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Originally published as JCO Early Release 10.1200/JCO.2005.02.2541 on August 8 2005 © 2005 American Society of Clinical Oncology. Differential Gene Expression in Ovarian Tumors Reveals Dusp 4 and Serpina 5 As Key Regulators for Benign Behavior of Serous Borderline Tumors
From the Department of Pathology, University of Maastricht, Maastricht; Department of Pathology, Leiden University Medical Centre, Leiden, the Netherlands; Department of Pathology, University College, London, United Kingdom; and the Department of Pathology, Autonomous University of Barcelona, Barcelona, Spain Address reprint requests to Nathalie L.G. Sieben MD, Department of Pathology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, the Netherlands; e-mail: nathalie.sieben{at}path.unimaas.nl.
PURPOSE: Ovarian serous borderline tumors (SBT) are characterized by arborizing papillae lined by stratified epithelial cells, varying atypia, and absence of stromal invasion. Originally, these tumors have been classified as borderline because they behaved in a remarkably indolent manner, even with widespread tumor deposits called implants and the presence of lymph node involvement. The molecular biology of these lesions has just begun to be explored. High prevalence of B-RAF/K-RAS mutations in SBTs in contrast to serous carcinomas (SCAs) indicates that the mitogenic RAS-RAF-MEK-ERK-MAP kinase pathway is crucial for the pathogenesis of SBTs. The purpose of this study was to further unravel the genetic pathways through which SBTs develop, with a special focus on explaining the generally benign SBT behavior. MATERIALS AND METHODS: We generated RNA expression profiles of 38 ovarian serous neoplasms. Global Test pathway analysis and significance analysis of microarrays (SAM) of the expression profiles was performed. RESULTS: SAM and Global Testing showed that although the mitogenic pathway is activated in SBTs, activation of downstream genes involved in extracellular matrix (ECM) degradation is absent, suggesting an uncoupling of both events. In addition, we show that two genes involved in regulating this uncoupling, ERK-inhibitor Dusp 4 and uPA-inhibitor Serpina 5, are downregulated in SCAs in contrast to SBTs. In SCAs, this was associated with downstream MMP-9 activation at both mRNA and protein level. CONCLUSION: We propose that the putative tumor suppressor genes Dusp 4 and Serpina 5 provide a major clue to the indolent behavior of SBTs.
Ovarian cancer represents the most lethal neoplasm of the female genital tract because most cases are diagnosed at an advanced stage. More than 80% of malignant tumors of the ovary are of epithelial origin. The large majority of carcinomas are serous and arise from the ovarian surface epithelium. Serous borderline tumors (SBTs) belong to an intermediate group between benign and malignant tumors. They are characterized by multiple fibrous papillae with extensive and complex hierarchical branching. The epithelial cells covering the papillae are crowded and form multilayered cellular tufts. The epithelial cells generally show only mild-to-moderate nuclear atypia.1,2 A variant within SBTs that may be encountered consists of a surface micropapillary pattern (micropapillary variant [MP-SBT]). This variant has not been described in large numbers of cases with long-term follow-up data and their significance is as yet unclear.1 SBTs were originally classified as borderline lesions because there appeared to be a major discrepancy between clinical presentation and behavior. Although 30% of SBT patients present with tumor deposits called implants on peritoneal surfaces; omental fat; and, in 7% to 23% of cases, even in lymph nodes; the 10-year survival in general remains excellent.1-3 From a clinical point of view, this is inconsistent with metastatic disease, leading to the classification of borderline. Although favorable in most cases, the biologic behavior of SBTs differs from that of the obviously benign tumors of the same cell type.4 Tumor-related deaths in SBT patients are typically associated with the development of invasive peritoneal implants, which occur in only 4% to 13% of patients with high-stage disease.1-3 Tumor biology studies of SBTs are in progress. Recently, we and others have identified activating mutations in B-RAF or K-RAS, both members of the ERK-MAP kinase pathway in 60% of the SBTs in contrast to less than 12% in serous carcinomas (SCAs).5,6 Activation of the mitogenic RAS-RAF-MEK-ERK-MAP kinase pathway constitutively activates uPA and uPAR expression with subsequent conversion of the proenzyme plasminogen to plasmin. Plasmin can now activate latent forms of matrix metalloproteases (MMPs), which results in degradation of extracellular matrix (ECM) molecules and promote the invasion of the healthy tissue interstitium by tumor cells.7 To unravel further the pathogenetic mechanism of serous borderline tumorigenesis, with a special focus on explaining the in general benign behavior, we performed mRNA expression profiling of 11 SBTs and two MP-SBTs, 10 low-grade (SCA I), and 15 high-grade carcinomas (SCA III) using Affymetrix focus array chips (Santa Clara, CA). Global testing and significance analysis of microarrays (SAM) indicates that in SBTs despite an activated mitogenic pathway, downstream ECM degradation is absent due to the presence of the extracellular receptor kinase (ERK) inhibitor Dusp 4 and the uPA inhibitor Serpina 5.
Tissue Samples A total of 38 surgically removed, snap-frozen tumor specimens were retrieved from the tumor bank of the Department of Pathology, Leiden University Medical Centre (Leiden, the Netherlands). The local institutional review board approved the use of human tissue material for research. The panel included 11 SBTs and two related MP-SBTs; 10 SCAs, grade I; and 15 SCA, grade III. The diagnoses of all cases were confirmed by two pathologists (G.J.F., N.S.) on both routine 3-µm hematoxylin (Klinipath, Duiven, the Netherlands) and eosin (Merck, Darmstadt, Germany) slides from paraffin-embedded material and 4-µm sections from frozen material. Within each group, a number of tissues were selected as internal control samples. We included nine technical replicates (six consisted of two sets of the same tissue block and three consisted of replicate samples generated by splitting samples after extracting total RNA); hence, 47 samples were generated for hybridization. mRNA expression profiles from 1,000 genes showing the highest variance across all 47 samples from 38 serous neoplasms were analyzed using Spotfire visualization software (Spotfire, Somerville, MA) by unsupervised hierarchical clustering using the unweighted pair group method with arithmetic mean (UPGMA) clustering method with correlation as a distance measure. The technical replicates clustered immediately adjacent to each other while branching close to the end points, underscoring the reproducibility of the Affymetrix system (Fig 1). For subsequent analyses, the expression values of the replicates for each sample were averaged.
RNA Isolation, cRNA Synthesis, and Gene Expression Profiling The percentage of tumor cells was estimated by visual examination of HE-stained, 5-µm frozen sections. For every frozen tissue block, areas that contained at least 60% viable tumor cells were selected. Areas consisting of cellular stroma were eliminated from the block by macrodissection, to enrich for epithelial cells. Subsequently, an HE-stained 5-µm section was prepared to verify tumor content after macrodissection and 30 tissue slides of 30 µm were cut, followed by one additional HE-stained 5µm section to verify the tumor content again. The whole procedure was performed by one of the pathologists (N.S.) to ensure optimal selection of the tumor cells. Because of these stringent input selection criteria, more than 75% of the frozen tissue blocks that were selected initially could not be included in this study. From the included cases, total RNA was extracted from frozen tissue slides with Trizol (Life Technologies Inc, Carlsbad, CA) and purified using RNeasy spin columns (Qiagen, Valencia, CA). The integrity and quantity of the total RNA and the labeled cRNA was assessed by spectrophotometric analysis and on the Agilent 2100 Bioanalyzer conductor using RNA 6000 Nano Assay Kit (Agilent Technologies, Palo Alto, CA), following the manufacturer's instructions. Whenever this analysis showed less-than-optimal results, the case was eliminated from the study. We used 20 µg of total RNA to synthesize double-stranded cDNA with the Superscript Choice system (Life Technologies, Rockville, MD). After purifying the resulting cDNA with the Gene Chip Sample Cleanup Module (Affymetrix, Santa Clara, CA), we applied the cDNA as a template for in vitro transcription with the RNA Transcript Labeling Kit (Enzo Diagnostics Inc, Farmingdale, NY). cRNA integrity and quantity were measured as described for the total RNA and again, whenever this test indicated suboptimal conditions, the case did not proceed to the next step. Subsequently, the labeled cRNA was purified using the columns provided in the Gene Chip Sample Cleanup Module (Affymetrix) and fragmentized by 5x RNA fragmentation buffer. Finally, 20 µg of labeled cRNA was hybridized on high-sensitivity oligonucleotide micro array chips (human genome focus array, Affymetrix, www.affymetrix.com/products/arrays/specific/focus.affx) at 45°C for 16 hours. The arrays were analyzed using a Gene Array Scanner (Affymetrix), following the manufacturer's instructions.8
B-RAF/K-RAS Mutation Analysis
Data Processing for Statistical Analysis
Statistical Analysis of Expression Arrays Since in the presence of an activated mitogenic RAS-RAF-MEK-ERK-MAP kinase pathway, an enhanced protease production such as uPA and MMPs is expected, we also selected the nine BioCarta genes involved in ECM degradation downstream of uPA in the Global Test.7 In addition, we used SAM to identify differentially expressed genes in the SBT and SCA group.12 SAM uses modified t test statistics for each gene of a dataset. A small fudge factor is added to the denominator in calculating the t value, thereby controlling for unrealistically low standard deviations in the tested gene. Furthermore SAM allows control of the false discovery rate (FDR) by setting a threshold to the difference between the actual test result and the result from repeated permutations of the tested groups.
MMP-9 Activity by Zymography
Quantitative Reverse Transcriptase PCR for Dusp 4 and Serpina 5
Hierarchical Clustering (unsupervised and related to B-RAF/K-RAS mutation status) Unsupervised hierarchical clustering (Fig 2 showed that the 11 SBTs and the two related MP-SBTs generated one tight cluster completely separate from all but one (NS21) of the SCAs. The grade I and III SCAs could not be further separated in identifiable clusters (Fig 2). K-RAS and B-RAF mutation analysis showed that five SBTs harbored a K-RAS codon 12-13 mutation, five SBTs harbored the common V599E B-RAF mutation and one SBT (NS16) had no mutation (Fig 2). None of the SBTs had mutations in both K-RAS and B-RAF. Concordant with previous publications, all of the SCAs were negative for B-RAF mutations and showed a low prevalence of K-RAS codon 12-13 mutations (two mutations in the SCA I group and three mutations in the SCA III group). Of the remaining cases, we detected one K-RAS mutation in a MP-SBT (Fig 2). Inclusion of the K-RAS and B-RAF mutation status did not influence the clustering results.
Global Test Analysis High prevalence of B-RAF and K-RAS mutations in SBTs, observed in the present study, as well as in previous studies, argues strongly in favor of the RAS-RAF-MEK-ERK-MAP kinase pathway's being crucial for SBT tumorigenesis in the majority of cases. To further unravel the molecular background of SBTs, we performed Global Test analysis for the Erk1/Erk2 MAPK signaling pathway genes and genes known to be involved in ECM degradation downstream of uPA. For the RAS-RAF-MEK-ERK-MAP kinase signaling pathway, the Global Test showed that the SBTs and the SCAs were significantly different (P = 4.4 x 10–5). In line with the presence of activating B-RAF/K-RAS mutations in 10 of 11 SBTs, gene plots for the RAS-RAF-MEK-ERK-MAP kinase signaling pathway indicated consistent upregulation in the SBTs compared with SCAs. No difference for SCAs I versus SCAs III could be observed. Global Test analysis for genes involved in the regulation of ECM degradation showed that SBTs and SCAs were significantly different (SBTs v SCAs, P = 4.8 x 10–5). However, gene plots indicated downregulation of all but one (tissue inhibitor of metalloproteinase 1 [TIMP1], a naturally occurring MMP inhibitor) MMPs in SBTs compared with SCAs.
MMP-9 mRNA and Activity by Zymography
To study the biologic relevance of the observed differences in expression levels for genes involved in ECM degradation, we performed MMP-9 zymography to assess the presence of functionally active MMP-9. Active MMP-9 was present in all stage III SCAs, and all but two (NS21, NS38) of the stage I SCAs (Fig 3).
SAM, Dusp 4 and Serpina 5 The results thus far indicated that in SBTs an uncoupling exists between an activated RAS-RAF-MEK-ERK-MAP kinase pathway and subsequent signaling leading to ECM degradation by overexpression of uPA and downstream MMPs. Therefore, we focused on unraveling the molecular mechanism that could explain what restrains these tumors from overexpressing tumor-associated proteases despite an activated mitogenic signal, reasoning that this could explain the typical indolent borderline behavior.12 From a SAM list of differentially expressed genes between SBTs and SCAs, we identified Dusp 4 and Serpina 5 as candidates that could explain this uncoupling in SBTs. Dusp 4 (MKP 2) is an ERK inhibitor and Serpina 5 (PAI 3) is an inhibitor of uPA. Both genes were consistently downregulated in the SCAs when compared with the SBTs. The mean mRNA Dusp 4 expression for SCAs was 3.1-fold decreased compared with SBTs. The mean Serpina 5 mRNA expression for SCAs was 5.3-fold decreased compared with SBTs (Table 1). The mean mRNA expression values for Dusp 4 and Serpina 5 in the MP-SBTs were between the mean values of SBTs and the SCAs. The microarray data on Dusp 4 and Serpina 5 were substantiated for all 38 cases by qualitative RT-PCR assays (Fig 4).
The distinction between SBTs and SCAs is one of the most common problems in ovarian tumor pathology, yet the literature on borderline tumors is confusing, particularly with regard to their diagnostic features, and treatment.4 Here, we present evidence on the molecular background of indolent behavior of SBTs based on expression profiling of 38 ovarian serous neoplasms. In addition, the results indicate that SBTs and SCAs represent separate pathogenetic entities. Unsupervised hierarchical clustering showed that all SBTs and related MP-SBTs, generated one tight cluster completely separate from all but one (NS21) of the SCAs, indicating that SBTs and SCAs represent separate pathogenetic entities. Clustering of the MP-SBT within the group of SBTs subscribes that this subtype is a morphologic variant of SBT, which has been suggested on the basis of similarity in the molecular genetic profile of these tumors.6 Our data are in agreement with recent work by Gilks et al, which also shows complete separation of SBTs and SCAs.14 In contrast, Meinhold-Heerlein et al describe similarities between SBT and low-grade SCA and could not identify separate clusters of SBTs and SCAs.15
In the last few years, there has been a shift in focus from studying the effects of single genes to studying effects of multiple, functionally related genes. Most of the current methods for studying pathways involve looking at increased proportions of differentially expressed genes in pathways of interest and test this by either performing a Hence, it became increasingly apparent that regulatory genes involved in uncoupling the mitogenic pathway and downstream genes involved in ECM might explain how a disease with pathologic features suggesting malignancy behaves in a generally nonprogressive way. SAM analysis identified two genes involved in regulating this uncoupling namely the ERK inhibitor Dusp 4 and uPA inhibitor Serpina 5, which are both downregulated in SCAs in contrast to SBTs. Dusp 4 mRNA is expressed at moderate levels in nearly all tissues and cells and encodes a phosphatase that inactivates MAPK in vitro and MAPK-dependent gene transcription in vivo.17 In healthy developing cells, MAPK phosphatases (MKPs) such as Dusp 4 dictate the choice of differentiation or proliferation. The correlation of Dusp 4 expression and lack of ERK activation in a human cancer was first demonstrated for pancreatic tumors that, like SBTs, frequently harbor K-RAS mutations.18 Recently, Dusp 4 was presented as a candidate tumor suppressor gene in breast cancer19 and Wang et al correlated breast cancer patients that have a favorable prognosis with a gene signature that includes Dusp 4.20 The second putative tumor suppressor gene we identified is Serpina 5 (protein C inhibitor), which is a member of the superfamily of serine protease inhibitors including alpha-1-antichymotrypsin alpha-1-antitrypsin, antithrombin III, and angiotensinogen. Serpina 5 was first isolated from human plasma. It functions as an inhibitor of several plasma proteases involved in blood coagulation and is a potent inhibitor of uPA. Significant quantities of Serpina 5 mRNA have been detected in numerous tissues, including the ovary.21 Given both the role of uPA in tumor cell metastasis and its inhibition by Serpina 5, the physiological relevance of Serpina 5 in tumor cell metastasis is apparent.21 The importance of Serpina 5 as a potential tumor suppressor gene through inhibition of serine proteases is underscored by Michael et al, who associated members of the human tissue kallikreins (KLK) family, consisting of 15 homologous serine protease genes, with different types of cancer.22 In addition, recent studies have shown that KLK5 is differentially regulated in a variety of hormone dependent malignancies, including ovarian, breast, prostate, and testicular cancers. In agreement with the results presented in the present study, a strong association was found between the presence of KLK5 expression and more aggressive forms of epithelial ovarian carcinoma, increased risk for relapse and death.23 This was substantiated by a report on prostate cancer in which loss of Serpina 5 expression was found in high-grade tumors.24 Additional evidence is presented by a report on the effect of MEK1/-2 inhibition in ovarian by CI-1040.25 This study shows that in cell lines from ovarian carcinoma, inactivation of MAPK results in a marked growth inhibition in tumors with mutations in K-RAS or B-RAF in contrast to only a modest effect in tumors with wild-type K-RAS and B-RAF. These findings support that uncoupling an activated mitogenic RAS-RAF-MEK-ERK-MAP kinase pathway and ECM activation provides a major clue for the indolent SBT behavior. Our observations, indicating that mutations in the K-RAS or B-RAF oncogene are thought to be insufficient to induce a malignant phenotype, are not unique for SBTs. Studies examining K-RAS mutations as an initiating event in cellular transformation have been hampered by the observation that mutant RAS induces primary senescence, which has been attributed to chronic hyperstimulation of the MAPK cascade.26 Illustrative examples are presented in the colon where hyperplastic polyps and adenomas are known to harbor activating mutations in B-RAF or K-RAS in more than 80% of the cases, and only a small fraction progress toward malignancy.27 These findings indicate that additional genetic events are needed to develop a frankly malignant phenotype. For SBTs, it remains intriguing how disease progression on a molecular genetic level by development of invasive (auto) implants occurs. Assuming a key regulatory function of Dusp 4 and Serpina 5, it is reasonable to speculate that destructive stromal invasion may be related to loss of ERK and uPA inhibition. Although presentation of solid proof is hampered by the low prevalence of invasive implants, we included one invasive implant case in the present array study. In addition to a B-RAF mutation, we found for this case suppressed mRNA expression values for Dusp 4 and Serpina 5 when compared to the mean SBT value. These data have been confirmed by qualitative RT-PCR Also, in this progressive case MMP-9 mRNA was in the range of the SCAs, and zymography confirmed the presence of active MMP-9 (data not shown). In conclusion, we present strong evidence that ERK inhibitor Dusp 4 and uPA-inhibitor Serpina 5 act as candidate key regulators responsible for indolent SBT behavior (Fig 5). These putative tumor suppressor genes act though uncoupling activation of the RAS-RAS-MEK-ERK-MAP kinase pathway and activation of downstream genes that regulate protease activity, such as MMP-9. In agreement with these results, we found downstream MMP-9 activation in SCAs in contrast to SBTs at both mRNA and protein level. In the future, Dusp 4 and Serpina 5 may have useful clinical application in ovarian cancer diagnosis and patient management.
The authors indicated no potential conflicts of interest.
We are grateful to Claudia Gaspar, PhD, for helping on the study design, and we thank Klaas van der Ham and Victor Thijssen for excellent technical assistance.
Supported by Grant No. 920-03-050 from ZON MW, the Netherlands. Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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5. Singer G, Oldt R, III, Cohen Y, et al: Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 95:484-486, 2003 6. Sieben NL, Macropoulos P, Roemen GMJM, et al: In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours. J Pathol 202:336-340, 2004[CrossRef][Medline] 7. Aguirre Ghiso JA, Alonso DF, Farias EF, et al: Deregulation of the signaling pathways controlling urokinase production: Its relationship with the invasive phenotype. Eur J Biochem 263:295-304, 1999[Medline] 8. Affymetrix GeneChip Expression Analysis Technical Manual. Santa Clara, CA, Affymetrix Inc, 2001
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12. Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116-5121, 2001 13. Cleutjens JP, Kandala JC, Guarda E, et al: Regulation of collagen degradation in the rat myocardium after infarction. J Mol Cell Cardiol 27:1281-1292, 1995[CrossRef][Medline] 14. Gilks CB, Vanderhyden BC, Zhu S, et al: Distinction between serous tumors of low malignant potential and serous carcinomas based on global mRNA expression profiling. Gynecol Oncol 96:684-694, 2005[CrossRef][Medline] 15. Meinhold-Heerlein I, Bauerschlag D, Hilpert F, et al: Molecular and prognostic distinction between serous ovarian carcinomas of varying grade and malignant potential. Oncogene 24:1-13, 2004
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17. Misra-Press A, Rim CS, Yao H, et al: A novel mitogen-activated protein kinase phosphatase: Structure, expression and regulation. J Biol Chem 270:14587-14596, 1995 18. Yip-Schneider MT, Lin A, Marshall MS: Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase Phosphatatse-2. Biochem Biophys Res Commun 280:992-997, 2001[CrossRef][Medline] 19. Armes JE, Hammet F, de Silva M, et al: Candidate tumor suppressor genes on chromosome 8p in early-onset and high grade breast cancers. Oncogene 23:5697-5702, 2004[CrossRef][Medline] 20. Wang Y, Klijn JGM, Sieuwerts AM, et al: Gene-expression profiles to predict distant metastasis of lymph node negative primary breast cancer. Lancet 365:671-678, 2005[Medline] 21. Jackson TP, Cooper ST, Church FC: Assessment of the interaction between urokinase and reactive site mutants of protein c inhibitor. J Protein Chem 16:819-828, 1997[CrossRef][Medline]
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25. Pohl G, Ho CL, Kurman RJ, et al: Inactivation of the mitogen-activated protein kinase pathway as a potential target based therapy in ovarian serous tumors with KRAS or BRAF mutations. Cancer Res 65:1994-2000, 2005
26. Lin AW, Barradas M, Stone JC, et al: Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes Dev 12:3008-3019, 1998 27. Yang S, Farraye FA, Mack CBS, et al: BRAF and KRAS mutations in hyperplastic polyps and serrated adenomas of the colorectum: Relationship to histology and CpG island methylation status. Am J Surg Pathol 28:1452-1459, 2004[Medline] Submitted April 5, 2005; accepted June 6, 2005.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test or a Fisher's exact test. These methods do not identify pathways where many genes have altered their expression in a small way. The Global Test was designed to address this issue.
