HSP90 is a promising target in gemcitabine and 5-luorouracil resistant pancreatic cancer
Tarik Ghadban1 · Judith L. Dibbern1 · Matthias Reeh1 · Jameel T. Miro1 · Tung Y. Tsui2 · Ulrich Wellner3 · Jakob R. Izbicki1 · Cenap Güngör1 · Yogesh K. Vashist1,4
Abstract
Chemotherapy (CT) options in pancreatic cancer (PC) are limited to gemcitabine and 5-luorouracil (5-FU). Several identiied molecular targets in PC represent client proteins of HSP90. HSP90 is a promising target since it interferes with many oncogenic signaling pathways simultaneously. The aim of this study was to evaluate the eicacy of diferent HSP90 inhibitors in gemcitabine and 5-FU resistant PC. PC cell lines 5061, 5072 and 5156 were isolated and brought in to culture from patients being operated at our institution. L3.6pl cell line served as a control. Anti-proliferative eicacy of three diferent HSP90 inhibitors (17-AAG, 17-DMAG and 17-AEPGA) was evaluated by the MTT assay. Alterations in signaling pathway efectors and apoptosis upon HSP90 inhibition were determined by western blot analysis and annexin V/PI staining. The cell lines 5061, 5072 and 5156 were resistant to gemcitabine and 5-FU. In contrast 17-AAG and the water-soluble derivates 17-DMAG and 17-AEPGA displayed high antiproliferative activity in all tested cell lines. The calculated IC50 was below 1 µM. Highly signiicant down regulation of epidermal-growth-factor-receptor, insulin-like-growthfactor-receptor-1, AKT and MAPK relected the intracellular molecular signaling-network disruption. Furthermore, besides HSP70 also HSP27 was upregulated in all cell lines. Apoptosis occurred early under HSP90 inhibition and was determined by annexin V/PI staining and CASPASE-3 and PARP assay. In contrast, gemcitabine treated cells did not show any apoptosis. HSP90 inhibition disrupts multiple signaling cascades in gemcitabine and 5-FU resistant PC simultaneously and promotes cancer cell apoptosis. Watersoluble 17-DMAG is equally efective as 17-AAG. HSP27, besides HSP70, may represent an efective response marker of successful HSP90 inhibition.
Keywords HSP90 · Pancreatic cancer · Anti-tumor therapy · Geldanamycin · Target therapy
Introduction
Pancreatic cancer (PC) has poor prognosis among gastrointestinal cancers with a 5-year survival rate of <5%. Median survival without surgical resection is <5 months and for patients undergoing surgery between 13 and 18 months only. Surgery is still considered as the only curative option [1, 2]. However, recent reports on histo-pathological work up of the operative specimen indicate that the majority (>80%) of all PC resections are in truth R1 resections [3, 4]. Hence, chemotherapy (CT) is essential, not only in metastatic PC, but also in patients who have undergone surgical resection in curative intention [5]. Currently used chemotherapeutic agents include DNA-damaging compounds such as gemcitabine, 5-luorouracil (5-FU) and oxaliplatin. The survival beneit is only modest due to high degree of intrinsic and acquired resistance [6–10]. Molecular proiling of PC has revealed several promising targets. The epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) as well as the SerineThreonine speciic protein kinases AKT and Mitogen-activated protein kinase (MAPK) signaling cascade seems to be essential for cell proliferation and survival in PC [11– 15]. Till date, only the EGFR kinase inhibitor Erlotinib has been evaluated in PC. Erlotinib has shown survival beneits in combination with gemcitabine though the overall efect remains modest [16]. Hence, a truly efective cytotoxic therapy for PC is still missing.
Heat shock proteins (HSP), a set of highly conserved proteins, are induced by diferent kinds of stress. They represent molecular chaperones and have strong cytoprotective attributes. HSP90 is one of the most abundant chaperone in the cytosol of all eukaryotic cells and maturation of a wide range of efectors, so called client proteins (CP), which are involved in signal transduction, cell growth, diferentiation and survival are dependent on HSP90 chaperone activity [17–19].
In cancer cells, HSPs ensure stabilization and protection of overexpressed or mutated signal transduction proteins that promote cell growth and survival [18, 20–22]. In recent years, among all known HSPs, HSP90 has emerged as a promising target in oncology particularly with regard to the availability of several HSP90 inhibitors including water-soluble formulations. In addition, as shown by Kamal et al., this inhibition nearly exclusively takes place in cancer cells only [22]. HSP90 is known to be overexpressed and highly active in various cancer cells including PC [23]. Many growth promoting anti-apoptotic factors and signaling transducers identiied in PC represent CP of HSP90 [21].
EGFR and IGF-1R inhibitors as well as AKT and MAPK cascade inhibitors are currently under investigation as potential drugs in PC [24, 25]. However, only EGFR inhibitors have been clinically tested so far. In addition, inhibition of EGFR tyrosine kinase does not necessarily exclude the activation of AKT and MAPK signaling pathways. Cancer cells are known to switch to a diferent molecular pathway to prevent apoptosis. Geitinib inhibits PC cell growth through EGFR dependent pathway but does not inluence IGF-1R mediated proliferation [26]. Taken the crosslinks into account we proposed that the inhibition of HSP90 may be valuable for targeting PC that is resistant to gemcitabine and/or 5-FU.
The best characterized HSP90 inhibitor is the geldanamycin derivate 17-allylamino-geldanamycin (17-AAG). However, the lack of solubility in physiological luids makes formulation for clinical delivery a challenge and limits its clinical use. Therefore, new water-soluble 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) and 17-[2-(Pyrrolidin-1-yl)ethyl]amino17-demethoxygeldanamycin (17-AEPGA) geldanamycin derivates have been developed [27, 28].
In the present study, we evaluated the antiproliferative efects of HSP90 inhibition on gemcitabine and 5-FU resistant PC cells in vitro. Furthermore, we focused on molecular alterations under HSP90 inhibition with special focus on key signaling nodules like growth receptors and signal transducer kinases.
Materials and methods
Ethical approval
The study was approved by the Ethics Committee of the Hamburg Chamber of Physicians, Germany, Approval-Nr PV3548.
Compounds
For in vitro experiments 17-DMAG (InvivoGen, ant-dgl) and 17-AEPGA (InvivoGen, ant-egl-1) were dissolved in water to a stock concentration of 1 mM and used at inal concentrations ranging from 0.1 to 10 μM. 17-AAG (InvivoGen, ant-agl) was dissolved in Dimethylsulfoxid (DMSO) to a stock concentration of 1 mM and the same inal concentrations (0.1–10 μM) were used. The stock solutions were stored at −20 °C.
Cell culture
The PC cell lines 5061, 5072 and 5156 were generated at our laboratory from patients that were treated for histological proven adenocarcinoma of the pancreas at the department of surgery. All patients were adjuvantly treated with gemcitabine and developed early recurrence. The overall survival ranged from 3 to 8 months.
Cell culture conditions and a detailed characterisation of the cell line 5061 have been published previously [29]. The cell lines 5072 and 5156 were established in the same way.Briely, small fragments of tumor tissue with a diameter of 1 mm were obtained by mincing the tumor specimen with a scalpel. The fragments were enzymatically disaggregated after incubation with 0.5% collagenase type IV (Sigma-Aldrich, Steinheim, Germany) solution at 37 °C on a rotary shaker. After 45 min, the solution was centrifuged at 700×g for 5 min, the pellet was collected, washed twice in cell culture medium (RPMI, Invitrogen, NY, USA) resuspended in complete medium (TUM), then plated into collagen-coated culture lasks (Becton Dickinson Labware, Bedford, MA, USA), and cultivated at 37 °C in a humidiied atmosphere with 5% CO2. Cells were grown in TUM medium [RPMI 1640 Medium (GIBCO) supplemented with 10% FBS (GIBCO), 1% Penicillin (GIBCO), 0.5% Transferrin (10 µmol/ml; SIGMA), 1% EGF (1 µg/ml;
Pepro Tech), 1% FGF (1 µg/ml; Pepro Tech), 1% Insulin (1 µg/ml; SIGMA) and 1% Gentamycin (10 mg/ml; Biochrom AG)]. Figure 1 shows the cell morphology of these cell lines.L3.6pl is a secondary cell line of an orthotopic mouse xenograft model, which we obtained from Prof. Bruns from the Ludwig-Maximilians-University Munich [30]. It was cultured in RPMI 1640 medium with 10% FCS (GIBCO), 1% Gentamycin (10 mg/ml; Biochrom AG) and 1% Penicillin/Streptomycin (GIBCO). All cells were incubated in humidiied atmosphere of 5% CO2 at 37 °C.
Evaluation of cell doubling time and determination of 50% cell growth inhibition (IC50)
Evaluation of the anti-proliferative eicacy of all three HSP90 inhibitors was determined in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric method, also known as the MTT reduction assay. This assay is based on the ability of viable cells to reduce MTT-tetrazolium salt into MTT-formazan by the mitochondrial enzyme succinate-dehydrogenase. Before initiating proliferation assay, cell doubling time of each tested cell line was evaluated with the MTT assay. Afterwards, 5061, 5072, 5156 and L3.6pl tumor cells were cultured in 96-well microtiter plates and seeded at densities predetermined. Ensuring cell conluence rates of >60%, fresh medium with or without each HSP90 inhibitor was added at diferent concentrations for 1, 2 or 3 doubling times. Then, 12.5 mL of an MTT solution in medium (5 mg/mL MTT; Sigma Chemical Co., St Louis, USA) was added for 3 h. The medium was removed and the MTTformazan crystals were solubilized by adding DMSO (100 mL/well). Absorbance (A562) was determined at 562 nm. Drug concentration inhibiting 50% (IC50) growth compared to untreated cells was calculated.
Proliferation assay
5156 and L3.6pl tumor cells were harvested by trypsinization, counted and 100 µL of cell suspension were illed in each well of 96 well plates at concentration of 50 × 103 cells/well. Ensuring cell conluence rates of >60%, fresh medium with or without 17-AAG, 17-DMAG and 17-AEPGA was added at concentrations from 0.1 to 5 µM for 72 or 96 h. MTT assay was processed as described above. Results were expressed as percentage of the control. The absorbance of the control (cell culture without any treatment) corresponds to 100% MTT reduction. Six independent experiments were performed for each cell line and data were presented as mean ± SD.
Western blotting for CP reduction and apoptosis
For Western blotting (WB), 5 ml of cell suspension with density of 0.6 × 106 cells/ml were transferred to dishes with 6 cm diameter and incubated overnight until >60% conluence was reached. Then cells were treated with 17-AAG, 17-AEPGA or 17-DMAG at concentration 1 and 5 µM for 48 h. Afterwards, cells were washed with DPBS (GIBCO 14190) and harvested with EDTA (Biochrom AG, L2113). Cells were lysated in a solution comprising lysis bufer, protease inhibitor cocktail and benzonase. Protein content was determined using the BCA Protein Assay Kit (PIERCE) and equal amounts of protein (30 µg/volume) were loaded onto a 12% Acrylamide gel containing H2O, 30% Acrylamide/Bis (Bio Rad Laboratories), 1.5 M tris bufer pH 8.8, 10% SDS (GIBCO), 10% APS and TEMED (Bio-Rad). For protein separation SDS-PAGE was used, followed by the transfer onto a nitrocellulose membrane [Bio-Rad, TransBlot Transfer Medium, Pure Nitrocellulose Membrane (0.45 µm)]. Afterwards, blots were incubated with antibodies and chemiluminescence was detected using Super Signal West Dura Extended Duration Substrate (Pierce). The following antibodies were used: EGFR1, IGF-1R, p44/42 MAPK (Erk1/2), AKT Cleaved Caspase-3, Cleaved PARP and HSP27 (Cell Signaling). HSP90 and HSP70 were obtained from Stressgen. Actin (Sigma) was used as loading control.
Detection and quantiication of apoptosis using Annexin V/PI staining
To further dissect the efects of HSP90 inhibitors on pancreatic cancer cell viability, we performed Annexin V/PI staining using the (FITC-conjugated) Annexin V/PI apoptosis detection kit according to the manufacturer (Abcam).Detection and quantiication of apoptosis was performed for the cell lines 5061, 5072, 5156 and L3.6pl following incubation with 17-DMAG (1 µM), 17-AAG (1 µM) and 17-AEPGA (1 µM) for 48 h.
Statistical analysis
The software Mirkowin 2000 (Mikrotek Laborsysteme GmbH, Overath, Germany) was used at Microplate Multimethod Reader to determine the values of the MTT assay.Mean values were calculated from the results of four wells with the same drug concentration to compensate pipetting errors. The blank value (value of only with medium illed well) was subtracted from the remaining amounts to correct the background absorbance produced by chemical interference of components of the medium. The resulting values are directly proportional to the number of living cells.Statistical signiicance was calculated by multivariable variance analysis including the efects of the drugs, the concentration and the measurement as factors.IC50 values were calculated with the software “R” (version 2.15.2, R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria) and the package “drc”.For each cell line, each measurement time and each drug at least three independent experiments were performed, in order to ensure reproducibility of the experiments.
Results
Cell doubling time
To determine a test conluence in which active cell proliferation is permitted during the experiments and also a suicient conluence for adding the drug, the cell doubling time of each tumor cell line was initially evaluated using diferent seeding densities and by following cell growth for 7 days (data not shown). The doubling time was between 72 and 96 h for the cell lines 5061 and 5072, and between 48 and 72 h for 5156 and L3.6pl cell lines.
According to this data, we selected a drug exposition time of 72 h for the cell lines 5156 and L3.6pl and a drug exposition time of 96 h for the cell lines 5061 and 5072. Drug exposition was the period of two cell cycles to assure that HSP90 inhibition could afect its CP during posttranslational process.
Efects of 5-FU and gemcitabine
We investigated cell proliferation and survival in the pancreatic adenocarcinoma cell lines 5061, 5072, 5156 and L3.6pl. All cell lines were exposed to varying concentrations of 5-FU or gemcitabine (Gemzar®) for two cell doubling times. Cell viability was subsequently detected by the MTT assay. Neither time nor dose-dependent reduction in cell proliferation was seen in 5061 and 5072 under gemcitabine and 5-FU (Fig. 2a, b).
In 5156 cell line, a cell reduction of 50% could be achieved upon a gemcitabine concentration of 0.2 µM, a further dose escalation could not achieve a greater reduction of cell growth. In contrast, under treatment with 5-FU only a cell reduction to a maximum of 70% could be attained at very high drug concentrations (Fig. 2c). However, L3.6pl displayed a high sensitivity for gemcitabine and maximal reduction of proliferation was already achieved after treatment with 0.05 µM. The sensitivity for 5-FU was lower, even at concentrations of 5 µM, a 50% growth inhibition could not be achieved (Fig. 2d).
Antiproliferative efects of HSP90-inhibition
To investigate the role of HSP90 in cell proliferation and survival, PC cells were treated with increasing concentrations of the HSP90-inhibitors 17-AAG, 17-AEPGA and 17-DMAG. After two cell doubling times, we determined the drug induced efect by the MTT assay. In contrast to gemcitabine and 5-FU, cell proliferation was inhibited signiicantly in all tested cell lines in a dose- and timedependent manner (Fig. 3). The mean IC50 values in all tested cell lines including the L3.6pl was below 1 µM. Comparison of the non-water-soluble 17-AAG with water-soluble drugs showed superiority for 17-DMAG. The diferences in their anti-proliferative efect were not signiicant (Table 1).
Efects of HSP90 inhibition on survival signaling pathways
To evaluate the drug induced efect on CP of HSP90, we performed WB-analysis after exposure to diferent concentrations of 17-AAG and 17-DMAG for 48 h. Inhibition of HSP90 consequently led to synchronic disruption of multiple signal transduction pathways by degradation of various CP already at 1 µM and after a short exposure of only 48 h. Expression of EGFR and IGF-1R was strongly diminished in HSP90 inhibited cells compared to untreated cells. In addition, AKT and MAPK expression was also down regulated. In 5156 cells however, AKT expression was not afected indicating presence of a mutated form or other chaperons that compensate HSP90 functional deiciency. Figures 4 and 5 summarize the results of the WB analyses.
Inhibition of HSP90 leads to apoptosis
Concerning the proliferation inhibitory mechanism, we performed CASPASE-3 and PARP assay and identiied apoptosis as the main factor of cell death. In WB analysis, a signiicant up regulation of PARP and cleaved Caspase-3 was evident in contrast to untreated or gemcitabine treated cells. Apoptosis was already present after 48 h indicating that disruption of intracellular molecular network is an early event under HSP90 inhibition (Fig. 6A). Annexin V/PI staining showed, a notable increase of pancreatic cancer cell death after 48 h of incubation in comparison to untreated cells (Fig. 6B). These results further support our hypothesis that the reduction of cell survival following HSP90 inhibition is the consequence of activated programmed cell death pathways mediated by the efector Caspase-3.
Expression of other HSPs
Since targeting HSP90 is a functional inhibition without altering the HSP90 protein levels, a prognostic indicator for successful inhibition have been established. As a hallmark of successful HSP90 inhibition, up regulation of HSP70 is a known phenomenon. In all tested cell lines HSP70 and HSP27 were signiicantly up regulated. The up regulation was already evident under 1 µM inhibitor concentration and after 48 h. No diference was noticed between the three HSP90 inhibitors and up regulation of HSP70 and HSP27 proteins (Fig. 7).
Discussion
Surgery has been proclaimed as a curative therapy in PC. Recent developments on the ield of histological work-up of the operative specimen have revealed that >80% of the so called R0 resections are truly R1 resections [3]. The impact of the modiied histological work-up of the operative specimen is tremendous and may be an explanation for the poor survival in PC despite radical surgery and adjuvant CT. The limitations of more radical surgery with unjustiiable morbidity and missing eicacy of adjuvant CT emphasize the necessity for development of more potent CT.
CT options for PC are very limited. Although since the ESPAC trials gemcitabine is considered as the standard CT, 5-FU has been proven to be equally eicient in PC [31]. Molecular proiling studies in PC have revealed several potential targets including growth receptors and kinases but till date only EGFR inhibitor Erlotinib has been clinically evaluated [16]. Target speciic therapies are mostly efective towards only one single factor or pathway and consequently acquired resistance during therapy is a common phenomenon. Oncogene switching or re-activation of signaling pathways by upstream activators leading to the re-activation of downstream signaling pathways, devastate all eforts to counteract tumor growth and progression [8, 32, 33]. An ideal drug should be only active in tumor cells and simultaneously afect several growth factors and signaling pathways to minimize the probability of resistance development.
Activation of EGFR induces a cascade of downstream signaling through several pathways, including MAPK and AKT, resulting in cellular proliferation, diferentiation and survival. EGFR is overexpressed or abnormally activated in several malignancies and many studies have suggested that the EGFR pathway is frequently activated and correlated with poor outcome in human PC [12, 14]. EGFR mutations with impaired sensitivity or even resistance to EGFR inhibitors are common. In cancer cells, tyrosine kinase growth factor receptors of diferent families including IGF-1R are often simultaneously activated and lead to activation of redundant and often overlapping signal transduction pathways that impact multiple cell functions and maintain cell survival by replacing EGFR function [14, 33, 34]. Signaling through the IGF-1R is an important alternative route for cell survival in EGFR inhibitor resistant cells. IGF-1R also transduces signals through AKT and MAPK pathway. IGF-1R is known to play a key role in cell growth, diferentiation and survival and is often over expressed and constitutively active in PC [35]. Studies with combined targeting of IGF-1R and EGFR showed enhanced apoptosis and reduced invasive potential in various cancer cells solidifying the crosslink between EGFR, IGF-IR, AKT and MAPK signal transduction pathways [36]. For the aforementioned growth factors and signaling pathways, target speciic therapies are available but application is still limited either for low eicacy or increased toxicity which parallels combination of several drugs [37, 38]. However, most of the growth factors and kinases have a common crucial point when passing through the post-translational modiication process. To attain the functional active “mature” protein structure, all of these proteins require HSP90 chaperone activity. Ogata et al. have reported on HSPs expression in PC. HSP90 was highly overexpressed in poor diferentiated adenocarcinoma of the pancreas compared to well diferentiated tumors and chronic pancreatitis. In addition, Ogata et al. demonstrated that tumors with overexpression of HSP90 displayed high proliferation activity veriied by PCNA mRNA detection [39]. Furthermore, the current focus of target therapy in PC like EGFR and IGF-1R represent CP of HSP90. Hence, HSP90 represents a very promising therapeutic target in PC inheriting the ability to simultaneously downregulate multiple cell growth, proliferation and survival signaling cascades by proteasomal degradation of various growth receptors and tyrosine kinases [21].
For our study, we have been able to bring gemcitabine and 5-FU resistant adenocarcinoma of the pancreas into culture and to demonstrate that all tested cell lines are highly sensitive towards all three HSP90 inhibitors. Though, 17-AEPGA was slightly less efective and the calculated IC50 for all three HSP90 inhibitors was below 1 µM.
Cao et al. demonstrated the anti-proliferative efect of Geldanamycin in pancreatic cancer cells [40]. Song et al. conirmed the potent anti-tumor activity of IPI-504, a formulation based on 17-AAG, in pancreatic cancer cell lines [41]. The natural Ansamycin antibiotic Geldanamycin cannot be used clinically because of unacceptable hepatotoxicity. The poor water-solubility of 17-AAG, leading to diiculties in clinical formulation, resulted in synthesis of new, water-soluble Geldanamycin derivates, such as 17-DMAG and 17-AEPGA. In our study, the watersoluble compounds showed equal potency compared to 17-AAG. These results are consistent with previous studies in PC and other tumor entities. However, there are only very few studies analyzing 17-DMAG and no data is available for 17-AEPGA till date.
The anti-proliferative efect of diferent HSP90 inhibitors seen in the MTT assay was the result of disruption of growth factor mediated signal transduction pathways due to depletion of EGFR, IGF-1R, MAPK and AKT as conirmed in the WB analyses. The molecular breakdown under HSP90 inhibition is an early event since these efects were seen already 48 h following drug application.
In addition, apoptosis was also already present in all cell lines as proven by annexin/PI staining and cleavage of Caspase-3 and PARP. In contrast, apoptosis in gemcitabine treated cells was less evident congruently to the results of the MTT assay since these cell lines are considered as gemcitabine resistant. All three cell lines were generated in our lab and the corresponding patients were operated at our department. All three patients received adjuvant gemcitabine CT and developed early recurrence and consequently died within 3–8 months postoperatively. The clinical course relects the aggressive biology of these cell lines and underlines the eicacy of HSP90 inhibition.
However, as anticipated not all cell lines responded equally to HSP90 inhibition as seen in the molecular response signature. The biological behavior of cancer cells remains unpredictable. In 5061 cells, AKT expression remained unaltered indicating either these cells have an HSP90 independent mutated protein or AKT is stabilized or compensated by other factors under HSP90 inhibition.
As a hallmark of successful HSP90 inhibition HSP70 expression was upregulated in all tested cell lines. Interestingly, we also found augmentation of the total amount of HSP90 and HSP27 in treated cells. The upregulation of the HSPs under drug exposure represents a counteraction of the cancer cell to compensate the loss of functional active HSP90 multichaperone complexes [42]. Despite our encouraging results with HSP90 inhibitors in PC, the upregulation of HSP27 and HSP70 remains a challenging problem of HSP90 inhibitors application in the clinic since anti-apoptotic properties are attributes of HSP70 and HSP27 [43]. Down regulation of HSP27 and HSP70 has been shown to induce apoptosis proving that endogenous level of these two chaperones control cell survival [44]. In cancer cells, depletion of HSP70 has been shown to provoke spontaneous apoptosis. HSP70 deiciency is known to induce Caspase-3 activation. Caspase activated DNAse that is responsible for chromosomal DNA digestion downstream of Caspase-3 activation are regulated by HSP70. Hence, HSP70 can rescue cells from a later phase of apoptosis than any known survival protein. However, HSP70 also inherits chaperone potential and is also active during absence of HSP90 and in contrast to HSP90 it does not exhibit any substrate speciicity resulting in stabilization of all proteins.
HSP27 has been described as an inhibitor of Caspase activation. It is associated with Cytochrome c in the cytosol thereby inhibiting the formation of a Caspase-3 activation complex [45]. In addition, HSP27 interacts with the microilament cell skeleton. In cancer cells, it binds to F-Actin and prevents cytoskeletal disruption, phosphorylation of HSP27 is dependent on AKT [46].
In our study we observed diferences between AKT and MAPK level under HSP90 inhibition in the investigated cell lines. This could be an efect of the compensatory up regulation of HSP70 and HSP27 leading to stabilization of these kinases under HSP90 inhibition. Interestingly, previous studies with various tumor types and HSP90 inhibition did not reported on up regulation of HSP27. However, HSP27 is a small HSP that is known to inherit the strongest chaperone activity under stress conditions [47].
Sato et al. reported on dependency of AKT upon HSP90 [48]. AKT forms complexes with HSP90 and deletion mutants of AKT are unable to form an active complex with HSP90 which leads to the dephosphorylation and inactivation of AKT. This results in an increased cell sensitivity to apoptosis-inducing stimuli. These results further indicate that HSP90 plays an important role in maintaining AKT activity. MAPK functions downstream of HSP90 and is dependent upon HSP90 as well. MAPK and AKT pathways are known to play a pivotal role in oncogenesis and progression of PC. Considering the function of HSP27 and HSP70 under HSP90 inhibition, a poly chemotherapy regimen would be more advisable in terms of avoidance of possible selection of more aggressive tumor cells with overexpression of HSP70 and HSP27 and compensatory activated signaling pathways. Hence, HSP70 and HSP27 represent druggable targets in PC.
In conclusion, our data suggest that HSP90 inhibition is a highly efective therapeutic option in PC convincing with its ability to inhibit anti-apoptotic and proliferative pathways by synchronous interruption of multiple key oncogenic signaling cascades in gemcitabine and 5-FU resistant PC. Importantly, water-soluble 17-DMAG is equally efective and may blaze a trail for clinical application. Other HSPs may serve as compensatory chaperones under HSP90 inhibition and should be considered as additional targets in PC.
References
1. Herreros-Villanueva M, Hijona E, Cosme A, Bujanda L (2012) Adjuvant and neoadjuvant treatment in pancreatic cancer. World J Gastroenterol 18(14):1565–1572
2. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ (2007) Cancer statistics, 2007. CA Cancer J Clin 57(1):43–66
3. Verbeke CS, Leitch D, Menon KV, McMahon MJ, Guillou PJ, Anthoney A (2006) Redeining the R1 resection in pancreatic cancer. Br J Surg 93(10):1232–1237
4. Verbeke CS (2008) Resection margins and R1 rates in pancreatic cancer–are we there yet? Histopathology 52(7):787–796
5. Adler G, Seuferlein T, Bischof SC, Brambs HJ, Feuerbach S, Grabenbauer G et al (2007) S3-guidelines “Exocrine pancreatic cancer” 2007. Z Gastroenterol 45(6):487–523
6. Bergman AM, Pinedo HM, Peters GJ (2002) Determinants of resistance to 2′,2′-diluorodeoxycytidine (gemcitabine). Drug Resist Updates 5(1):19–33
7. Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y et al (2011) FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med 364(19):1817–1825
8. Conroy T, Gavoille C, Adenis A (2011) Metastatic pancre-atic cancer: old drugs, new paradigms. Curr Opin Oncol 23(4):390–395
9. Li J, Saif MW (2009) Any progress in the management of advanced pancreatic cancer? JOP 10(4):361–365
10. Makrilia N, Syrigos KN, Saif MW (2011) Updates on treat-ment of gemcitabine-refractory pancreatic adenocarcinoma. JOP 12(4):351–354
11. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M (1988) Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53(4):549–554
12. Ahsan A, Ramanand SG, Whitehead C, Hiniker SM, Rehemtulla A, Pratt WB et al (2012) Wild-type EGFR is stabilized by direct interaction with HSP90 in cancer cells and tumors. Neoplasia 14(8):670–677
13. Lang SA, Moser C, Gaumann A, Klein D, Glockzin G, Popp FC et al (2007) Targeting heat shock protein 90 in pancreatic cancer impairs insulin-like growth factor-I receptor signaling, disrupts an interleukin-6/signal-transducer and activator of transcription 3/hypoxia-inducible factor-1alpha autocrine loop, and reduces orthotopic tumor growth. Clin Cancer Res 13(21):6459–6468
14. Talar-Wojnarowska R, Malecka-Panas E (2006) Molecular pathogenesis of pancreatic adenocarcinoma: potential clinical implications. Med Sci Monit 12(9):RA186–RA93
15. Samuel N, Hudson TJ (2012) The molecular and cellular het-erogeneity of pancreatic ductal adenocarcinoma. Nat Rev Gastroenterol Hepatol 9(2):77–87
16. Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gall-inger S et al (2007) Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 25(15):1960–1966
17. Burrows F, Zhang H, Kamal A (2004) Hsp90 activation and cell cycle regulation. Cell Cycle 3(12):1530–1536
18. Schmitt E, Gehrmann M, Brunet M, Multhof G, Garrido C (2007) Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol 81(1):15–27
19. Wandinger SK, Richter K, Buchner J (2008) The Hsp90 chaperone machinery. J Biol Chem 283(27):18473–18477
20. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70
21. Neckers L (2007) Heat shock protein 90: the cancer chaperone. J Biosci 32(3):517–530
22. Kamal A, Thao L, Sensintafar J, Zhang L, Boehm MF, Fritz LC et al (2003) A high-ainity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 425(6956):407–410
23. Calderwood SK, Khaleque MA, Sawyer DB, Ciocca DR (2006) Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci 31(3):164–172
24. Xue M, Cao X, Zhong Y, Kuang D, Liu X, Zhao Z et al (2012) Insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitors in cancer therapy: advances and perspectives. Curr Pharm Des 18(20):2901–2913
25. Ryu YL, Jung KH, Son MK, Yan HH, Kim SJ, Shin S et al (2014) Anticancer activity of HS-527, a novel inhibitor targeting PI3-kinase in human pancreatic cancer cells. Cancer Lett 353(1):68–77
26. Li J, Kleef J, Giese N, Buchler MW, Korc M, Friess H (2004) Geitinib (‘Iressa’, ZD1839), a selective epidermal growth factor receptor tyrosine kinase inhibitor, inhibits pancreatic cancer cell growth, invasion, and colony formation. Int J Oncol 25(1):203–210
27. Hollingshead M, Alley M, Burger AM, Borgel S, PaculaCox C, Fiebig HH et al (2005) In vivo antitumor eicacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative. Cancer Chemother Pharmacol 56(2):115–125
28. Smith V, Sausville EA, Camalier RF, Fiebig HH, Burger AM (2005) Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: efects on Hsp90 and client proteins in melanoma models. Cancer Chemother Pharmacol 56(2):126–137
29. Kalinina T, Gungor C, Thieltges S, Moller-Krull M, Penas EM, Wicklein D et al (2010) Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung. BMC Cancer 10:295
30. Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ (1999) In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia 1(1):50–62
31. Neoptolemos JP, Stocken DD, Bassi C, Ghaneh P, Cunningham D, Goldstein D et al (2010) Adjuvant chemotherapy with luorouracil plus folinic acid vs gemcitabine following pancreatic cancer resection: a randomized controlled trial. Jama 304(10):1073–1081
32. Ohhashi S, Ohuchida K, Mizumoto K, Fujita H, Egami T, Yu J et al (2008) Down-regulation of deoxycytidine kinase enhances acquired resistance to gemcitabine in pancreatic cancer. Anticancer Res 28(4B):2205–2212
33. El Maalouf G, Le Tourneau C, Batty GN, Faivre S, Raymond E (2009) Markers involved in resistance to cytotoxics and targeted therapeutics in pancreatic cancer. Cancer Treat Rev 35(2):167–174
34. Adjei AA (2001) Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 93(14):1062–1074
35. Nair PN, De Armond DT, Adamo ML, Strodel WE, Freeman JW (2001) Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2. Oncogene 20(57):8203–8214
36. van der Veeken J, Oliveira S, Schifelers RM, Storm G, van Bergen En, Henegouwen PM, Roovers RC (2009) Crosstalk between epidermal growth factor receptor- and insulin-like growth factor-1 receptor signaling: implications for cancer therapy. Curr Cancer Drug Targets 9(6):748–760
37. Saridaki Z, Georgoulias V, Souglakos J (2010) Mechanisms of resistance to anti-EGFR monoclonal antibody treatment in metastatic colorectal cancer. World J Gastroenterol 16(10):1177–1187
3 8. Sawai A, Chandarlapaty S, Greulich H, Gonen M, Ye Q, Arteaga CL et al (2008) Inhibition of Hsp90 down-regulates mutant epidermal growth factor receptor (EGFR) expression and sensitizes EGFR mutant tumors to paclitaxel. Cancer Res 68(2):589–596
39. Ogata M, Naito Z, Tanaka S, Moriyama Y, Asano G (2000) Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma. J Nippon Med Sch 67(3):177–185
40. Cao X, Bloomston M, Zhang T, Frankel WL, Jia G, Wang B et al (2008) Synergistic antipancreatic tumor efect by simultaneously targeting hypoxic cancer cells with HSP90 inhibitor and glycolysis inhibitor. Clin Cancer Res 14(6):1831–1839
41. Song D, Chaerkady R, Tan AC, Garcia-Garcia E, Nalli A, Suarez-Gauthier A et al (2008) Antitumor activity and molecular efects of the novel heat shock protein 90 inhibitor, IPI-504, in pancreatic cancer. Mol Cancer Ther 7(10):3275–3284
42. Garrido C, Arrigo A, Solary E (2000) Hsp27: a small heat shock protein with protective and tumorigenic efects. Recent Res Dev 2:105–114
43. Lanneau D, de Thonel A, Maurel S, Didelot C, Garrido C (2007) Apoptosis versus cell diferentiation: role of heat shock proteins HSP90, HSP70 and HSP27. Prion 1(1):53–60
44. Lee CH, Hong HM, Chang YY, Chang WW (2012) Inhibi-tion of heat shock protein (Hsp) 27 potentiates the suppressive efect of Hsp90 inhibitors in targeting breast cancer stem-like cells. Biochimie 94(6):1382–1389
45. Garrido C, Gurbuxani S, Ravagnan L, Kroemer G (2001) Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286(3):433–442
46. Lavoie JN, Lambert H, Hickey E, Weber LA, Landry J (1995) Modulation of cellular thermoresistance and actin ilament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Mol Cell Biol 15(1):505–516
47. Samali A, Orrenius S (1998) Heat shock proteins: regula-tors of stress response and apoptosis. Cell Stress Chaperones 3(4):228–236
48. Sato S, Fujita N, Tsuruo T (2000) Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA 97(20):10832–10837