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Nerve growth factor and its high-affinity receptor trkA participate in the control of vascular endothelial growth factor expression in epithelial ovarian cancer
Gynecologic Oncology 104 (2007) 168 – 175 www.elsevier.com/locate/ygyno
Nerve growth factor and its high-affinity receptor trkA participate in the control of vascular endothelial growth factor expression in epithelial ovarian cancer Ximena Campos a,1 , Yenny Muñoz a,1 , Alberto Selman b , Roberto Yazigi c , Leonor Moyano d , Caroline Weinstein-Oppenheimer e , Hernán E. Lara f , Carmen Romero a,b,⁎ a
f
Laboratorio de Endocrinología y Biología Reproductiva, Hospital Clinico Universidad de Chile, Chile b Departamento de Obstetricia y Ginecología, Facultad de Medicina, Universidad de Chile, Chile c Clinica Las Condes, Chile d Servicio de Anatomía Patológica, Hospital Clínico Universidad de Chile, Chile e Departamento de Bioquímica, Facultad de Farmacia, Universidad de Valparaíso, Chile Laboratorio de Neurobioquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile Received 29 March 2006 Available online 28 August 2006
Abstract Objectives. To compare the expression of nerve growth factor (NGF) and its high-affinity receptor trkA in normal ovaries and in epithelial ovarian carcinomas. Given NGF acts as an angiogenic factor through a vascular endothelial growth factor (VEGF)-mediated mechanism in several types of tissues, we examined whether NGF regulates the expression of VEGF isoforms in epithelial ovarian cancer (EOC). Methods. The expression and localization of NGF and tyrosine kinase receptor A (trkA) in normal ovarian samples and in ovarian cancer samples were analyzed by RT-PCR and immunohistochemistry. NGF regulates the expression of three VEGF isoforms (VEGF121, VEGF165 and VEGF189); these were examined using RT-PCR in explants of EOC and ELISA in culture media. Results. TrkA mRNA levels were over-expressed in ovarian cancer compared to normal ovarian samples, whereas NGF mRNA levels remained unchanged. NGF and trkA proteins were absent or found in very low levels in normal ovarian surface epithelium (OSE), whereas they were highly expressed in epithelial cells of EOC. Additionally, NGF stimulated the expression of VEGF isoforms in cancer explants. The effect was dose-dependent and inhibited by a NGF antibody and by K252a, a trk receptor inhibitor. Conclusion. The abundance of NGF and trkA receptors in epithelial cells of EOC, together with the ability of NGF to increase VEGF expression strongly suggests an autocrine role of NGF in EOC. These findings suggest that blocking neurotrophin action could be a therapeutic target in treating ovarian cancer. © 2006 Elsevier Inc. All rights reserved. Keywords: NGF; trkA; VEGF; Epithelial ovarian cancer
Introduction Each year, more women die from ovarian cancer than from any other gynecologic malignancy. The lifetime risk for a ⁎ Corresponding author. Laboratorio de Endocrinologia y Biologia Reproductiva, Departamento de Obstetricia y Ginecologia, Hospital Clinico Universidad de Chile, Santos Dumont 999, Santiago, Chile. Fax: +56 (2)7374555. E-mail address: [email protected] (C. Romero). 1 Both authors contributed equally to this study. 0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2006.07.007
woman to be affected by ovarian cancer is 1.7% and the annual incidence approaches 61.3 per 100,000 in women 75 to 79 years of age [1]. Ovarian cancer is a deadly insidious disease because typically it is asymptomatic until the malignancy has reached beyond the ovaries. Unfortunately, screening for the early detection of ovarian malignancies has not proven effective. Several mechanisms may explain the etiology of ovarian cancer: Fathalla's theory of “incessant ovulation” [2], excessive stimulation by gonadotropins and estrogens [3–5],
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and local stimulation of androgens [6]. Several authors postulate that most ovarian cancers arise from the transformation of a surface epithelial affected by ovulation: the integrity of the DNA on surface cells circumjacent to the ovarian rupture site is compromised during the ovulatory process and clonal expansion of an epithelial cell with damaged (unrepaired) DNA is a putative factor in carcinogenesis [7,8]. However, it has not been established whether this malignancy evolves from benign or borderline tumors, or whether malignancy develops de novo from surface epithelial cells, or inclusion epithelial cysts. Mok et al. [8] provide evidence supporting both progressions. Although many studies try to explain the progression of epithelial cancer, none have proposed the mechanisms underlying these changes, probably because the sequence of events that leads to ovarian cancer is multifactorial and not adequately understood [9]. Local increases in growth factors may be responsible for angiogenesis and proliferation of ovarian surface epithelial cells (OSE) could participate in the initial stages of cancer progression [10]. Ovarian cancer is highly angiogenic and VEGF is the main factor responsible for angiogenesis, vascular permeability and metastasis [11]. VEGF is secreted by various tumors, including epithelial tumors of the ovary, and is involved in tumor progression and maintenance [11]. The VEGF gene encodes five different protein isoforms (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206) that are generated by alternative splicing from a single gene [12–14]. VEGF121 is the only isoform that does not bind heparin and is efficiently secreted from the cell [11,15]. VEGF165 is partially retained on the cell surface as a result of binding to heparin-sulfate proteoglycans [11,15]. The other VEGF isoforms are primarily retained on the surface [14,16]. Recently, the dynamic and selective expression of VEGF isoforms in the corpus luteum during the luteal life span in the natural menstrual cycle of macaques was established [17]. The functional role of VEGF165 in ovarian cyst formation associated with ovarian cancer was also established [18]. Other growth factors, such as nerve growth factor (NGF), which is involved in angiogenesis [19], is also expressed in fetal mammalian ovaries [20,21] including human ovaries [22,23]. NGF is required for early follicular development in the mammalian ovary [20,21]. NGF is involved in the growth of primordial ovarian follicles, a process known to occur independently from pituitary gonadotropins [21]. Also, NGF and its high-affinity receptor trkA are localized in granulosa cells from secondary and pre-antral follicles in the human ovary [22–24]. Interestingly, VEGF has been found in the same type of cells from human follicles [25] as it has been observed in other species [26–28]. The expression and production of VEGF inside the ovaries are important for normal reproductive function [27,28]. Similarly, NGF acts as a direct angiogenic factor or by stimulating VEGF expression in the ischemic hindlimb [29,30]. Defects in angiogenesis can contribute to a variety of gynecological disorders, including
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anovulation, infertility, miscarriage, ovarian hyperstimulation syndrome and ovarian cancer [11]. Tyrosine kinase A receptors (trkA) are high-affinity receptors for NGF, and have been widely linked to non neural tumor genesis, primarily in carcinomas [31–35]. Expression of trkA receptors was documented in several epithelial malignancies including ovarian carcinoma [31,32,36], yet trkA receptors were not found in normal ovarian surface epithelium [32,37]. Recent studies in murine models have demonstrated a role of neurotrophin signaling in tumor growth and metastasis [34,35]. However, the biological roles of NGF and trkA receptors in ovarian carcinoma are still poorly defined. Recent data suggest that NGF could be involved in the etiology of ovarian cancer because: (a) NGF acts as an angiogenic factor in endothelial cells of human umbilical veins (HUVEC) [19], (b) NGF induces the expression of inflammatory markers in skin vessels [38], (c) NGF stimulates angiogenesis and arteriogenesis in the setting of ischemia, thereby accelerating hemodynamic recovery [30], and (d) NGF plays a functional role in reparative neovascularization through a VEGF-mediated mechanism [29].
Fig. 1. Normal ovary and epithelial ovarian cancer express the mRNAs encoding both NGF and its high-affinity receptor trkA as determined by RT-PCR. The ethidium bromide-stained gels show the presence of PCR products amplified from reversed-transcribed total RNA extracted from normal ovaries, and epithelial ovarian cancer amplified with primers recognizing specific regions of NGF (upper panel) and trkA (lower panel) mRNAs. While NGF mRNA contents were similar in normal ovaries to contents in epithelial ovarian cancer, trkA mRNA levels increased significantly in epithelial ovarian cancer. Amplification of a segment from β-actin mRNA (lower panel) demonstrates that these differences are not due to procedural variability. Each lane shows the PCR products amplified from total RNA derived from two samples of normal ovaries and epithelial ovarian cancer, representative of a total of 10 samples each. Panel A: L: ladder; C+: positive control corresponding to a human pheochromocytoma; C−: negative control for PCR; N: normal ovaries, and Ca: epithelial ovarian cancer. Panel B: C+: positive control corresponding to human pheochromocytoma; C−: negative control for PCR; L: ladder; N: normal ovaries and Ca: epithelial ovarian cancer.
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Overall, due to their angiogenic properties, both VEGF and NGF may act synergistically in angiogenesis of the ovaries. The aim of this study is to investigate (a) the expression and localization of NGF and trkA in normal human ovaries as well as in epithelial ovarian cancer, and (b) whether NGF and the trkA receptors are involved in the regulation of VEGF expression in epithelial ovarian cancer. Material and methods Sample collection Samples from twenty normal ovarian specimens and from 50 epithelial ovarian cancer (45 serous ovarian carcinomas and 5 mucinous ovarian carcinomas) were collected from the Department of Obstetrics and Gynecology, Hospital Clinico Universidad de Chile and from Clinica Las Condes, Santiago, Chile, following informed consent. Normal ovarian samples were collected from total hysterectomies in women 38–58 years old, undergoing elective pelvic surgery for non-ovarian indications. This work was approved by the Institutional Review Board. After surgery, the pathologist separated a portion of the tissue that was immediately frozen in liquid nitrogen and kept frozen until used for RNA extraction. The remnant tissues were fixed and paraffin-embedded for morphological and immunohistochemical analysis. In 18 samples (16 serous ovarian carcinomas and 2 mucinous ovarian carcinomas), a third portion was immediately put in culture media, sent to the laboratory and used for tissue culture (explants) analysis. NGF and trkA expression levels were evaluated by immunohistochemistry and RT-PCR in normal and pathological samples. NGF-induced VEGF expression was examined in explants of epithelial ovarian cancer specimens. The VEGF mRNA levels were evaluated by RT-PCR. VEGF protein contents were determined by ELISA.
Epithelial ovarian cancer samples Ovarian cancer samples were cut in pieces (100 mg approximately) and incubated for 2 h at 37°C with NGF (10, 50 and 100 ng/ml) in a serum-free
HAM F-12 Dulbecco medium (Sigma, Saint Louis, MO, USA) supplemented with 50 mg/l penicillin/streptomycin, and 80 mg/l gentamicin in 24-well plates. To neutralize the biological effect of NGF, other explants were treated with polyclonal antibody against NGF (kindly provided by Dr. H.F. Urbanski, Oregon Regional Primate Research Centre, USA) diluted 1:1000, and in NGF (100 ng/ml) plus the antibody. Simultaneously, other pieces of ovarian cancer were cultured with NGF (100 ng/ml) and NGF (100 ng/ml) plus K252a (Calbiochem, La Jolla, CA, USA) used as a tyrosine receptor inhibitor. After incubation the samples were stored at −80°C until total RNA was extracted. The reverse transcription reaction and PCR were performed as described below. Culture media were stored at − 80°C until VEGF was detected by ELISA (R&D System, Minneapolis, MN, USA).
Human VEGF immunoassay VEGF levels were measured in spent culture media from 10 different experiments described above using a Quantikine kit from R&D Systems (Abingdon, UK). VEGF levels (pg/ml) were normalized by wet ovarian tissue weight. The results were expressed in relation to these control conditions. For the VEGF immunoassay, the inter-assay coefficient of variance was 8.5%; assay sensitivity was 5 pg/ml. This assay detected secretion of two VEGF isoforms: VEGF121 and VEGF165.
RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA from normal ovaries, and epithelial ovarian cancer samples were isolated using TrizolR (Invitrogen, Life Technologies, Carlsbad, CA). 5 μg of total RNA was reversed transcribed in a 20 μl volume using Superscript II (Invitrogen, Life Technologies, Carlsbad, CA). The primers we used to amplify human VEGF, NGF and trkA receptor mRNA, were those previously reported by others authors [13,24]. The forward primer for NGF (5′ TAA AAA GCG GCG ACT CCG TT 3′) is complementary to 405–424 nucleotides in the human NGF mRNA (accession No. BC032517); the reverse primer (5′ ATT CGC CCC TGT GGA AGA TG 3′) is complementary to nucleotides 552–571 in the mRNA. The forward trkA primer (5′ CCA TCG TGA AGA GTG GTC TC 3′) corresponds to nucleotides 289–308 in trkA mRNA (Accession No.
Fig. 2. Detection of NGF and trkA immunoreactivity in epithelial cells from human ovaries and epithelial ovarian cancer. (A) Morphology of normal ovarian surface epithelial cells with Artheta staining. (B) Low NGF immunoreactivity in ovarian surface epithelial cells (OSE). (C) OSE cells from normal ovaries contain very low detectable levels of trkA protein. (D) Lack of staining in sections incubated with pre absorbed antibodies to NGF. (E) Morphology of transformed epithelial cells in epithelial ovarian cancer sample. (F) High NGF immunoreactivity in the transformed epithelial cells from epithelial ovarian cancer. (G) High trkA immunoreactivity in the transformed epithelial cells from epithelial ovarian cancer. (H) Lack of staining in sections incubated with pre absorbed antibodies. Scale bars in panels A–H = 30 μm (100×). Inner panel (10×).
X. Campos et al. / Gynecologic Oncology 104 (2007) 168–175 BC062580); the reverse primer (5′ GGT GAC ATT GGC CAG GGT CA 3′) is complementary to nucleotides 745–764 in trkA mRNA. These primers amplify NGF cDNA of 167 bp, and trkA cDNA of 475 bp. The forward primer for VEGF (5′ AGG CCA GCA CAT AGG AGA GA 3′) corresponds to nucleotides 1370–1389 in the VEGF mRNA (VEGF189 Accession No. NM 003376, VEGF165 Accession No. NM 001025368 and VEGF121 Accession No. NM 001025370), the reverse primer (5′ ACC GCC TCG GCT TGT CAC AT 3′) is complementary to nucleotides 1658–1677 with a 307-bp product (VEGF189), to nucleotides 1586–1605 with a 236-bp product (VEGF165) and to nucleotides 1454–1473 with a 104-bp amplified product (VEGF121). The PCR reaction consisted of 27 cycles for VEGF, 35 cycles for NGF and 40 cycles for trkA with denaturation at 94°C for 30 s, annealing at 62°C for 1 min, and the first extension at 72°C for 1 min, followed by a final extension of 7 min at 72°C. The PCR products were sized fractioned on a 2% agarose gel and the signals were visualized using an UV Transilluminator UVP with Doc-it Software Image Acquisition and 1 D Analysis (UVP, Inc. Laboratory Products, Upland, CA, USA). For NGF and VEGF, but not for trkA, the PCR reaction also contained primers to amplify a segment of the βactin gene, which is constitutively expressed and thus can be used for signal normalization. The β-actin forward primer used (5′ TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA 3′) corresponds to nucleotides 543–572 in human β-actin mRNA (Accession. No. BC013380); the antisense primer (5′ CTA GAA GCA TTG CGG TGG ACG ATG GAG GG 3′) is complementary to nucleotides 1174–1203 in this mRNA. The resulting PCR product was 661 bp in length.
Immunohistochemistry All specimens were fixed in 10% buffered formalin. Formalin-fixed ovarian tissues were cut in 4- to 6-μm sections and deparaffinized. One in every five slides was selected for morphological study (Artheta staining). Immunohistochemical identification of NGF and trkA in human ovarian samples was carried out by the following protocol: four sections for each molecule in study (NGF and trkA) were washed twice for 3 min in water, the sections were then incubated for 40 min at 90°C in 10 mM sodium citrate buffer pH 6.0 and then for 20 min at room temperature for antigen retrieval. The sections were then washed three times for 5 min in 0.01 M PBS buffer pH 7.3. The slides were then incubated at room temperature for 10 min in peroxidase blocking reagent (DakoCytomation, Inc., CA, USA) to inhibit endogenous peroxidases. The sections were washed three times for 10 min each time in 0.01 M PBS buffer pH 7.3. The sections were then incubated for 10 min at RT with 10% skim milk to block non-specific binding and incubated overnight at 4°C with rabbit polyclonal antibody against NGF in a 1:500 dilution in 1% PBS-BSA. The use of this NGF antibody for immunohistochemical detection of NGF has been previously reported [39–41]. Rabbit polyclonal antibody to trkA was used in 1:500 dilutions in 1% PBS-BSA (sc-14024 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The next day, sections were rinsed twice with PBS for 10 min at room temperature and incubated with the corresponding secondary antibody (peroxidase-labeled affinity purified antibody to rabbit IgG) (KPL Kirkegaard & Perry Laboratories Inc, Maryland, USA) in a 1:300 dilution in 1% PBS-BSA for 30 min at 37°C. To visualize the immunoreactive cells, the sections were incubated for 15 min at room temperature with liquid 3,3′diaminobenzidine substrate (DAB) (DakoCytomation, Inc., CA, USA) washed with water and counterstained with 10% hematoxylin (Lerner Laboratores, Pittsburgh, PA, USA). Sections were then dehydrated in ethanol and cleared in xylene, coverslipped and examined in a photomicroscope (Zeiss Axioskop Germany and Nikon Coolpix 995 Tokyo, Japan. digital camera). Each immunoreaction was repeated a minimum of three times. Negative controls lacked primary or secondary antibodies. To confirm specificity of NGF staining, selected sections were run with pre-absorbed antibodies using excess NGF (5.000 ng/ml) (Sigma, St. Louis, MO, USA).
Statistical analysis Differences between groups were analyzed using the Kruskal Wallis test. We used this non-parametric test because the sample number was small and showed an asymmetric distribution. P-values less than 0.05 were considered significant.
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Results TrkA, NGF mRNA, and protein expression in the normal ovary and in epithelial ovarian cancer RT-PCR analysis of NGF mRNA showed no differences between samples of EOC and normal ovarian samples (Fig. 1, panel A). However, levels of trkA mRNA were higher in EOC when compared to normal ovarian samples (Fig. 1, panel B). Immunohistochemical analysis for NGF and its receptor in normal ovarian surface epithelium showed their presence in only 10% (2/20) of the samples analyzed and we found low levels of NGF and very low levels of trkA expression as showed in Figs. 2A–D. It is interesting to observe an heterogeneous NGF expression in OSE, because some of these cells are negative for NGF staining (Fig. 2B, arrow head) and others are positive (Fig. 2B, arrow). High positive staining for trkA and NGF was found in 100% (22/22) of the epithelial cells from serous and mucinous ovarian carcinomas (Figs. 2E–H). This staining was remarkably higher compared with the normal surface epithelium.
Fig. 3. Normal ovaries and epithelial ovarian cancer express the mRNAs encoding VEGF isoforms determined by RT-PCR. The ethidium bromidestained gels show the presence of PCR products amplified from reversedtranscribed total RNA extracted from human ovaries and epithelial ovarian cancer amplified with primers recognizing specific regions of VEGF isoforms (VEGF189; VEGF165 and VEGF121) representative of a total of six samples each. Panel A: L: ladder; C+: positive control corresponding to human pheochromocytoma; C−: negative control for the PCR; N: represents two samples of normal ovaries and Ca: two samples of epithelial ovarian cancer. Panel B: VEGF isoforms mRNA levels expressed as arbitrary units of VEGF mRNA with respect to the β-actin expression as a housekeeping gene. The isoforms of VEGF165 and VEGF121 are abundant in both normal and pathological samples. When comparing the isoforms content between normal and pathological samples, both VEGF189 and VEGF165 contents were higher in epithelial ovarian cancer. The results represent the mean ± standard error (SE) from six normal ovaries and six epithelial ovarian cancers in duplicate. *p < 0.05 and **p < 0.01.
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VEGF mRNA expression in normal ovary and in epithelial ovarian cancer In this study, we found the presence of three of the five VEGF mRNA isoforms in normal and cancer ovarian samples by RT-PCR. Fig. 3A shows the three bands of VEGF (308 bp for VEGF189, 236 bp for VEGF165 and 104 pb for VEGF121) and the β-actin band to 661 bp. We found higher levels of the VEGF121 and VEGF165 isoforms in both epithelial ovarian cancer and in normal ovaries, compared to mRNA levels for VEGF189. Levels of both VEGF165 and VEGF189 were significantly higher in EOC samples compared with normal samples (Fig. 3B). NGF increases VEGF expression levels in cultured explants of epithelial ovarian cancer When EOC explants were incubated with increasing doses of NGF, we found that 2 h in culture was enough to produce a significant increase in VEGF121, VEGF165 and VEGF189 mRNA levels in a dose-dependent manner, with a maximal effective dose at 100 ng/ml NGF (Figs. 4A, B, C). The stimulatory effects of NGF were also observed in the VEGF protein (Fig. 4D). The increase in VEGF mRNA induced by NGF was blocked using either an antibody against NGF, or an inhibitor of tyrosine kinase receptors (K252a), indicating that the
effect was specific and mediated by the activation of NGF highaffinity receptor, trkA. (Figs. 5A, B, C). Similar results were obtained using spent culture media by ELISA for the VEGF protein (Fig. 5D). VEGF detected by this assay was a mixture of VEGF121 and VEGF165 because both isoforms are secreted in the culture media. Discussion The role of neurotrophins in the development and function of the mammalian ovaries is well established. NGF, a member of this family, is required for early follicular development and ovulation [21,39]. Recent studies demonstrated that NGF and its high-affinity receptor trkA are localized in granulosa cells of the secondary and pre-antral follicles in the human ovary [22,24]. This study found, furthermore, low expression of NGF and very low expression of trkA in epithelial cells surrounding normal ovaries. The presence of NGF high-affinity receptor trkA was reported in several epithelial malignancies, including ovarian carcinoma [28,31]. In our work, we found a very strong presence of this neurotrophin and its receptor in the epithelial cells in all ovarian cancers studied. It would be important to study further whether the initiation of trkA expression in ovarian surface epithelial cells is a relevant factor in the transformation of normal cells into malignant cells. If this were
Fig. 4. NGF increases VEGF expression in epithelial ovarian cancer in vitro. VEGF mRNA levels from epithelial ovarian cancer explants cultured for 2 h with 10, 50 and 100 ng/ml of NGF. A significant increase of VEGF isoforms was observed with 100 ng/ml of NGF. (A) VEGF121, the results represent the mean ± SE from n = 10 (*p < 0.05). (B) VEGF165, the results represent the mean ± SE from n = 10 (* p < 0.05). (C) VEGF189, the results represent the mean ± SE from n = 10. (***p < 0.005). (D) Represents the amount of VEGF protein secreted in spent culture media from epithelial ovarian cancer explants cultured with 10, 50 and 100 ng/ml of NGF. The VEGF protein was expressed in arbitrary units with respect to the control condition (mean ± SE). VEGF protein assays were carried out by ELISA which detected both VEGF121 and VEGF165. The results represent the mean ± SE from n = 6. (*p < 0.05).
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Fig. 5. The Increase in VEGF mRNA and protein induced by NGF is blocked with an antibody against NGF or by the presence of a tyrosine kinase receptors inhibitor (K252a). (A) VEGF121 mRNA levels from epithelial ovarian cancer explants, showed a significant increase with 100 ng/ml of NGF (**p < 0.01). Inhibition of NGF action with an antibody against NGF (NGF-Ab) and K252a (NGF versus NGF plus NGF-Ab = ++p < 0.01; NGF versus NGF plus K252a #p < 0.05). The results represent the mean ± SE from n = 8. (B) VEGF165 mRNA levels from epithelial ovarian cancer explants, showed a significant increase with 100 ng/ml of NGF (*p < 0.05), and a significant inhibition of NGF action using NGF-Ab (++p < 0.01). No significant inhibition was observed with K252a. The results represent the mean ± SE from n = 8. (C) Significant increase in VEGF189 mRNA levels from epithelial ovarian cancer explants in culture with 100 ng/ml of NGF (**p < 0.01) as well as an important inhibition of NGF action using NGF-Ab and with K252a (+p < 0.05). The results represent the mean ± SE from n = 8. (D) VEGF protein levels detected by ELISA from spent culture media of epithelial ovarian cancer explants showed a significant increase with 100 ng/ml of NGF (*p < 0.05) and a significant inhibition using NGF-Ab and K252a (NGF versus NGF plus NGF-Ab = ++p < 0.01; NGF versus NGF plus K252a = #p < 0.05). The VEGF secreted corresponds to the VEGF121 and VEGF165 isoforms. The results represent the mean ± SE from n = 8.
the case, the expression of trkA receptors in the ovarian surface epithelium could be used as an early predictive tool for epithelial ovarian cancer. Angiogenesis, and the production of angiogenic factors, are essential for tumor growth and metastasis. One angiogenic factor, VEGF, has been implicated in the neovascularization of a wide variety of tumors [42]. VEGF may up-regulate this pathogenic process. High expression of angiogenic factors may be associated with advanced tumor stages in human ovarian cancer [43]. Several studies have documented the positive correlations between the extent of tumor vascularization, with metastasis formation, as well as with decreased patient survival rates in ovarian carcinoma [44–47]. Also, high levels of VEGF protein were found in ovarian cyst fluid and have been associated with ovarian malignancy [48]. NGF acts either as a direct angiogenic factor or by upregulating the expression of VEGF, as demonstrated in pheochromocytoma cells (PC12) [49]. Phosphorylated trkA (p-trkA) expression has also been reported in endothelial cells in serous ovarian carcinomas suggesting that the proangiogenic role attributed to NGF in vivo and in vitro may be relevant in clinical cancer [36]. Our results showed that NGF up-regulates
the expression of VEGF in ovarian epithelial cancer, supporting the idea that NGF may be directly involved in the in vivo regulation of VEGF-mediated angiogenesis in this type of cancer. The VEGF gene encodes five different isoforms that are generated by alternative splicing from a single gene [12–14]. Functional evidence shows that VEGF165 plays a role in ovarian cyst formation associated with ovarian cancer [18]. We found that NGF increases three isoforms of VEGF; VEGF121, VEGF165 and VEGF189. This effect was achieved by activation of trkA receptors. The co-localization of NGF and VEGF in epithelial cells from serous ovarian carcinoma [36], suggests that NGF-mediated VEGF increase may be an autocrine action. NGF may play a dual role during ovarian angiogenesis, acting as (1) a direct factor in the endothelial cells of the serous ovarian carcinoma vasculature [36] and (2) indirectly through the regulation of VEGF expression in epithelial ovarian cells, as we found in this study. These results suggest that the expression of NGF and trkA in ovarian surface epithelium is not only important for the initial stages of angiogenesis, but may also play a role in the progression of epithelial ovarian cancer, by mediating an
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increase in VEGF expression levels, a known tumor-induced angiogenic factor that facilitates the transition from a small noninvasive tissue into a rapidly expanding tumor [44]. This supports the hypothesis that the pro-angiogenic role attributed to NGF in vitro and in vivo is a key factor in the process of malignant transformation in epithelial ovarian cancer. Acknowledgments This work has been supported by Grants Fondo Nacional de Ciencia y Tecnologia (Fondecyt #1030661 to CR). Ximena Campos is a PhD student in the Biochemistry Graduate Program of University of Chile, supported by a Fellowship from Comision Nacional de Ciencia y Tecnologia (Conicyt #102051 and AT-4040058 to X.C.). References [1] Ries LA, Kosary CL, Hankey BF, Miller BA, Edwards BK, editors. SEER Cancer Statistics. Review 1973–1995. Bethesda, MD: National Cancer Institute; 1998. [2] Fathalla MF. Incessant ovulation-a factor in ovarian neoplasia? Lancet 1971;2(7716):163. [3] Cramer DW, Welch WR. Determinants of ovarian cancer risk: II. Inferences regarding pathogenesis. J Natl Cancer Inst 1983;71(4):717–21. [4] Syed V, Ulinsky G, Mok SC, Yiu GK, Ho SM. Expression of gonadotrophin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cell. Cancer Res 2001;61:6768–76. [5] Helzlsouer KJ, Alberg AJ, Gordon GB, Longcope C, Bush TL, Hoffman SC, et al. Serum gonadotropins and steroid hormones and the development of ovarian cancer. JAMA 1995;274(24):1926–30. [6] Risch HA. Hormonal etiology of epithelial ovarian cancer, with a hypothesis concerning the role of androgens and progesterone. J Natl Cancer Inst 1998;90(23):1774–86. [7] Jacobs IJ, Kohler MF, Wiseman RW, Marks JR, Whitaker R, Kerns BA, et al. Clonal origin of epithelial ovarian carcinoma: analysis by loss of heterozycocity, p53 mutation, and X-chromosome inactivation. J Natl Cancer Inst 1992;84:1793–8. [8] Mok CH, Tsao SW, Knapp RC, Fishbaugh PM, Lau CC. Unifocal origin of advanced human epithelial ovarian cancers. Cancer Res 1992;52:5119–22. [9] Hamilton TC, Davis P, Criffiths K. Steroid hormone receptor status of the normal and neoplastic ovarian surface germinal epithelium. In: Greenwald GS, Terranova PF, editors. Factors Regulating Ovarian Function. New York: Raven Press; 1983. p. 81–5. [10] Vanderhyden BC, Shaw TJ, Garson K, Tonary AM. Ovarian carcinogenesis. In: Leung PCK, Adashi EY, editors. The Ovary. 2nd ed. San Diego: Elsevier Academic Press; 2004. p. 591–612. [11] Neufeld G, Cohen T, Gengrinivich S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13(11):9–22. [12] Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 1991;5:1806–14. [13] Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991;266:11947–54. [14] Poltorak Z, Cohen T, Sivan R, Kandelis Y, Spira G, Vlodavsky I, et al. VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem 1997;272:7151–8. [15] Grunstein J, Masbad JJ, Hickey R, Giordano F, Johnson RS. Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature. Mol Cell Biol 2000;20(19):7282–91.
[16] Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 1992;267:26031–7. [17] Tesone M, Stouffer RL, Borman SM, Hennebold JD, Molskness TA. Vascular endothelial growth factor (VEGF) production by the monkey corpus luteum during the menstrual cycle: isoform-selective messenger RNA expression in vivo and hypoxia-regulated protein secretion in vitro. Biol Reprod 2005;73:927–34. [18] Duyndam MCA, Hilhorst MCGW, Schluper HMM, Verheul HMW, van Diest PJ, Kraal G, et al. Vascular endothelial growth factor-165 overexpression stimulates angiogenesis and induces cyst formation and macrophage infiltration in human ovarian cancer xenografts. Am J Pathol 2002;160(2):537–48. [19] Cantarella G, Lempereur L, Presta M, Ribatti D, Lombardo G, Lazarovici P, et al. Nerve growth factor-endothelial cell interaction leads to angiogenesis in vitro and in vivo. FASEB J 2002;16:1307–9. [20] Dissen GA, Newman Hirshfield A, Malamed S, Ojeda SR. Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis. Endocrinology 1995;136:4681–92. [21] Dissen GA, Romero C, Newman-Hirshfield A, Ojeda SR. Nerve growth factor is required for early follicular development in the mammalian ovary. Endocrinology 2001;142:2078–86. [22] Abir R, Fisch B, Jin S, Barnnet M, Ben-Haroush A, Felz C, et al. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol Hum Reprod 2005;11(4):229–36. [23] Anderson RA, Robinson LL, Brooks J, Spears N. Neurotropins and their receptors are expressed in the human fetal ovary. Mol Hum Reprod 2005;1(2):79–85. [24] Salas C, Julio-Pieper M, Valladares M, Pommer R, Vega M, Mastronardi C, et al. Nerve growth factor-dependent activation of trkA receptors in the human ovary results in synthesis of FSH receptors and estrogen secretion. J Clin Endocrinol Metab 2006;91:2396–403. [25] Ferrara N, Frantz G, LeCouter J, Dillard-Telm L, Pham T, Draksharapu A, et al. Differential expression of the angiogenic factor genes vascular endothelial growth factor (VEGF) and endocrine gland-derived VEGF in normal and polycystic human ovaries. Am J Pathol 2003;162(6):1881–93. [26] Dissen GA, Lara HE, Fahrenbach WH, Costa ME, Ojeda SR. Immature rat ovaries become revascularized rapidly alter autotransplantation and show a gonadotrophin-dependent increase in angiogenic factor gene expression. Endocrinology 1999;134(3):1146–54. [27] Fraser HM, Wu C. Angiogenesis in the primate ovary. Reprod Fertil Dev 2001;13:557–66. [28] Lee A, Christenson LK, Patton PE, Burry KA, Stouffer RL. Vascular endothelial growth factor production by human luteinized granulosa cells in vitro. Hum Reprod 1997;12:2756–61. [29] Calza L, Giardino L, Guiliani A, Aloe L, Levi-Montalcini R. Nerve growth factor control of neuronal expression of angiogenic and vasoactive factors. Proc Natl Acad Sci U S A 2001;98:4160–5. [30] Emanueli C, Salis MB, Pinna A, Graiani G, Manni L, Madeddu P. Nerve growth factor promotes angiogenesis and arteriogenesis in ischemic hindlimbs. Circulation 2002;106:2257–62. [31] Davidson B, Lazarovici P, Ezersky A, Nesland JM, Risberg B, Trope CG, et al. Expression levels of the NGF receptors trkA and p75 in effusions and solid tumors of serous ovarian carcinoma patients. Clin Cancer Res 2001;7:3457–64. [32] Shibayama E, Koizumi H. Cellular localization of the trk neurotrophin receptor family in human non-neuronal tissues. Am J Pathol 1996;148:1807–18. [33] McGregor LM, McCune BK, Graff JR, McDowell PR, Romans KE, Yancopoulos GD, et al. Roles of trk family neurotrophin receptors in medullary thyroid carcinoma development and progression. Proc Natl Acad Sci U S A 1999;96:4540–5. [34] Miknyoczki SJ, Wan W, Chang H, Dobrzanski P, Ruggeri BA, Dionne CA, et al. The neurotrophin-trk receptor axes are critical for the growth and progression of human prostatic carcinoma and pancreatic ductal adenocarcinoma in nude mice. Clin Cancer Res 2002;8:1924–31. [35] Weeraratna AT, Dalrymple SL, Lamb JC, Denmeade SR, Miknyoczki S,
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Nerve growth factor and its high-affinity receptor trkA participate in the control of vascular endothelial growth factor expression in epithelial ovarian cancer Ximena Campos a,1 , Yenny Muñoz a,1 , Alberto Selman b , Roberto Yazigi c , Leonor Moyano d , Caroline Weinstein-Oppenheimer e , Hernán E. Lara f , Carmen Romero a,b,⁎ a
f
Laboratorio de Endocrinología y Biología Reproductiva, Hospital Clinico Universidad de Chile, Chile b Departamento de Obstetricia y Ginecología, Facultad de Medicina, Universidad de Chile, Chile c Clinica Las Condes, Chile d Servicio de Anatomía Patológica, Hospital Clínico Universidad de Chile, Chile e Departamento de Bioquímica, Facultad de Farmacia, Universidad de Valparaíso, Chile Laboratorio de Neurobioquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile Received 29 March 2006 Available online 28 August 2006
Abstract Objectives. To compare the expression of nerve growth factor (NGF) and its high-affinity receptor trkA in normal ovaries and in epithelial ovarian carcinomas. Given NGF acts as an angiogenic factor through a vascular endothelial growth factor (VEGF)-mediated mechanism in several types of tissues, we examined whether NGF regulates the expression of VEGF isoforms in epithelial ovarian cancer (EOC). Methods. The expression and localization of NGF and tyrosine kinase receptor A (trkA) in normal ovarian samples and in ovarian cancer samples were analyzed by RT-PCR and immunohistochemistry. NGF regulates the expression of three VEGF isoforms (VEGF121, VEGF165 and VEGF189); these were examined using RT-PCR in explants of EOC and ELISA in culture media. Results. TrkA mRNA levels were over-expressed in ovarian cancer compared to normal ovarian samples, whereas NGF mRNA levels remained unchanged. NGF and trkA proteins were absent or found in very low levels in normal ovarian surface epithelium (OSE), whereas they were highly expressed in epithelial cells of EOC. Additionally, NGF stimulated the expression of VEGF isoforms in cancer explants. The effect was dose-dependent and inhibited by a NGF antibody and by K252a, a trk receptor inhibitor. Conclusion. The abundance of NGF and trkA receptors in epithelial cells of EOC, together with the ability of NGF to increase VEGF expression strongly suggests an autocrine role of NGF in EOC. These findings suggest that blocking neurotrophin action could be a therapeutic target in treating ovarian cancer. © 2006 Elsevier Inc. All rights reserved. Keywords: NGF; trkA; VEGF; Epithelial ovarian cancer
Introduction Each year, more women die from ovarian cancer than from any other gynecologic malignancy. The lifetime risk for a ⁎ Corresponding author. Laboratorio de Endocrinologia y Biologia Reproductiva, Departamento de Obstetricia y Ginecologia, Hospital Clinico Universidad de Chile, Santos Dumont 999, Santiago, Chile. Fax: +56 (2)7374555. E-mail address: [email protected] (C. Romero). 1 Both authors contributed equally to this study. 0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2006.07.007
woman to be affected by ovarian cancer is 1.7% and the annual incidence approaches 61.3 per 100,000 in women 75 to 79 years of age [1]. Ovarian cancer is a deadly insidious disease because typically it is asymptomatic until the malignancy has reached beyond the ovaries. Unfortunately, screening for the early detection of ovarian malignancies has not proven effective. Several mechanisms may explain the etiology of ovarian cancer: Fathalla's theory of “incessant ovulation” [2], excessive stimulation by gonadotropins and estrogens [3–5],
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and local stimulation of androgens [6]. Several authors postulate that most ovarian cancers arise from the transformation of a surface epithelial affected by ovulation: the integrity of the DNA on surface cells circumjacent to the ovarian rupture site is compromised during the ovulatory process and clonal expansion of an epithelial cell with damaged (unrepaired) DNA is a putative factor in carcinogenesis [7,8]. However, it has not been established whether this malignancy evolves from benign or borderline tumors, or whether malignancy develops de novo from surface epithelial cells, or inclusion epithelial cysts. Mok et al. [8] provide evidence supporting both progressions. Although many studies try to explain the progression of epithelial cancer, none have proposed the mechanisms underlying these changes, probably because the sequence of events that leads to ovarian cancer is multifactorial and not adequately understood [9]. Local increases in growth factors may be responsible for angiogenesis and proliferation of ovarian surface epithelial cells (OSE) could participate in the initial stages of cancer progression [10]. Ovarian cancer is highly angiogenic and VEGF is the main factor responsible for angiogenesis, vascular permeability and metastasis [11]. VEGF is secreted by various tumors, including epithelial tumors of the ovary, and is involved in tumor progression and maintenance [11]. The VEGF gene encodes five different protein isoforms (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206) that are generated by alternative splicing from a single gene [12–14]. VEGF121 is the only isoform that does not bind heparin and is efficiently secreted from the cell [11,15]. VEGF165 is partially retained on the cell surface as a result of binding to heparin-sulfate proteoglycans [11,15]. The other VEGF isoforms are primarily retained on the surface [14,16]. Recently, the dynamic and selective expression of VEGF isoforms in the corpus luteum during the luteal life span in the natural menstrual cycle of macaques was established [17]. The functional role of VEGF165 in ovarian cyst formation associated with ovarian cancer was also established [18]. Other growth factors, such as nerve growth factor (NGF), which is involved in angiogenesis [19], is also expressed in fetal mammalian ovaries [20,21] including human ovaries [22,23]. NGF is required for early follicular development in the mammalian ovary [20,21]. NGF is involved in the growth of primordial ovarian follicles, a process known to occur independently from pituitary gonadotropins [21]. Also, NGF and its high-affinity receptor trkA are localized in granulosa cells from secondary and pre-antral follicles in the human ovary [22–24]. Interestingly, VEGF has been found in the same type of cells from human follicles [25] as it has been observed in other species [26–28]. The expression and production of VEGF inside the ovaries are important for normal reproductive function [27,28]. Similarly, NGF acts as a direct angiogenic factor or by stimulating VEGF expression in the ischemic hindlimb [29,30]. Defects in angiogenesis can contribute to a variety of gynecological disorders, including
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anovulation, infertility, miscarriage, ovarian hyperstimulation syndrome and ovarian cancer [11]. Tyrosine kinase A receptors (trkA) are high-affinity receptors for NGF, and have been widely linked to non neural tumor genesis, primarily in carcinomas [31–35]. Expression of trkA receptors was documented in several epithelial malignancies including ovarian carcinoma [31,32,36], yet trkA receptors were not found in normal ovarian surface epithelium [32,37]. Recent studies in murine models have demonstrated a role of neurotrophin signaling in tumor growth and metastasis [34,35]. However, the biological roles of NGF and trkA receptors in ovarian carcinoma are still poorly defined. Recent data suggest that NGF could be involved in the etiology of ovarian cancer because: (a) NGF acts as an angiogenic factor in endothelial cells of human umbilical veins (HUVEC) [19], (b) NGF induces the expression of inflammatory markers in skin vessels [38], (c) NGF stimulates angiogenesis and arteriogenesis in the setting of ischemia, thereby accelerating hemodynamic recovery [30], and (d) NGF plays a functional role in reparative neovascularization through a VEGF-mediated mechanism [29].
Fig. 1. Normal ovary and epithelial ovarian cancer express the mRNAs encoding both NGF and its high-affinity receptor trkA as determined by RT-PCR. The ethidium bromide-stained gels show the presence of PCR products amplified from reversed-transcribed total RNA extracted from normal ovaries, and epithelial ovarian cancer amplified with primers recognizing specific regions of NGF (upper panel) and trkA (lower panel) mRNAs. While NGF mRNA contents were similar in normal ovaries to contents in epithelial ovarian cancer, trkA mRNA levels increased significantly in epithelial ovarian cancer. Amplification of a segment from β-actin mRNA (lower panel) demonstrates that these differences are not due to procedural variability. Each lane shows the PCR products amplified from total RNA derived from two samples of normal ovaries and epithelial ovarian cancer, representative of a total of 10 samples each. Panel A: L: ladder; C+: positive control corresponding to a human pheochromocytoma; C−: negative control for PCR; N: normal ovaries, and Ca: epithelial ovarian cancer. Panel B: C+: positive control corresponding to human pheochromocytoma; C−: negative control for PCR; L: ladder; N: normal ovaries and Ca: epithelial ovarian cancer.
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Overall, due to their angiogenic properties, both VEGF and NGF may act synergistically in angiogenesis of the ovaries. The aim of this study is to investigate (a) the expression and localization of NGF and trkA in normal human ovaries as well as in epithelial ovarian cancer, and (b) whether NGF and the trkA receptors are involved in the regulation of VEGF expression in epithelial ovarian cancer. Material and methods Sample collection Samples from twenty normal ovarian specimens and from 50 epithelial ovarian cancer (45 serous ovarian carcinomas and 5 mucinous ovarian carcinomas) were collected from the Department of Obstetrics and Gynecology, Hospital Clinico Universidad de Chile and from Clinica Las Condes, Santiago, Chile, following informed consent. Normal ovarian samples were collected from total hysterectomies in women 38–58 years old, undergoing elective pelvic surgery for non-ovarian indications. This work was approved by the Institutional Review Board. After surgery, the pathologist separated a portion of the tissue that was immediately frozen in liquid nitrogen and kept frozen until used for RNA extraction. The remnant tissues were fixed and paraffin-embedded for morphological and immunohistochemical analysis. In 18 samples (16 serous ovarian carcinomas and 2 mucinous ovarian carcinomas), a third portion was immediately put in culture media, sent to the laboratory and used for tissue culture (explants) analysis. NGF and trkA expression levels were evaluated by immunohistochemistry and RT-PCR in normal and pathological samples. NGF-induced VEGF expression was examined in explants of epithelial ovarian cancer specimens. The VEGF mRNA levels were evaluated by RT-PCR. VEGF protein contents were determined by ELISA.
Epithelial ovarian cancer samples Ovarian cancer samples were cut in pieces (100 mg approximately) and incubated for 2 h at 37°C with NGF (10, 50 and 100 ng/ml) in a serum-free
HAM F-12 Dulbecco medium (Sigma, Saint Louis, MO, USA) supplemented with 50 mg/l penicillin/streptomycin, and 80 mg/l gentamicin in 24-well plates. To neutralize the biological effect of NGF, other explants were treated with polyclonal antibody against NGF (kindly provided by Dr. H.F. Urbanski, Oregon Regional Primate Research Centre, USA) diluted 1:1000, and in NGF (100 ng/ml) plus the antibody. Simultaneously, other pieces of ovarian cancer were cultured with NGF (100 ng/ml) and NGF (100 ng/ml) plus K252a (Calbiochem, La Jolla, CA, USA) used as a tyrosine receptor inhibitor. After incubation the samples were stored at −80°C until total RNA was extracted. The reverse transcription reaction and PCR were performed as described below. Culture media were stored at − 80°C until VEGF was detected by ELISA (R&D System, Minneapolis, MN, USA).
Human VEGF immunoassay VEGF levels were measured in spent culture media from 10 different experiments described above using a Quantikine kit from R&D Systems (Abingdon, UK). VEGF levels (pg/ml) were normalized by wet ovarian tissue weight. The results were expressed in relation to these control conditions. For the VEGF immunoassay, the inter-assay coefficient of variance was 8.5%; assay sensitivity was 5 pg/ml. This assay detected secretion of two VEGF isoforms: VEGF121 and VEGF165.
RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA from normal ovaries, and epithelial ovarian cancer samples were isolated using TrizolR (Invitrogen, Life Technologies, Carlsbad, CA). 5 μg of total RNA was reversed transcribed in a 20 μl volume using Superscript II (Invitrogen, Life Technologies, Carlsbad, CA). The primers we used to amplify human VEGF, NGF and trkA receptor mRNA, were those previously reported by others authors [13,24]. The forward primer for NGF (5′ TAA AAA GCG GCG ACT CCG TT 3′) is complementary to 405–424 nucleotides in the human NGF mRNA (accession No. BC032517); the reverse primer (5′ ATT CGC CCC TGT GGA AGA TG 3′) is complementary to nucleotides 552–571 in the mRNA. The forward trkA primer (5′ CCA TCG TGA AGA GTG GTC TC 3′) corresponds to nucleotides 289–308 in trkA mRNA (Accession No.
Fig. 2. Detection of NGF and trkA immunoreactivity in epithelial cells from human ovaries and epithelial ovarian cancer. (A) Morphology of normal ovarian surface epithelial cells with Artheta staining. (B) Low NGF immunoreactivity in ovarian surface epithelial cells (OSE). (C) OSE cells from normal ovaries contain very low detectable levels of trkA protein. (D) Lack of staining in sections incubated with pre absorbed antibodies to NGF. (E) Morphology of transformed epithelial cells in epithelial ovarian cancer sample. (F) High NGF immunoreactivity in the transformed epithelial cells from epithelial ovarian cancer. (G) High trkA immunoreactivity in the transformed epithelial cells from epithelial ovarian cancer. (H) Lack of staining in sections incubated with pre absorbed antibodies. Scale bars in panels A–H = 30 μm (100×). Inner panel (10×).
X. Campos et al. / Gynecologic Oncology 104 (2007) 168–175 BC062580); the reverse primer (5′ GGT GAC ATT GGC CAG GGT CA 3′) is complementary to nucleotides 745–764 in trkA mRNA. These primers amplify NGF cDNA of 167 bp, and trkA cDNA of 475 bp. The forward primer for VEGF (5′ AGG CCA GCA CAT AGG AGA GA 3′) corresponds to nucleotides 1370–1389 in the VEGF mRNA (VEGF189 Accession No. NM 003376, VEGF165 Accession No. NM 001025368 and VEGF121 Accession No. NM 001025370), the reverse primer (5′ ACC GCC TCG GCT TGT CAC AT 3′) is complementary to nucleotides 1658–1677 with a 307-bp product (VEGF189), to nucleotides 1586–1605 with a 236-bp product (VEGF165) and to nucleotides 1454–1473 with a 104-bp amplified product (VEGF121). The PCR reaction consisted of 27 cycles for VEGF, 35 cycles for NGF and 40 cycles for trkA with denaturation at 94°C for 30 s, annealing at 62°C for 1 min, and the first extension at 72°C for 1 min, followed by a final extension of 7 min at 72°C. The PCR products were sized fractioned on a 2% agarose gel and the signals were visualized using an UV Transilluminator UVP with Doc-it Software Image Acquisition and 1 D Analysis (UVP, Inc. Laboratory Products, Upland, CA, USA). For NGF and VEGF, but not for trkA, the PCR reaction also contained primers to amplify a segment of the βactin gene, which is constitutively expressed and thus can be used for signal normalization. The β-actin forward primer used (5′ TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA 3′) corresponds to nucleotides 543–572 in human β-actin mRNA (Accession. No. BC013380); the antisense primer (5′ CTA GAA GCA TTG CGG TGG ACG ATG GAG GG 3′) is complementary to nucleotides 1174–1203 in this mRNA. The resulting PCR product was 661 bp in length.
Immunohistochemistry All specimens were fixed in 10% buffered formalin. Formalin-fixed ovarian tissues were cut in 4- to 6-μm sections and deparaffinized. One in every five slides was selected for morphological study (Artheta staining). Immunohistochemical identification of NGF and trkA in human ovarian samples was carried out by the following protocol: four sections for each molecule in study (NGF and trkA) were washed twice for 3 min in water, the sections were then incubated for 40 min at 90°C in 10 mM sodium citrate buffer pH 6.0 and then for 20 min at room temperature for antigen retrieval. The sections were then washed three times for 5 min in 0.01 M PBS buffer pH 7.3. The slides were then incubated at room temperature for 10 min in peroxidase blocking reagent (DakoCytomation, Inc., CA, USA) to inhibit endogenous peroxidases. The sections were washed three times for 10 min each time in 0.01 M PBS buffer pH 7.3. The sections were then incubated for 10 min at RT with 10% skim milk to block non-specific binding and incubated overnight at 4°C with rabbit polyclonal antibody against NGF in a 1:500 dilution in 1% PBS-BSA. The use of this NGF antibody for immunohistochemical detection of NGF has been previously reported [39–41]. Rabbit polyclonal antibody to trkA was used in 1:500 dilutions in 1% PBS-BSA (sc-14024 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The next day, sections were rinsed twice with PBS for 10 min at room temperature and incubated with the corresponding secondary antibody (peroxidase-labeled affinity purified antibody to rabbit IgG) (KPL Kirkegaard & Perry Laboratories Inc, Maryland, USA) in a 1:300 dilution in 1% PBS-BSA for 30 min at 37°C. To visualize the immunoreactive cells, the sections were incubated for 15 min at room temperature with liquid 3,3′diaminobenzidine substrate (DAB) (DakoCytomation, Inc., CA, USA) washed with water and counterstained with 10% hematoxylin (Lerner Laboratores, Pittsburgh, PA, USA). Sections were then dehydrated in ethanol and cleared in xylene, coverslipped and examined in a photomicroscope (Zeiss Axioskop Germany and Nikon Coolpix 995 Tokyo, Japan. digital camera). Each immunoreaction was repeated a minimum of three times. Negative controls lacked primary or secondary antibodies. To confirm specificity of NGF staining, selected sections were run with pre-absorbed antibodies using excess NGF (5.000 ng/ml) (Sigma, St. Louis, MO, USA).
Statistical analysis Differences between groups were analyzed using the Kruskal Wallis test. We used this non-parametric test because the sample number was small and showed an asymmetric distribution. P-values less than 0.05 were considered significant.
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Results TrkA, NGF mRNA, and protein expression in the normal ovary and in epithelial ovarian cancer RT-PCR analysis of NGF mRNA showed no differences between samples of EOC and normal ovarian samples (Fig. 1, panel A). However, levels of trkA mRNA were higher in EOC when compared to normal ovarian samples (Fig. 1, panel B). Immunohistochemical analysis for NGF and its receptor in normal ovarian surface epithelium showed their presence in only 10% (2/20) of the samples analyzed and we found low levels of NGF and very low levels of trkA expression as showed in Figs. 2A–D. It is interesting to observe an heterogeneous NGF expression in OSE, because some of these cells are negative for NGF staining (Fig. 2B, arrow head) and others are positive (Fig. 2B, arrow). High positive staining for trkA and NGF was found in 100% (22/22) of the epithelial cells from serous and mucinous ovarian carcinomas (Figs. 2E–H). This staining was remarkably higher compared with the normal surface epithelium.
Fig. 3. Normal ovaries and epithelial ovarian cancer express the mRNAs encoding VEGF isoforms determined by RT-PCR. The ethidium bromidestained gels show the presence of PCR products amplified from reversedtranscribed total RNA extracted from human ovaries and epithelial ovarian cancer amplified with primers recognizing specific regions of VEGF isoforms (VEGF189; VEGF165 and VEGF121) representative of a total of six samples each. Panel A: L: ladder; C+: positive control corresponding to human pheochromocytoma; C−: negative control for the PCR; N: represents two samples of normal ovaries and Ca: two samples of epithelial ovarian cancer. Panel B: VEGF isoforms mRNA levels expressed as arbitrary units of VEGF mRNA with respect to the β-actin expression as a housekeeping gene. The isoforms of VEGF165 and VEGF121 are abundant in both normal and pathological samples. When comparing the isoforms content between normal and pathological samples, both VEGF189 and VEGF165 contents were higher in epithelial ovarian cancer. The results represent the mean ± standard error (SE) from six normal ovaries and six epithelial ovarian cancers in duplicate. *p < 0.05 and **p < 0.01.
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VEGF mRNA expression in normal ovary and in epithelial ovarian cancer In this study, we found the presence of three of the five VEGF mRNA isoforms in normal and cancer ovarian samples by RT-PCR. Fig. 3A shows the three bands of VEGF (308 bp for VEGF189, 236 bp for VEGF165 and 104 pb for VEGF121) and the β-actin band to 661 bp. We found higher levels of the VEGF121 and VEGF165 isoforms in both epithelial ovarian cancer and in normal ovaries, compared to mRNA levels for VEGF189. Levels of both VEGF165 and VEGF189 were significantly higher in EOC samples compared with normal samples (Fig. 3B). NGF increases VEGF expression levels in cultured explants of epithelial ovarian cancer When EOC explants were incubated with increasing doses of NGF, we found that 2 h in culture was enough to produce a significant increase in VEGF121, VEGF165 and VEGF189 mRNA levels in a dose-dependent manner, with a maximal effective dose at 100 ng/ml NGF (Figs. 4A, B, C). The stimulatory effects of NGF were also observed in the VEGF protein (Fig. 4D). The increase in VEGF mRNA induced by NGF was blocked using either an antibody against NGF, or an inhibitor of tyrosine kinase receptors (K252a), indicating that the
effect was specific and mediated by the activation of NGF highaffinity receptor, trkA. (Figs. 5A, B, C). Similar results were obtained using spent culture media by ELISA for the VEGF protein (Fig. 5D). VEGF detected by this assay was a mixture of VEGF121 and VEGF165 because both isoforms are secreted in the culture media. Discussion The role of neurotrophins in the development and function of the mammalian ovaries is well established. NGF, a member of this family, is required for early follicular development and ovulation [21,39]. Recent studies demonstrated that NGF and its high-affinity receptor trkA are localized in granulosa cells of the secondary and pre-antral follicles in the human ovary [22,24]. This study found, furthermore, low expression of NGF and very low expression of trkA in epithelial cells surrounding normal ovaries. The presence of NGF high-affinity receptor trkA was reported in several epithelial malignancies, including ovarian carcinoma [28,31]. In our work, we found a very strong presence of this neurotrophin and its receptor in the epithelial cells in all ovarian cancers studied. It would be important to study further whether the initiation of trkA expression in ovarian surface epithelial cells is a relevant factor in the transformation of normal cells into malignant cells. If this were
Fig. 4. NGF increases VEGF expression in epithelial ovarian cancer in vitro. VEGF mRNA levels from epithelial ovarian cancer explants cultured for 2 h with 10, 50 and 100 ng/ml of NGF. A significant increase of VEGF isoforms was observed with 100 ng/ml of NGF. (A) VEGF121, the results represent the mean ± SE from n = 10 (*p < 0.05). (B) VEGF165, the results represent the mean ± SE from n = 10 (* p < 0.05). (C) VEGF189, the results represent the mean ± SE from n = 10. (***p < 0.005). (D) Represents the amount of VEGF protein secreted in spent culture media from epithelial ovarian cancer explants cultured with 10, 50 and 100 ng/ml of NGF. The VEGF protein was expressed in arbitrary units with respect to the control condition (mean ± SE). VEGF protein assays were carried out by ELISA which detected both VEGF121 and VEGF165. The results represent the mean ± SE from n = 6. (*p < 0.05).
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Fig. 5. The Increase in VEGF mRNA and protein induced by NGF is blocked with an antibody against NGF or by the presence of a tyrosine kinase receptors inhibitor (K252a). (A) VEGF121 mRNA levels from epithelial ovarian cancer explants, showed a significant increase with 100 ng/ml of NGF (**p < 0.01). Inhibition of NGF action with an antibody against NGF (NGF-Ab) and K252a (NGF versus NGF plus NGF-Ab = ++p < 0.01; NGF versus NGF plus K252a #p < 0.05). The results represent the mean ± SE from n = 8. (B) VEGF165 mRNA levels from epithelial ovarian cancer explants, showed a significant increase with 100 ng/ml of NGF (*p < 0.05), and a significant inhibition of NGF action using NGF-Ab (++p < 0.01). No significant inhibition was observed with K252a. The results represent the mean ± SE from n = 8. (C) Significant increase in VEGF189 mRNA levels from epithelial ovarian cancer explants in culture with 100 ng/ml of NGF (**p < 0.01) as well as an important inhibition of NGF action using NGF-Ab and with K252a (+p < 0.05). The results represent the mean ± SE from n = 8. (D) VEGF protein levels detected by ELISA from spent culture media of epithelial ovarian cancer explants showed a significant increase with 100 ng/ml of NGF (*p < 0.05) and a significant inhibition using NGF-Ab and K252a (NGF versus NGF plus NGF-Ab = ++p < 0.01; NGF versus NGF plus K252a = #p < 0.05). The VEGF secreted corresponds to the VEGF121 and VEGF165 isoforms. The results represent the mean ± SE from n = 8.
the case, the expression of trkA receptors in the ovarian surface epithelium could be used as an early predictive tool for epithelial ovarian cancer. Angiogenesis, and the production of angiogenic factors, are essential for tumor growth and metastasis. One angiogenic factor, VEGF, has been implicated in the neovascularization of a wide variety of tumors [42]. VEGF may up-regulate this pathogenic process. High expression of angiogenic factors may be associated with advanced tumor stages in human ovarian cancer [43]. Several studies have documented the positive correlations between the extent of tumor vascularization, with metastasis formation, as well as with decreased patient survival rates in ovarian carcinoma [44–47]. Also, high levels of VEGF protein were found in ovarian cyst fluid and have been associated with ovarian malignancy [48]. NGF acts either as a direct angiogenic factor or by upregulating the expression of VEGF, as demonstrated in pheochromocytoma cells (PC12) [49]. Phosphorylated trkA (p-trkA) expression has also been reported in endothelial cells in serous ovarian carcinomas suggesting that the proangiogenic role attributed to NGF in vivo and in vitro may be relevant in clinical cancer [36]. Our results showed that NGF up-regulates
the expression of VEGF in ovarian epithelial cancer, supporting the idea that NGF may be directly involved in the in vivo regulation of VEGF-mediated angiogenesis in this type of cancer. The VEGF gene encodes five different isoforms that are generated by alternative splicing from a single gene [12–14]. Functional evidence shows that VEGF165 plays a role in ovarian cyst formation associated with ovarian cancer [18]. We found that NGF increases three isoforms of VEGF; VEGF121, VEGF165 and VEGF189. This effect was achieved by activation of trkA receptors. The co-localization of NGF and VEGF in epithelial cells from serous ovarian carcinoma [36], suggests that NGF-mediated VEGF increase may be an autocrine action. NGF may play a dual role during ovarian angiogenesis, acting as (1) a direct factor in the endothelial cells of the serous ovarian carcinoma vasculature [36] and (2) indirectly through the regulation of VEGF expression in epithelial ovarian cells, as we found in this study. These results suggest that the expression of NGF and trkA in ovarian surface epithelium is not only important for the initial stages of angiogenesis, but may also play a role in the progression of epithelial ovarian cancer, by mediating an
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