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Heat shock protein 90 is a putative therapeutic target in patients with recurrent advanced-stage ovarian carcinoma with serous effusions
Human Pathology (2012) 43, 529–535
www.elsevier.com/locate/humpath
Original contribution
Heat shock protein 90 is a putative therapeutic target in patients with recurrent advanced-stage ovarian carcinoma with serous effusions☆ Mari Bunkholt Elstrand MD a , Helene Tuft Stavnes MSc b , Claes G. Tropé MD, PhD a,c , Ben Davidson MD, PhD b,c,⁎ a
Department of Gynecological Oncology, Oslo University Hospital, Norwegian Radium Hospital, N-0424 Oslo, Norway Division of Pathology, Oslo University Hospital, Norwegian Radium Hospital, N-0424 Oslo, Norway c Division of Clinical Medicine, the Medical Faculty, University of Oslo, N-0316 Norway b
Received 8 April 2011; revised 23 May 2011; accepted 24 May 2011
Keywords: HSP90; Ovarian carcinoma; Serous effusion; Chemotherapy; Survival
Summary Heat shock protein 90 (HSP90) has anti-apoptotic properties exerted through its cytoprotective function of chaperone activity and increased expression in response to stress. The present study analyzed the clinical role of HSP90 in effusions from patients with advanced-stage ovarian carcinoma. HSP90 protein expression was investigated in 265 effusions using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response and survival. The correlation between HSP90 and a panel of previously-studied antiapoptotic proteins was additionally investigated. HSP90 was expressed in the cytoplasm and nucleus of tumor cells in 97% and 18% of specimens, respectively. Nuclear HSP90 expression was significantly higher in post-chemotherapy compared to pre-chemotherapy effusions (P = .005), significantly related to previous treatment with both platinol (P = .024) and paclitaxel (P = .007). Cytoplasmic HSP90 expression was significantly higher in effusions from patients with complete compared to incomplete/no response after second-line chemotherapy (P = .016). No association was found between HSP90 expression and other clinicopathologic parameters or survival. Cytoplasmic HSP90 expression was significantly associated with that of Bcl-2 in pre-chemotherapy effusions (P = .04), and marginally associated with cytoplasmic Survivin expression in post-chemotherapy effusions (P = .05). HSP90 is upregulated along tumor progression from primary diagnosis to recurrent effusion. HSP90 does not provide prognostic data in patients with advanced ovarian carcinoma effusions. However, HSP90 may be of predictive value as to who will benefit from treatment with HSP90 inhibitors to potentiate the effectiveness of platinol and paclitaxel in patients with recurrent advanced ovarian carcinoma effusions. We propose HSP90 as a potential therapeutic target in this patient group. © 2012 Elsevier Inc. All rights reserved.
☆
Funding: This work was supported by grants from the Norwegian Cancer Society and the Inger and John Fredriksen Foundation for Ovarian Cancer Research. Mari Bunkholt Elstrand is the receiver of a research stipend from the South-East Health Region of Norway. ⁎ Corresponding author. Division of Pathology, Oslo University Hospital, Norwegian Radium Hospital Montebello N-0310 Oslo Norway. E-mail address: [email protected] (B. Davidson). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.05.022
1. Introduction Ovarian carcinoma (OC) is the most lethal gynecologic malignancy in developed countries [1]. Contributing to the poor survival is that most patients (N60%) present with advanced-stage (FIGO III or IV) disease at the time of
530 diagnosis. Pleural effusion is the most common determinant of stage IV disease, and ascites is present in the majority of patients [2]. The tumor microenvironment has a crucial role in modifying the cancer cell phenotype [3], and the survival of metastatic OC cells in effusions is the result of their striking ability to adjust to their new environment [4]. While solid tumors can be removed surgically, pleural and peritoneal effusions are only amenable to elimination by chemotherapy. Therapy failure in these patients is largely due to primary or acquired drug resistance, giving rise to recurrent disease and tumor progression [5]. This clinical scenario dictates further study of the molecular characteristics of these cells, directed at understanding their mechanisms of sustaining cell survival and resisting apoptosis. Heat shock proteins (HSP) are a class of functionally related proteins present in virtually all eukaryotic cells. HSP90 is one of the most common HSP family members, and is expressed in different isoforms primarily in the cytosol, but also in the nucleus, mitochondria and endoplasmatic reticulum [6]. Among its nuclear effects, HSP90 regulates the activity of heat shock transcription factor 1 (HSF1), which in turn upregulates the expression of heat shock proteins in response to proteotoxic stress, as well as that of steroid hormone receptors, including glucocorticoid, androgen and estrogen receptors. Binding of HSP90 to Bcl-6 represses Bcl-6 target genes, such as ATR and TP53, in diffuse B cell large cell lymphomas [7]. In addition, HSP90 is able to translocate to the extracellular matrix where it mediates tumor cell invasiveness and immunological functions, thus being a candidate for anticancer vaccines [8,9]. HSP90 is a key mediator of cellular homeostasis and is required for housekeeping functions such as protein folding during nascent polypeptide-chain synthesis, preventing protein aggregation, intracellular disposition, translocation of proteins across membranes, and proteolysis through the proteasome [6,7]. In response to various physical, environmental and chemical stress conditions (eg, hypoxia, heat, chemotherapeutics etc.) the intracellular expression of HSP90 increases in an attempt to restore cellular homeostasis, allowing the cells to survive potentially lethal conditions [7,10]. In normal cells, HSP90 possesses general protective chaperone activity upon client proteins, and requires various co-chaperones to facilitate activation of these substrates. Together they constitute the dynamic complex known as the HSP90 chaperone machinery [6]. HSP90 associates with numerous signaling proteins, including ligand-dependent transcription factors such as steroid receptors, or ligandindependent transcription factors such as signal-transducing kinases, many of which are implicated in apoptosis [7,10,11]. The cytoprotective function of chaperone activity and increased expression in response to stress insults can largely be explained by the anti-apoptotic properties of HSP90, including the above-mentioned effect on HSF1 [11]. In malignant cells, HSP90 modulates anti-apoptotic activity,
M. B. Elstrand et al. mediated through effects on AKT, tumor necrosis factor (TNF) receptors and nuclear factor-κB (NF-κB) function [6]. It also prevents the formation of an active apoptosome complex by inhibiting oligomerization of Apaf-1 [12]. Moreover, Bcl-2 associates with HSP90β in mast cells, thus preventing the release of cytochrome c from mitochondria and subsequent activation of caspase-3 [13]. In addition, the HSP90 homologue tumor necrosis factor receptorassociated protein 1 (TRAP-1) is solely localized to mitochondria in tumor cells, where it regulates the mitochondrial membrane permeabilization and cytochrome c release [14]. HSP90 also has a role in metastasis-related events by assisting induction of vascular endothelial growth factor (VEGF) through hypoxia-inducible factor (HIF)-1 protein, which is important for angiogenesis and consequently for tumor growth. Both extracellular and intracellular HSP90 promotes invasion and metastasis by activation of matrix metalloproteinase-2 (MMP-2) [8,9]. Three previous reports have evaluated the clinical role of HSP90 in patients with OC [15-17]. In a study of 52 solid tumors using immunohistochemistry (IHC), HSP90 expression correlated only with FIGO stage, and was unrelated to survival [15]. In serological screening, HSP90 auto-antibodies were frequently found in late-stage ovarian cancer [16]. The expression of mRNA coding for HSP90 was reported to increase at OC of advanced stage compared to normal ovaries, but no relationship was observed between HSP90 mRNA abundance and the receptor status of epidermal growth factor, estrogen and progesterone [17]. To the best of our knowledge, the expression and clinical role of HSP90 in pleural and peritoneal effusions from patients with advanced-stage OC has not been studied to date. The aim of the present study was to investigate the expression of HSP90 in malignant effusions, and to determine its prognostic role in a retrospective analysis of a large cohort of OC patients. We additionally analyzed the correlation between HSP90 immunostaining and the expression of a panel of anti-apoptotic proteins previously studied by our group [18-21].
2. Materials and methods 2.1. Patients and material Effusions and clinical data were obtained from the Department of Gynecological Oncology at the Norwegian Radium Hospital from 1998-2005 (Table 1). The Regional Committee for Medical Research and Ethics in Norway approved the study. Fresh, non-fixed effusions (n = 265; 220 peritoneal, 45 pleural) were collected from 265 patients diagnosed with OC (n = 228), primary peritoneal serous carcinoma (n = 27) or tubal carcinoma (n = 10). All will be referred to as OC henceforth. Effusions were submitted for
Heat shock protein 90 is a putative therapeutic target Table 1
Clinicopathologic data (261 patients a)
Characteristics
Primary Disease diagnosis recurrence (n = 152) (n = 109)
Age Effusion site
63; 38-87 120 32 84 65 3 10 34 79 29 132 20 59 59 34 77 63 12 12 96 44
(Mean; Range) Peritoneal Pleural FIGO III IV NA b Grade I II III NA c Histological type Serous Non-serous d Residual tumor ≤1 cm N1 cm NA e Response to first-line Complete chemotherapy Incomplete f ND g Response to second-line Complete chemotherapy Incomplete f ND g
60; 34-88 97 12 77 26 6 9 22 73 5 91 18 39 55 15 58 48 3 28 70 11
a One effusion was obtained from each patient. Four of the 265 patients in this study had no information regarding chemotherapy status. b NA= non available. c Including effusions from inoperable patients where biopsy was too small for grading and patients operated in other hospitals, for which the primary tumor could not be accessed for assessment of grade. d Including clear cell, endometrioid and undifferentiated adenocarcinomas, adenocarcinomas of mixed histology, and patients operated on at other hospitals, for which the primary tumor could not be accessed for review of histological type. e Including patients who were inoperable and patients with no record. f Partial response/stable disease/progression/allergic or adverse reaction. g ND = non determined, including patients who received no chemotherapy and patients who died before chemoresponse could be assessed.
routine diagnostic purposes to the Division of Pathology at the Norwegian Radium Hospital. Effusions were centrifuged immediately after tapping and cell blocks were prepared using the thrombin clot method. Diagnoses were established based on morphology and IHC [22]. All effusions were morphologically evaluated, as were the primary carcinomas from patients operated at the Norwegian Radium Hospital. Immunohistochemistry was applied to primary carcinoma diagnosis when morphology was not unequivocal with respect to histological type and/or organ of origin, and was applied to effusions whenever the differential diagnosis with reactive mesothelial cells and malignant mesothelioma was deemed relevant, as well as when patients have been operated on at other hospitals. The FIGO classification was used for staging and grading. Previously, our research group has analyzed effusions from this patient cohort for expression of the anti-apoptotic
531 proteins Bcl-2, Bcl-XL, Bag-1, Bag-4, HSP27, HSP70, NF-κB, AKT and mTOR, as well as the inhibitor of apoptosis (IAP) family members XIAP and Survivin [18-21]. Staining results from these studies were analyzed for possible correlation to HSP90 expression in patientmatched samples.
2.2. Immunohistochemistry Formalin-fixed paraffin-embedded sections from 265 effusions were analyzed for protein expression of HSP90 using IHC. Staining was performed manually using the Dako En Vision™+ system-HRP (Dako A/S, Glostrup, Denmark). Deparaffinized and re-hydrated sections underwent antigen retrieval in Tris EDTA buffer (pH = 9.0) in microwave. Sections were treated with peroxidase block for 5 minutes at room temperature, and were then incubated for 30 minutes with a mouse monoclonal IgG2a anti-HSP90 antibody diluted 1:200 (Novocastra™ Lyophilized Mouse Monoclonal Antibody Heat Shock Protein 90; Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK). Sections were subsequently incubated with a peroxidase-labeled antimouse polymer for 30 minutes. Visualization was achieved using liquid DAB substrate-chromogen solution, with counterstaining with Hematoxylin. Positive controls consisted of a tissue microarray previously shown to be immunoreactive for the studied antigen, and were satisfactory in every run. Negative controls underwent similar staining procedures with a nonrelevant Mouse IgG2a in the same concentration as the primary antibody. Staining was considered positive when nuclear and/or cytoplasmic. Staining extent was scored on a scale of 0 to 4, as follows: 0 = no staining, 1 = 1% to 5%, 2 = 6% to 25%, 3 = 26% to 75% and 4 = 76% to 100% of tumor cells. Slides were scored by a surgical pathologist experienced in effusion cytology (BD) and by another author (MBE).
2.3. Statistical analysis Statistical analyses were performed applying the SPSSPC package (Version 16.0, Chicago, IL). All statistical tests were two-tailed and probability of b.05 was considered statistically significant. Descriptive analyses of clinicopathologic parameters were performed for patients with pre-chemotherapy and post-chemotherapy effusions. The relationship between the expression of HSP90 and clinicopathologic characteristics was assessed using the Mann-Whitney U test, as variables were not normally distributed. For the purpose of statistical analysis, clinicopathologic characteristics were grouped as follows: Age: ≤60 vs. N60 years; grade: 1-2 vs. 3; effusion site: peritoneal vs. pleural; FIGO stage: IIIc vs. IV; chemotherapy status: pre- vs. post-chemotherapy specimens; residual disease volume (≤1cm vs. N1cm);
532 response to chemotherapy for primary disease and for disease recurrence: complete vs. partial response/stable disease/progression. The association between expression of HSP90 and previously studied anti-apoptotic proteins was similarly analyzed with the Mann-Whitney U test, separately for pre-chemotherapy and post-chemotherapy effusions. Overall survival (OS) and progression free survival (PFS) were estimated using the Kaplan-Meier method, and the log-rank test was performed on the whole cohort, and separately for patients with pre-chemotherapy and postchemotherapy effusions. For survival analyses, expression categories were grouped as focal (≤25% of cells) or diffuse (N25% of cells).
M. B. Elstrand et al.
3. Results 3.1. HSP90 expression in OC effusions is related to chemotherapy status and response to chemotherapy Staining for HSP90 was most frequently present in the cytoplasm with staining score=4 registered in 45% of cases. Nuclear staining was observed in 18% of specimens, none of which had staining score=4. Seven specimens were negative at both the cytoplasm and nucleus (Fig. 1; Table 2). No significant difference was found in protein expression in cancer cells from pleural compared to peritoneal effusions. Nuclear HSP90 expression was significantly higher in post-chemotherapy compared to pre-chemotherapy
Fig. 1 Examples of HSP90 immunostaining in 4 effusions, showing carcinoma cells with cytoplasmic (A, D), cytoplasmic and focal nuclear (B) and nuclear (C) localization of this protein. Reactive mesothelial cells and macrophages are HSP90-negative in D.
Heat shock protein 90 is a putative therapeutic target Table 2
533
HSP90 immunohistochemistry results in 265 effusions
Molecule
Localization
HSP90
Cytoplasm Nucleus
Staining extent (% of cancer cells) 0%
1-5%
6-25%
26-75%
76-100%
8 (3%) 217 (82%)
28 (10%) 35 (13%)
36 (14%) 9 (3%)
74 (28%) 4 (2%)
119 (45%) 0 (0%)
effusions (P = .005), and showed significant association with previous exposure to platinol (P = .024) and paclitaxel (P = .007). Cytoplasmic HSP90 expression was significantly higher in effusions from patients with complete response after second-line chemotherapy compared to those with partial response/stable disease/progression/allergic or adverse reaction (P = .016). No associations were found between nuclear or cytoplasmic HSP90 expression and the remaining clinicopathologic parameters, including age, tumor grade, FIGO stage, residual tumor volume and response to chemotherapy after first-line treatment.
3.2. HSP90 expression in OC effusions is unrelated to survival Survival data were available for all 265 patients with OC effusions. The follow up period for these patients ranged from 1-187 months (mean = 31 months, median = 26 months). At last follow-up, 4 patients were alive without disease, 17 were alive with disease and 242 were dead of disease. Two patients died of unrelated causes. PFS ranged from 0-82 months (mean = 8 months, median = 4 months). HSP90 cytoplasmic or nuclear staining extent was unrelated to OS and PFS in analysis of the entire cohort (P N .05; data not shown). Similar findings were observed in separate analyses of patients with pre-chemotherapy and postchemotherapy effusions (P N .05; data not shown).
3.3. HSP90 expression in OC effusions is related to the expression of proteins involved in cell survival The correlation between HSP90 expression and previously-studied proteins involved in cell survival and inhibition of apoptosis was studied, including other members of the HSP family, Bag proteins, Bcl-2 family members, IAP proteins, NF-κB, AKT and mTOR. In prechemotherapy effusions, a correlation was found between expression of cytoplasmic HSP90 and Bcl-2 (P = .04), whereas in post-chemotherapy effusions, a marginally significant association was observed between cytoplasmic HSP90 and cytoplasmic Survivin (P = .05). None of the other proteins were significantly related to HSP90 expression at the nucleus or cytoplasm (P N .05; data not shown).
4. Discussion Malignant effusions are most often seen in advancedstage disease, and are associated with resistance to chemotherapy, progressive disease and poor prognosis, all of which are clinical manifestations of cancer cell survival [2,5]. In recent years, our group has focused on characterizing survival mechanisms enabling cancer cells in effusions to survive in this unique microenvironment. In the present study, we investigated the expression of HSP90 in malignant effusions and its relation to clinical outcome in patients with advanced OC. In addition, we analyzed the correlation between the expression of HSP90 and previously studied anti-apoptotic proteins [18-21]. Expression of HSP90 was abundant in the cytoplasm and less frequent in the nucleus of OC cells. This is in agreement with Elpek et al., who studied 52 solid OC of all stages and observed immunoreactivity for HSP90 in both the cytoplasm and the nucleus, with scarce expression in the latter subcellular location [15]. These authors also found HSP90 to be significantly less expressed in FIGO stage I-II compared to stage III-IV. Similarly, Mileo et al. reported on low expression of mRNA coding for HSP90 in normal and benign ovarian tumors, and increased expression with more advanced stages of OC [17]. Furthermore, in serological screening, HSP90 autoantibodies were more frequently found in stage III-IV compared to stage I-II OC (32% vs. 10%, respectively) [16]. In general, these three reports suggest increased expression of HSP90 to be associated with more aggressive disease. Our study included only patients diagnosed at FIGO stages III and IV, and no significant difference was found in the expression of HSP90 between these two stages. Standard treatment of patients with OC is primary surgery followed by platinol and paclitaxel combination chemotherapy [2]. Chemotherapy is primarily effective against proliferating cells, and the mechanisms of action of these drugs are known to involve induction of apoptosis [5]. Indeed, the apoptotic response in OC cell lines correlates with cisplatin and paclitaxel sensitivity [23]. However, OC cells in malignant effusions have been shown to undergo little apoptosis, reflecting the fact that this condition is often characterized by cancer cell survival and chemotherapy resistance [19]. The molecular mechanisms involved in chemotherapy resistance arise from alterations in multiple signaling and apoptotic pathways, often as a consequence of prior chemotherapy or in response to the microenvironment
534 [5,24]. Our group has highlighted this in previous studies, showing different expression and clinical roles of cancerassociated molecules in effusions obtained at diagnosis compared to effusions obtained at disease recurrence [25,26]. In accordance with previous results, we found that nuclear HSP90 expression in carcinoma cells was higher in post-chemotherapy compared to pre-chemotherapy effusions, and that it was significantly related to previous exposure to platinol and paclitaxel. Only nuclear HSP90 expression was increased in postchemotherapy compared to pre-chemotherapy effusions, while cytoplasmic HSP90 expression was comparable in these two groups. HSP90 is present at several intracellular locations and may well be differentially affected by chemotherapy at these various sites. In addition to the nuclear effects of HSP90 mentioned in the introduction, this protein modulates chromatin remodeling and the activity of DNA polymerase-η in the nucleus [9]. These activities may change in response to cellular stress (ie, chemotherapy exposure), as well as during the acquisition of chemoresistance. In support of these differences between pre- and post-chemotherapy effusions, we previously reported on the role of the chromatin remodeling protein Rsf-1 as a prognostic factor associated with poor outcome in post-chemotherapy, but not pre-chemotherapy OC [27]. The relevance of HSP90 in chemotherapy response may further be reflected in our finding that cytoplasmic HSP90 expression was higher in patients with complete response after second-line chemotherapy compared to those with less favorable response. Similar to cellular activity induced by increased cytoplasmic HSP90 expression, elevated nuclear HSP90 expression in postchemotherapy effusions could be interpreted as a heat-shock response in an attempt to restore cellular homeostasis after exposure to chemotherapy [6,8,9]. Of note, elevated expression of nuclear HSP90 was recently reported to be associated with more advanced TNM stage in breast carcinoma [28], supporting its role as a marker of more aggressive disease in cancer. The postulated cytoprotective and anti-apoptotic properties of HSP90 in post-chemotherapy effusions suggest a role in chemotherapy resistance and tumor progression. Elpek et al. found no correlation between the expression of HSP90 in primary OC and clinicopathologic characteristics, including age, histological subtype and tumor grade, nor was there a correlation with survival [15]. Our results in analysis of metastatic OC cells in effusions are in agreement with this report. Failure to detect an association with survival in our cohort may result from the opposing biological roles of HSP90, i.e. mediating chemotherapy resistance and tumor progression while promoting an immune response which may be growth-limiting [8]. Additionally, the presence of HSP90 is virtually universal in eukaryotic cells, a fact which may render difficult the detection of differences in survival among patients. Overexpression of HSP90 prevents apoptosis triggered by various stimuli, such as hyperthermia or anticancer drugs,
M. B. Elstrand et al. and inhibition of HSP90 has been shown to sensitize tumor cells to apoptosis, suggesting that this chaperone interacts with key proteins of the apoptotic signaling pathways [6,7,10,11]. In this context we found a significant positive correlation between the expression levels of the antiapoptotic and pro-survival protein Bcl-2 and cytoplasmic HSP90 in pre-chemotherapy OC effusions. The formation of a Bcl-2/HSP90β complex has previously been described to prevent the release of cytochrome c from the mitochondria, caspase 3 activation and apoptosis in mast cells [13]. Bcl-2 has also been shown to regulate HIF-1α protein stabilization in hypoxic melanoma cells with the involvement of HSP90β [29]. HSP90 isoforms have different affinity for client proteins, and also exert different survival effect on various tumor cells, as illustrated for TRAP1 gene expression which was significantly upregulated in platinol-resistant compared to platinol-sensitive OC cell lines [30]. The correlation between Bcl-2 and cytoplasmic HSP90 expression levels in our cohort might indicate a supportive role for Bcl-2 in assisting HSP90 in metastasis-related events and cancer cell survival in the hypoxic environment of pre-chemotherapy effusions. Cancer cells in effusions already exposed to chemotherapy are adapted to an unfavorable microenvironment which again determines the expression of pro-survival molecules [3-5]. In post-chemotherapy effusions we found marginal association between cytoplasmic HSP90 and cytoplasmic Survivin. Survivin is a member of the IAP family, and has a dual function as an inhibitor of apoptosis and as a key regulator of cell proliferation by stabilizing the microtubules during mitosis, thus controlling both cell death and cell growth [31]. Our group has previously found Survivin to be expressed in the nucleus and the cytoplasm of OC cells in effusions, and cytoplasmic Survivin was associated with more aggressive disease as it was more highly expressed in high-grade than in low-grade tumors, and in solid metastases compared to primary tumors and effusions [20]. HSP90 was shown to associate with and stabilize Survivin, and inhibition of HSP90 resulted in proteasomal degradation of Survivin, induction of apoptosis and inhibition of cell proliferation in a previous study [32]. However, to make the picture more complex, a recent study found HSP90 inhibitors to induce overexpression of Survivin in certain cell lines, suggesting that dual inhibition of these proteins is necessary [33]. The versatile properties of HSP90 imply a potential role as therapeutic target, and HSP90 inhibitors have already entered phase II and III clinical trials [7,34]. The first HSP90 inhibitor, Tanespimycin (17-AAG), is extensively studied, and carboplatin and 17-AAG have shown additive growth inhibitory effects in OC cell lines in vitro [35]. Furthermore, 17-AAG may sensitize a subset of OC to paclitaxel, particularly in those tumors in which resistance is driven by ERBB2 and/or p-AKT [36]. Identification of cancer-associated molecules in effusions may improve our understanding of cancer cell survival and disease progression, and be a tool for application of targeted therapeutics. We found increased expression of HSP90 in
Heat shock protein 90 is a putative therapeutic target post-chemotherapy effusions and in carcinoma cells exposed to carboplatin and paclitaxel, confirming molecular alterations in cancer cells along tumor progression. Previous studies with HSP90 inhibitor in combination with carboplatin and paclitaxel have shown a synergistic effect on growth inhibition and apoptosis, respectively [35,36]. Our results might indicate a potential role for HSP90 inhibitors in combination with carboplatin and paclitaxel as treatment for patients with recurrent advanced OC.
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Original contribution
Heat shock protein 90 is a putative therapeutic target in patients with recurrent advanced-stage ovarian carcinoma with serous effusions☆ Mari Bunkholt Elstrand MD a , Helene Tuft Stavnes MSc b , Claes G. Tropé MD, PhD a,c , Ben Davidson MD, PhD b,c,⁎ a
Department of Gynecological Oncology, Oslo University Hospital, Norwegian Radium Hospital, N-0424 Oslo, Norway Division of Pathology, Oslo University Hospital, Norwegian Radium Hospital, N-0424 Oslo, Norway c Division of Clinical Medicine, the Medical Faculty, University of Oslo, N-0316 Norway b
Received 8 April 2011; revised 23 May 2011; accepted 24 May 2011
Keywords: HSP90; Ovarian carcinoma; Serous effusion; Chemotherapy; Survival
Summary Heat shock protein 90 (HSP90) has anti-apoptotic properties exerted through its cytoprotective function of chaperone activity and increased expression in response to stress. The present study analyzed the clinical role of HSP90 in effusions from patients with advanced-stage ovarian carcinoma. HSP90 protein expression was investigated in 265 effusions using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response and survival. The correlation between HSP90 and a panel of previously-studied antiapoptotic proteins was additionally investigated. HSP90 was expressed in the cytoplasm and nucleus of tumor cells in 97% and 18% of specimens, respectively. Nuclear HSP90 expression was significantly higher in post-chemotherapy compared to pre-chemotherapy effusions (P = .005), significantly related to previous treatment with both platinol (P = .024) and paclitaxel (P = .007). Cytoplasmic HSP90 expression was significantly higher in effusions from patients with complete compared to incomplete/no response after second-line chemotherapy (P = .016). No association was found between HSP90 expression and other clinicopathologic parameters or survival. Cytoplasmic HSP90 expression was significantly associated with that of Bcl-2 in pre-chemotherapy effusions (P = .04), and marginally associated with cytoplasmic Survivin expression in post-chemotherapy effusions (P = .05). HSP90 is upregulated along tumor progression from primary diagnosis to recurrent effusion. HSP90 does not provide prognostic data in patients with advanced ovarian carcinoma effusions. However, HSP90 may be of predictive value as to who will benefit from treatment with HSP90 inhibitors to potentiate the effectiveness of platinol and paclitaxel in patients with recurrent advanced ovarian carcinoma effusions. We propose HSP90 as a potential therapeutic target in this patient group. © 2012 Elsevier Inc. All rights reserved.
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Funding: This work was supported by grants from the Norwegian Cancer Society and the Inger and John Fredriksen Foundation for Ovarian Cancer Research. Mari Bunkholt Elstrand is the receiver of a research stipend from the South-East Health Region of Norway. ⁎ Corresponding author. Division of Pathology, Oslo University Hospital, Norwegian Radium Hospital Montebello N-0310 Oslo Norway. E-mail address: [email protected] (B. Davidson). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.05.022
1. Introduction Ovarian carcinoma (OC) is the most lethal gynecologic malignancy in developed countries [1]. Contributing to the poor survival is that most patients (N60%) present with advanced-stage (FIGO III or IV) disease at the time of
530 diagnosis. Pleural effusion is the most common determinant of stage IV disease, and ascites is present in the majority of patients [2]. The tumor microenvironment has a crucial role in modifying the cancer cell phenotype [3], and the survival of metastatic OC cells in effusions is the result of their striking ability to adjust to their new environment [4]. While solid tumors can be removed surgically, pleural and peritoneal effusions are only amenable to elimination by chemotherapy. Therapy failure in these patients is largely due to primary or acquired drug resistance, giving rise to recurrent disease and tumor progression [5]. This clinical scenario dictates further study of the molecular characteristics of these cells, directed at understanding their mechanisms of sustaining cell survival and resisting apoptosis. Heat shock proteins (HSP) are a class of functionally related proteins present in virtually all eukaryotic cells. HSP90 is one of the most common HSP family members, and is expressed in different isoforms primarily in the cytosol, but also in the nucleus, mitochondria and endoplasmatic reticulum [6]. Among its nuclear effects, HSP90 regulates the activity of heat shock transcription factor 1 (HSF1), which in turn upregulates the expression of heat shock proteins in response to proteotoxic stress, as well as that of steroid hormone receptors, including glucocorticoid, androgen and estrogen receptors. Binding of HSP90 to Bcl-6 represses Bcl-6 target genes, such as ATR and TP53, in diffuse B cell large cell lymphomas [7]. In addition, HSP90 is able to translocate to the extracellular matrix where it mediates tumor cell invasiveness and immunological functions, thus being a candidate for anticancer vaccines [8,9]. HSP90 is a key mediator of cellular homeostasis and is required for housekeeping functions such as protein folding during nascent polypeptide-chain synthesis, preventing protein aggregation, intracellular disposition, translocation of proteins across membranes, and proteolysis through the proteasome [6,7]. In response to various physical, environmental and chemical stress conditions (eg, hypoxia, heat, chemotherapeutics etc.) the intracellular expression of HSP90 increases in an attempt to restore cellular homeostasis, allowing the cells to survive potentially lethal conditions [7,10]. In normal cells, HSP90 possesses general protective chaperone activity upon client proteins, and requires various co-chaperones to facilitate activation of these substrates. Together they constitute the dynamic complex known as the HSP90 chaperone machinery [6]. HSP90 associates with numerous signaling proteins, including ligand-dependent transcription factors such as steroid receptors, or ligandindependent transcription factors such as signal-transducing kinases, many of which are implicated in apoptosis [7,10,11]. The cytoprotective function of chaperone activity and increased expression in response to stress insults can largely be explained by the anti-apoptotic properties of HSP90, including the above-mentioned effect on HSF1 [11]. In malignant cells, HSP90 modulates anti-apoptotic activity,
M. B. Elstrand et al. mediated through effects on AKT, tumor necrosis factor (TNF) receptors and nuclear factor-κB (NF-κB) function [6]. It also prevents the formation of an active apoptosome complex by inhibiting oligomerization of Apaf-1 [12]. Moreover, Bcl-2 associates with HSP90β in mast cells, thus preventing the release of cytochrome c from mitochondria and subsequent activation of caspase-3 [13]. In addition, the HSP90 homologue tumor necrosis factor receptorassociated protein 1 (TRAP-1) is solely localized to mitochondria in tumor cells, where it regulates the mitochondrial membrane permeabilization and cytochrome c release [14]. HSP90 also has a role in metastasis-related events by assisting induction of vascular endothelial growth factor (VEGF) through hypoxia-inducible factor (HIF)-1 protein, which is important for angiogenesis and consequently for tumor growth. Both extracellular and intracellular HSP90 promotes invasion and metastasis by activation of matrix metalloproteinase-2 (MMP-2) [8,9]. Three previous reports have evaluated the clinical role of HSP90 in patients with OC [15-17]. In a study of 52 solid tumors using immunohistochemistry (IHC), HSP90 expression correlated only with FIGO stage, and was unrelated to survival [15]. In serological screening, HSP90 auto-antibodies were frequently found in late-stage ovarian cancer [16]. The expression of mRNA coding for HSP90 was reported to increase at OC of advanced stage compared to normal ovaries, but no relationship was observed between HSP90 mRNA abundance and the receptor status of epidermal growth factor, estrogen and progesterone [17]. To the best of our knowledge, the expression and clinical role of HSP90 in pleural and peritoneal effusions from patients with advanced-stage OC has not been studied to date. The aim of the present study was to investigate the expression of HSP90 in malignant effusions, and to determine its prognostic role in a retrospective analysis of a large cohort of OC patients. We additionally analyzed the correlation between HSP90 immunostaining and the expression of a panel of anti-apoptotic proteins previously studied by our group [18-21].
2. Materials and methods 2.1. Patients and material Effusions and clinical data were obtained from the Department of Gynecological Oncology at the Norwegian Radium Hospital from 1998-2005 (Table 1). The Regional Committee for Medical Research and Ethics in Norway approved the study. Fresh, non-fixed effusions (n = 265; 220 peritoneal, 45 pleural) were collected from 265 patients diagnosed with OC (n = 228), primary peritoneal serous carcinoma (n = 27) or tubal carcinoma (n = 10). All will be referred to as OC henceforth. Effusions were submitted for
Heat shock protein 90 is a putative therapeutic target Table 1
Clinicopathologic data (261 patients a)
Characteristics
Primary Disease diagnosis recurrence (n = 152) (n = 109)
Age Effusion site
63; 38-87 120 32 84 65 3 10 34 79 29 132 20 59 59 34 77 63 12 12 96 44
(Mean; Range) Peritoneal Pleural FIGO III IV NA b Grade I II III NA c Histological type Serous Non-serous d Residual tumor ≤1 cm N1 cm NA e Response to first-line Complete chemotherapy Incomplete f ND g Response to second-line Complete chemotherapy Incomplete f ND g
60; 34-88 97 12 77 26 6 9 22 73 5 91 18 39 55 15 58 48 3 28 70 11
a One effusion was obtained from each patient. Four of the 265 patients in this study had no information regarding chemotherapy status. b NA= non available. c Including effusions from inoperable patients where biopsy was too small for grading and patients operated in other hospitals, for which the primary tumor could not be accessed for assessment of grade. d Including clear cell, endometrioid and undifferentiated adenocarcinomas, adenocarcinomas of mixed histology, and patients operated on at other hospitals, for which the primary tumor could not be accessed for review of histological type. e Including patients who were inoperable and patients with no record. f Partial response/stable disease/progression/allergic or adverse reaction. g ND = non determined, including patients who received no chemotherapy and patients who died before chemoresponse could be assessed.
routine diagnostic purposes to the Division of Pathology at the Norwegian Radium Hospital. Effusions were centrifuged immediately after tapping and cell blocks were prepared using the thrombin clot method. Diagnoses were established based on morphology and IHC [22]. All effusions were morphologically evaluated, as were the primary carcinomas from patients operated at the Norwegian Radium Hospital. Immunohistochemistry was applied to primary carcinoma diagnosis when morphology was not unequivocal with respect to histological type and/or organ of origin, and was applied to effusions whenever the differential diagnosis with reactive mesothelial cells and malignant mesothelioma was deemed relevant, as well as when patients have been operated on at other hospitals. The FIGO classification was used for staging and grading. Previously, our research group has analyzed effusions from this patient cohort for expression of the anti-apoptotic
531 proteins Bcl-2, Bcl-XL, Bag-1, Bag-4, HSP27, HSP70, NF-κB, AKT and mTOR, as well as the inhibitor of apoptosis (IAP) family members XIAP and Survivin [18-21]. Staining results from these studies were analyzed for possible correlation to HSP90 expression in patientmatched samples.
2.2. Immunohistochemistry Formalin-fixed paraffin-embedded sections from 265 effusions were analyzed for protein expression of HSP90 using IHC. Staining was performed manually using the Dako En Vision™+ system-HRP (Dako A/S, Glostrup, Denmark). Deparaffinized and re-hydrated sections underwent antigen retrieval in Tris EDTA buffer (pH = 9.0) in microwave. Sections were treated with peroxidase block for 5 minutes at room temperature, and were then incubated for 30 minutes with a mouse monoclonal IgG2a anti-HSP90 antibody diluted 1:200 (Novocastra™ Lyophilized Mouse Monoclonal Antibody Heat Shock Protein 90; Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK). Sections were subsequently incubated with a peroxidase-labeled antimouse polymer for 30 minutes. Visualization was achieved using liquid DAB substrate-chromogen solution, with counterstaining with Hematoxylin. Positive controls consisted of a tissue microarray previously shown to be immunoreactive for the studied antigen, and were satisfactory in every run. Negative controls underwent similar staining procedures with a nonrelevant Mouse IgG2a in the same concentration as the primary antibody. Staining was considered positive when nuclear and/or cytoplasmic. Staining extent was scored on a scale of 0 to 4, as follows: 0 = no staining, 1 = 1% to 5%, 2 = 6% to 25%, 3 = 26% to 75% and 4 = 76% to 100% of tumor cells. Slides were scored by a surgical pathologist experienced in effusion cytology (BD) and by another author (MBE).
2.3. Statistical analysis Statistical analyses were performed applying the SPSSPC package (Version 16.0, Chicago, IL). All statistical tests were two-tailed and probability of b.05 was considered statistically significant. Descriptive analyses of clinicopathologic parameters were performed for patients with pre-chemotherapy and post-chemotherapy effusions. The relationship between the expression of HSP90 and clinicopathologic characteristics was assessed using the Mann-Whitney U test, as variables were not normally distributed. For the purpose of statistical analysis, clinicopathologic characteristics were grouped as follows: Age: ≤60 vs. N60 years; grade: 1-2 vs. 3; effusion site: peritoneal vs. pleural; FIGO stage: IIIc vs. IV; chemotherapy status: pre- vs. post-chemotherapy specimens; residual disease volume (≤1cm vs. N1cm);
532 response to chemotherapy for primary disease and for disease recurrence: complete vs. partial response/stable disease/progression. The association between expression of HSP90 and previously studied anti-apoptotic proteins was similarly analyzed with the Mann-Whitney U test, separately for pre-chemotherapy and post-chemotherapy effusions. Overall survival (OS) and progression free survival (PFS) were estimated using the Kaplan-Meier method, and the log-rank test was performed on the whole cohort, and separately for patients with pre-chemotherapy and postchemotherapy effusions. For survival analyses, expression categories were grouped as focal (≤25% of cells) or diffuse (N25% of cells).
M. B. Elstrand et al.
3. Results 3.1. HSP90 expression in OC effusions is related to chemotherapy status and response to chemotherapy Staining for HSP90 was most frequently present in the cytoplasm with staining score=4 registered in 45% of cases. Nuclear staining was observed in 18% of specimens, none of which had staining score=4. Seven specimens were negative at both the cytoplasm and nucleus (Fig. 1; Table 2). No significant difference was found in protein expression in cancer cells from pleural compared to peritoneal effusions. Nuclear HSP90 expression was significantly higher in post-chemotherapy compared to pre-chemotherapy
Fig. 1 Examples of HSP90 immunostaining in 4 effusions, showing carcinoma cells with cytoplasmic (A, D), cytoplasmic and focal nuclear (B) and nuclear (C) localization of this protein. Reactive mesothelial cells and macrophages are HSP90-negative in D.
Heat shock protein 90 is a putative therapeutic target Table 2
533
HSP90 immunohistochemistry results in 265 effusions
Molecule
Localization
HSP90
Cytoplasm Nucleus
Staining extent (% of cancer cells) 0%
1-5%
6-25%
26-75%
76-100%
8 (3%) 217 (82%)
28 (10%) 35 (13%)
36 (14%) 9 (3%)
74 (28%) 4 (2%)
119 (45%) 0 (0%)
effusions (P = .005), and showed significant association with previous exposure to platinol (P = .024) and paclitaxel (P = .007). Cytoplasmic HSP90 expression was significantly higher in effusions from patients with complete response after second-line chemotherapy compared to those with partial response/stable disease/progression/allergic or adverse reaction (P = .016). No associations were found between nuclear or cytoplasmic HSP90 expression and the remaining clinicopathologic parameters, including age, tumor grade, FIGO stage, residual tumor volume and response to chemotherapy after first-line treatment.
3.2. HSP90 expression in OC effusions is unrelated to survival Survival data were available for all 265 patients with OC effusions. The follow up period for these patients ranged from 1-187 months (mean = 31 months, median = 26 months). At last follow-up, 4 patients were alive without disease, 17 were alive with disease and 242 were dead of disease. Two patients died of unrelated causes. PFS ranged from 0-82 months (mean = 8 months, median = 4 months). HSP90 cytoplasmic or nuclear staining extent was unrelated to OS and PFS in analysis of the entire cohort (P N .05; data not shown). Similar findings were observed in separate analyses of patients with pre-chemotherapy and postchemotherapy effusions (P N .05; data not shown).
3.3. HSP90 expression in OC effusions is related to the expression of proteins involved in cell survival The correlation between HSP90 expression and previously-studied proteins involved in cell survival and inhibition of apoptosis was studied, including other members of the HSP family, Bag proteins, Bcl-2 family members, IAP proteins, NF-κB, AKT and mTOR. In prechemotherapy effusions, a correlation was found between expression of cytoplasmic HSP90 and Bcl-2 (P = .04), whereas in post-chemotherapy effusions, a marginally significant association was observed between cytoplasmic HSP90 and cytoplasmic Survivin (P = .05). None of the other proteins were significantly related to HSP90 expression at the nucleus or cytoplasm (P N .05; data not shown).
4. Discussion Malignant effusions are most often seen in advancedstage disease, and are associated with resistance to chemotherapy, progressive disease and poor prognosis, all of which are clinical manifestations of cancer cell survival [2,5]. In recent years, our group has focused on characterizing survival mechanisms enabling cancer cells in effusions to survive in this unique microenvironment. In the present study, we investigated the expression of HSP90 in malignant effusions and its relation to clinical outcome in patients with advanced OC. In addition, we analyzed the correlation between the expression of HSP90 and previously studied anti-apoptotic proteins [18-21]. Expression of HSP90 was abundant in the cytoplasm and less frequent in the nucleus of OC cells. This is in agreement with Elpek et al., who studied 52 solid OC of all stages and observed immunoreactivity for HSP90 in both the cytoplasm and the nucleus, with scarce expression in the latter subcellular location [15]. These authors also found HSP90 to be significantly less expressed in FIGO stage I-II compared to stage III-IV. Similarly, Mileo et al. reported on low expression of mRNA coding for HSP90 in normal and benign ovarian tumors, and increased expression with more advanced stages of OC [17]. Furthermore, in serological screening, HSP90 autoantibodies were more frequently found in stage III-IV compared to stage I-II OC (32% vs. 10%, respectively) [16]. In general, these three reports suggest increased expression of HSP90 to be associated with more aggressive disease. Our study included only patients diagnosed at FIGO stages III and IV, and no significant difference was found in the expression of HSP90 between these two stages. Standard treatment of patients with OC is primary surgery followed by platinol and paclitaxel combination chemotherapy [2]. Chemotherapy is primarily effective against proliferating cells, and the mechanisms of action of these drugs are known to involve induction of apoptosis [5]. Indeed, the apoptotic response in OC cell lines correlates with cisplatin and paclitaxel sensitivity [23]. However, OC cells in malignant effusions have been shown to undergo little apoptosis, reflecting the fact that this condition is often characterized by cancer cell survival and chemotherapy resistance [19]. The molecular mechanisms involved in chemotherapy resistance arise from alterations in multiple signaling and apoptotic pathways, often as a consequence of prior chemotherapy or in response to the microenvironment
534 [5,24]. Our group has highlighted this in previous studies, showing different expression and clinical roles of cancerassociated molecules in effusions obtained at diagnosis compared to effusions obtained at disease recurrence [25,26]. In accordance with previous results, we found that nuclear HSP90 expression in carcinoma cells was higher in post-chemotherapy compared to pre-chemotherapy effusions, and that it was significantly related to previous exposure to platinol and paclitaxel. Only nuclear HSP90 expression was increased in postchemotherapy compared to pre-chemotherapy effusions, while cytoplasmic HSP90 expression was comparable in these two groups. HSP90 is present at several intracellular locations and may well be differentially affected by chemotherapy at these various sites. In addition to the nuclear effects of HSP90 mentioned in the introduction, this protein modulates chromatin remodeling and the activity of DNA polymerase-η in the nucleus [9]. These activities may change in response to cellular stress (ie, chemotherapy exposure), as well as during the acquisition of chemoresistance. In support of these differences between pre- and post-chemotherapy effusions, we previously reported on the role of the chromatin remodeling protein Rsf-1 as a prognostic factor associated with poor outcome in post-chemotherapy, but not pre-chemotherapy OC [27]. The relevance of HSP90 in chemotherapy response may further be reflected in our finding that cytoplasmic HSP90 expression was higher in patients with complete response after second-line chemotherapy compared to those with less favorable response. Similar to cellular activity induced by increased cytoplasmic HSP90 expression, elevated nuclear HSP90 expression in postchemotherapy effusions could be interpreted as a heat-shock response in an attempt to restore cellular homeostasis after exposure to chemotherapy [6,8,9]. Of note, elevated expression of nuclear HSP90 was recently reported to be associated with more advanced TNM stage in breast carcinoma [28], supporting its role as a marker of more aggressive disease in cancer. The postulated cytoprotective and anti-apoptotic properties of HSP90 in post-chemotherapy effusions suggest a role in chemotherapy resistance and tumor progression. Elpek et al. found no correlation between the expression of HSP90 in primary OC and clinicopathologic characteristics, including age, histological subtype and tumor grade, nor was there a correlation with survival [15]. Our results in analysis of metastatic OC cells in effusions are in agreement with this report. Failure to detect an association with survival in our cohort may result from the opposing biological roles of HSP90, i.e. mediating chemotherapy resistance and tumor progression while promoting an immune response which may be growth-limiting [8]. Additionally, the presence of HSP90 is virtually universal in eukaryotic cells, a fact which may render difficult the detection of differences in survival among patients. Overexpression of HSP90 prevents apoptosis triggered by various stimuli, such as hyperthermia or anticancer drugs,
M. B. Elstrand et al. and inhibition of HSP90 has been shown to sensitize tumor cells to apoptosis, suggesting that this chaperone interacts with key proteins of the apoptotic signaling pathways [6,7,10,11]. In this context we found a significant positive correlation between the expression levels of the antiapoptotic and pro-survival protein Bcl-2 and cytoplasmic HSP90 in pre-chemotherapy OC effusions. The formation of a Bcl-2/HSP90β complex has previously been described to prevent the release of cytochrome c from the mitochondria, caspase 3 activation and apoptosis in mast cells [13]. Bcl-2 has also been shown to regulate HIF-1α protein stabilization in hypoxic melanoma cells with the involvement of HSP90β [29]. HSP90 isoforms have different affinity for client proteins, and also exert different survival effect on various tumor cells, as illustrated for TRAP1 gene expression which was significantly upregulated in platinol-resistant compared to platinol-sensitive OC cell lines [30]. The correlation between Bcl-2 and cytoplasmic HSP90 expression levels in our cohort might indicate a supportive role for Bcl-2 in assisting HSP90 in metastasis-related events and cancer cell survival in the hypoxic environment of pre-chemotherapy effusions. Cancer cells in effusions already exposed to chemotherapy are adapted to an unfavorable microenvironment which again determines the expression of pro-survival molecules [3-5]. In post-chemotherapy effusions we found marginal association between cytoplasmic HSP90 and cytoplasmic Survivin. Survivin is a member of the IAP family, and has a dual function as an inhibitor of apoptosis and as a key regulator of cell proliferation by stabilizing the microtubules during mitosis, thus controlling both cell death and cell growth [31]. Our group has previously found Survivin to be expressed in the nucleus and the cytoplasm of OC cells in effusions, and cytoplasmic Survivin was associated with more aggressive disease as it was more highly expressed in high-grade than in low-grade tumors, and in solid metastases compared to primary tumors and effusions [20]. HSP90 was shown to associate with and stabilize Survivin, and inhibition of HSP90 resulted in proteasomal degradation of Survivin, induction of apoptosis and inhibition of cell proliferation in a previous study [32]. However, to make the picture more complex, a recent study found HSP90 inhibitors to induce overexpression of Survivin in certain cell lines, suggesting that dual inhibition of these proteins is necessary [33]. The versatile properties of HSP90 imply a potential role as therapeutic target, and HSP90 inhibitors have already entered phase II and III clinical trials [7,34]. The first HSP90 inhibitor, Tanespimycin (17-AAG), is extensively studied, and carboplatin and 17-AAG have shown additive growth inhibitory effects in OC cell lines in vitro [35]. Furthermore, 17-AAG may sensitize a subset of OC to paclitaxel, particularly in those tumors in which resistance is driven by ERBB2 and/or p-AKT [36]. Identification of cancer-associated molecules in effusions may improve our understanding of cancer cell survival and disease progression, and be a tool for application of targeted therapeutics. We found increased expression of HSP90 in
Heat shock protein 90 is a putative therapeutic target post-chemotherapy effusions and in carcinoma cells exposed to carboplatin and paclitaxel, confirming molecular alterations in cancer cells along tumor progression. Previous studies with HSP90 inhibitor in combination with carboplatin and paclitaxel have shown a synergistic effect on growth inhibition and apoptosis, respectively [35,36]. Our results might indicate a potential role for HSP90 inhibitors in combination with carboplatin and paclitaxel as treatment for patients with recurrent advanced OC.
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