1Department of Pathology, The Norwegian Radium Hospital, University of Oslo, ... Hebrew University of Jerusalem, Jerusalem, Israel; 3Department of Pathology, ...
Breast Cancer Research and Treatment 83: 119–128, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
Report
Altered expression and activation of the nerve growth factor receptors TrkA and p75 provide the first evidence of tumor progression to effusion in breast carcinoma Ben Davidson1 , Reuven Reich2,4 , Philip Lazarovici2,4 , Vivi Ann Flørenes1 , Søren Nielsen3, and Jahn M. Nesland1 1 Department
of Pathology, The Norwegian Radium Hospital, University of Oslo, Montebello, Oslo, Norway; of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel; 3 Department of Pathology, Aalborg University, Aalborg, Denmark; 4 David R. Bloom Center for Pharmacy, Hebrew University, Jerusalem, Israel
2 Department
Key words: breast cancer, neurotrophins, pleural effusion, tyrosine kinase receptors
Summary The aim of this study was to characterize phenotypic alterations along the progression of breast carcinoma from primary tumor to pleural effusion through analysis of the expression of nerve growth factor (NGF) and its receptors phospho-TrkA (p-TrkA activated receptor) and p75. Sections from 42 malignant pleural effusions from breast cancer patients and 65 corresponding solid tumors (34 primary, 31 metastatic) were evaluated for protein expression of the activated p-TrkA receptor. The majority of lesions were additionally studied for NGF and p75 expression. Six effusions and four breast carcinoma cell lines were studied for expression of p-TrkA using immunoblotting (IB). Membrane expression of p-TrkA was high in carcinoma cells in effusions (39/42, 93%) and locoregional recurrences (12/13, 92%), with significantly lower expression in both primary tumors (14/34, 41%) and lymph node metastases (8/18, 44%), respectively (p < 0.001 for effusions vs. primary tumors; p = 0.001 for effusions vs. lymph nodes). In contrast, p75 expression was less frequent in effusions compared to both primary tumors and lymph node metastases, significantly so for the latter (p = 0.019). NGF expression was comparable at all sites, but its expression in tumor cells in effusions (7/21 cases) was limited to cases in which time to progression (TTP) to effusion occurred within 5 years or less from primary operation. In univariate analysis of survival, mean and median TTP were 6.3 and 6 years for NGF-negative effusions, compared to 3 and 4 years for NGF-positive cases (p = 0.013). IB confirmed expression of p-TrkA in five of six effusions, while all four breast cancer cell lines were p-TrkA-negative. Our data provide the first documented evidence of molecular events that occur along tumor progression of breast carcinoma from primary tumors to effusion. The almost universal expression of p-TrkA in cancer cells in effusions and late recurrences is in full agreement with our recent report linking this factor with poor prognosis in ovarian cancer. Furthermore, the rapid progression to effusion in cases showing NGF expression in tumor cells underscores the aggressive clinical behavior of tumors that are able to utilize this pathway in an autocrine manner.
Introduction Neurotrophins are a family of growth factors, consisting at present of the prototype compound nerve growth factor (NGF), and additional members such as brain-derived neurotrophic factor (BDNF), and the
neurotrophins NT-3, NT-4 and NT-6 [1–4]. Neurotrophins bind to the TrkA, TrkB and TrkC receptors. NGF binds to the specific high-affinity tyrosine kinase receptor TrkA [5]. NGF ligand binding to TrkA receptors activates TrkA autophosphorylation at several sites such as tyrosine 490, promoting SHC binding
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and phosphorylation, coupling of GRB2-SOS complexes and activation of Ras [6, 7]. The net result in the central nervous system is differentiation and survival of neuronal cells [2, 7]. Ras is able to activate two major intracellular signal transduction pathways – the mitogen activated protein kinase (MAPK) pathway and the phosphoinositol-3-kinase (PI3K)/AKT pathway [3, 7]. NGF signaling is able to induce survival and proliferation, but can also induce differentiation without proliferation [8], through increased nuclear expression of p53 and direct activation of the cyclindependent kinase inhibitor p21WAF1/CIP1 [9–11]. TrkA itself has been shown to associate with p53, and was phosphorylated in presence of the latter even in the absence of NGF [12–13]. Though originally discovered as a protooncogene of the nervous system, TrkA isoforms are involved in tumorigenesis in non-neural tumors, primarily carcinomas, and have been generally associated with disease progression and poor outcome [14–16]. Recently, it was also experimentally demonstrated in nude mice that overexpression of TrkA receptor increased the tumorogenicity of pheochromocytoma [17], as well as that of prostatic and pancreatic adenocarcinoma [18]. Conversely, the expression of TrkA as a protooncogene has been associated with better prognosis in neuroblastoma [19, 20]. In addition to the alteration in structure and expression of Trk receptors, intracellular signaling appears to be dysregulated in cancer cells [14]. p75, an additional neutrophin receptor, belongs to the tumor necrosis receptor family, has a different structure, lacks intrinsic catalytic activity and is able to bind all neurotrophins [1]. The evidence regarding the biological role of p75 in human cancer is more limited, but a large study has recently evaluated the expression of this receptor in a variety of human tumors and benign tissues [21]. Frequent expression was seen in some neural and soft tissue tumors, while most carcinomas were negative [21]. Work by the Djakiew group has shown that p75 expression suppresses growth and metastasis of prostate cancer cells in vitro [22], and that expression of this receptor reduces NGF-mediated growth in this malignancy through the induction of apoptosis [23]. Furthermore, p75 expression has been found to be reduced in carcinomas compared to non-neoplastic tissue [24], although the latter finding was not confirmed in an additional study [25]. We have recently reported on marked differences in the expression of the NGF receptor TrkA, its acti-
vated form p-TrkA and the low-affinity neurotrophin receptor p75 at different anatomic sites in serous ovarian carcinoma [26, 27]. Specifically, TrkA and p-TrkA expression was down-regulated in effusions, while the opposite was true for p75. Furthermore, we observed co-expression of p-TrkA with angiogenic molecules, as well as its expression on endothelial cells in the vicinity of tumor cells [27]. Finally, p-TrkA expression in solid tumors predicted poor outcome in our cohort [27]. Interestingly, in a recent study of malignant mesotheliomas, we found opposite trends in the comparison of effusions to solid tumors, namely higher p-TrkA and lower p75 expression in effusions (Davidson et al., submitted). These and the above-mentioned studies suggest a novel emerging concept in cancer, that NGF and other neurotrophins may be involved in the growth of certain non-neural tumors by paracrine and/or autocrine regulation via humoral–tumoral–stromal Trk interaction. Breast cancer is the most common malignancy in women, second in mortality only to lung cancer [28]. Breast cancer metastasizes most frequently to axillary lymph nodes, but any organ may be involved. Metastatic spread to serosal surfaces involves primarily the pleural cavity, where breast cancer is the etiology of approximately 25% of malignant effusions [29, 30]. However, breast carcinoma metastasis may be found infrequently in the pericardial and peritoneal cavity as well [31]. Involvement of the pleural cavity by breast carcinoma may occur at any phase of the clinical course, and may be the sole manifestation of metastatic disease [32]. It is associated with poor prognosis, with a median survival of 5 [32] and 11 [33] months in two series. Despite the magnitude of the clinical problem, studies of the biology of breast cancer have focused exclusively on primary tumors and solid metastases. Consequently, the biological characteristics of breast carcinoma cells in effusions have been poorly characterized at both the phenotypic and genotypic level, and their potential differences from primary and metastatic solid tumors are entirely unknown. Identification of these potential differences may aid in explaining why the appearance of pleural effusion in breast cancer is associated with a rapidly fatal disease. Several studies have analyzed the expression and role of NGF and its receptors in breast carcinoma. NGF has been shown to be produced by breast cancer cells [34] and was mitogenic in breast cancer cell lines, an effect mediated through TrkA and MAPK signaling [35]. Furthermore, the pro-mitogenic role
NGF, TrkA and p75 in breast carcinoma
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Table 1. p-TrkA expression results in the studied material (42 effusions, 34 primary tumors, 18 lymph node metastases, 13 LRa ) Site
Cellular location
Effusion Primary Lymph node LRa Effusion Primary Lymph node LRa Effusion Primary Lymph node LRa
Membraneb Membrane Membrane Membrane Cytoplasm Cytoplasm Cytoplasm Cytoplasm Nucleus Nucleus Nucleus Nucleus
Percentage of stained carcinoma cells 0%
1–5%
6–25%
26–75%
76–100%
3 (7%) 20 (59%) 10 (56%) 1 (8%) 25 (59%) 8 (23%) 5 (27%) 1 (8%) 23 (55%) 15 (44%) 12 (66%) 11 (84%)
6 (14%) 12 (35%) 7 (39%) 7 (54%) 4 (10%) 5 (15%) 3 (17%) 1 (8%) 5 (12%) 4 (12%) 1 (6%) 0 (0%)
6 (14%) 1 (3%) 0 (0%) 2 (15%) 4 (10%) 2 (6%) 3 (17%) 4 (30%) 10 (24%) 1 (3%) 1 (6%) 1 (8%)
14 (34%) 1 (3%) 1 (5%) 1 (8%) 6 (14%) 6 (18%) 3 (17%) 0 (0%) 3 (7%) 4 (12%) 2 (11%) 0 (0%)
13 (31%) 0 (0%) 0 (0%) 2 (15%) 3 (7%) 13 (38%) 4 (22%) 7 (54%) 1 (2%) 10 (29%) 2 (11%) 1 (8%)
a Locoregional recurrences. b p < 0.001 for the difference in membrane expression between effusions and primary tumors,
p = 0.001 for effusions vs. lymph nodes.
was independent of p75 activity, whereas the antiapoptotic effect of NGF was mediated through p75 and the NF-kB transcription factor [36], specifically through interaction with the tumor necrosis-associated death domain protein (TRADD) [37]. NGF effect mediated through TrkA has been shown to be prevented by Tamoxifen in MCF-7 cells [38]. Clinical studies of NGF receptor expression in primary breast carcinoma showed correlation with better survival and with clinicopathologic parameters that are associated with less aggressive disease [39, 40]. The objective of the current study was to investigate the expression of NGF and its receptors in breast cancer cells in effusions and solid tumors, as well as to study the possible correlation between these markers and the interval between primary intervention and relapse in the form of pleural effusion.
Material and methods Patients and clinicopathologic data. The study cohort consisted of 39 female patients with histologically verified breast cancer. Patient age ranged from 35 to 85 years at diagnosis. Thirty-five women were diagnosed with infiltrating duct carcinoma, three with lobular carcinoma and one with a tumor combining both histologic types. Tumor grade was available for all 35 ductal carcinomas and was as follows: 1 grade one tumor, 26 grade two tumors and 8 grade three tu-
mors. The period of time from primary diagnosis to the sampling of pleural effusion ranged from 0 (simultaneous) to 17 years. For the purpose of this study, this period was defined as time to progression (TTP). Effusion specimens. The material consisted of 42 pleural effusions from the above-mentioned 39 patients, all submitted for routine diagnostic purposes to the Section of Cytology, Department of Pathology, The Norwegian Radium Hospital, and the Section of Cytology, Department of Pathology, Aalborg Hospital, during the period of January 1998–September 2002. Submitted specimens arrived within minutes after tapping and were processed immediately. Cellblocks were prepared using the thrombin clot method. Additional material, available for 20 effusions, was suspended and frozen in RPMI + DMSO at −70◦C. Smears and cellblock sections from all specimens underwent morphological evaluation by three experienced cytopathologists, and were further characterized using immunocytochemistry with broad antibody panels against cancer and mesothelial epitopes, as previously detailed [41, 42]. Tumor specimens. Thirty-four corresponding primary tumors, 18 lymph node metastases and 13 locoregional recurrence specimens from the above patients were available for comparative analyses. Formalin-fixed paraffin-embedded tissue blocks were obtained from archival material at the above-
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NGF, TrkA and p75 in breast carcinoma mentioned departments of Pathology. All tissue specimens underwent microscopic confirmation of diagnosis, tumor type and histological grade, following established criteria. Immunohistochemical analysis. Sections from all 42 malignant effusions and 65 solid tumors were stained using a p-TrkA antibody isolated from a serum-free hybridoma culture medium [43]. The characteristics of this antibody and the staining procedure have been previously described [26, 27]. Positive immunostaining with this antibody indicates that the TrkA receptor was autophosphorylated at tyrosine 490, as shown in our previous reports [26, 27]. Staining for p75 was performed in 31 effusions and 44 solid tumors (18 primary, 13 lymph node metastases, 13 locoregional recurrences) using an antibody (clone NGFR5, diluted 1:200) purchased from NeoMarkers (Fremont, CA). Staining for NGF was performed in 21 effusions and 20 solid tumors (nine primary, four lymph node metastases, seven locoregional recurrences) using an antibody previously described [44]. This rabbit polyclonal antibody selectively cross-reacts with proNGF and mature NGF but lacks any cross-reactivity with other neurotrophins such as BDNF, CNTF, NT-3 or NT-4 as verified by ELISA assay. Pretreatment using all three antibodies consisted of microwave oven antigen retrieval for 4 × 5 min in citrate buffer. Staining using both antibodies was done using the Envision peroxidase system (Dako, ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1. Protein expression of NGF and its receptors p-TrkA and p75 in breast cancer. (A)–(C): Examples of expression of each of the markers in the studied material. (A) Cytoplasmic staining for NGF in a primary grade II infiltrating duct carcinoma. This patient suffered from a malignant pleural effusion already at diagnosis. (B) Membrane immunoreactivity for p75 in the same tumor. (C) p-TrkA membrane expression in cancer cells in a pleural effusion. This patient was diagnosed with a grade III infiltrating duct carcinoma and relapsed with pleural effusion 1 year after her primary operation. (D)–(F): Comparative example of p-TrkA expression in a primary tumor, lymph node metastasis and pleural effusion from one patient diagnosed with a grade III infiltrating duct carcinoma who relapsed with pleural effusion 2 years after her primary operation. p-TrkA expression is absent in both primary tumor (D) and lymph node metastasis (E), but is distinctly expressed at the cell membrane of all cells in the pleural effusion of that patient (F). (G)–(I): Comparative example of p75 expression in the same primary tumor, lymph node metastasis and pleural effusion shown in (D)–(F). Expression of p75 in the primary tumor is limited to small vessels in the tumor vicinity, but tumor cells are negative (G). In contrast, the majority of cells show membrane expression in the lymph node metastasis (H). No expression is seen in the pleural effusion (I).
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Glostrup, Denmark). Positive controls for these antibodies consisted of an ovarian carcinoma shown to be positive in our earlier studies. Evaluation of IHC results. p-TrkA expression was scored at the membrane, cytoplasm and nucleus. Membrane and cytoplasm immunoreactivity was scored for p75 and NGF, respectively. The extent of staining was scored using the following scale: 0 = no staining, 1 = staining of 0–5% of tumor cells, 2 = staining of 6–25% of tumor cells, 3 = staining of 26–75% of tumor cells, 4 = staining of 76–100% of tumor cells. A minimum of 500 cells, when present, was evaluated. Evaluation was done without knowledge of patient clinical data. Immunoblotting (IB). Six malignant effusions containing a large fraction (>50%) of tumor cells and the breast carcinoma cell lines MCF-7, T47-D, SKBR3 and MDA-MD-231 were studied. Blotting procedure was as previously described [27]. Filters were hybridized with the antibody against p-TrkA used for IHC. A mouse monoclonal antibody against α-tubulin (clone 57) (Oncogene, Cambridge, MA) was used as loading control. The specificity of the p-TrkA reaction was confirmed using a specific blocking antibody, as previously described [27]. Statistical analysis. Statistical analysis was performed applying the SPSS-PC package (Version 10.1, SPSS, Chicago, 2001). Probability of
