17-Allylamino-17-demethoxygeldanamycin induces downregulation of critical Hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells
© Karkoulis et al; licensee BioMed Central Ltd. 2010
Received: 4 May 2010
Accepted: 9 September 2010
Published: 9 September 2010
17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin antibiotic, specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in cellular signaling. In this study, we have investigated the effect of 17-AAG on the regulation of Hsp90-dependent signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cell lines.
We have used MTT-based assays, FACS analysis, Western blotting, semi-quantitative RT-PCR, immunocytochemistry and scratch-wound assay in RT4, RT112 and T24 human urinary bladder cancer cell lines.
We have demonstrated that, upon 17-AAG treatment, bladder cancer cells are arrested in the G1 phase of the cell cycle and eventually undergo apoptotic cell death in a dose-dependent manner. Furthermore, 17-AAG administration was shown to induce a pronounced downregulation of multiple Hsp90 protein clients and other downstream effectors, such as IGF-IR, Akt, IKK-α, IKK-β, FOXO1, ERK1/2 and c-Met, resulting in sequestration-mediated inactivation of NF-κB, reduced cell proliferation and decline of cell motility.
In total, we have clearly evinced a dose-dependent and cell type-specific effect of 17-AAG on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of multiple Hsp90 clients and subsequent disruption of signaling integrity.
Urinary bladder cancer is the fifth most common malignancy in the industrialized world and the second most frequent malignancy of the genitourinary tract, demonstrating high heterogeneity and differential response to clinical treatment [1, 2]. Bladder cancer incidence, morbidity and mortality rates vary by genetic background, country, gender and age . The most prevalent type of bladder cancer in the developed world is urothelial carcinoma (UC), representing over 90% of all bladder cancers, followed by squamous cell carcinoma (5%) and adenocarcinoma (2%) . A high percentage of bladder cancer patients (20-30%) present with an aggressive muscle-invasive tumor of low differentiation, whereas the rest develop superficial, highly differentiated, non-invasive papillary tumors, 30% of which, nevertheless, are estimated to recur to invasive. Unfortunately, more than half of the patients with invasive tumors will develop distant metastases over a time period of two years , while the five-year survival rate for metastatic disease is as low as 6%. This apparent heterogeneity in bladder cancer is thought to be mainly due to discrete genetic alterations involved in tumor development and progression. Thus, since established systemic chemotherapy protocols for metastatic urothelial carcinoma are associated with significant toxicities, new clinical protocols designed for higher efficiency, while reducing the adverse side effects, are urgently needed.
Relatively recently, heat shock protein 90 (Hsp90) has emerged as an important target in cancer therapy. Hsp90 normally accounts for approximately 1-2% of the total cytosolic protein content, while under stress conditions, its levels increase up to 4-6% of the whole proteomic load of the cell [6–8]. The Hsp90 chaperone activity relies on its transient NH2-terminal dimerization, which facilitates its intrinsic ATPase activity . The Hsp90 chaperone complex maintains the correct folding, cellular localization and activity of a broad range of protein clients that are implicated in various signal transduction pathways involved, among others, in cell proliferation, differentiation and survival [7, 10]. There is evidence that Hsp90 is a major facilitator of cellular response to extracellular signals, particularly required for normal cell growth, proliferation and development . On the other hand, over-expression and/or presence of mutations in a variety of Hsp90 protein clients during cancer initiation is associated with a requirement for increased Hsp90 levels in order to maintain the active conformations and thus functional integrities of these oncogenic molecules. In this frame, Hsp90 is a key molecule in the conformational maturation of several bona fide oncogenic signaling proteins, such as HER2/ErbB2, Akt, Met, Raf1, p53 and HIF-1α [10, 12]. Therefore, due to the dependence of cancer cells upon specific Hsp90 oncogenic protein clients, inhibition of Hsp90 was shown to be able to negatively interfere with a number of important signaling pathways involved in cell development, proliferation, survival and motility, arousing significant interest in the field of cancer therapeutics .
Thus, a diverse group of molecules that target Hsp90 have been discovered or synthesized over the past several years. These include natural products, such as geldanamycin, radicicol and derivatives; synthetic purine-based inhibitors, such as PU3, PU24FCI and PU29FCI; and compounds that bind to Hsp90 on a secondary ATP-binding site, such as novobiocin and cisplatin . The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) possesses an allylamino group at position 17 of the scaffold structure of geldanamycin . Compared to the parental compound, 17-AAG demonstrates reduced toxicity, with enhanced biological activity and metabolic stability, retaining the Hsp90-related therapeutic characteristics. 17-AAG exerts its anti-tumor potency through its high affinity binding to the NH2-terminal ATP-interacting domain of Hsp90, thus inhibiting its ability to form transient, active homodimers, and to consequently participate in chaperone-client complexes, with a subsequent hindering of client maturation and stabilization.
In this context, here, we have thoroughly studied the effects of Hsp90 inhibition by 17-AAG on the Hsp90-assisted signaling repertoire associated with cell cycle progression, apoptosis and motility in three human urinary bladder cancer cell lines of different malignancy grade, namely RT4 (grade I), RT112 (grade I-II) and T24 (grade III).
Drugs and reagents
17-AAG chemotherapeutic reagent was obtained from InvivoGen (San Diego, California, USA). Polyclonal and monoclonal antibodies against Caspase-8, Caspase-9, Caspase-3, PARP, Lamin A/C, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-IGF-ΙRβ (Tyr1131), IGF-ΙRα, FOXO, phospho-FOXO, phospho-IKKα/β (Ser180/Ser181), IKKα, IKKβ, phospho-p44/42 (Thr202/Tyr204), p44/42, α-tubulin, phospho-c-Met (Tyr1234/Tyr1235), c-Met, CHIP and pan-actin were purchased from Cell Signaling Technology Inc. (Hertfordshire, UK), whereas antibodies against Hsp90α/β, Hsp70, Cdk4, pRb, E2F1 and NF-κB (p65) were supplied by Santa Cruz Biotechnology Inc. (California, USA). Enhanced Chemilluminescence (ECL) Western blot detection reagents were obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Oligonucleotide primers were synthesized by Operon (California, USA). All other chemicals were of analytical grade from Sigma-Aldrich (Missouri, USA), Fluka (Hannover, Germany) and AppliChem GmbH (Darmstadt, Germany).
Cell lines and culture conditions
The present study was performed on three human urinary bladder cancer cell lines, namely RT4, RT112 and T24, all originating from urothelial carcinomas. RT4 cells are derived from grade I tumor and were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK); RT112 cells are derived from a grade I→II tumor, whereas T24 cells are derived from a grade III tumor. RT112 and T24 cells were a generous gift from Professor J. R. Masters (Prostate Cancer Research Centre, Institute of Urology, University College London, UK). Cells were maintained in DMEM, supplemented with 10% heat inactivated FBS, at 37°C in a humidified 5% CO2 atmosphere. All cell culture media and reagents were supplied by Biochrom AG (Berlin, Germany).
Cell viability assay
Urinary bladder cancer cells were seeded at a density of 15-20 × 103 per well into 48-well plates and treated with various drug concentrations for 24 h. The next day, cells were incubated in methylthiazole tetrazolium (MTT) solution. The spectrophotometric absorbance was measured in an ELISA microtiter plate reader (Dynatech MR5000, Dynatech Laboratories, Virginia, USA) at 550 nm, using measurement at 630 nm as reference. Absorbance rates obtained by untreated cells were considered as 100% cell survival. Each assay was repeated at least three times, using three wells per drug concentration in each experimental condition.
Cell cycle analysis
Bladder cancer cells were seeded at a density of approximately 5 × 105 in 100 mm plates and drug treatments of different concentrations of 17-AAG were applied for 24 h. Cells were collected, fixed in 1% methanol-free formaldehyde for 20 min and subsequently suspended in a 70% ethanol solution and stored at -20°C to dehydrate. Twenty four hours later, cells were suspended in 1 ml of 0.1% Triton X-100 solution and incubated in 500 μl of propidium iodide solution (50 μg/ml) containing 250 μg of DNase-free RNase A. Cells were analyzed with a Beckton Dickinson's FACScalibur (California, USA) at 542 nm and results were processed with the Modfit software program. Each assay was repeated three times.
Whole cell protein extracts were prepared as previously described . Approximately 30 μg of total protein preparations were resolved by SDS-polyacrylamide gel electrophoresis and subsequently electro-transferred overnight onto nitrocellulose membranes of 0.45 μm pore size (Schleicher and Schuell GmbH, Dassel, Germany). Membrane blocking was performed in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl and 0.1% Tween-20) containing 5% non-fat dry milk (or 5% BSA grade V, where appropriate) and membranes were incubated with the appropriate antibodies at room temperature for 90 min, followed by an overnight incubation at 4°C. The next day, membranes were incubated with the suitable anti-mouse or anti-rabbit HRP-conjugated secondary antibody and immunoreacting proteins were detected using an ECL Western blotting kit according to the manufacturer's instructions. All immunoblotting experiments were repeated three times.
Total RNA from both control and treated cells was extracted as previously described . 1 μl of cDNA solution was amplified by PCR in a total volume of 25 μl, using cDNA-specific primers corresponding to the various mRNA species examined in the present study. Most gene-specific cDNA primer sequences and associated PCR information have been previously described [14, 15], whereas additional genes amplified for the purpose of this study were: Cyclin D1 (forward: 5'-GTGTCCTACTTCAAATGTGTGC-3', reverse: 5'-GGAGTTGTCGGTGTAGATGC-3', Ta: 57°C, 30 cycles), Hsp90α (forward: 5'-CCAAGATGCCTGAGGAAAC-3', reverse: 5'-TCATACCGGATTTTGTCCAAT-3' Ta: 53°C, 30 cycles) and Hsp90β (forward: 5'-TCCTTTTCTTTTCAAGATGCC-3', reverse: 5'-TGTCCAACTTCGAAGGGTCT-3', Ta: 54°C, 30 cycles). The obtained PCR fragments were resolved in 2% agarose gels, according to standard procedures. All RT-PCR experiments were repeated three times.
T24 urinary bladder cancer cells were seeded on poly-L-lysine coated slides (Thermo Fisher Scientific Inc., Minnesota, USA) and treated with a 17-AAG concentration of 10 μM for 24 h. After treatment, slides were fixed with a paraformaldehyde solution (3% in 1 × PBS) for 15 min at room temperature. Cell permeabilization was achieved by administration of a Triton X-100 solution (0.5% in 1 × PBS) for 20 min. Subsequently, slides were blocked with a 1% BSA solution for 60 min and then incubated with an NF-κB anti-p65 antibody overnight at 4°C. The next day, slides were incubated with a FITC-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories Inc., Pennsylvania, USA), while nuclear staining of cells was obtained by incubation with propidium iodide solution (1 μg/ml in 1 × PBS containing RNase A). Finally, cells were observed under a Nikon EZ-C1 confocal microscope (Nikon Instruments Inc., Japan). Images taken were processed with the support of the Nikon EZ-C1 software program. Immunofluorescence experiments were repeated three times.
Human urinary bladder cancer cells were seeded at a density of 5 × 105 per 100 mm diameter Petri dish and incubated overnight. The day after, the surfaces of the dishes were mildly scratched with a sterile Pasteur pipette and images were taken under a Carl Zeiss Axiovert 25 (Thornwood New York, USA) inverted microscope with the use of a Cannon Powershot G9 digital camera and a PS-Remote software program. Then, cells were treated with a 10 μΜ 17-AAG solution and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. Twenty four hours later, treated and untreated cells were observed under the inverted microscope at the scratch-wounded areas. Scratch-wound assays were repeated three times.
In order to study the cause of the 17-AAG-induced response pattern of Hsp90 in the three cell lines, we decided to analyze the expression of another member of the Hsp90 chaperone complex, namely Carboxyl terminus of Hsp70 interacting protein (CHIP), an E3 ubiquitin ligase, which regulates the turnover of Hsp90 protein clients in mammalian cells, but also Hsp90 itself, via ubiquitination of specific residues of the chaperone, therefore making it a suitable candidate for proteasomal degradation [16–18]. In the bladder cancer cell lines used in this study, CHIP showed a dose-dependent and cell type-specific decrease in response to 17-AAG administration, with RT4 and RT112 cells exhibiting the most notable reduction, whereas CHIP protein control levels were found to steadily increase from RT4 to RT112 and then T24 cells. Pan-actin was used as protein of reference in all experiments performed herein, whereas α-tubulin, interestingly, appeared to follow an expression pattern very similar to that of Hsp90. This is consistent with the recently discovered association between tubulin and the Hsp90 chaperone complex .
Finally, we examined the transcriptional profiles of Hsp90α and Hsp90β genes in response to the drug, in order to identify a possible association of 17-AAG-induced Hsp90 downregulation with transcriptional repression of Hsp90 genes. In this frame, Hsp90 mRNA expression levels were tested and found to remain unaffected in all the cell lines used here (Figure 6B), thus excluding any type of transcriptional control involvement in the 17-AAG-induced downregulation of Hsp90 protein.
A prominent downstream target of the IGF-I receptor and distinguished protein member of the Hsp90 clientele is the Akt kinase, a critical regulatory component in many signaling pathways. Upon administration of 17-AAG, Akt proved to be downregulated in all three bladder cancer cell lines, in a dose-dependent manner (Figure 7). Interestingly, in RT112 cells we were able to observe the formation of a lower molecular weight fragment, possibly representing a cleavage product of the intact Akt kinase (55 kDa, approximately), specifically generated after exposure to 1 and 10 μM 17-AAG. Next, we tested the presence of phosphorylated Akt in the same cells before and after 17-AAG treatment. Even though phosphorylation of Akt on serine residue at position 473 (Ser473) could not be detected in RT4, it was marginal in RT112, and highly activated in T24 cells. In RT112 and T24 cell lines, Akt phosphorylation showed a dose-dependent decrease, resulting in an almost total elimination of the active form of the protein from drug concentrations higher than 0.1 μM for RT112 and 1 μM for T24 cells. Akt phosphorylation on threonine residue at position 308 (Thr308) ranged from absent (RT4 and T24) to marginal (RT112) levels.
Furthermore, we studied the effect of 17-AAG on the Ras-Raf-MEK-ERK pathway (known to usually cross-talk with Akt) in bladder cancer cells, by detection of total and phosphorylated p44/42 (Erk1/2) kinase protein levels. As illustrated in Figure 9, upon 17-AAG administration, total p44/42 levels in both RT4 and RT112 cell lines exhibited a pattern reminiscent of the one previously encountered in Hsp90, α-tubulin and FOXO4. More precisely, in RT4 cells, p44/42 protein levels displayed a pattern of dose-dependent downregulation up to 1 μM concentration of the drug, whereas a significant increase could be observed in the highest dose (10 μM). In RT112 cells, the pattern was similar, but shifted to lower concentrations. Thus, total p44/42 protein levels were found to manifest a drug-mediated reduction in the lower concentrations only, whereas in the higher ones a clear increase could be observed. On the contrary, in T24 cells, a slight but notable dose-dependent decrease of p44/42 expression levels was observed. In order to evaluate the potency of p44/42 signal transduction upon exposure to 17-AAG, the active form of the protein (phosphorylated on threonine and tyrosine residues at positions 202 and 204, respectively) was analyzed. As shown in Figure 9, all three cell lines demonstrated a severe dose-dependent reduction of active p44/42, therefore causing the downregulation of a variety of downstream targets, mainly involved in cell proliferation and survival. In toto, 17-AAG proved to induce a prominent inhibitory effect upon multiple Hsp90 clients, affecting both the NF-κB and the FOXO axes of the IGF-IR/Akt signaling repertoire, as well as the p44/p42-dependent pathway, likely promoting the downregulation of downstream targets and finally leading to decreased cell proliferation and survival.
To further illuminate the effect of 17-AAG on the efficiency of bladder cancer cell motility, we have conducted scratch-wound assays on all the cell lines examined herein (Figure 10B). As clearly demonstrated in this work, the low malignancy grade cell lines RT4 and RT112 presented with reduced proliferation and motility potency, unable to heal the "wounds" during a 24-hours incubation period, either under high 17-AAG concentration (10 μΜ) or control conditions (Figure 10B, RT4: panels i-iv, RT112: panels v-viii). In contrast, the highly aggressive (grade III) T24 cells were characterized by a prominent efficiency in motility, being able to successfully "heal the wound" in an incubation period of 24 hours, creating a compact monolayer of cells (Figure 10B, panels ix and x). Although administration of 10 μΜ 17-AAG was not able to abrogate T24 proliferation and motility responses, it is clear that the scratch-wound "healing" mechanism in these cells has been significantly impaired due to the effect of the drug, since cells appeared to maintain the gap without being tightly condensed as initially observed under control conditions (Figure 10B, panels xi and xii).
Human urinary bladder cancer is considered an increasingly significant public health issue in the industrialized countries, with a worldwide estimate of about two million patients . Due to the importance of Hsp90 molecular chaperone on client protein maturation and function, along with its voluminous and highly diverse clientele of cancer-related proteins, a variety of Hsp90 inhibitors have emerged as promising anticancer agents [6, 12]. In the present study, we have comparatively examined the effects of 17-AAG-induced Hsp90 inhibition on multiple protein targets implicated in signaling pathways critically regulating cell proliferation, apoptosis and motility, in RT4 (grade I), RT112 (grade I-II) and T24 (grade III) human urothelial bladder cancer cells. The data presented herein clearly demonstrate that, upon 17-AAG treatment, cell type-specific downregulation of multiple signaling molecules is followed by cell cycle arrest, finally resulting in Caspase-mediated cell death.
Depending on the cellular context and malignancy grade, 17-AAG has been shown to facilitate arrest in all checkpoints of the cell cycle, as for example, in human malignant pleural mesothelioma (G1 or G2/M block)  and breast cancer cells (G1 block) overexpressing HER2 . In all human bladder cancer cell lines examined in this study, apoptotic cell death was found to be preceded predominantly by a drug dose-dependent G1/S cell cycle block, with arrest in other phases of the cell cycle appearing in a cell type-specific manner. The unpredictability of cell cycle arrest induced by 17-AAG in bladder cancer cells is in agreement with previous reports and might be related to differences in client protein repertoires and cellular contexts . To elucidate the 17-AAG-induced block of the cell cycle, we undertook analysis of expression and/or activation profiles of several key-modulators of cell cycle progression. This demonstrated that, in response to 17-AAG exposure, the drug-dependent protein downregulation patterns correlate well with the observed G1 arrest of the cell cycle, as well as with the reduction in cell proliferation capacity.
Implementation of apoptosis, due to the effect of 17-AAG, has previously been reported in glioblastoma  and colon cancer . In the bladder cancer cell lines used in this study, cell type-specific and drug dose-dependent activation of a Caspase-induced cell death program proved to be initiated upon 17-AAG administration. These findings are in accordance with the survival rates observed in the cytotoxicity tests, although, in these experiments, 17-AAG-induced cell death percentages in the three bladder cancer cell lines were not found to differ significantly. In contrast, the cell-type specific profile of Caspase repertoire activation, and especially the diminished levels of processed Caspase-3 in RT112 and T24 cells, could possibly implicate other types of executive Caspases not studied here (i.e. Caspase-6 or -7) or even Caspase-independent cell death mechanisms such as autophagy [25, 26].
Hsp90 expression levels seem to be upregulated in cancer, resulting in addiction of tumor cells to multiple oncogenic pathways in which Hsp90 clients play a critical role. In bladder cancer, Hsp90 was found to be expressed in more than 90% of human tumor specimens, with high-grade and muscle invasive tumors expressing significantly higher levels of Hsp90 than low-grade and superficial tumours . Nevertheless, in 10% of the tumor samples Hsp90 expression was found to be downregulated and this was associated with infiltrating recurrences and poor prognosis [28, 29], most likely due to the overall molecular profile of the individual tumors. Besides the importance of Hsp90 expression levels, specific conformations of the chaperone have been implicated in cancer versus normal cell sensitivity to Hsp90 inhibitors: Hsp90 was shown to display higher binding affinity for 17-AAG exclusively in cancer cells , leading to the formation of 17-AAG-sensitive Hsp90-containing "superchaperone" complexes in malignant cells, whereas normal cells bearing a predominantly uncomplexed Hsp90 are significantly less sensitive to these types of inhibitors [13, 30]. This feature is likely exploited by Hsp90 targeting with the use of 17-AAG and subsequent effects on multiple Hsp90 targets.
Hsp90 inhibition and subsequent Hsp70 and Hsp27 upregulation, due to 17-AAG, have been reported in human colon , prostate  and cervical cancer cells . As presented in this study, even though a 17-AAG-induced Hsp90 downregulation was detected in all bladder cancer cell lines over a 24-hours treatment period, a cell type-specific pattern of inhibition was observed. In RT4 and RT112 cells, after exposure to the highest dose of the drug, an additional protein band was generated, whereas no such band could be detected in T24 cells. This novel finding in relation to Hsp90 structural integrity, upon high dose of 17-AAG administration, is presented herein for the first time. We suggest that this fragment may well be a product of Hsp90 proteolytic processing by Granzyme B [14, 15]. Use of the GrabCas algorithm has revealed a putative Granzyme B recognition and cleavage site in the amino acid sequence of both Hsp90α and Hsp90β protein isoforms, indicating that Hsp90 must be a bona fide substrate of Granzyme B. On the contrary, no Caspase cleavage site could be identified, with the help of GrabCas, fitting to the molecular weight of the possible Hsp90 cleavage fragment under discussion. Interestingly, Hsp90 cleavage has been reported previously, as a response to oxidative stress factors , arsenic-based compounds  and exposure to doxorubicin and cisplatin chemotherapeutic agents [14, 15]. Yet, it is not known whether the putative cleavage product is associated, somehow, with malignancy grade or p53 genetic status of the cells, since RT4 and RT112 are grade I and I-II, respectively, harboring a wild-type p53, whereas T24 are grade III, bearing a mutant p53 (Figure 6A). Intriguingly, the RT4- and RT112-specific production of a ~ 65 kDa putative proteolytic fragment could further enhance the functional amputation effect of 17-AAG on Hsp90, likely acting as a putative dominant negative component able to severely impair Hsp90 chaperoning properties. Thus, despite the Hsp90 upregulation observed in response to the highest 17-AAG concentration in grade I (RT4) and I-II (RT112) cell lines, the protein, due to its functional titration by the ~ 65 kDa processed product, seems unable to support its numerous clients thoroughly analyzed here. Therefore, we suggest that the chaperosomes containing these Hsp90 truncated forms are most likely inefficient to exert their cellular tasks.
The three bladder cancer cell lines seemed to follow a distinct and cell type-dependent downregulation profile of the Hsp90 molecular chaperone. However, as shown herein, despite 17-AAG administration, gene expression at the level of transcription remained unaffected for both isoforms of Hsp90 (α and β), clearly indicating that the regulation of Hsp90 is beyond transcriptional control, but occurs more likely at the post-translational level, via ubiquitination and subsequent proteasomal degradation or autophagy.
Hsp90 inhibition was suggested to be tightly associated with a compensatory upregulation of Hsp70  and/or Hsp27 protein levels, likely inducing resistance to 17-AAG . In this work, upon exposure to 17-AAG, total Hsp70 expression levels proved to exhibit a dose-dependent increase and generation of an ~ 65 kDa protein fragment in all three cell lines, reaching peak value at dose 10 μΜ. Using the GrabCas software, we propose that, similarly to Hsp90, the lower molecular weight band could likely represent a product derived from Hsp70 proteolytic processing by 17-AAG-induced Granzyme B activity, but not Caspase protease function.
CHIP was studied in order to illuminate the intriguing pattern of Hsp90 protein level alterations after 17-AAG treatment. CHIP levels were found to be downregulated in a dose-dependent manner in all three bladder cancer cell lines, suggesting a CHIP-regulated effect on proteasomal degradation of associated target proteins, such as Hsp90 and its clients. However, the higher dose-dependent upregulation of Hsp90 and α-tubulin implies a likely redundant, or non-essential, role of CHIP and, therefore, other ubiquitin ligases must be critically implicated in this type of response. An alternative scenario is that affinity threshold phenomena are at play here, with CHIP, although downregulated, still being able to implement its ubiquitin ligase activities regarding Hsp90 clients, but not Hsp90 itself.
The critical role of IGF-IR/Akt signaling pathway deregulation in tumor cell proliferation, survival and migration has been well documented . It has been previously reported that 17-AAG administration causes severe inhibition of the Akt-dependent signaling pathways in osteosarcoma  and gastric cancer . As demonstrated here, in human urinary bladder cancer cells, 17-AAG-induced inhibition of Hsp90 resulted in a cell type-specific downregulation of several proteins involved in Akt-dependent signaling, critically contributing to the negative regulation of proliferation, survival and motility. As a consequence, NF-κB transcription null activation potential was significantly compromised, mainly due to the sequestration of the factor into the cytoplasm, as clearly illustrated in Figure 8A. Reduced NF-κB activity was indirectly assessed by measuring the mRNA expression levels of Survivin and cIAP1, two well known bona fide NF-κB target genes. Thus, we have demonstrated that 17-AAG-dependent inhibition of NF-κB activity is tightly associated with transcriptional repression of Survivin and cIAP1 anti-apoptotic genes, thus decisively contributing to the cytotoxic potency of 17-AAG by decreasing the required "apoptotic threshold" in bladder cancer cells .
Moreover, 17-AAG-mediated Hsp90 inhibition resulted in alterations of the phosphorylation status of members of the Forkhead family of transcription factors (FOXO), immediate downstream substrates of Akt kinase, in bladder cancer cells. As shown in this study, FOXO factors proved to be strongly phosphorylated in the highly malignant T24 cells, whereas extremely low, but detectable, levels were also observed in RT112 cells. Administration of 17-AAG caused a notable downregulation of phosphorylated FOXO1 and FOXO3 family members, likely inducing an enhancement of their apoptotic activity.
Interestingly, the undetectable phosphorylation of the IGF-I-dependent downstream mediators (i.e. IGF-IR, Akt, IKKs and FOXOs) in RT4 cells strongly suggests the deactivated character of the pathway under the particular growth conditions, whereas, on the contrary, in T24 cells the IGF-IR/Akt pathway seems to be constitutively activated. RT112 cells proved to display an intermediate pattern of signaling potency, with the IGF-IR/Akt pathway being activated at very low levels. This novel finding of cell type-specific activation of the IGF-IR/Akt-dependent signaling repertoire, herein demonstrated for the first time, could be tightly associated with the underlying differences in various features of the malignant phenotype observed in the three bladder cancer cell lines examined.
Hsp90 inhibition and ensuing Akt inactivation in bladder cancer cells was accompanied by downregulation of Erk1/2-dependent signaling. Exposure to 17-AAG has been previously reported to cause inhibition of the Raf/MEK/ERK signaling cascade in Hodgkin's lymphoma  and leukemia . Although total Erk1/2 protein levels exhibited a cell type-specific and drug dose-dependent response similar to the one of α-tubulin and Hsp90, phosphorylated p44/42 levels were severely downregulated in all bladder cancer cell lines, implying the differential control between total and phosphorylated protein destabilization processes in response to the high drug dose treatments.
Invasion and metastasis are one of the hallmark traits of cancer involved in the advanced stages of tumor progression. Hsp90 inhibition by ansamycins has been reported to suppress cancer cell motility and invasion through depletion of the HGF/c-Met signaling pathway in both leiomyosarcoma and glioblastoma cell lines . Another novel finding of the present study is the notable expression and constitutive activation of c-Met receptor in T24 bladder cancer cells, whereas in RT4 and RT112 cells total c-Met protein levels were either absent (RT4) or barely detectable (RT112). Finally, in this study, we have clearly demonstrated that T24 cell line expresses high amounts of phosphorylated IGF-IR, Akt, FOXOs, p44/42 and c-Met proteins and exhibits strong migration dynamics, which could well be associated with a more invasive and metastatic potency, exactly as a result of this over-activated signaling network. Nevertheless, we have shown that the inhibitory effect of 17-AAG on T24 cells is reflected on the significant decrease of both total and phosphorylated c-Met protein levels, with subsequent suppression of other oncogenic parameters, such as increased cell proliferation and motility, hence critically contributing to the impairment of aggressive cancer cell phenotype [41–43].
We are deeply grateful to E.G. Konstantakou, Ph.D. candidate (Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou, Athens, Greece) for her valuable assistance in the maintenance of cell cultures and scientific advice. We would like to thank Dr. Vasiliki Labropoulou (Laboratory of Molecular Genetics of Insects and Biotechnology, Institute of Biology, NCSR "Demokritos", Athens, Greece) for the provision of the anti-p65 (NF-κB) antibody. Many thanks to Dr. Harris Pratsinis (Laboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR "Demokritos", Athens, Greece) for his assistance in cell cycle analysis and to Dr. Dimitrios Kletsas (Head Researcher of the Laboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR "Demokritos", Athens, Greece) for his valuable suggestions during the implementation of this study. P.K. Karkoulis is financially supported by a fellowship awarded from the Institute of Biology, NCSR "Demokritos", Athens, Greece (Ph.D. fellowship). Financial support was provided from the Greek Ministry of Health and Social Solidarity (ΔY2β/OIK.98909/17-07-2008) and the Hellenic Society of Medical Oncology (HESMO) (1804/20-03-2009).
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