Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells
© Cheung et al. 2010
Received: 5 January 2010
Accepted: 15 April 2010
Published: 15 April 2010
Survivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression.
Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by Western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced proteasomal activity while inhibition of 26S proteasome activity further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines.
We showed for the first time that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. Instead, targeting Hsp90 with small molecule inhibitors will induce the over-expression of survivin in certain cancer cell lines and subsequently enhances the ability of cell survival in drug-treated situations. The current study suggests that dual inhibition of Hsp90 and survivin may be warranted.
- 17-AAG :
- Hsp90 :
heat-shock protein 90
- siR-C :
scramble siRNA oligos
- siR-S :
survivin-targeted siRNA oligos
Heat shock protein 90 (Hsp90) is a molecular chaperone that assists the correct folding and stabilization of various proteins in cells. During the last decade, Hsp90 has emerged as an exciting target for cancer therapy. The over-expression of Hsp90 has been shown in various cancers such as non-small cell lung cancer, oesophageal squamous cell carcinoma, pancreatic carcinoma and advanced malignant melanoma [1–4]. In addition, studies showed that Hsp90 stabilizes various key oncogenic proteins such as survivin, Akt, Erb-2 and HIF-1α in cancer cells [5–7]. Therefore, targeting hsp90 gives therapeutic advantages over other target-therapies as multiple Hsp90-related oncogenic proteins can be targeted simultaneously .
Survivin is a member of the inhibitors of apoptosis (IAPs) family. Unlike other IAPs, survivin is a bifunctional protein that functions as a key regulator of mitosis and inhibitor of programmed cell death. It is well-demonstrated that the over-expression of survivin induces resistance to various anti-cancer therapies such as chemotherapy and radiation therapy in cancer cells [8–12]. For example, over-expression of survivin has been shown to induce drug resistance against anti-mitotic compounds by stabilizing microtubule network in vincristine/colchicine-resistant oral cancer cells and down-regulation of it restores drug sensitivity to those compounds in the same cell line . In addition, literature revealed that over-expression of survivin attenuated both tamoxifen and cisplatin-induced apoptosis in human breast cancer cells and gastric cancer cells respectively [10, 12]. Interestingly, a recent report suggests that over-expression of survivin may also enhance DNA double-strand breaks (DBD) repair capability in radiation-treated oral cancer cells by up-regulating the molecular sensor of DNA damage, Ku70 . In clinical situations, the level of survivin expression was shown to be inversely related to the levels of apoptosis and positively related to the risk of local tumor recurrence in rectal cancer patients treated with radiotherapy . Furthermore, patients with gastric tumors that express lower level of survivin seems to have a longer mean survival time than patients with higher survivin expression level after cisplatin treatment . It has also been shown that survivin expression is associated with human prostate cancer bone metastasis . Thus, survivin plays an important role in tumorigenesis, tumor metastasis and may act as an indicator of therapeutic effectiveness.
It is widely believed that Hsp90 physically interacts and stabilizes survivin in cells [5, 15, 16]. Although Hsp90 is a molecular chaperone that assists the correct folding of various proteins in cells, it does not bind to unfolded survivin . Instead, Hsp90 binds to the mature form of survivin . Structurally, the amino acid sequence Lys-70-Lys-90 of survivin is important for the binding to the N-terminal domain (ATP-binding site) of Hsp90 . Various studies have investigated the possibility of targeting survivin using Hsp90 inhibitors, based on the fact that survivin is important for cancer survival and progression. Hsp90 inhibitors such as geldanamycin, 17-AAG and shepherdin have been shown effective in targeting the Hsp90/survivin complex and subsequently inducing proteasomal degradation of survivin [5, 16–18].
Although it is widely believed that Hsp90 inhibitors induce cancer cell death through indirect down-regulation of survivin as one of its multiple therapeutic functions, a study demonstrated that 17-AAG treatment slightly increased the amount of survivin present in the human DU145 prostate cancer cells . However, the mechanism of the over-expression of survivin in such cell line was unknown. Interestingly, we also observed an up-regulation of survivin in 17-AAG and geldanamycin-treated human A549, HONE-1 and HT-29 cancer cells. Since Hsp90 interferes with multiple molecules such as sp1, sp3 (both transcriptional factors of survivin), and 26S proteasome (negative regulator of survivin protein level) simultaneously [19, 20], we hypothesize that targeting Hsp90 will affect the expression of survivin at various stages. We also hypothesize that the use of Hsp90 inhibitors may not be able to down-regulate survivin expression in certain cancer cells. Therefore, the purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression.
Targeting Hsp90 induces the over-expression of survivin in cancer cells
To determine whether Hsp90 inhibitors were able to down-regulation survivin in other cancer cell lines, human A549, HT-29 and HONE-1 cancer cells were used. In A549 cells, targeting Hsp90 with 17-AAG and geldanamycin slightly induced the baseline expression of Hsp90 as previously reported (Figure 1B) . The same treatment also induced down-regulation of both Akt and phosporylated-Erk in human A549 (p53-mutant) lung carcinoma cells as expected (Figure 1B) [7, 25]. Together, these results indicated that both Hsp90 inhibitors were functioning normally at the molecular level. Surprisingly, targeting Hsp90 with 17-AAG and geldanamycin did not induce survivin down-regulation in A549 cancer cells. Instead, Western blot analysis revealed that survivin expression was induced by Hsp90 inhibitors in A549 cells in a concentration-dependent manner (Figure 1B). In addition, over-expression of survivin was shown in cells treated with 17-AAG in a time-dependent manner (Figure 1C). Since clinical study of 17-AAG revealed that the maximum peak serum level of this compound could reach 2-3 μM, A549 cells were further treated with high concentrations of 17-AAG and the expression of survivin was determined. Over-expression of survivin was also found in A549 cells treated with high concentrations (1 μM and 2 μM) of 17-AAG (Figure 1D). Moreover, Western blot analysis revealed that the expression of survivin was up-regulated in three-dimensionally cultured A549 cells treated with 1 μM of 17-AAG for 24 h (Additional file 1).
Targeting Hsp90 induces the over-expression of survivin through a cell-cycle independent mechanism
Targeting Hsp90 affects survivin expression at the transcriptional level
Targeting Hsp90 induces survivin expression through post-transcriptional mechanisms
To determine whether 17-AAG may interfere with the stability of survivin protein in A549 cells, the rate of survivin protein degradation was determined in cells treated with/without 17-AAG. Cells were pre-incubated with 500 nM of cyclohexmide for one hour and subsequently co-incubated with 17-AAG for various time. Western blot analysis revealed that the degradation rate of survivin protein was slightly slower in 17-AAG treated A549 cells than the untreated cells (Figure 5B). The amount of survivin protein presented in 17-AAG untreated-cells was reduced by 70% after 4 h of protein synthesis inhibition (Figure 5C). In contrast, the amount of survivin presented in 17-AAG-treated cells was reduced by only 20% at the same time point (Figure 5C). Thus, the reduced rate of protein degradation might also contribute to the increased amount of survivin protein presented in 17-AAG treated A549 cancer cells.
Targeting Hsp90 with 17-AAG reduces proteasomal activity in cancer cells
Targeting survivin increases drug sensitivity to 17-AAG in cancer cells
It is widely believed that targeting Hsp90 with small molecule inhibitors is able to directly interfere with the physical interaction between Hsp90 and survivin, leading to the decrease of survivin protein level and induction of cancer cell death [5, 15]. Interestingly, this study demonstrated for the first time that targeting Hsp90 with small molecule inhibitors will affect the expression of survivin at various stages, resulting in an increase of the amount of survivin protein presented in cancer cells. Furthermore, this study demonstrated that survivin plays an important role in the sensitivity to the Hsp90 inhibitor, 17-AAG, in cancer cells.
Here, we showed that targeting Hsp90 with small molecule inhibitor affected the amount of survivin mRNA transcript presented in cancer cells. It is not surprising that targeting Hsp90 induces different effect at the level of gene transcription in different cancer cells. Literatures revealed that the rate of survivin gene transcription is positively regulated by molecules such as sp1, sp3 and Myc [29, 30]. In contrast, the gene transcription process of survivin is negatively regulated by molecules such as p53, retinoblastoma (Rb) and prostate-derived Ets transcription factor (PDEF) [31–33]. Importantly, Hsp90 interferes with sp1, sp3, p53 and Rb simultaneously [34, 35]. Hence, differences in the response of survivin gene transcription may reflect different dependencies of various Hsp90-interfered and Hsp90-unrelated transcriptional factors on the expression of survivin in different cell types. Therefore, depending on the cellular context, targeting Hsp90 might indirectly up-regulate/down-regulate the process of survivin gene transcription through the interference with various survivin-related transcriptional factors.
Interestingly, our data also demonstrated that decreases at the mRNA level did not translate into decreases in survivin protein level in 17-AAG treated A549 cells. Together with results from the translation inhibition experiment, the protein degradation experiment and the examination of the survivin-related 26S proteasome, the current study strongly indicates that Hsp90 also interferes with survivin expression at the post-transcriptional level. Thus, Hsp90-targeted treatment interferes with the process of survivin gene transcription, protein translation and protein degradation simultaneously. In fact, Hsp90 plays an important role in the assembly and maintenance of the 26S proteasome [20, 36]. The activity of 26S proteasome was shown to be reduced by the addition of the Hsp90 inhibitor, geldanaymicin, in vitro . Reduced proteasomal activity was also shown previously in Hsp90-inhibited multiple myeloma cells . On the other hand, previous studies demonstrated that indomethacin (NASID) and chlamydocin (HDAC inhibitor) enhanced survivin degradation through ubiquitin proteasome machinery in cells [37, 38]. In our study, the use of proteasome inhibitor MG-132 was shown effective in increasing the amount of survivin present in our tested cancer cell lines, indicating that the activity of proteasome was important for survivin regulation. Therefore, the level of activity of proteasome might be one of the determinants of the amount of survivin present in Hsp90-inhibited cancer cells. However, it is hard to determine whether the interference with proteasome plays the most important role in the up-regulation of survivin. Further investigations are needed to determine the relative importance of transcription, translation and proteasome-related protein degradation in different Hsp90-targeted cancer cells. It is also worth noting that both 17-AAG and geldanamycin treatment reduced the amount of survivin presented in HeLa cells and this result was consistent with other studies. In contrast, results of the 3D-culture model revealed that 17-AAG treatment (1 μM) was also able to induce the over-expression of survivin in three dimensional cultured A549, HONE-1 and HT-29 cells (Additional file 1). Thus, the current study indicates that targeting Hsp90 may induce cell line-specific responses in the expression of survivin.
Importantly, results of the current study raise the concern that Hsp90 inhibitors might not function in a way as we previously thought. Indeed, literature reported that 17-AAG promoted formation of osteolytic lesions and bone metastases in murine breast cancer model, even though the drug reduced tumor growth at the orthotopic site . Furthermore, Kayani et al. demonstrated that 17-AAG treatment was able to enhance the expression of Hsp70 in C2C12 muscle fiber cells and the recovery of extensor digitorum longus (EDL) following lengthening contraction-induced damage in animal model . Thus, targeting Hsp90 with small molecular inhibitors may not be able to induce cell death in certain circumstances.
In conclusion, the current study reveals the complex interaction between Hsp90 and survivin in cancer cells. Besides stabilizing the survivin protein through simple physical interaction, Hsp90 also indirectly interferes with survivin expression through transcription, translation and proteasome-related protein degradation. These novel findings suggest a model in which gene transcription, together with protein translation and proteasomal degradation, constitute a platform capable of modulating the amount of survivin expressed in Hsp90-targeted cancer cells. Our findings suggest that down-regulation of survivin is not a definitive therapeutic function of Hsp90 inhibitors and that dual inhibition of Hsp90 and survivin may be warranted.
Materials and methods
Cell lines, antibodies and reagents
The human lung carcinoma (A549), nasopharyngeal carcinoma (HONE-1) and colorectal adenocarcinoma (HT-29) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). A549 cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (0.29 mg/mL), at 37°C. HONE-1 and HT-29 cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY), supplemented with 5% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (0.29 mg/mL), at 37°C. The antibodies used in this study included a mouse anti-Actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit anti-Survivin antibody (R&D Systems, Minneapolis, MN), a rabbit anti-Akt antibody (Cell Signaling Technology, Danvers, MA) and a mouse anti-26S proteasome antibody (abcam, Cambridge, UK). Hsp90 inhibitors used in this study included: 17-AAG (Calbiochem, Darmstadt, Germany), geldanamycin (Calbiochem, Darmstadt, Germany) and cycloheximide (Calbiochem, Darmstadt, Germany).
Real-time reverse transcription-polymerase chain reaction (Real-time PCR)
Expression level of survivin transcript was determined by real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) using a LightCycler instrument (Roche, Indianapolis, IN). Primers and Taqman probes were designed by Probe Finder™ http://www.universalprobelibrary.com. Taqman probes were from the Universal Probe Library: survivin and hGAPDH. Specific primers with following sequences were used: survinin forward, 5' GCCCAGTGTTTCTTCTGCTT; Survivin reverse, 5'CCGGACGAATGCTTTTTATG; hGAPDH forward, 5' AGCCACATCGCTCAGACAC and hGAPDH reverse, 5' GCCCAATACGACCAAATCC. The real-time PCR condition was as follows: 1 cycle of initial denaturation at 95°C for 10 min, 45 cycles of amplification at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s, with a single fluorescence acquisition. hGAPDH gene was used as an internal control. All experiments have been repeated twice.
SDS-PAGE and Western blot analysis
Cells were lysed with ice-cold lysis buffer (10 mM Tris, 1 mM EDTA, 1 mM DTT, 60 mM KCl, 0.5% NP-40 and protease inhibitors). Total cell lysates, fractions of supernatant or pellet were resolved on 10% and 12% polyacrylamide SDS gels under reducing conditions. The resolved-proteins were electrophoretically transferred to PVDF membranes (Amersham Life Science, Amersham, U.K.) for Western blot analysis. The membranes were blocked with 5% non-fat milk powder at room temperature for two hours, washed twice with PBST (1% Tween) and then incubated with primary antibody for 90 minutes at room temperature. The membranes were washed twice with PBST then subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (dilution at 1:10000, Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactivity was detected by Enhanced Chemiluminescence (Amersham International, Buckingham, U.K.) and autoradiography. All experiments have been repeated twice.
Proteasome activity assay
Cells exposed to various concentrations of 17-AAG and MG-132 for 24 h were washed twice with PBS and lysed with TNESV buffer [50 mM Tris-HCl (pH 7.5), 1% NP40 detergent, 2 mM EDTA, 100 mM NaCl, 10 mM sodium orthovanadate] without protease inhibitors. Cell lysate were assayed for proteasome chymotrypsin activity using the synthetic fluorogenic peptide chymotrypsin substrate, N-Succinyl-Leu-Leu-Val-Tyr-AMC. Fluorescent signals were measured with a 96-well plate reader with an excitation wavelength of 380 nm and emission wavelength of 460 nm. All experiments have been performed as triplicate and repeated twice.
Target-validated siRNA oligos (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into cells using the Lipofectamine-2000 reagent (Invitrogen, Carlsbad, CA). Briefly, cells were seeded onto 96-well plates or chamber-slides, and cultured overnight in 100 μl of antibiotic-free RPMI media. siRNA oligomers (8 pmol in 0.4 μl) were diluted in 25 μl of Opti-MEM® I medium (Invitrogen, Carlsbad, CA) without serum, and then mixed with 0.2 μl of Lipofectamine-2000 transfection reagent for 25 min at room temperature. Cells were overlaid with the transfection mixture, and incubated for various times.
MTT cell viability assay
Cells seeded onto 96-well plates were transfected with/without survivin-specific siRNA oligomer for 48 h and subsequently treated with 17-AAG for 24 h. 25 μl of MTT (5 mg/mL) was added to each sample and incubated for 4 hours, under 5% CO2 and 37°C. 100 μl of lysis buffer (20% SDS, 50% DMF) was subsequently added into each sample and further reacted for 16 hours.
C. H. A. Cheung, Ph.D. (Post-doctoral research fellow, molecular biologist)
H. H. Chen, Ph.D. (Post-doctoral research fellow, molecular biologist)
L.T. Cheng, Ph.D. (Post-doctoral research fellow, molecular biologist)
K.W. Lyu, M.Sc. (Research student)
J.R. Kanwar, Ph.D. (Principle investigator, Assistance professor, molecular biologist)
J. Y. Chang, M.D. (Distinguished investigator, Professor, medical oncologist)
This work was supported by intramural grants NSC98-2323-B-400-004 from the National Science Council, Taiwan R.O.C.; DOH99-TD-C-111-004 from the Department of Health, Taiwan R.O.C. and CA-097-PP-02 from the National Health Research Institutes, R.O.C.
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