN1 and data not shown). Although AsPC1 cells were resistant to its radiosensitizing actions, treatment with 17DMAG resulted in a significant decrease in the levels of Raf, Akt, and ErbB2 proteins in this cell line (Fig. 2A). Because loss of ErbB2 correlated with 17DMAG-induced radiosensitization in the other cell lines (8) and because ErbB2 is a member of the ErbB family, which is dependent on heterodimer formation for activity (22, 23), we compared the expression of ErbB family members in the radiosensitized cell lines with those in AsPC1 (Fig. 2B). Whereas the levels of ErbB1 and ErbB2 were similar among the four cell lines, in contrast to the DU145, MiaPaCa, and PSN1, which are sensitized by 17DMAG, AsPC1 expressed significant levels of ErbB3. These results suggested that ErbB3 might play a role in the resistance of AsPC1 to 17DMAG-induced radiosensitization. As a first test of this hypothesis, two additional cell lines, NCI/ADR-res and T47D, were investigated. Whereas T47D expresses ErbB4 and a relatively low level of ErbB1, in common with AsPC1, both the T47D and NCI/ADR-res cell lines express readily detectable ErbB3 (Fig. 2B). As shown in Fig. 2C and D, as for AsPC1, the radiosensitivity of the two additional ErbB3-expressing cell lines was also not affected by 17DMAG.
To more specifically address the hypothesized inhibitory role of ErbB3 in 17DMAG-induced radiosensitization, we focused on MiaPaCa and AsPC1 as representatives of sensitized and unsensitized cell lines, respectively. As shown in Fig. 3A, exposure of both pancreatic carcinoma cell lines to 17DMAG (16 hours, 50 nmol/L) reduced ErbB2 levels, but had no significant effect on ErbB1 or ErbB3, which is consistent with previous reports of Hsp90 inhibitors on ErbB family members (24, 25). Because signaling through the ErbB pathway is primarily dependent on heterodimer formation, the effects of 17DMAG on ErbB heterodimers were determined using a coimmunoprecipitation assay. As shown in Fig. 3B heterodimers of ErbB1 and ErbB2 were detected in MiaPaCa cells under untreated conditions. Exposure to 17DMAG reduced the ErbB1/ErbB2 heterodimers; the levels of ErbB1 homodimers and/or of monomers were not significantly affected. In AsPC1 cells, in addition to the ErbB1/ErbB2 heterodimer, ErbB1/ErbB3, and ErbB2/ErbB3 heterodimers, each of which has signaling activity (26), were detected under untreated conditions (Fig. 3B). Although 17DMAG exposure reduced the levels of ErbB1/ErbB2 and ErbB3/ErbB2 heterodimers in AsPC1 cells, the levels of ErbB1/ErbB3 heterodimers were not affected. The effects of 17DMAG on ErbB heterodimers were thus consistent with the selective reduction in ErbB2 protein.
To determine whether ErbB3 expression was causally involved in the resistance to 17DMAG-induced radiosensitization, siRNA was used to knock down ErbB3 protein in AsPC1 cells. The immunoblots shown in Fig. 4A illustrate the effects of siRNA to ErbB3 and/or 17DMAG on ErbB protein levels in AsPC1 cells. Treatment of cells with 17DMAG selectively reduced the level of ErbB2 and treatment with siRNA to ErbB3 selectively reduced ErbB3. Exposure of cells to the combination of 17DMAG and siRNA to ErbB3 resulted in the reduction of both ErbB2 and ErbB3 with no effect on ErbB1 levels. A clonogenic assay was also done on AsPC1 cells combining these treatments with radiation (Fig. 4B and C). In cells exposed to only17DMAG or siRNA to ErbB3, the surviving fractions at 4 Gy were essentially the same as that obtained in cells receiving 4 Gy only (Fig. 4B). However, in cells that had been exposed to the combination of 17DMAG and siRNA to ErbB3 there was a significant increase in the radiation-induced cell killing. Complete survival curves for the single and combined treatments are shown in Fig. 4C. Consistent with the results obtained for 4 Gy only, 17DMAG alone had no effect on AsPC1 radiosensitivity nor did siRNA to ErbB3. However, the combination of 17DMAG and siRNA to ErbB3 enhanced AsPC1 radiosensitivity with a dose enhancement factor of 1.35. These data indicate that ErbB3 plays a causal role in resistance to 17DMAG-induced radiosensitization.
ErbB1 activity, which is dependent on heterodimer formation (22), is generally considered to provide protection against radiation-induced cell death (27). To address the role of ErbB1 signaling in 17DMAG-induced radiosensitization, the levels of phospho-ErbB1, which is indicative of kinase activity (28, 29), were determined after treatment of MiaPaCa and AsPC1 cells with 17DMAG and/or siRNA to ErbB3 (Fig. 5A). In MiaPaCa cells, phospho-ErbB1 was significantly decreased after exposure to 17DMAG, consistent with losses of ErbB2 and the ErbB1/ErbB2 heterodimer. In contrast, 17DMAG had no effect on phospho-ErbB1 levels in AsPC1 cells nor did exposure to siRNA to ErbB3. However, phospho-ErbB1 was significantly reduced in AsPC1 cells exposed to the combination of 17DMAG and siRNA to ErbB3. This study was then extended to the direct measure of ErbB1 tyrosine kinase activity (Fig. 5B). Exposure of MiaPaCa cells to 17DMAG resulted in a significant decrease in ErbB1 kinase activity. In contrast, this Hsp90 inhibitor had no effect on ErbB1 kinase activity in AsPC1 cells. Furthermore, treatment of AsPC1 cells with siRNA to ErbB3 had no effect on the kinase activity. However, the combination of 17DMAG and siRNA to ErbB3 resulted in a significant loss of ErbB1 kinase activity in AsPC1 cells. Thus, the changes in ErbB1 kinase activity were consistent with the modifications of phospho-ErbB1 levels. These results, combined with the heterodimer data presented in Fig. 3B, suggest that whereas 17DMAG decreases ErbB1 kinase activity in MiaPaCa cells through the loss of ErbB2 and the ErbB1/ErbB2 heterodimer, the kinase activity is maintained in 17DMAG-treated AsPC1 cells because of the presence of the ErbB1/ErbB3 heterodimer. Moreover, these data suggest that a decrease ErbB1 activity is necessary for 17DMAG-induced radiosensitization.
Although data generated to this point indicated that the loss of ErbB activity plays a causal role, it was unclear whether this event was sufficient for 17DMAG-induced radiosensitization. To address this issue, MiaPaCa cells, which express only ErbB1 and ErbB2 and not ErbB3 (Fig. 2B), and are radiosensitized by 17DMAG (Fig. 1), were exposed to siRNA to ErbB2 and their radiosensitivity determined by clonogenic assay. As shown in Fig. 6A and B, treatment of cells with siRNA to ErbB2 significantly reduced ErbB2 levels and ErbB1 kinase activity. However, this loss of ErbB2 had no detectable effect on the radiosensitivity of MiaPaCa cells (Fig. 6C). Similar results were obtained for DU145 and PSN1 (data not shown), which also do not express ErbB3 (Fig. 2B) and are radiosensitized by 17DMAG (Fig. 1). These data suggest that for the loss of ErbB1 activity is necessary but not sufficient 17DMAG-induced radiosensitization.
Discussion
It is now well-established that cellular radiosensitivity reflects the end result of a combinatorial process comprised of a wide variety of signaling and effector molecules. However, it is also clear that the contribution of an individual molecule to this process is often determined by the genetic and epigenetic circumstances of a given cell. This cell type specificity combined with the inter- and intratumor heterogeneity that exists in most solid neoplasms suggests that the ability to target multiple radioresponse regulatory proteins may provide a significant advantage as an approach to tumor cell radiosensitization. Towards this end, we have been investigating the use of compounds that inhibit Hsp90, which serves as a molecular chaperone for a variety of signaling proteins previously associated with tumor cell radioresponse. Indeed, Hsp90 inhibitors have been shown to significantly enhance the radiosensitivity of a number of tumor cell lines initiated from a variety of histologies (8–12). However, using 17DMAG as a representative of clinically applicable Hsp90 inhibitors, the results presented here indicate that even this multitarget approach to radiosensitization is subject to tumor cell type specificity. Thus, as for other molecularly targeted radiosensitizers, the potential clinical application of an Hsp90 inhibitor would significantly benefit from an ability to predict tumor susceptibility.
Although Hsp90 inhibition results in the loss of a number of radioresponse associated proteins, because the loss of ErbB2 best correlated with radiosensitization after treatment with 17DMAG or 17AAG (8, 9) and because of the putative significance of ErbB signaling in radioresponse (27, 30–32), we have initially focused on this protein. ErbB2 is a member of the ErbB tyrosine kinase receptor family, which also includes ErbB1 (epidermal growth factor receptor), ErbB3, and ErbB4 (22). Whereas signaling through this receptor family is dependent on the formation of homodimeric and heterodimeric combinations (22), the biological activity of the ErbB receptors is primarily dependent on ErbB heterodimers with homodimers having significantly less activity (26, 33–35). It should be pointed out that in contrast to other family members, ErbB3 lacks intrinsic kinase activity and only contributes to signaling when complexed with other ErbB receptors (22). The data presented here (Figs. 3 and 5) indicate that the loss of ErbB2 after 17DMAG treatment of MiaPaCa cells results in a loss of ErbB1/ErbB2 heterodimers and a reduction in ErbB1 kinase activity, which is consistent with the critical role of heterodimer formation in ErbB1 signaling. However, in AsPC1 cells, although ErbB2 is lost after 17DMAG exposure, ErbB3/ErbB1 heterodimers were not affected, consequently, there was no reduction in ErbB1 kinase activity. Although there remains the potential for differential effects of ErbB1/ErbB3 and ErbB1/ErbB2 heterodimers on downstream pathways, these data suggest that the presence of ErbB3 provides a redundant mechanism for maintaining ErbB1 activity.
A role for ErbB3 was initially suggested by data showing that whereas 17DMAG enhanced radiation-induced cell killing in a disparate group of human tumor cell lines, the Hsp90 inhibitor had no effect on the radiosensitivity of ErbB3 expressing tumor cells. Importantly, a causal relationship between ErbB3 expression and the lack of radiosensitization was established by experiments showing that 17DMAG treatment of AsPC1 cells in which ErbB3 protein levels had been knocked-down with siRNA resulted in a significant increase in radiosensitivity. Although the specific downstream events remain to be determined, these data indicate that ErbB3 serves as a molecular determinant of tumor cell susceptibility to 17DMAG-induced radiosensitization. Thus, with respect to the potential design of clinical protocols combining an Hsp90 inhibitor and radiotherapy, these results suggest that ErbB3 expression may be useful in patient selection. That is, patients with tumors that do not express ErbB3 would be predicted to respond best to this combined modality.
The data generated from the ErbB3 knockdown studies suggested that a reduction in ErbB1 activity was necessary for 17DMAG-induced radiosensitization. These results thus suggested that loss of ErbB2 protein and consequent reduction in ErbB1 kinase activity in non-ErbB3–expressing cell lines then plays a causative role in 17DMAG-induced radiosensitization However, the selective reduction in ErbB2 protein by siRNA and the accompanying reduction in ErbB1 activity had no effect on the radiosensitivity of a panel of non-ErbB3 expressing tumor cells. Thus, taken together, these data suggest that the loss of ErbB2 resulting in a decrease ErbB1 activity is necessary but not sufficient for 17DMAG-induced radiosensitization. The failure of a single protein to account for the effects of 17DMAG is actually consistent with the rationale for the multitarget approach to tumor cell radiosensitization mediated by Hsp90 inhibitors. Initial studies of Hsp90 and radiosensitization focused on ErbB2, Raf and Akt and, as shown here, have identified the loss of ErbB1 activity as necessary for 17DMAG-induced radiosensitization. However, recent reports have identified additional Hsp90-dependent proteins such as Chk1 (36) and survivin (37), which have also been associated with the regulation of radiosensitivity, albeit in a cell type–dependent manner (3, 38–41). Moreover, in a study using yeast, Zhao et al. recently identified >600 putative Hsp90 substrates and cofactors, which linked this chaperone to, among other functions, transcriptional regulation and chromatin remodeling (42). Thus, future studies will use the cellular context of reduced ErbB1 activity in an attempt to define the combination of critical protein targets mediating radiosensitization induced by Hsp90 inhibitors. In essence, these studies will attempt to apply an approach analogous to synthetic lethality to radiosensitization. That is, rather than cell death as an end point, the goal will be to identify molecules that in combination with ErbB1 inhibition results in an enhancement of tumor cell radiosensitivity.
Acknowledgments
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