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Expression of lysophosphatidic acid receptors and vascular endothelial growth factor mediating lysophosphatidic acid in the development of human ovarian cancer
Cancer Letters 192 (2003) 161–169 www.elsevier.com/locate/canlet
Expression of lysophosphatidic acid receptors and vascular endothelial growth factor mediating lysophosphatidic acid in the development of human ovarian cancer Takuji Fujitaa, Shingo Miyamotoa,*, Ichiro Onoyamaa, Kenzo Sonodaa, Eisuke Mekadab, Hitoo Nakanoa a
Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan b Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received 2 October 2002; received in revised form 26 November 2002; accepted 2 December 2002
Abstract Lysophosphatidic acid (LPA) receptors including LPA1, LPA2, and LPA3 mediate lysophosphatidic acid signals. We analyzed the expression of LPA receptors, vascular endothelial growth factor (VEGF), and interleukin-8 in 97 patients from normal ovary to ovarian cancer, using reverse transcription polymerase chain reaction. LPA2, LPA3, and VEGF expression ratios significantly increased in cancer, compared to those in non-cancerous state ðP , 0:05Þ. A significant correlation in the expression ratios between LPA2 or LPA3 and VEGF was found (g ¼ 0:617, P , 0:0001; g ¼ 0:431, P , 0:001) in patients with cancer. These results suggested that LPA2 and LPA3 may be involved in VEGF expression mediated by LPA signals in human ovarian oncogenesis. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lysophosphatidic acid receptors; Vascular endothelial growth factor; Interleukin-8; Reverse transcription polymerase chain reaction
1. Introduction Abbreviations: NIH, National Institutes of Health; LPA, lysophosphatidic acid; VEGF, vascular endothelial growth factor; IL-8, interleukin-8; RT-PCR, reverse transcriptase-polymerase chain reaction; FIGO, International Federation of Gynecology and Obstetrics; OCT, optimum cutting temperature; PCR, polymerase chain reaction; HIF-a1, hypoxia-inducible factor-a1; VEGF/VPF, vascular endothelial growth factor or vascular permeability factor; AP-1, activator protein-1; NF-kB, nuclear factor kappa B. * Corresponding author. Tel.: þ81-92-642-5395; fax: þ 81-92642-5414. E-mail address: [email protected] (S. Miyamoto).
Lysophosphatidic acid (LPA), which is generated from precursors in membranes, has numerous cellular effects including growth promotion, calcium homeostasis, cytoskeletal organization, and secretion of peptide growth factors [1 – 4]. In ovarian cancer, LPA is found to be elevated in plasma and acsites from patients with ovarian cancer in all stages [5,6]. In ovarian cancer cell lines, LPA also has a variety of properties including the enhancement of cell adhesion/attachment, production of angiogenetic fac-
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 1 3 - 9
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tors such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), secretion of urokinase, prevention of cell apoptosis, and a decrease of cis-diamminedichloroplatinum-induced cell death [7 – 12]. According to this evidence, LPA is attributable to tumor behavior in ovarian cancer. Specific G-protein coupled transmembrane receptors including LPA1, LPA2, and LPA3, which share 50 –54% identical amino acids, can mediate LPA signaling in a variety of cells [1 –4]. Northern blot analyses revealed that LPA1 is expressed in oligodendrocytes and peripheral tissues including testis, intestine, heart, lung, kidney, spleen, thymus, muscle, and stomach [13 – 15]. LPA2 mRNA is found in testis and peripheral blood leukocytes, whereas LPA3 mRNA is localized in the prostate, testis, pancreas, kidney, and heart [15 –17]. LPA1 is mostly associated with activation of Gia pathways [13,14], whereas LPA2 and LPA3 are most prominently associated with Gq/11a pathways [16,17]. Therefore, LPA1, LPA2, and LPA3 have the distinct biological property of LPA receptor. In addition, LPA1, not LPA2 or LPA3, is dominantly expressed in immortalized normal ovary cell lines, whereas LPA2 and LPA3 expression significantly increases in ovarian cancer cell lines [11,12,18]. LPA1 may function as a negative regulator for ovarian epithelial cell growth and metastasis [19]. LPA2 plays a role in LPA stimulation of ovarian tumor growth, accompanied by the production of VEGF [12,18]. LPA3 is possibly involved in mediating the proliferation/transformation signals in ovarian cancer ascites [11,18]. In preliminary results, the expression of LPA2 and LPA3 mRNAs increased in patients with ovarian cancer, compared to those in patients with normal ovary [20]. However, there have been few reports concerning the LPA receptors including LPA1, LPA2, and LPA3 expression in detail and VEGF and IL-8 expression mediated by LPA signals in ovarian oncogenesis from normal ovary to ovarian cancer. To identify which LPA receptor contributes to transmitting LPA signals during human ovarian oncogenesis, we examined the expression of LPA1, LPA2, LPA3, and angiogenetic factors including VEGF and IL-8 in tissue specimens dissected from 97 patients with normal ovary (12 cases), tumor of low malignant potential (11 cases), ovarian cancer with stage I (19 cases), stage II (eight cases), stage III
(32 cases), and stage IV (15 cases), using reverse transcription polymerase chain reaction (RT-PCR).
2. Materials and methods
2.1. Patients and surgical specimens All the 85 patients in this study with tumor of low malignant potential and ovarian cancer had undergone surgery between January 1994 and September 2000 at the Department of Obstetrics and Gynecology, Kyushu University Hospital. All tissue samples were obtained from 85 women, consisting of 11 cases with tumor of low malignant potential, 19 cases with International Federation of Gynecology and Obstetrics (FIGO) Stage I disease, eight cases with FIGO Stage II disease, 32 cases with FIGO Stage III disease, and 15 cases with FIGO Stage IV disease. After dissection, half of all fresh tumor tissue specimens were immediately snap-frozen in liquid nitrogen and then stored at 2 858C until use. The other half of the fresh tissue specimens was immediately embedded for frozen section and paraffin section. Diagnosis was based on conventional morphological examination of the paraffin-embedded specimens, and tumors were classified according to the World Health Organization classification [21]. Histological grade was determined using the grading systems [22]. As for histological subtype, 11 cases with tumor of low malignant potential consisted of three cases with serous tumor of borderline malignancy, five cases with mucinous tumor of borderline malignancy, and three cases with mixed epithelial tumor of borderline malignancy. In ovarian cancer, 48 of the 71 cases indicated serous papillary adenocarcinoma, and the remaining 26 cases consisted of seven cases with endometrioid adenocarcinoma, seven cases with mucinous adenocarcinoma, six cases with clear cell adenocarcinoma, and five cases with undifferentiated carcinoma. Normal ovarian tissue specimens were also obtained at surgery for benign disease from 12 patients consisting of six cases in the premenopausal state and six cases in the postmenopausal state. In this study, informed consent was obtained from all the patients.
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2.2. Preparation of RNA and performance of RT-PCR for the LPA receptors, VEGF, and IL-8 expression To ascertain the presence of cancer cells, half of each fresh tumor tissue specimen was immediately embedded in optimum cutting temperature (OCT)compound (Miles, Kankakee, IL). Frozen sections were cut on a cryostat to a thickness of 4 mm and immediately stained with hematoxyline and eosin. More than 80% of a given tumor specimen, which contained cancer cells, was used for RT-PCR. Total cellular RNA was purified from frozen tumor tissues by the acid guanidinium thiocyanate procedure [23]. First-strand cDNA synthesis was performed with 0.8 mg of total RNA using a cDNA synthesis kit (GIBCOBRLeII) and 10 pmol of random primers, following the manufacturer’s protocol. For PCR amplification, we used a 1 mL aliquot of the reaction mixture. To obtain reproducible quantitative performance of the RT-PCR assay for LPA1, LPA2, LPA3, VEGF, and IL-8, we titrated the amount of starting cDNA and the number of amplification cycles. Within 35 cycles of PCR, the expression of these genes was found to increase in a linear range. All subsequent assays were performed using the parameters that yielded amplification (30 cycles of PCR) of LPA1, LPA2, LPA3, VEGF, IL-8, and b-actin DNA (the internal control) [24 – 27]. On the basis of the nucleotide sequences of LPA1 cDNA, LPA2 cDNA, LPA3 cDNA, VEGF cDNA, and IL-8 cDNA, 50 -AATCGAGAGGCACATTACGG-30 was used as the sense primer of LPA1 cDNA and 50 TGTGGACAGCACACGTCTAG-30 as the antisense primer of LPA1 cDNA, 50 -CATCATGCTTCCCGAGAACG-30 as the sense primer of LPA2 cDNA and 50 GGGCTTACCAAGGATACGCAG-30 as the antisense primer of LPA2 cDNA, 50 -AGGATGCGGGTCCATAGCAA-30 as the sense primer of LPA3 cDNA and 50 -GATGATGGGGTTCACGACGG-30 as the antisense primer of LPA3 cDNA, 50 -AGGCCAGCACATAGGAGAGA-30 as the sense primer of VEGF cDNA and 50 -ACCGCCTCGGCTTGTCACAT-30 as the antisense primer of VEGF cDNA, and 50 TTCTGCAGCTCTGTGTGAAGG-30 as the sense primer of IL-8 and 50 -GAAGAGGGCTGAGAATTCAT-30 as the antisense primer of IL-8. Each reaction of mixture was subjected to 30 PCR amplification cycles of 60 s at 948C, 60 s at 618C, and 60 s at 748C
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for the amplification of LPA1 cDNA and LPA3 cDNA, 60 s at 948C, 60 s at 608C, and 60 s at 748C for the amplification of LPA2 cDNA, 60 s at 948C, 60 s at 588C, and 60 s at 748C for the amplification of VEGF cDNA, and 60 s at 948C, 60 s at 598C, and 60 s at 748C for the amplification of IL-8 cDNA. b-actin DNA amplification was used as the internal PCR control; the sense primer was 5 0 -GGCATCGTGATGGACTCCG-30 , and the antisense primer was 50 GCTGGAAGGTGGACAGCGA-30 . The reaction of the mixture was also subjected to 25 amplification cycles of 60 s at 948C, 60 s at 608C, and 60 s at 748C for the amplification of b-actin cDNA. The PCR products were electrophoresed on 3% agarose gels, and the bands visualized with ethidium bromide and photographed with a camera (Funakoshi, Tokyo, Japan). Densitometric analysis of the photography was used for band quantification using a NIH image. As a representative of VEGF expression, the band of VEGF165 expression was measured [24]. The densitometric values obtained for LPA1, LPA2, LPA3, VEGF165, and IL-8 bands of a given tumor tissue sample were divided by that of b-actin, and referred to as LPA1, LPA2, LPA3, VEGF, and IL-8 expression ratios. When the value was not zero, it was regarded as a positive expression of LPA1, LPA2, LPA3, VEGF, and IL-8. Concerning the expression of all genes, the same PCR procedures were performed at least twice and the mean ratio was evaluated as the value for each specimen. As for negative expression of LPA1, LPA2, LPA3, VEGF, and IL-8, the first-strand cDNA was synthesized at least twice and the PCR procedure for each cDNA product was performed twice. 2.3. Statistical analysis The Mann –Whitney test was performed to test the equality of the distribution of age among patients with tumor of low malignant potential, and ovarian cancer. The statistical significance of differences in the expression ratios of genes was assessed using Bonferroni’s test. The correlation in expression ratios of genes was analyzed using Pearson’s correlation analysis. Statistical significance (P ) was based on two-tailed statistical analyses; P less than 0.05 was considered as statistically significant.
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3. Results
VEGF or IL-8, or between LPA2 and LPA3 in patients with ovarian cancer.
3.1. Detection of the LPA receptors, VEGF, IL-8 expression using RT-PCR in patients with normal ovary, tumor of low malignant potential and ovarian cancer LPA1 was expressed in all patients with normal ovary and tumor of low malignant potential, and few of these cases indicated the expression of LPA2, LPA3, VEGF, or IL-8 (Fig. 1 and Table 1). In ovarian cancer, most of the patients had a positive expression of LPA1, LPA2, LPA3, VEGF and IL-8 (Fig. 1 and Table 1). The number of patients with a positive expression of LPA1, LPA2, LPA3, VEGF, and IL-8 was almost the same positive expression in ovarian cancer with each stage. There were strikingly significant differences in the expression ratios of LPA2, LPA3, and VEGF among the patients with normal ovary, tumor of low malignant potential, and ovarian cancer (Fig. 2; P , 0:05). No significant differences in the expression ratios of LPA1 and IL-8 were found in any of the three groups, although the expression ratios of IL-8 in patients with ovarian cancer were quite elevated, compared to those in patients with normal ovary or tumor of low malignant potential (P , 0:05) (Fig. 2). The values for age (mean ^ SD) were 50.8 ^ 16.4, 50.4 ^ 17.3, and 54.7 ^ 11.4 in patients with normal ovary, tumor of low malignant potential, and ovarian cancer, respectively. There was no significant difference in the age distribution among the three groups. 3.2. The correlation coefficiency in the expression ratios between the LPA receptors and VEGF or IL-8 in patients with ovarian cancer A significant correlation was found in the expression ratios between LPA 2 and VEGF (g ¼ 0:613, P , 0:0001) (Fig. 3) The correlation coefficient in the expression ratios of LPA3 and VEGF was 0.434 ðP , 0:001Þ (Fig. 3). In comparison with the expression ratios between IL-8 and LPA2 or LPA3, the correlation coefficients of 0.341 and 0.25, respectively, (P , 0:01, P , 0:05), were not statistically significant. There were no significant correlations in the expression ratios between LPA1 and
Fig. 1. Expression of the LPA receptors including LPA1, LPA2, and LPA3, VEGF, and IL-8 in patients with normal ovary, tumor of low malignant potential, and ovarian cancer. Lane 1, size marker; lanes 2–4, cases with normal ovary; lanes 5 and 6, cases with tumor of low malignant potential; lanes 7–9, cases with early ovarian cancer; lanes 10 –12, cases with advanced ovarian cancer. (A) Agarose gel electrophoresis of RT-PCR amplified 432 bp LPA1 DNA. Lanes 2– 10, cases with positive LPA1 gene expression; lanes 11 and 12, cases with negative LPA1 gene expression. (B) Agarose gel electrophoresis of RT-PCR amplified 352 bp LPA2 DNA. Lanes 2–6 and 9, cases with negative LPA2 gene expression; lanes 7, 8 ,and 10–12, cases with positive LPA2 gene expression. (C) Agarose gel electrophoresis of RT-PCR amplified 481 bp LPA3 DNA. Lanes 2–7, cases with negative LPA3 gene expression; lanes 8,9, and 11, cases with positive LPA3 gene expression. (D) Agarose gel electrophoresis of RT-PCR amplified 104 bp (VEGF121), 236 bp (VEGF165), 308 bp (VEGF189), and 359 bp (VEGF206) VEGF DNA. Lanes 2 –6 and 9, cases with negative VEGF gene expression; lanes 7,8, 10 and 11, cases with positive VEGF gene expression. (E) Agarose gel electrophoresis of RT-PCR amplified 254 bp IL8DNA. Lanes 2–6, 10, and 11, cases with negative IL-8 gene expression; lanes 7 –9 and 12, cases with positive IL-8 gene expression. (F) Agarose gel electrophoresis of RT-PCR amplified bactin DNA (internal PCR control) of each specimen.
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Table 1 Expression of LPA receptors, VEGF, and IL-8 during human oncogenesis of ovarian cancer
Normal ovary (N ¼ 12) Tumor of low Malignant potential (N ¼ 11) Ovarian cancer Stage I (N ¼ 19) Stage II (N ¼ 8) Stage III (N ¼ 32) Stage IV (N ¼ 1 5) a
LPA1
LPA2
LPA3
VEGF
IL-8
b-Actin
12/12a (100%)
1/12 (8.3%)
2/12 (16.7%)
2/12 (16.7%)
2/12 (16.7%)
12/12 (100%)
11/11 (100%)
2/11 (14.3%)
2/11 (14.3%)
2/11 (14.3%)
2/11 (14.3%)
11/11 (100%)
16/19 (84.2%) 6/8 (75.0%) 25/32 (78.1%) 11/15 (73.3%)
16/19 (84.2%) 4/8 (50.0%) 24/32 (75.0%) 7/15 (46.7%)
14/19 (64.3%) 6/8 (75.0%) 22/32 (68.8%) 12/15 (80.0%)
16/19 (84.2%) 5/8 (62.5%) 29/32 (90.6%) 11/15 (73.3%)
9/19 (47.4%) 2/8 (25.0%) 16/32 (50.0%) 7/15 (46.7%)
19/19 (100%) 8/8 (100%) 32/32 (100%) 15/15 (100%)
No. of cases with positive expression of LPA receptors, VEGF, IL-8, or b-actin/no. of cases analyzed.
4. Discussion In this study, we demonstrated that LPA2 and LPA3 were abundantly expressed in most patients with ovarian cancer, but not in patients with normal ovary or tumor of low malignant potential. The expression of LPA1 did not always decrease in patients with ovarian cancer, compared to that in patients with normal ovary or tumor of low malignant potential. These results almost coincided with previous data on ovarian cancer cell lines [11,12,18]. However, it remains unclear why LPA2 and LPA3 expression
occurred during ovarian oncogenesis. Some possibilities are as follows: (1) gene amplification, (2) mutation of genes, and (3) activated transcriptional regulation of genes. LPA1, LPA2, and LPA3 genes are located on human chromosomes 9q31.3-32, 19p12, and 7, respectively [16, 28 –30]. In ovarian cancer, the most frequent sites of amplification are 8q24, 3q26.3, and 20q13.3 [31]. In addition, allelotyping studies of ovarian cancer also reveal that the highest loss of heterozygosity is located on 17p, 17q, 18q, 6q, 11q, and 11p [32]. According to these results, LPA2 and LPA3 expression in ovarian cancer may not be due to
Fig. 2. Expression ratios of the LPA receptors including LPA1, LPA2, and LPA3, VEGF, and IL-8 in patients with normal ovary, tumor of low malignant potential, and ovarian cancer. Each figure represents the values of (A) LPA1, (B) LPA2, (C) LPA3, (D) VEGF and (E) IL-8 in patients with normal ovary (NO), tumor of low malignant potential (LMP), and ovarian cancer (OV). Values are the mean ^ SE. (standard error). Asterisk indicates P , 0:05.
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Fig. 3. Pearson’s correlation in expression ratios between LPA2 or LPA3 and VEGF in patients with ovarian cancer. (A) The ordinate and abscissas indicate the expression ratios of VEGF165 and LPA2, respectively. (B) The ordinate and abscissas indicate the expression ratios of VEGF165 and LPA3, respectively. g indicates the Pearson’ correlation coefficient.
genetic alteration. Although reports have been made on mutated LPA2 cDNA with four replaced and 31 additional amino acids at the position of the Cterminal cytoplasmic tail in the ovarian cancer cell line [30], there have been no reports concerning the mutation of LPA2 and LPA3 in ovarian cancer. This mutated LPA2 protein may contribute to sustained receptor activation, leading to cellular transformation [30]. In this study, approximately 70% of the patients with ovarian cancer indicated a significant expression of both LPA2 and LPA3. These two genes seem to have principally a similar function in cancer progression. It is unlikely that the mutations of both LPA2 and LPA3 genes simultaneously occur with an incidence rate of 70%. However, the mutations of these genes should be evaluated in future studies. The final possible explanation for the occurrence of LPA2 and LPA3 expression lies in the transcriptional regulation of both genes. In this study, the expression statuses of LPA2 and LPA3 were correlated with that of VEGF. On the basis of previous reports, VEGF expression is mediated by LPA or hypoxia in ovarian cancer [12,33]. In rat cardiac myocytes, hypoxia and adrenergic agonists induce the increased expression of the LPA receptors, and then LPA signaling through
the up-regulated LPA receptors, protects cardiac myocytes from apoptosis induced by hypoxia and/or adrenergic stimulation [4,34]. In ovarian cancer, therefore, hypoxia induced by tumor growth might modulate LPA2 and LPA3 expression, which results in increased LPA signaling. In our study, however, LPA2 expression was not always associated with LPA3 expression in patients with ovarian cancer. Thus, further study is required in order to clarify the molecular mechanism behind LPA 2 and LPA3 expression induced in ovarian cancer. VEGF and IL-8 relate to several kinds of angiogenetic factors [35,36]. Both VEGF and IL-8 expression are induced by LPA stimulation as well as hypoxia in ovarian cancer [8,12,33,37]. Transcriptional up-regulation of the VEGF gene during hypoxia is dependent on transactivation by the transcriptional factor HIF-a1, which binds to a HIF-a1 consensus site located in the 50 flanking region of the vascular endothelial growth factor or vascular permeability factor (VEGF/VPF) gene [37 – 39]. On the other hand, no HIF-a1-binding motif appears in the published sequence of the IL-8 promoter region, and the induction of IL-8 expression is mediated by transactivation of the IL-8 promoter by the transcriptional factors activator protein-1 (AP-1)
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and nuclear factor kappa B (NF-kB) [37,40]. Therefore, the transcriptional regulation for VEGF expression is different from that of IL-8 expression. In our study, VEGF expression, not IL-8 expression, was correlated with LPA2 or LPA3 expression, suggesting that the expression of VEGF, LPA2, and LPA3 might be mediated by a similar transcriptional regulation and that the transcriptional regulation of IL8 expression might be different from that of LPA2 or LPA3 expression. Ovarian cancer activating factor is composed of various types of LPA, containing saturated and unsaturated fatty acyl chains [5]. It has been recognized that LPA with unsaturated fatty acids is more biologically active than that with saturated fatty acid chains [41]. An increased presence of unsaturated fatty acids in plasma LPA is found in patients with late-stage or recurrent ovarian cancer [42]. In general, LPA1, LPA2, and LPA3 are differentially activated by various LPA types [43]. LPA2 responds to both saturated and unsaturated LPA at lower concentrations, compared to LPA1 and LPA3 [43]. LPA3 is the most highly reactivated by unsaturated fatty acid, whereas LPA1 shows broad ligand specificities at low affinity [43]. LPA2 is regarded as a powerful mediator for LPA signaling and LPA3 expression also has the advantage of transmitting signals induced by unsaturated LPA. Ovarian cancer cells constitutively produce increased amounts of LPA as compared with normal ovarian epithelium, the precursor of ovarian epithelial cancer, or breast cancer cells [44]. Therefore, it is plausible that the occurrence of LPA2 and LPA3 expression may imply the existence of properties which promote ovarian cancer progression. Our study demonstrates that the increase of LPA2 and LPA3 expression occur in vivo cancerous state and that these expression ratios are correlated with the expression ratio of VEGF. These results suggested that the LPA2 and LPA3 expression may associate the activation of angiogenetic signals for constitutive LPA stimulation in human ovarian cancer.
Acknowledgements We thank Mrs L. Saza for helpful discussions. This
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work was supported in part by a grant-in-aid for cancer research from the Ministry of Health and Welfare of Japan (numbers: 13671727, 13671728, and 14571568). This work was in part done at the Station for Collaborative Research and at the Morphology Core, Graduate School of Medical Sciences, Kyushu University.
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Expression of lysophosphatidic acid receptors and vascular endothelial growth factor mediating lysophosphatidic acid in the development of human ovarian cancer Takuji Fujitaa, Shingo Miyamotoa,*, Ichiro Onoyamaa, Kenzo Sonodaa, Eisuke Mekadab, Hitoo Nakanoa a
Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan b Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received 2 October 2002; received in revised form 26 November 2002; accepted 2 December 2002
Abstract Lysophosphatidic acid (LPA) receptors including LPA1, LPA2, and LPA3 mediate lysophosphatidic acid signals. We analyzed the expression of LPA receptors, vascular endothelial growth factor (VEGF), and interleukin-8 in 97 patients from normal ovary to ovarian cancer, using reverse transcription polymerase chain reaction. LPA2, LPA3, and VEGF expression ratios significantly increased in cancer, compared to those in non-cancerous state ðP , 0:05Þ. A significant correlation in the expression ratios between LPA2 or LPA3 and VEGF was found (g ¼ 0:617, P , 0:0001; g ¼ 0:431, P , 0:001) in patients with cancer. These results suggested that LPA2 and LPA3 may be involved in VEGF expression mediated by LPA signals in human ovarian oncogenesis. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lysophosphatidic acid receptors; Vascular endothelial growth factor; Interleukin-8; Reverse transcription polymerase chain reaction
1. Introduction Abbreviations: NIH, National Institutes of Health; LPA, lysophosphatidic acid; VEGF, vascular endothelial growth factor; IL-8, interleukin-8; RT-PCR, reverse transcriptase-polymerase chain reaction; FIGO, International Federation of Gynecology and Obstetrics; OCT, optimum cutting temperature; PCR, polymerase chain reaction; HIF-a1, hypoxia-inducible factor-a1; VEGF/VPF, vascular endothelial growth factor or vascular permeability factor; AP-1, activator protein-1; NF-kB, nuclear factor kappa B. * Corresponding author. Tel.: þ81-92-642-5395; fax: þ 81-92642-5414. E-mail address: [email protected] (S. Miyamoto).
Lysophosphatidic acid (LPA), which is generated from precursors in membranes, has numerous cellular effects including growth promotion, calcium homeostasis, cytoskeletal organization, and secretion of peptide growth factors [1 – 4]. In ovarian cancer, LPA is found to be elevated in plasma and acsites from patients with ovarian cancer in all stages [5,6]. In ovarian cancer cell lines, LPA also has a variety of properties including the enhancement of cell adhesion/attachment, production of angiogenetic fac-
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 1 3 - 9
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tors such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), secretion of urokinase, prevention of cell apoptosis, and a decrease of cis-diamminedichloroplatinum-induced cell death [7 – 12]. According to this evidence, LPA is attributable to tumor behavior in ovarian cancer. Specific G-protein coupled transmembrane receptors including LPA1, LPA2, and LPA3, which share 50 –54% identical amino acids, can mediate LPA signaling in a variety of cells [1 –4]. Northern blot analyses revealed that LPA1 is expressed in oligodendrocytes and peripheral tissues including testis, intestine, heart, lung, kidney, spleen, thymus, muscle, and stomach [13 – 15]. LPA2 mRNA is found in testis and peripheral blood leukocytes, whereas LPA3 mRNA is localized in the prostate, testis, pancreas, kidney, and heart [15 –17]. LPA1 is mostly associated with activation of Gia pathways [13,14], whereas LPA2 and LPA3 are most prominently associated with Gq/11a pathways [16,17]. Therefore, LPA1, LPA2, and LPA3 have the distinct biological property of LPA receptor. In addition, LPA1, not LPA2 or LPA3, is dominantly expressed in immortalized normal ovary cell lines, whereas LPA2 and LPA3 expression significantly increases in ovarian cancer cell lines [11,12,18]. LPA1 may function as a negative regulator for ovarian epithelial cell growth and metastasis [19]. LPA2 plays a role in LPA stimulation of ovarian tumor growth, accompanied by the production of VEGF [12,18]. LPA3 is possibly involved in mediating the proliferation/transformation signals in ovarian cancer ascites [11,18]. In preliminary results, the expression of LPA2 and LPA3 mRNAs increased in patients with ovarian cancer, compared to those in patients with normal ovary [20]. However, there have been few reports concerning the LPA receptors including LPA1, LPA2, and LPA3 expression in detail and VEGF and IL-8 expression mediated by LPA signals in ovarian oncogenesis from normal ovary to ovarian cancer. To identify which LPA receptor contributes to transmitting LPA signals during human ovarian oncogenesis, we examined the expression of LPA1, LPA2, LPA3, and angiogenetic factors including VEGF and IL-8 in tissue specimens dissected from 97 patients with normal ovary (12 cases), tumor of low malignant potential (11 cases), ovarian cancer with stage I (19 cases), stage II (eight cases), stage III
(32 cases), and stage IV (15 cases), using reverse transcription polymerase chain reaction (RT-PCR).
2. Materials and methods
2.1. Patients and surgical specimens All the 85 patients in this study with tumor of low malignant potential and ovarian cancer had undergone surgery between January 1994 and September 2000 at the Department of Obstetrics and Gynecology, Kyushu University Hospital. All tissue samples were obtained from 85 women, consisting of 11 cases with tumor of low malignant potential, 19 cases with International Federation of Gynecology and Obstetrics (FIGO) Stage I disease, eight cases with FIGO Stage II disease, 32 cases with FIGO Stage III disease, and 15 cases with FIGO Stage IV disease. After dissection, half of all fresh tumor tissue specimens were immediately snap-frozen in liquid nitrogen and then stored at 2 858C until use. The other half of the fresh tissue specimens was immediately embedded for frozen section and paraffin section. Diagnosis was based on conventional morphological examination of the paraffin-embedded specimens, and tumors were classified according to the World Health Organization classification [21]. Histological grade was determined using the grading systems [22]. As for histological subtype, 11 cases with tumor of low malignant potential consisted of three cases with serous tumor of borderline malignancy, five cases with mucinous tumor of borderline malignancy, and three cases with mixed epithelial tumor of borderline malignancy. In ovarian cancer, 48 of the 71 cases indicated serous papillary adenocarcinoma, and the remaining 26 cases consisted of seven cases with endometrioid adenocarcinoma, seven cases with mucinous adenocarcinoma, six cases with clear cell adenocarcinoma, and five cases with undifferentiated carcinoma. Normal ovarian tissue specimens were also obtained at surgery for benign disease from 12 patients consisting of six cases in the premenopausal state and six cases in the postmenopausal state. In this study, informed consent was obtained from all the patients.
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2.2. Preparation of RNA and performance of RT-PCR for the LPA receptors, VEGF, and IL-8 expression To ascertain the presence of cancer cells, half of each fresh tumor tissue specimen was immediately embedded in optimum cutting temperature (OCT)compound (Miles, Kankakee, IL). Frozen sections were cut on a cryostat to a thickness of 4 mm and immediately stained with hematoxyline and eosin. More than 80% of a given tumor specimen, which contained cancer cells, was used for RT-PCR. Total cellular RNA was purified from frozen tumor tissues by the acid guanidinium thiocyanate procedure [23]. First-strand cDNA synthesis was performed with 0.8 mg of total RNA using a cDNA synthesis kit (GIBCOBRLeII) and 10 pmol of random primers, following the manufacturer’s protocol. For PCR amplification, we used a 1 mL aliquot of the reaction mixture. To obtain reproducible quantitative performance of the RT-PCR assay for LPA1, LPA2, LPA3, VEGF, and IL-8, we titrated the amount of starting cDNA and the number of amplification cycles. Within 35 cycles of PCR, the expression of these genes was found to increase in a linear range. All subsequent assays were performed using the parameters that yielded amplification (30 cycles of PCR) of LPA1, LPA2, LPA3, VEGF, IL-8, and b-actin DNA (the internal control) [24 – 27]. On the basis of the nucleotide sequences of LPA1 cDNA, LPA2 cDNA, LPA3 cDNA, VEGF cDNA, and IL-8 cDNA, 50 -AATCGAGAGGCACATTACGG-30 was used as the sense primer of LPA1 cDNA and 50 TGTGGACAGCACACGTCTAG-30 as the antisense primer of LPA1 cDNA, 50 -CATCATGCTTCCCGAGAACG-30 as the sense primer of LPA2 cDNA and 50 GGGCTTACCAAGGATACGCAG-30 as the antisense primer of LPA2 cDNA, 50 -AGGATGCGGGTCCATAGCAA-30 as the sense primer of LPA3 cDNA and 50 -GATGATGGGGTTCACGACGG-30 as the antisense primer of LPA3 cDNA, 50 -AGGCCAGCACATAGGAGAGA-30 as the sense primer of VEGF cDNA and 50 -ACCGCCTCGGCTTGTCACAT-30 as the antisense primer of VEGF cDNA, and 50 TTCTGCAGCTCTGTGTGAAGG-30 as the sense primer of IL-8 and 50 -GAAGAGGGCTGAGAATTCAT-30 as the antisense primer of IL-8. Each reaction of mixture was subjected to 30 PCR amplification cycles of 60 s at 948C, 60 s at 618C, and 60 s at 748C
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for the amplification of LPA1 cDNA and LPA3 cDNA, 60 s at 948C, 60 s at 608C, and 60 s at 748C for the amplification of LPA2 cDNA, 60 s at 948C, 60 s at 588C, and 60 s at 748C for the amplification of VEGF cDNA, and 60 s at 948C, 60 s at 598C, and 60 s at 748C for the amplification of IL-8 cDNA. b-actin DNA amplification was used as the internal PCR control; the sense primer was 5 0 -GGCATCGTGATGGACTCCG-30 , and the antisense primer was 50 GCTGGAAGGTGGACAGCGA-30 . The reaction of the mixture was also subjected to 25 amplification cycles of 60 s at 948C, 60 s at 608C, and 60 s at 748C for the amplification of b-actin cDNA. The PCR products were electrophoresed on 3% agarose gels, and the bands visualized with ethidium bromide and photographed with a camera (Funakoshi, Tokyo, Japan). Densitometric analysis of the photography was used for band quantification using a NIH image. As a representative of VEGF expression, the band of VEGF165 expression was measured [24]. The densitometric values obtained for LPA1, LPA2, LPA3, VEGF165, and IL-8 bands of a given tumor tissue sample were divided by that of b-actin, and referred to as LPA1, LPA2, LPA3, VEGF, and IL-8 expression ratios. When the value was not zero, it was regarded as a positive expression of LPA1, LPA2, LPA3, VEGF, and IL-8. Concerning the expression of all genes, the same PCR procedures were performed at least twice and the mean ratio was evaluated as the value for each specimen. As for negative expression of LPA1, LPA2, LPA3, VEGF, and IL-8, the first-strand cDNA was synthesized at least twice and the PCR procedure for each cDNA product was performed twice. 2.3. Statistical analysis The Mann –Whitney test was performed to test the equality of the distribution of age among patients with tumor of low malignant potential, and ovarian cancer. The statistical significance of differences in the expression ratios of genes was assessed using Bonferroni’s test. The correlation in expression ratios of genes was analyzed using Pearson’s correlation analysis. Statistical significance (P ) was based on two-tailed statistical analyses; P less than 0.05 was considered as statistically significant.
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3. Results
VEGF or IL-8, or between LPA2 and LPA3 in patients with ovarian cancer.
3.1. Detection of the LPA receptors, VEGF, IL-8 expression using RT-PCR in patients with normal ovary, tumor of low malignant potential and ovarian cancer LPA1 was expressed in all patients with normal ovary and tumor of low malignant potential, and few of these cases indicated the expression of LPA2, LPA3, VEGF, or IL-8 (Fig. 1 and Table 1). In ovarian cancer, most of the patients had a positive expression of LPA1, LPA2, LPA3, VEGF and IL-8 (Fig. 1 and Table 1). The number of patients with a positive expression of LPA1, LPA2, LPA3, VEGF, and IL-8 was almost the same positive expression in ovarian cancer with each stage. There were strikingly significant differences in the expression ratios of LPA2, LPA3, and VEGF among the patients with normal ovary, tumor of low malignant potential, and ovarian cancer (Fig. 2; P , 0:05). No significant differences in the expression ratios of LPA1 and IL-8 were found in any of the three groups, although the expression ratios of IL-8 in patients with ovarian cancer were quite elevated, compared to those in patients with normal ovary or tumor of low malignant potential (P , 0:05) (Fig. 2). The values for age (mean ^ SD) were 50.8 ^ 16.4, 50.4 ^ 17.3, and 54.7 ^ 11.4 in patients with normal ovary, tumor of low malignant potential, and ovarian cancer, respectively. There was no significant difference in the age distribution among the three groups. 3.2. The correlation coefficiency in the expression ratios between the LPA receptors and VEGF or IL-8 in patients with ovarian cancer A significant correlation was found in the expression ratios between LPA 2 and VEGF (g ¼ 0:613, P , 0:0001) (Fig. 3) The correlation coefficient in the expression ratios of LPA3 and VEGF was 0.434 ðP , 0:001Þ (Fig. 3). In comparison with the expression ratios between IL-8 and LPA2 or LPA3, the correlation coefficients of 0.341 and 0.25, respectively, (P , 0:01, P , 0:05), were not statistically significant. There were no significant correlations in the expression ratios between LPA1 and
Fig. 1. Expression of the LPA receptors including LPA1, LPA2, and LPA3, VEGF, and IL-8 in patients with normal ovary, tumor of low malignant potential, and ovarian cancer. Lane 1, size marker; lanes 2–4, cases with normal ovary; lanes 5 and 6, cases with tumor of low malignant potential; lanes 7–9, cases with early ovarian cancer; lanes 10 –12, cases with advanced ovarian cancer. (A) Agarose gel electrophoresis of RT-PCR amplified 432 bp LPA1 DNA. Lanes 2– 10, cases with positive LPA1 gene expression; lanes 11 and 12, cases with negative LPA1 gene expression. (B) Agarose gel electrophoresis of RT-PCR amplified 352 bp LPA2 DNA. Lanes 2–6 and 9, cases with negative LPA2 gene expression; lanes 7, 8 ,and 10–12, cases with positive LPA2 gene expression. (C) Agarose gel electrophoresis of RT-PCR amplified 481 bp LPA3 DNA. Lanes 2–7, cases with negative LPA3 gene expression; lanes 8,9, and 11, cases with positive LPA3 gene expression. (D) Agarose gel electrophoresis of RT-PCR amplified 104 bp (VEGF121), 236 bp (VEGF165), 308 bp (VEGF189), and 359 bp (VEGF206) VEGF DNA. Lanes 2 –6 and 9, cases with negative VEGF gene expression; lanes 7,8, 10 and 11, cases with positive VEGF gene expression. (E) Agarose gel electrophoresis of RT-PCR amplified 254 bp IL8DNA. Lanes 2–6, 10, and 11, cases with negative IL-8 gene expression; lanes 7 –9 and 12, cases with positive IL-8 gene expression. (F) Agarose gel electrophoresis of RT-PCR amplified bactin DNA (internal PCR control) of each specimen.
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Table 1 Expression of LPA receptors, VEGF, and IL-8 during human oncogenesis of ovarian cancer
Normal ovary (N ¼ 12) Tumor of low Malignant potential (N ¼ 11) Ovarian cancer Stage I (N ¼ 19) Stage II (N ¼ 8) Stage III (N ¼ 32) Stage IV (N ¼ 1 5) a
LPA1
LPA2
LPA3
VEGF
IL-8
b-Actin
12/12a (100%)
1/12 (8.3%)
2/12 (16.7%)
2/12 (16.7%)
2/12 (16.7%)
12/12 (100%)
11/11 (100%)
2/11 (14.3%)
2/11 (14.3%)
2/11 (14.3%)
2/11 (14.3%)
11/11 (100%)
16/19 (84.2%) 6/8 (75.0%) 25/32 (78.1%) 11/15 (73.3%)
16/19 (84.2%) 4/8 (50.0%) 24/32 (75.0%) 7/15 (46.7%)
14/19 (64.3%) 6/8 (75.0%) 22/32 (68.8%) 12/15 (80.0%)
16/19 (84.2%) 5/8 (62.5%) 29/32 (90.6%) 11/15 (73.3%)
9/19 (47.4%) 2/8 (25.0%) 16/32 (50.0%) 7/15 (46.7%)
19/19 (100%) 8/8 (100%) 32/32 (100%) 15/15 (100%)
No. of cases with positive expression of LPA receptors, VEGF, IL-8, or b-actin/no. of cases analyzed.
4. Discussion In this study, we demonstrated that LPA2 and LPA3 were abundantly expressed in most patients with ovarian cancer, but not in patients with normal ovary or tumor of low malignant potential. The expression of LPA1 did not always decrease in patients with ovarian cancer, compared to that in patients with normal ovary or tumor of low malignant potential. These results almost coincided with previous data on ovarian cancer cell lines [11,12,18]. However, it remains unclear why LPA2 and LPA3 expression
occurred during ovarian oncogenesis. Some possibilities are as follows: (1) gene amplification, (2) mutation of genes, and (3) activated transcriptional regulation of genes. LPA1, LPA2, and LPA3 genes are located on human chromosomes 9q31.3-32, 19p12, and 7, respectively [16, 28 –30]. In ovarian cancer, the most frequent sites of amplification are 8q24, 3q26.3, and 20q13.3 [31]. In addition, allelotyping studies of ovarian cancer also reveal that the highest loss of heterozygosity is located on 17p, 17q, 18q, 6q, 11q, and 11p [32]. According to these results, LPA2 and LPA3 expression in ovarian cancer may not be due to
Fig. 2. Expression ratios of the LPA receptors including LPA1, LPA2, and LPA3, VEGF, and IL-8 in patients with normal ovary, tumor of low malignant potential, and ovarian cancer. Each figure represents the values of (A) LPA1, (B) LPA2, (C) LPA3, (D) VEGF and (E) IL-8 in patients with normal ovary (NO), tumor of low malignant potential (LMP), and ovarian cancer (OV). Values are the mean ^ SE. (standard error). Asterisk indicates P , 0:05.
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Fig. 3. Pearson’s correlation in expression ratios between LPA2 or LPA3 and VEGF in patients with ovarian cancer. (A) The ordinate and abscissas indicate the expression ratios of VEGF165 and LPA2, respectively. (B) The ordinate and abscissas indicate the expression ratios of VEGF165 and LPA3, respectively. g indicates the Pearson’ correlation coefficient.
genetic alteration. Although reports have been made on mutated LPA2 cDNA with four replaced and 31 additional amino acids at the position of the Cterminal cytoplasmic tail in the ovarian cancer cell line [30], there have been no reports concerning the mutation of LPA2 and LPA3 in ovarian cancer. This mutated LPA2 protein may contribute to sustained receptor activation, leading to cellular transformation [30]. In this study, approximately 70% of the patients with ovarian cancer indicated a significant expression of both LPA2 and LPA3. These two genes seem to have principally a similar function in cancer progression. It is unlikely that the mutations of both LPA2 and LPA3 genes simultaneously occur with an incidence rate of 70%. However, the mutations of these genes should be evaluated in future studies. The final possible explanation for the occurrence of LPA2 and LPA3 expression lies in the transcriptional regulation of both genes. In this study, the expression statuses of LPA2 and LPA3 were correlated with that of VEGF. On the basis of previous reports, VEGF expression is mediated by LPA or hypoxia in ovarian cancer [12,33]. In rat cardiac myocytes, hypoxia and adrenergic agonists induce the increased expression of the LPA receptors, and then LPA signaling through
the up-regulated LPA receptors, protects cardiac myocytes from apoptosis induced by hypoxia and/or adrenergic stimulation [4,34]. In ovarian cancer, therefore, hypoxia induced by tumor growth might modulate LPA2 and LPA3 expression, which results in increased LPA signaling. In our study, however, LPA2 expression was not always associated with LPA3 expression in patients with ovarian cancer. Thus, further study is required in order to clarify the molecular mechanism behind LPA 2 and LPA3 expression induced in ovarian cancer. VEGF and IL-8 relate to several kinds of angiogenetic factors [35,36]. Both VEGF and IL-8 expression are induced by LPA stimulation as well as hypoxia in ovarian cancer [8,12,33,37]. Transcriptional up-regulation of the VEGF gene during hypoxia is dependent on transactivation by the transcriptional factor HIF-a1, which binds to a HIF-a1 consensus site located in the 50 flanking region of the vascular endothelial growth factor or vascular permeability factor (VEGF/VPF) gene [37 – 39]. On the other hand, no HIF-a1-binding motif appears in the published sequence of the IL-8 promoter region, and the induction of IL-8 expression is mediated by transactivation of the IL-8 promoter by the transcriptional factors activator protein-1 (AP-1)
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and nuclear factor kappa B (NF-kB) [37,40]. Therefore, the transcriptional regulation for VEGF expression is different from that of IL-8 expression. In our study, VEGF expression, not IL-8 expression, was correlated with LPA2 or LPA3 expression, suggesting that the expression of VEGF, LPA2, and LPA3 might be mediated by a similar transcriptional regulation and that the transcriptional regulation of IL8 expression might be different from that of LPA2 or LPA3 expression. Ovarian cancer activating factor is composed of various types of LPA, containing saturated and unsaturated fatty acyl chains [5]. It has been recognized that LPA with unsaturated fatty acids is more biologically active than that with saturated fatty acid chains [41]. An increased presence of unsaturated fatty acids in plasma LPA is found in patients with late-stage or recurrent ovarian cancer [42]. In general, LPA1, LPA2, and LPA3 are differentially activated by various LPA types [43]. LPA2 responds to both saturated and unsaturated LPA at lower concentrations, compared to LPA1 and LPA3 [43]. LPA3 is the most highly reactivated by unsaturated fatty acid, whereas LPA1 shows broad ligand specificities at low affinity [43]. LPA2 is regarded as a powerful mediator for LPA signaling and LPA3 expression also has the advantage of transmitting signals induced by unsaturated LPA. Ovarian cancer cells constitutively produce increased amounts of LPA as compared with normal ovarian epithelium, the precursor of ovarian epithelial cancer, or breast cancer cells [44]. Therefore, it is plausible that the occurrence of LPA2 and LPA3 expression may imply the existence of properties which promote ovarian cancer progression. Our study demonstrates that the increase of LPA2 and LPA3 expression occur in vivo cancerous state and that these expression ratios are correlated with the expression ratio of VEGF. These results suggested that the LPA2 and LPA3 expression may associate the activation of angiogenetic signals for constitutive LPA stimulation in human ovarian cancer.
Acknowledgements We thank Mrs L. Saza for helpful discussions. This
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work was supported in part by a grant-in-aid for cancer research from the Ministry of Health and Welfare of Japan (numbers: 13671727, 13671728, and 14571568). This work was in part done at the Station for Collaborative Research and at the Morphology Core, Graduate School of Medical Sciences, Kyushu University.
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