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Phosphorylated 4E Binding Protein 1: A Hallmark of Cell Signaling That Correlates With Survival in Ovarian Cancer Josep Castellvi, MD1 Angel Garcia, MD1 Federico Rojo, MD1 Carmen Ruiz-Marcellan, MD1 Antonio Gil, MD2 Jose Baselga, MD3 Santiago Ramon y Cajal, MD, PhD1
BACKGROUND. Growth factor receptors and cell signaling factors play a crucial role in human carcinomas and have been studied in ovarian tumors with varying results. Cell signaling involves multiple pathways and a myriad of factors that can be mutated or amplified. Cell signaling is driven through the mammalian target of rapamycin (mTOR) and extracellular regulated kinase (ERK) pathways and by some downstream molecules, such as 4E binding protein 1 (4EBP1), eukaryotic initiation factor 4E, and p70 ribosomal protein S6 kinase (p70S6K). The objectives of this study were to analyze the real role that these pathways play in
1
Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain.
ovarian cancer, to correlate them with clinicopathologic characteristics, and to
2
identify the factors that transmit individual proliferation signals and are associated with pathologic grade and prognosis, regardless specific oncogenic altera-
3
METHODS. One hundred twenty-nine ovarian epithelial tumors were studied,
Department of Gynecology, Vall d’Hebron University Hospital, Barcelona, Spain. Department of Oncology, Vall d’Hebron University Hospital, Barcelona, Spain.
tions upstream. including 20 serous cystadenomas, 7 mucinous cystadenomas, 11 serous borderline tumors, 16 mucinous borderline tumors, 29 serous carcinomas, 16 endometrioid carcinomas, 15 clear cell carcinomas, and 15 mucinous carcinomas. Tissue microarrays were constructed, and immunohistochemistry for the receptors epidermal growth factor receptor (EGFR) and c-erb-B2 was performed and with phosphorylated antibodies for protein kinase B (AKT), 4EBP1, p70S6K, S6, and ERK.
RESULTS. Among 129 ovarian neoplasms, 17.8% were positive for c-erb-B2, 9.3% were positive for EGFR, 47.3% were positive for phosphorylated AKT (p-AKT), 58.9% were positive for p-ERK, 41.1% were positive for p-4EBP1, 26.4% were positive for p70S6K, and 15.5% were positive for p-S6. Although EGFR, p-AKT, and p-ERK expression did not differ between benign, borderline, or malignant tumors, c-erbB2, p-4EBP1, p-p70S6K, and p-S6 were expressed significantly more often in malig-
Supported by Grant SAF2002-02184 (to S.R.y.C.) from the Spanish Ministry of Education.
nant tumors. Only p-4EBP1 expression demonstrated prognostic significance (P ¼ .005), and only surgical stage and p-4EBP1 expression had statistical significance in the multivariate analysis.
CONCLUSIONS. In patients with ovarian carcinoma, significant expression of p-4EBP1 We thank Dr. Carlos Cordon-Cardo from Memorial Sloan-Kettering Cancer Center (New York, NY) and Dr. Jaime Prat from Hospital de Sant Pau (Barcelona, Spain) for their critical review of the article. We also thank Jose Jimenez and Sonia Rodriguez for technical assistance. Address for reprints: Santiago Ramon y Cajal, MD, PhD, Department of Pathology, Vall d’Hebron University Hospital, Pg. Vall d’Hebron, 119-129, 08035 Barcelona, Spain; Fax: (011) 34 932746818; E-mail:
[email protected] Received March 9, 2006; revision received June 1, 2006; accepted July 5, 2006.
ª 2006 American Cancer Society
was associated with high-grade tumors and a poor prognosis, regardless other oncogenic alterations upstream. This finding supports the study of this factor as a hallmark or pivotal factor in cell signaling in ovarian carcinoma that may crucial in the transmission of the proliferation cell signal and may reflect the real oncogenic role of this pathway in ovarian tumors. Cancer 2006;107:1801–11. 2006 American Cancer Society.
KEYWORDS: cell signaling, ovarian neoplasms, prognostic factors, 4E binding protein 1.
M
ost human neoplasms have broad clinical, pathologic, and molecular heterogeneity; and, in nearly all malignant solid tumors, there is oncogenic activation of cell immortalization, cell
DOI 10.1002/cncr.22195 Published online 18 September 2006 in Wiley InterScience (www.interscience.wiley.com).
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signaling, cell cycle, apoptosis, and tumor-invasive pathways.1 To date, > 350 cancer genes have been identified, and hundreds of epigenetic alterations have been described in tumors.2 In recent years, with the study of messenger RNA (mRNA) arrays, new sets of genetic alterations have been proposed in tumors, some of them related to histologic type, prognosis, and response to anticancer treatments. Ovarian carcinoma is the gynecologic malignancy associated with the highest mortality in industrialized countries, with a 5-year survival rate of < 30% reported in some series.3 The prognosis for patients with ovarian cancer is determined by conventional factors, such as histologic type, histologic grade, and surgical stage; however, to our knowledge to date, no single molecular profile has helped to identify the most aggressive tumors. In addition, little is known regarding the molecular mechanisms involved in malignant transformation or the biochemical alterations of the cell cycle, cell signaling, apoptosis, and angiogenesis-related pathways affected. The genes involved in these pathways show great redundancy, and many genetic alterations have been described. All carcinomas show activated growth factor receptor-cell signaling pathways with genetic alterations that involve either the receptor, or some of the factors that drive the proliferation signal downstream, or both. Although normal cell growth depends on 2 types of stimuli, 1 type from growth factors and another type from the presence of nutrients, malignant cells acquire so-called ‘‘self sufficiency in growth signals,’’ which means that malignant cells harbor mutation or gene amplification either in the growth factor receptor family (Ras, phosphatase and tensin homolog, phosphoinositide-3 kinase [PI3K]) or in other factors involved in the labyrinthine routes of cellular pathways downstream.4 Most stimuli follow different pathways that converge in the mammalian target of rapamycin (mTOR),5 which plays a central role in cell growth control (Fig. 1), and this factor can phosphorylate p70 ribosomal protein S6 kinase (p70S6K) and 4EBP1, which clearly are related to protein synthesis and cell proliferation. 4EBP1 dimerizes with eukaryotic initiation factor 4E (eIF4E), blocking the formation of the initiation complex. When 4EBP1 is phosphorylated, eIF4E is released, and translation can begin. The principal substrate of the kinase p70S6K is ribosomal protein S6, which is part of the 40S ribosomal unit. It is involved in the translation of proteins encoded by 50 terminal oligopyrimide genes. Most of these proteins are regulators of protein synthesis and can act as protooncogenes.6 It is noteworthy that 4EBP1 has 8 phosphorylation sites7 and also can be phosphorylated by other kinases, including PI3K, ataxia telangiec-
FIGURE 1. The signaling pathways involved in cell growth control are illustrated in this diagram, which shows the main cell signaling pathways studied. 4E binding protein 1 (4EBP1) is proposed as a central factor in which diverse oncogenic signals may converge, providing a possible explanation for the high percentage of positive tumors and its significant clinical correlation. TK indicates tyrosine kinase, PI3K, phosphoinositide-3 kinase; PTEN, phosphatase and tensin homolog; MEK, mitogen-activated protein kinase/ERK kinase; AKT, protein kinase B; ATM, ataxia telangiectasia mutated; PLD, phospholipase D; Aa, amino acids; mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase; MNK, mitogen-activated protein kinaseinteracting kinase; eIF4E, eukaryotic initiation factor 4E; 50 TOP, 50 terminal oligopyrimide; CAP, a structure that stabilizes the RNA; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor.
tasia mutated kinase, mitogen-activated protein kinase/ extracellular signal-regulated kinase (MEK), and others that remain unknown. Wild-type p53 can modulate 4EBP1 through cdc2 or other unidentified phosphatases.8 Other p70S6K targets also include transcription factors or translation regulators.9 Moreover, p70S6K and eIF4E can be activated by pathways that are independent of mTOR, including the Ras-Raf-MEK-ERK pathway.10 In previous studies, it was observed that epidermal growth factor receptor (EGFR), c-erb-B2, protein kinase B (AKT), and ERK were expressed frequently in ovarian neoplasms11–14; however, downstream effectors have not been characterized well in these tumors. We hypothesized that very few factors should exist downstream that could relay the oncogenic signal to the ribosomes or nucleus, regardless of the individual oncogenic alterations upstream. Then, these final factors would be transmitters of the proliferation signal, regardless the specific oncogene alteration upstream, and could be like a choke point for these pathways.
Phospo-4EBP1 in Epithelial Ovarian Neoplasms/Castellvi et al.
To investigate this hypothesis, we studied activation of the receptors EGFR and c-erb-B2, the 2 main biochemical pathways AKT and mitogen-activated protein kinase ERK, and the downstream factors 4EBP1, p70S6K, and S6 by immunohistochemistry using specific phosphorylated antibodies. Expression of these factors was measured in ovarian tumors, and the results were correlated with patient survival and tumor recurrence to determine which, if any, of these factors fulfilled the concept of a ‘‘hallmark factor’’ of the pathway and was associated with clinical prognosis regardless the oncogenic alterations upstream.
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FIGURE 2. Western blot validation of immunohistochemical stainings showed a good correlation between Western blot analysis and immunohistochemical analysis for phosphorylated 4E binding protein 1 in serous carcinoma (SC), endometrioid carcinoma (EC), clear cell carcinoma (CCC), serous
MATERIALS AND METHODS Patients and Tissue Characteristics Over 5 years, from January 1994 to December 1998, 129 patients underwent surgery for primary epithelial ovarian tumors at Hospital Vall d’Hebron (Barcelona, Spain). The tumors included 20 serous cystadenomas, 7 mucinous cystadenomas, 11 serous borderline tumors, 16 mucinous borderline tumors, 29 serous adenocarcinomas, 15 mucinous adenocarcinomas, 16 endometrioid adenocarcinomas, and 15 clear cell adenocarcinomas. The patients ranged in age from 20 years to 87 years (mean age, 55 years). All International Federation of Gynecology and Obstetrics (FIGO) stages of ovarian carcinomas were represented; however, because of the small number of patients in some subgroups and the uneven distribution of patients, only the 4 major groups (Stages I through IV) were used in the analysis. Thus, the series included 29 Stage I tumors, 5 Stage II tumors, 37 Stage III tumors, and 4 Stage IV tumors. Follow-up information included disease-free survival, overall survival, and cancer-related death. All data collected were entered prospectively into the ovarian cancer registry data base. The mean follow-up was 31 months (range, 24–80 months). The tumors had been fixed in neutral formalin and embedded in paraffin. In all patients, the diagnosis was based on a light microscopy examination using conventional hematoxylin and eosin stain. For immunohistochemical staining, tissue microarrays from all patients were constructed, including 3 cores that measured 2 mm in greatest dimension for each patient. Cell Lines and Western Blot Analysis The human breast MDA-MB-435 lung-2 cell line15 was grown in Dulbecco Medium Essential Medium (GibcoBRL) containing 10% fetal calf serum (BioWhittaker Europe). To validate the specificity of the
borderline tumor (SBLT), and mucinous borderline tumor (MBLT). The positive control (C þ) for Western blot analysis was the breast carcinoma cell line MDA-MB-435.
antibodies used in this study, Western blot analysis was performed in the cell line and in lysates of frozen tissue from 15 human ovarian tumors (Fig. 2). Protein was quantified (Biorad assay; Biorad, Munchen, Germany), and lysates were processed for Western blot analysis as reported previously16,17 by using the same antibodies that were used for immunohistochemistry (Table 1). Immunocomplexes were washed extensively and resuspended in sample buffer 5. Antibody detection was achieved by using enhanced chemiluminescence (Amersham Pharmacia, Uppsala, Sweden). Antibody expression also was studied in paraffin embedded histologic specimens from the same tumors (Fig. 2).
Antibodies and Immunohistochemistry Immunohistochemical staining using the avidin-biotin-peroxidase technique was carried out for each antibody. Five-micrometer sections were cut from the tissue specimens and placed on poly-L-lysinecoated glass slides. Sections were deparaffined in xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked by immersing the sections in 0.1% hydrogen peroxide in absolute methanol for 20 minutes. For antigen retrieval, the tissue sections were heated in a pressure cooker in 10 mM citric acid monohydrate (pH 6.0) for 5 minutes and then incubated with the primary antibody at room temperature. The primary antibodies, dilutions, and incubation times used are shown in Table 1. Immunostaining was performed with the EnVision system (DakoCytomation, Glostrup, Denmark). All slides were counterstained with hematoxylin, dehydrated, and mounted. Negative controls were performed by
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TABLE 1 Primary Antibodies and Dilutions Used Protein
Phosphorylation site
Source
Antibody type
Dilution
Incubation time, minutes
c-erb-B2 EGFR p70S6K S6 AKT 4EBP1 ERK1/ERK2
— — Thr 389 Ser 240/Ser 244 Ser 473 Thr 70 Thr 202/Tyr 204
Biogenex DakoCytomation Cell Signaling Tech. Cell Signaling Tech. Cell Signaling Tech. Cell Signaling Tech. Cell Signaling Tech.
Monoclonal Monoclonal Monoclonal Polyclonal Polyclonal Polyclonal Polyclonal
1/20 1/100 1/100 1/50 1/50 1/100 1/50
60 60 60 30 120 60 120
EGFR indicates epidermal growth factor receptor; Thr, threonine; p70S6K, p70 ribosomal protein S6 kinase; AKT, protein kinase B; Ser, serine; 4EBP1, 4E binding protein P1; ERK, extracellular signal-regulating kinase; Tyr, tyrosine.
omitting the primary antibody. All primary antibodies were tested first by Western blot to evaluate the specificity of the staining in both the cell lines and the tumor samples. To evaluate immunohistochemical staining, we scored the percentage of positive cells and intensity of the staining, which was assessed semiquantitatively. Samples that showed no immunostaining were considered negative, and samples that showed any positivity were grouped together for statistical purposes. Some markers, such as phosphorylated 4EBP1 (p-4EBP1) and p-p70S6K, showed 2 types of positive expression: a weak, uniform nuclear staining (Fig. 3A) and a rough, moderate, or intense staining (Fig. 3D). Moderate-to-strong positivity was identified easily at low magnification ( 40), whereas weak positivity was visible at high magnification ( 400). Some of the samples that had intense nuclear staining also showed cytoplasmic, granular positivity. Because weak positivity was observed in benign ovarian tumors, normal skin samples, and other normal tissues, such as lobular breast epithelium, we considered this intensity pattern for p-4EBP1 and p-p70S6K, as well as the absence of staining, as negative. For EGFR, we considered samples positive if they had membrane staining in > 25% of cells; and, for c-erb-B2, we used the same scoring system that is used commonly for breast tumors and only considered samples positive if they showed complete membrane staining (2 þ and 3 þ). When the 3 cores from the same patient showed different positivity results, then the highest score was considered valid.
Statistical Analysis Statistical analysis was performed with the Statistical Package for Social Science (SPSS version 13.0; SPSS Inc., Chicago, IL). Categorical variables were analyzed by using cross-tabulation, and differences were evaluated by using the chi-square test. A 2-sided P
FIGURE 3. Immunohistochemical staining for phosphorylated 4E binding protein 1 is observed in benign, low-grade, and high-grade tumors, including (A) ovarian mucinous cystadenoma, (B) serous cystadenoma, (C) mucinous borderline tumor with mild nuclear staining, and (D) serous carcinoma with intense nuclear and cytoplasmic staining (original magnification 400 in A-D).
value .05 was considered indicative of a statistically significant difference. The effects of the various parameters on survival, including histologic grade and type, FIGO stage, and immunostaining results, were tested with the Kaplan–Meier method, and differences were compared by using the log-rank test. Multivariate analysis for overall survival was performed with the Cox proportional hazards method.
RESULTS Antibody Validation by Western Blot Analysis To validate the paraffin results, we correlated the immunostaining obtained in paraffin sections with the intensity and specificity obtained by Western blot analysis of fresh tumors. In the 15 tumors that were
Phospo-4EBP1 in Epithelial Ovarian Neoplasms/Castellvi et al.
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showed c-erb-B2 expression more frequently than low-grade carcinomas, although no differences were observed among surgical stages. When associations between the expression of c-erb-B2 and the expression the other markers were assessed, we observed no significant statistical correlation with p-AKT (P ¼ .162) or p-ERK (P ¼ .807), and we observed significant correlations with p-4EBP1 (P ¼ .016) and pp70S6K (P ¼ .030). EGFR expression did not correlate with the expression of any other marker.
FIGURE 4. These photomicrographs illustrate immunohistochemical staining for c-erb-B2, epidermal growth factor receptor (EGFR), phosphorylated protein kinase B (p-AKT), phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated p70 ribosomal protein S6 kinase (p-p70S6K), and phosphorylated ribosomal protein S6 (p-S6) in ovarian tumors. (A) c-erb-B2 membrane positivity is shown in a high-grade clear cell carcinoma. (B) EGFR
p-AKT expression patterns p-AKT was expressed in either the nucleus or the cytoplasm, and both staining patterns were considered positive. Most tumors with nuclear positivity also had some cytoplasmic expression of p-AKT. Approximately 50% of tumors were positive for pAKT, and there were no differences in the percentage of positive cells or the intensity of expression between benign, borderline, or malignant tumors. Mucinous carcinomas and low-grade carcinomas were positive for p-AKT less often. Some tumors showed intense nuclear positivity of stromal cells with no relation to epithelial expression or morphology. With regard with downstream effectors, p-AKTpositive tumors showed positivity for p-4EBP1 in 34 patients (55%; P ¼ .002) and for p-p70S6K in 24 patients (39%; P ¼ .002). In 23 patients (37%), both markers were negative (P ¼ .003).
expression is shown in a serous carcinoma. (C) This endometrioid carcinoma has moderate nuclear and cytoplasmic expression of p-AKT. (D) This highgrade serous carcinoma has strong cytoplasmic and nuclear staining for ERK. (E) This high-grade serous carcinoma has nuclear expression of pp70S6K. Some cytoplasmic staining also is observed. (F) Diffuse cytoplasmic expression of p-S6 is observed in a high-grade endometrioid carcinoma (original magnification 280 (A,C,E,F); 400 (D).
studied, a good correlation was found between the intensity of the band on Western blot analysis and the immunoreactivity of paraffin tissue (Fig. 2).
EGFR and c-erb-B2 expression patterns Both EGFR and c-erb-B2 proteins were expressed in tumor cell membranes, and granular cytoplasmic positivity also was observed in some tumors (Fig. 4). EGFR was observed in < 10% of tumors and without significant differences between benign, borderline, and malignant tumors, although benign tumors rarely expressed the protein (Tables 2 and 3). No correlation was observed with histologic type, grade, or surgical stage. Conversely, only malignant tumors were positive for c-erb-B2. High-grade carcinomas
p-ERK expression patterns Immunohistochemistry was positive for p-ERK in either the nucleus or the cytoplasm, although only nuclear staining positive was considered positive (Fig. 4). Like p-AKT, no significant differences were observed in p-ERK expression between benign, borderline, or malignant tumors. Clear cell carcinomas were positive more often. Some tumors had strong stromal cell cytoplasmic and nuclear staining. Tumors that expressed p-ERK were positive for p-4EBP1 in 40 patients (53%; P ¼ .002) and positive for p-p70S6K in 25 patients (33%; P ¼ .067). In 22 patients (29%), both markers were negative (P ¼ .011). p-4EBP1 expression patterns p-4EBP1 immunostaining was mainly nuclear and was expressed in 53 tumors (41.1%), with significantly lower expression observed in mucinous tumors. Malignant tumors were positive for 4EBP1 more often than benign or borderline tumors, and there was a significant correlation with histologic grade (P < .0001) (Fig. 5). Cytoplasmic staining was observed in 18 tumors (25%) and was correlated with higher histologic
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TABLE 2 Summary of Immunohistochemical Results Comparing Benign, Borderline, and Malignant Ovarian Tumors No. of patients (%)
Tumor type Benign Borderline Malignant Total P
Total no. 27 27 75 129
c-erb-B2
EGFR
p-AKT
p-ERK
p-4EBP1
p-p70S6K
p-S6
0 (0.0) 0 (0.0) 23 (30.7) 23 (17.8) < .0001
1 (3.7) 3 (11.1) 8 (10.7) 12 (9.3) .529
9 (33.3) 13 (48.1) 39 (52.0) 61 (47.3) .248
14 (51.9) 16 (59.3) 46 (61.3) 76 (58.9) .691
5 (18.5) 7 (25.9) 41 (54.7) 53 (41.1) .001
2 (7.4) 4 (14.8) 28 (37.3) 34 (26.4) .003
1 (3.7) 1 (3.7) 18 (24.0) 20 (15.5) .007
EGFR indicates epidermal growth factor receptor; p-AKT, phosphorylated protein kinase B; p-ERK, phosphorylated ERK; p-4EBP1, phosphorylated 4E binding protein 1; p-p70S6K, phosphorylated p70 ribosomal protein S6 kinase; p-S6, phosphorylated ribosomal protein S6.
TABLE 3 Immunohistochemical Results and Clinicopathologic Characteristics in Patients with Epithelial Ovarian Carcinoma No. of patients (%)
Characteristic Age < 60 y > 60 y Histologic type Serous carcinoma Mucinous carcinoma Endometrioid carcinoma Clear cell carcinoma Histologic grade Grade 1 Grade 2 Grade 3 Disease stage Stage I-II Stage III-IV
Total no.
c-erb B2
EGFR
p-AKT
p-ERK
p-4EBP1
p-p70S6K
p-S6
34 41
11 (34.4) 12 (27.9)
3 (9.4) 5 (11.6)
20 (62.5) 19 (44.2)
24 (75.0) 22 (51.2)
22 (68.8) 19 (44.2)
14 (43.8) 14 (32.6)
8 (25.0) 10 (23.3)
29 15 16 15
8 (27.6) 1 (6.7) 7 (43.8) 7 (46.7)
3 (10.3) 1 (6.7) 3 (18.8) 1 (6.7)
15 (51.7) 3 (20.0) 10 (62.5) 11 (73.3)
19 (65.5) 6 (40.0) 8 (50.0) 13 (86.7)
18 (62.1) 2 (13.3) 9 (56.3) 12 (80.0)
10 (34.5) 2 (13.3) 7 (43.8) 9 (60.0)
5 (17.2) 4 (26.7) 4 (25.0) 5 (33.3)
11 13 51
1 (9.1) 3 (23.1) 19 (37.3)
0 (0.0) 4 (30.8) 4 (7.8)
2 (18.2) 7 (53.8) 30 (58.8)
4 (36.4) 7 (53.8) 35 (68.6)
1 (9.1) 5 (38.5) 35 (68.6)
2 (18.2) 3 (23.1) 23 (45.1)
2 (18.2) 5 (38.5) 11 (21.6)
34 41
7 (20.6) 16 (39.0)
3 (8.8) 5 (12.2)
17 (50.0) 22 (53.7)
18 (52.9) 28 (68.3)
16 (47.1) 25 (61.0)
14 (41.2) 14 (34.1)
8 (23.5) 10 (24.4)
EGFR indicates epidermal growth factor receptor; p-AKT, phosphorylated protein kinase B; p-ERK, phosphorylated ERK; p-4EBP1, phosphorylated 4E binding protein 1; p-p70S6K, phosphorylated p70 ribosomal protein S6 kinase; p-S6, phosphorylated ribosomal protein S6.
grades (P ¼ .037). Moreover, cytoplasmic staining was observed only in malignant tumors. No correlation was observed between nuclear or cytoplasmic expression of the protein and the surgical stage of the tumor. Tumors that were positive for p-4EBP1 expressed p-AKT in 34 tumors (64%; P ¼ .002) and expressed p-ERK in 40 tumors (75%; P ¼ .001). Only 10 tumors (19%) were negative for both p-AKT and p-ERK (P < .0001).
p-p70S6K expression patterns Nuclear immunohistochemistry for p-p70S6K was positive in 30% of tumors, and additional cytoplasmic expression was found in 15% of tumors. Malignant
tumors showed greater nuclear expression of p-p70S6K than benign or borderline tumors. Cytoplasmic staining was observed only in malignant tumors, although there was no correlation with histologic grade. Among the ovarian carcinomas, high-grade tumors showed stronger nuclear immunostaining, although the difference was not significant (P ¼ .109). Clear cell carcinomas were the most frequently positive tumors, and mucinous carcinomas were the least frequently positive tumors. Tumors that were positive for p-p70S6K also were positive for p-ERK in 25 tumors (73.5%; P ¼ .067) and were positive for p-AKT in 24 tumors (70.6%; P ¼ .002). Only 5 tumors (14.7%) did not express either
Phospo-4EBP1 in Epithelial Ovarian Neoplasms/Castellvi et al.
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FIGURE 6. Overall survival curves are shown in relation to phosphorylated 4E binding protein 1 (p-4EBP1) expression.
FIGURE 5. Phosphorylated 4E binding protein 1 (p-4EBP1)-positive tumors are illustrated according to histologic grade in patients with ovarian carcinomas. Most tumors with p-4EBP1 expression were high-grade carcinomas, whereas only 1 well differentiated carcinoma (2%) was positive.
TABLE 4 Overall Survival Results of the Signaling Proteins Studied Mean survival, months Signaling protein
Negative patients
Positive patients
P (Log-rank)
c-erb-B2 EGFR p-AKT p-ERK p-4EBP1 p-p70S6K p-S6
44.87 52.84 54.04 61.12 70.55 58.75 56.35
55.13 55.53 53.27 49.47 46.87 43.38 43.97
.808 .820 .370 .151 .005* .197 .906
EGFR indicates epidermal growth factor receptor; p-AKT, phosphorylated protein kinase B; p-ERK, phosphorylated extracellular signal-regulated kinase; p-4EBP1, phosphorylated 4E binding protein 1; p-p70S6K, phosphorylated p70 ribosomal protein S6 kinase; p-S6, phosphorylated ribosomal protein S6. * p-4EBP1 was the only marker that had clear prognostic significance.
protein (P ¼ 0.010). Conversely, 12 tumors (35%) that were positive for p-p70S6K expressed p-S6 (P ¼ .001).
p-S6 expression patterns p-S6 was expressed in cytoplasm, and immunohistochemical results were positive in only 15.5% of tumors. Benign and borderline tumors mostly were negative (P ¼ .004), although there were no differences among the histologic types. Sixty percent of p-S6-positive tumors showed p-p70S6K expression (P ¼ .009).
Because p70S6K and 4EBP1 can be activated by both the AKT and ERK pathways, we determined the effect of activation of either or both pathways on expression of the phosphorylated forms of these proteins. We observed that p-p70S6K expression was more frequent in tumors in which both AKT and ERK were phosphorylated (58%) compared with tumors in which only 1 protein was expressed (26%) or in which both proteins were negative (14%; P ¼ .026). These findings were more significant for 4EBP1 expression (i.e., 58% of tumors that were positive for 4EBP1 demonstrated coexpression of p-AKT and pERK, 22% were positive only for 1 protein, and 19% were negative for both proteins [P < .0001]).
Survival Analysis Of 75 patients with ovarian carcinoma, 22 died of disease, and 14 survived with progressive disease. One of 27 patients who had borderline tumors had died of disease at the time of last follow-up, and another was alive with progressive disease. The mean survival was 55 months (range, 13–75 months). Eight patients (5 patients with carcinomas and 3 patients with borderline tumors) died of unrelated causes. A survival analysis of patients with carcinoma in relation to activation of the proteins studied showed a significant log-rank value only for p-4EBP1 nuclear expression (Table 4) when overall survival was assessed (Fig. 6); however, similar to the other markers, p-4EBP1 was not significant for progression-free survival (P ¼ .052). Tumors that were negative for
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p-4EBP1 accounted for only 7.4% of deaths, whereas tumors that were positive for p-4EBP1 represented 43.9% of deaths. In the multivariate analysis with Cox regression that included histologic grade, surgical stage, and p-4EBP1 expression, only surgical stage (P ¼ .010) and p-4EBP1 expression (P ¼ .028) were statistically significant.
DISCUSSION Study of the signaling pathways involved in cell growth, the cell cycle, and apoptosis has contributed greatly to our understanding of the molecular mechanisms of carcinogenesis in human tumors. The interest in this line of study is enhanced further when there are potential or known therapeutic targets involved, prognostic factors, or factors predictive of resistance to therapy. These signaling pathways are crucial in many malignancies, including breast, prostate, and gastric carcinomas, lymphomas, and multiple myelomas.18–20 Few studies have evaluated the role of these molecules in ovarian carcinomas, and most of the work has been done in cell lines. For this reported, we evaluated the main components of the cell signaling pathways, including the epithelial growth factor receptors EGFR and c-erb-B2, the ERK and AKT pathways, and their downstream factors, p70S6K, S6 and 4EBP1, in a large series of ovarian tumors. The primary objective of this study was to correlate the expression of these factors with pathologic features and the patient’s clinical prognosis and, ultimately, to identify factors that may reflect the real oncogenic role of these important biochemical pathways in ovarian tumors regardless the oncogenic alterations present upstream. With regard to c-erb-B2 and EGFR, we observed their expression in 17.8% and 9.3% of ovarian tumors, respectively. In other studies, the percentage of c-erbB2-positive tumors ranged from 15% to 30%.12,21,22 It is noteworthy that c-erb-B2 overexpression in our series was observed only in carcinomas: mostly in highgrade carcinomas. Surprisingly, despite this correlation with aggressive morphology, results of the survival study did not indicate statistical significance. Although the predictive value of c-erb-B2 in ovarian carcinomas is not clear, some studies have shown a significantly worse prognosis for patients with positive tumors,11,12,21 whereas others did not observe this correlation.23–25 Similarly, EGFR reportedly was overexpressed in 30% to 77% of ovarian carcinomas, and its association with a poor prognosis, like in our series, was not verified.11,25–27 Downstream of the growth factor receptors, there are at least 2 main biochemical routes. It is known
that the PI3K-AKT-mTOR pathway and the Ras-RafMEK-ERK pathways are activated in ovarian carcinomas. Regarding the PI3K-AKT-mTOR pathway, p-AKT is found in 36% to 68% of tumors, and 55% of tumors express mTOR.13,28 Our current results indicated that AKT was activated in 47% of carcinomas, but no significant differences were observed between benign or borderline tumors or between histologic grades. Moreover, the results did not demonstrate that p-AKT expression had prognostic significance. These results contrast with other reports, which reported the prognostic value of AKT activation in certain tumors, such as melanomas29 and lung30 or prostate carcinomas,31 but they are in agreement with studies in breast carcinoma in which no correlation was observed.32 The downstream effectors of this pathway, 4EBP1 and p70S6K, frequently are overexpressed in ovarian carcinomas. p-4EBP1 is expressed in 55% of carcinomas and is more frequent in high-grade tumors. It is worth noting that, in the current study, the overexpression of p-4EBP1 was correlated with a poor prognosis, even in the multivariate analysis, regardless of the surgical stage. In contrast, p-p70S6K was overexpressed in 37% of carcinomas but did not correlate with histologic grade, surgical stage, or survival. Only 35% of tumors that were positive for p-p70S6K were also positive for pS6 positivity in our series, supporting the finding that p70S6K has substrates other than the S6 ribosomal protein. The 3 downstream molecules were expressed more often in malignant tumors than in benign or borderline tumors. It has been demonstrated that the Ras-Raf-MEKERK pathway also is important in ovarian carcinomas, particularly in serous carcinomas, in which it carries prognostic significance.14,33 K-ras and BRAF mutations often are found in serous borderline tumors and in low-grade serous carcinomas.34 These mutations support the existence of 2 different carcinogenic pathways for serous carcinomas: a pathway for low-grade carcinomas, which probably results from progression of a borderline tumor with K-ras and BRAF mutations, and a pathway for high-grade serous carcinomas with a low incidence of mutations in these molecules.35 K-ras and BRAF mutations also are frequent in mucinous tumors.36 The effector of these molecules is ERK, and the phosphorylated form of the protein can be an indicator of the overall status of this pathway. Moreover, it has been demonstrated that active ERK is a good prognostic marker of high-grade serous carcinomas by other pathways that have not been identified well.14 In the current study, no significant differences in p-ERK expression were observed between borderline and high-grade serous carcinomas. Activated ERK can
Phospo-4EBP1 in Epithelial Ovarian Neoplasms/Castellvi et al.
phosphorylate p70S6K at a different site than mTOR.7 In multiple myeloma cells, the phosphorylation of both sites reportedly showed a synergistic effect.37 In our series, positivity for p-p70S6K was stronger in tumors with p-ERK and p-AKT coexpression than in tumors in which only 1 or neither was expressed. Similarly, 4EBP1 can be activated by the ERK pathway.38,39 Moreover, for p-4EBP1 expression, we observed the same synergistic effect when ERK and AKT were expressed (Fig. 7), suggesting that these 2 pathways involved in translational control are interrelated. This effect was not observed in relation to receptor status (Fig. 7). In this sense, in a recent study, it was observed that, by blocking both pathways simultaneously, there was a synergistic effect on the inhibition of cell proliferation.40 Because p-4EBP1 was the only studied markers that was associated with prognosis and histologic grade, it may represent a hallmark of the activation status of at least 2 signaling pathways. Our results and conclusions regarding 4EBP1 are supported by the oncogenic role of eIF4E, which is released when 4EBP1 is phosphorylated. Previous data have described 4EBP1-eIF4E in human tumors and animal models.41–44 In fact, eIF4E is an essential component of the malignant phenotype in breast carcinoma,45 and hyperphosphorylation of 4EBP1 is crucial in this effect. The high cytoplasmic level of 4EBP1 observed in a subset of high-grade tumors may reflect a hyperphosphorylated state and, indirectly, concomitant oncogenic alterations upstream. This means that 4EBP1 can be phosphorylated by mTOR, which can be activated by AKT,5 phospholipase D,46 and other kinases and by other PI3K kinases of the Ras-RafMEK-ERK pathway.39 It is noteworthy that, because p53 mediates dephosphorylation of p-4EBP1,8 nonfunctional or mutated p53 also can contribute toward maintaining a hyperphosphorylated 4EBP1 protein and, thus, an activated eIF4E. Moreover, colocalization of p-4EBP1 in the nucleus and cytoplasm of high-grade carcinomas suggests a true oncogenic role in these tumors, in which associated biochemical and molecular factors need to be investigated. eIF4E forms nuclear bodies and has an important function in the translation of a subset of growth-promoting mRNAs, including cyclin D1, Myc, ornithine decarboxylase, fibroblastic growth factor, and vascular endothelial growth factor.47 Future immunohistochemical studies will be needed to validate eIF4E expression in human tumors as a true prognostic factor and to correlate it with 4EBP1. In the setting of cell signaling and in an attempt to identify molecules that clearly reflect the oncogenic role of a specific pathway in ovarian cancer,
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FIGURE 7. Percentages of ovarian neoplasms with phosphorylated 4E binding protein 1 (p-4EBP1) expression are illustrated relative to upstream markers. Note that, among p-4EBP1-positive (p-4EBP1 þ) tumors, there was a marked predominance of c-erb-B2 overexpression compared with epidermal growth factor receptor (EGFR)-positive (EGFR þ) tumors. Conversely, phosphorylated protein kinase B (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) appear to contribute to 4EBP1 activation. It is noteworthy that there is a synergistic effect on 4EBP1 when both pathways are activated.
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we found that 4EBP1 may represent a hallmark of cell signaling and may act as a pivotal factor in the oncogenic pathways. We propose the concept of 4EBP1 as a choke point at which many oncogenic signals converge and at which, after their phosphorylation, many transcription and phosphorylation factors are activated. The findings that p-4EBP1 expression is associated with tumor progression and an adverse prognosis and that it can be detected by using immunohistochemistry allows us to suggest its study as a new molecular marker in ovarian tumors, regardless of the status of other possible oncogenic alterations. Investigation into factors like 4EBP1 in human tumors may lead to the identification of molecular markers in each oncogenic pathway that, in turn, may complement the pathology report with a small set of factors that are hallmarks of the functional molecular signature of the tumor. Even more important, 4EBP1 and eIF4E may be molecular targets for clinical treatment.
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