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Gene expression in fibroadenomas of the rat mammary gland in contrast to spontaneous adenocarcinomas and normal mammary gland
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 58 (2006) 145–150
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Gene expression in fibroadenomas of the rat mammary gland in contrast to spontaneous adenocarcinomas and normal mammary gland Heike Marxfelda,b,, Frank Staedtlerc, Johannes H. Harlemana a
Preclinical Safety, Novartis Pharma AG, Basel, Switzerland Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany c Biomarker Development, Novartis Pharma AG, Basel, Switzerland b
Received 17 March 2006; accepted 14 June 2006
Abstract Fibroadenomas are considered a benign lesion in rodent carcinogenicity studies. However, the entity adenocarcinoma arising in fibroadenoma does exist and in humans there is evidence of certain forms of fibroadenomas to confer greater risk of subsequent breast cancer. In this study, we aim to elucidate the molecular features of both spontaneous fibroadenomas and adenocarcinomas. The gene expression of the two tumour types is examined and compared to mammary gland in the same developmental state and examined for similarities which might indicate common molecular pathways. In the present study no similarities were discovered. We conclude that in the tumours examined here, no progression to adenocarcinoma is likely. Further studies are needed, examining a greater number of tumours and including cases of adenocarcinoma arising in fibroadenoma. r 2006 Elsevier GmbH. All rights reserved. Keywords: Gene expression profiling; Rat; Spontaneous mammary tumours; Adenocarcinoma; Fibroadenoma
Introduction Fibroadenomas are the most common neoplasm of the human breast and the rat mammary gland (Lester, 2004; Chandra et al., 1992; McMartin et al., 1992). It has been noted in both humans and rats that carcinoma can develop in the area of a fibroadenoma (e.g., Stafyla et al., 2004; Diaz et al., 1991; Bader et al., 1993). In humans, the risk factors are not yet clearly defined; it seems possible that only a certain form of fibroadenoma (complex fibroadenoma) confers greater risk (Dupont et al., 1994). In rats, no specification for Corresponding author. Present address: Charles River Laboratories, Tranent, Edinburgh, EH33 2NE, Scotland, UK. Tel.: +44 1875 618512; fax: +44 1875 614555. E-mail address: [email protected] (H. Marxfeld).
0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.06.007
certain histologic types conferring greater risk are known. So far, fibroadenomas are counted among benign lesions in toxicology studies, while adenocarcinoma and adenocarcinoma arising in fibroadenoma are recorded as malignant. The aberrations in the gene expression of fibroadenomas are not well characterized in humans or rats. Recently, gene expression profiling has been successfully applied to distinguish benign from malignant tumours (e.g., thyroid tumours: Rosen et al., 2005, adrenocortical neoplasms: Velazquez-Fernandez et al., 2005). Thus, we hypothesized that gene expression profiling could be used to elucidate the relationship of adenocarcinoma and fibroadenoma of the rat mammary gland on a molecular level and whether the gene expression profile of the fibroadenomas contained any of the genes characterizing adenocarcinomas.
ARTICLE IN PRESS 146
H. Marxfeld et al. / Experimental and Toxicologic Pathology 58 (2006) 145–150
Materials and methods
Microarray experiment
Female Sprague–Dawley rats were obtained from the following suppliers:
Total RNA was obtained by acid guanidinium isothiocyanate–phenol–chloroform extraction (Trizol; Invitrogen Life Technologies, San Diego, CA, USA) and purified on an affinity resin (RNeasy; Qiagen, Hilden, Germany) according to manufacturer’s instructions. DNA microarray experiments were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Inc. 2002). Rat-specific RAE230A gene expression probe arrays (Affymetrix, Inc., Santa Clara, CA, USA) were used containing 15,923 probe sets interrogating primarily annotated genes. Per tissue and per animal, one chip was used. The resulting image files (.dat files) were processed using the Microarray Analysis Suite 5 (MAS5) software (Affymetrix, Inc.). Tab delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).
Sixty retired breeding animals from Hsd, Harlan (Netherlands), at the age of 8 months (spontaneous fibroadenoma and adenocarcinoma) were used. Three 12–14-week-old post lactating Sprague–Dawley rats (normal mammary gland) were obtained from Taconic (Borup, Denmark). Animals were housed in MUT-2881, Muttenz, Switzerland. Room temperature was 20–24 1C with a humidity of 40–70%. For 12 h a day, fluorescent light was turned on in the animal quarters. Animals were kept in groups of three in a type IV Macrolon cage on sterilized softwood particle bedding (Rettenmaier & So¨hne, Ellwangen-Holzmu¨hle, Germany) under optimal hygenic conditions. KLIBA-NAFAG no. 3893.0.25 pelleted standard diet was fed ad libitum (Provimi Kliba AG, Kaiseraugst, Switzerland). Tap water from the local supply was available ad libitum from polyethylene bottles. Animals were identified by electronic transducers (DATAMARS SA RF-identification technology, Bedano-Lugano, Switzerland).
Permission for the animal experiment was obtained from Kantonales Veterina¨ramt, Basel (Permission no.: Tierversuchsbewilligung 5087). Animals were palpated weekly for occurence of mammary tumours and were daily observed for signs of illness. If tumours reached a size of 1 cm3 or the animal seemed moribund, it was euthanized. Tumours were cut into two halves, one half was immediately snap-frozen in liquid nitrogen and stored at 80 1C until RNA extraction. The other half was fixed in phosphate-buffered 4% formaldehyde. After embedding in paraffin, sections were cut at nominally 3 mm and stained with haematoxylin and eosin for histological evaluation. Three samples from each normal mammary gland, fibroadenoma and adenocarcinoma were chosen for microarray analysis.
Table 1.
Data analysis To determine genes which were diffentially expressed between the tumour groups and their control group, a one-way analysis of variance (ANOVA) was applied to genes that had a present call in all of the samples. Genes with a p-valueo0.05 were considered statistically significant. Group average 3-fold changes were calculated by using the average of the tumour groups compared with the control group. A filter on raw data (raw data 4100) was applied to the genes 3-fold upregulated, and this filter was omitted in the 3-fold downregulated genes (Silicongenetics, Redwood City, CA 94063, USA). Immunohistochemistry was performed with the following antibodies. Buffer: Ci6: 10 mM citrate buffer pH 6.0 (Milan analytica AG, La Roche, Switzerland). Borat: 5 ml stock solution added to 250 ml aqua dest. Stock solution: 31 g boric acid (Sigma B 0396) in 500 ml aqua dest, calibrated to pH 7 by adding NaOH. 5 ml of stock solution.
Primary antibodies, source of antibodies, pre-treatments, dilutions and secondary antibody
Antibody
Source
Pre-treatment
Dilution
Secondary antibody
Pan-cytokeratin Vimentin Actin Thy-1
Sigma C-1801 DAKO DAKO Research diagnostics
1:10000 1:100 1:800 1:20
Goat Goat Goat Goat
NSE
DAKO
Borat 20 Ci6 MW 100 98 1C Ci6 MW 100 98 1C Ci6 MW 450 98 1C ampli EDTA MW 100 98 1C
1:50
Goat anti mouse
anti anti anti anti
mouse mouse mouse mouse
Ci6: citrate buffer pH 6, Borat: borate buffer, EDTA: ethylenediaminetetraacetic acid TEC: Tris–EDTA–citrate buffer, MW: microwave
ARTICLE IN PRESS H. Marxfeld et al. / Experimental and Toxicologic Pathology 58 (2006) 145–150
EDTA: 25 ml stock solution in 250 ml aqua dest, adjusted to pH 8. Stock solution:14.6 g EDTA (Sigma) in 500 ml aqua dest. MW: microwave with temperature sensor (H2500 microwave processor, Energy Beam Sciences, Inc.). Borat 2: slides cooked in a pressure cooker for 2 min, then left to cool for 20 min in buffer. The secondary antibody goat anti mouse (JacksonImmunoResearch, West Grove, PA, USA) was used in a dilution of 1: 1000 (diluted in Ventana antibody diluent (Hauptman–Woodward Medical Research Inst., Inc., Buffalo, NY, USA). Slides were dewaxed in graded alcohol, pre-treated according to the information given in Table 1 and stained with an automated staining system (Ventana Medical Systems, 6 Buffalo, NY, USA).
Table 2.
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Results In fibroadenomas, genes related to fibroblasts were found to be overexpressed compared to both normal post-lactating mammary gland and adenocarcinomas. These could be divided into genes directly involved in collagen synthesis, fibrosis and growth factors/ transcription factors (Table 2).
Collagen synthesis Extracellular steps in collagen synthesis require the presence of procollagen C-proteinase and procollagen Nproteinase as well as lysyl oxidase. Procollagen C proteinase, its enhancer protein Pcolce and lysyl oxidase were up-regulated. The collagens found are col1a1,
Genes related to fibroblasts, genes also reported on myoepithelial cells or myofibroblasts marked with*
Affymetrix identifier
Short name
Gene name
Post-lactating control mean (range)
Fibroadenoma mean (range)
Extracellular matrix Collagen synthesis 1370864_at 1387854_at 1369955_at 1368172_a_at 1368171_at
Col1a1 Col1a2 Col5a1 Lox Lox
Collagen alpha1
0.833 0.822 0.892 0.568 0.354
3.269 3.427 4.915 2.934 1.793
1367749_at 1367563_at 1386912_at
Lum Sparc* Pcolce
1368237_at
Tnmd
Fibroblast-related genes 1369422_at 1369651_at 1368860_at 1369415_at
Fap* Thy1* Tdag Bhlhb2
1387306_a_at 1370341_at
Egr2 NSE*
Extracellular matrix homeostasis 1367571_a_at Igf2 1387180_at 1369947_at
Il1r2 Ctsk
Growth factors 1367571_a_at
Igf2
1387306_a_at 1386884_at
Egr2 Prss11*
Collagen, type V, alpha 1 Lysyl oxidase (an H-rev gene with its expression downregulated in HRAStransformed rat 208F fibroblasts) Lumican Osteonectin Procollagen C-proteinase enhancer protein Myodulin Fibroblast activation protein Thymus cell surface antigen T-cell death-associated gene Basic helix-loop-helix domain containing, class B2 Early growth response 2 Neuron-specific enolase Insulin-like growth factor II (somatomedin A) Interleukin 1 receptor, type II Cathepsin K
Insulin-like growth factor II (somatomedin A) Early growth response 2 Protease, serine, 11
(0.431–1.343) (0.475–1.169) (0.411–1.788) (0.393–0.76) (0.226–0.452)
(3.081–3.628) (3–4.033) (3.733–6.194) (1.522–5.015) (1.245–2.187)
0.802 (0.559–1.427) 0.673 (0.387–1.071) 0.814 (0.471–1.627)
2.726 (2.257–3.268) 2.456 (2.164–2.758) 4.613 (4.039–5.645)
1.152 (1.034–1.383)
15.75 (9.527–22.69)
1.447 (0.853–2.206) 0.825 (0.496–1.304) 0.5 (0.344–0.68) 0.271 (0.219–0.317)
7.648 5.579 2.331 1.056
0.977 (0.778–1.198) 0.8 (0.7–0.9)
6.4 (5.848–6.816) 2.98 (1.89–4.01)
1.968 (1.513–2.368)
8.044 (5.841–12.58)
0.557 (0.346–1) 1.062 (0.607–1.645)
2.869 (2.571–3.237) 5.522 (4.616–7.61)
1.968 (1.513–2.368)
8.044 (5.841–12.58)
0.977 (0.778–1.198) 0.867 (0.564–1.205)
6.4 (5.848–6.816) 2.702 (2.193–3.361)
(6.041–8.772) (3.554–7.338) (1.639–3.496) (0.912–1.152)
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col1a2, col5a1 which occur in skin and form fibrils with each other (Alberts et al., 2002). Lumican is a major component of dermal connective tissues and necessary for the development of orderly collagen fibrils (Chakravarti et al., 1998). Osteonectin is a downstream target of TGFbeta1 and can induce the expression of collagen1a2 (Zhou et al., 2005). TNMD has been found in dense connective tissue of tendons and is involved in the maturation of collagen fibrils (Docheva et al., 2005). In adenocarcinoma, only coll2a1 was found.
Fibroblasts Expression of fibroblast activation protein is highly specific for tumour fibroblasts in reactive stroma of cancer (Garin-Chesa et al., 1990). Thymus cell surface antigen 1 expression on fibroblasts promotes focal adhesion and actin stress fibre formation and inhibits migration. These fibroblasts maintain continuity with the surrounding layer and spread rather than migrate (Barker et al., 2004a, b). Tdag51 is implicated in the senescence of mammary fibroblasts (Hardy et al., 2005). DEC1 (BHLHB2)/Stra13/Sharp2, a basic helix-loophelix (bHLH) transcription factor, has been suggested to be involved in the control of proliferation and/or differentiation of several cells including fibroblasts. (OMIM). EGR-2 is regulated by fibroblast mitogens (Joseph et al., 1988a, b). IGF-2 has been described as overexpressed in the stroma of human fibroadenomas (Sawyer et al., 2003). PRSS11 is a serine protease, which can cleave insulin growth factor binding proteins, and thus causes the release of IGFs (Zumbrunn and Trueb, 1996). Another set of genes which was overexpressed in fibroadenomas were genes related to a putative myoepithelial or myofibroblast differentiation of tumour cells. Neuron-specific enolase has been described as a marker for neuroendocrine differentiation in breast cancer. The occurrence of neurocrine markers in fibroadenomas is under debate, in normal breast, only myoepithelial cells are positive for NSE (Nesland et al., 1986). Osteonectin has been described as a marker for myoepithelial cells as well as on fibroblasts (Jones et al., 2004). THY-1 has been described on myoepithelial-like cells in the rat mammary gland (Barraclough et al., 1987). Rudland and colleagues described THY-1 on
Table 3.
both myoepithelial cells and fibroblasts (Rudland et al., 1982). Fibroblast activation protein and PRSS11 have been found in myofibroblasts of aggressive fibromatosis (Skubitz and Skubitz, 2004). Only two genes related to epithelial cells have been found: GAS6 and CDRAP. CDRAP and the transcription factor BHLHB2 were the only genes which were expressed in both adenocarcinoma and fibroadenoma where it was expressed in lower levels than in adenocarcinomas (Table 3). GAS6 has anti-apoptotic functions and signals via PI3K and Src as well as inducing beta-catenin stabilization, T-cell factor activation and growth in C57 mammary cells (Goruppi et al., 2001). A detailed report about the gene expression in spontaneous adenocarcinoma will be published elsewhere (Marxfeld et al., 2006). Immunohistochemistry was performed to locate the signal of NSE and THY-1 as both antigens have been described on fibroblasts and myoepithelial cells. Vimentin and pan-cytokeratin staining was performed to determine the nature of the stromal tumour cells. Tumour cells stained positive with vimentin and were negative for pan-cytokeratin, consistent with fibroblasts (data not shown). These cells also showed positive staining with both NSE (Fig. 1) and THY-1 (Fig. 2). No staining was observed in stromal cells of adenocarcinoma or in densely fibrotic areas of fibroadenomas.
Fig. 1. Immunohistochemical staining of NSE antigen (Dako) in cytoplasm of fibroblasts in fibroadenoma (DAB method) surrounding glandular elements.
Genes found in both fibroadenoma and adenocarcinoma
Genes in both adenocarcinoma and fibroadenoma 1369320_at 1369415_at
Cdrap Bhlhb2
Cartilage-derived retinoic acid sensitive protein Basic helix-loop-helix domain containing, class B2
6.346 (4.834–9.108) 0.271 (0.219–0.317)
19.32 (12.3–28.29) 1.056 (0.912–1.152)
ARTICLE IN PRESS H. Marxfeld et al. / Experimental and Toxicologic Pathology 58 (2006) 145–150
Fig. 2. Immunohistochemical staining of Thy-1 antigen (Research diagnostics) in cytoplasm of fibroblasts in fibroadenoma (DAB method) surrounding glandular elements.
Discussion In our study, we detected only CDRAP in both fibroadenoma and adenocarcinoma. This gene is detected in breast cancer and transient endogenous expression of CDRAP in the mammary bud is found (Xie et al., 2000). The rest of the gene expression profile differed as most genes in fibroadenoma seemed to be expressed by fibroblasts. This led to the question if the stromal component might be the neoplastic part of the tumour on the one hand and whether it is possible that this stroma still could provide a permissible milieu for the adjacent epithelium to form cancerous lesions on the other. It has become increasingly noticed that only an interplay of stroma and epithelium lead to neoplastic conversion (Wiseman and Werb, 2002; Barcellos-Hoff and Ravani, 2000). A factor which could contribute to the growth stimulation of epithelial cells we detected is IGF-2. IGF-2 was described in the stroma of breast cancer and was suspected to have a growth promoting effect on adjacent epithelial tumour cells (Cullen et al., 1991). But, as already menitioned, IGF-2 is also expressed in the stroma of human fibroadenomas without known association with epithelial conversion (Sawyer et al., 2003). Furthermore, lumican, FAP and osteonectin have been reported in breast cancer stroma. The role of these genes should be investigated in adenocarcinomas, fibroadenomas and adenocarcinomas arising in fibroadenomas for their contribution to tumourigenesis before fibroadenoma can be unequivocally be termed a benign tumour without risk for cancer development. It has, however, to be noted that in the case of the adenocarcinomas and fibroadenomas we investigated, the stroma of the two tumour types seemed to be
149
differently composed: in fibroadenoma, we found dermal collagens and an activated type of fibroblast, which was able to express NSE and THY-1, while the adenocarcinomas expressed RNA for a collagen found in cartilage and vitreous body and showed in their stromal cells no staining for THY-1 or NSE. It is possible that the stroma represents the neoplastic part of the tumour. In human fibroadenomas, it has been reported that the stromal part of the tumour was clonal in origin, while the epithelial part was polyclonal. It has also been proposed that fibroadenoma could progress to both phyllodes tumour and adenocarcinoma (Kuijper et al., 2002a, b). Phyllodes tumour is a biphasic tumour of the human breast, which contains a malignant stromal component. This entity, however, is not known in the rat. In the course of time most rat fibroadenomas seem rather to develop into a fibrous mass with no or atrophic epithelium. In our analysis, we found TDAG51 a gene participating in senescence of fibroblasts, which might participate in this process. In human cases, this has also been associated with cessation of hormonal stimulation after menopause. Further studies are needed to characterize the stroma of fibroadenoma in a time course during tumour development. In this context, the roles of NSE and THY-1 could be investigated as well, as staining with both antibodies was lost in areas of fibroadenomas containing dense fibrotic tissue. Concluding, there is up to now little evidence that fibroadenoma plays a role as precursor lesion for adenocarcinoma, although further studies should be initiated to investigate the rarely occuring adenocarcinoma arising in fibroadenoma.
References Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. Garland Science 2002;1:1096–8. Bader R, Gembard C, Kaufmann W, et al. 5. Integumentary system In: Mohr U, editor. International classification of rodent tumours Part 1: the rat. International Agency for Research on Cancer Scientific Publications No. 122; 1993. p. 34–5. Barcellos-Hoff MH, Ravani SA. Irradiated mammary gland stroma promotes the expression of tumourigenic potential by unirradiated epithelial cells. Cancer Res 2000;60: 1254–60. Barker TH, Grenett HE, Macewen MW, et al. Thy-1 regulates fibroblast focal adhesions, cytoskeletal organization and migration through modulation of p190 RhoGAP and Rho GTPase activity. Exp Cell Res 2004a;295:488–96. Barker TH, Pallero MA, Macewen MW, et al. Thrombospondin-1-induced focal adhesion disassembly in fibroblasts requires Thy-1 surface expression, lipid raft integrity, and Src activation. J Biol Chem 2004b;279:23510–6 Epub 2004 Mar 18.
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Barraclough R, Kimbell R, Rudland PS. Differential control of mRNA levels for Thy-1 antigen and laminin in rat mammary epithelial and myoepithelial-like cells. J Cell Physiol 1987;131:393–401. Chakravarti S, Magnuson T, Lass JH, et al. Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican. J Cell Biol 1998;141:1277–86. Chandra M, Riley MG, Johnson DE. Spontaneous neoplasms in aged Sprague–Dawley rats. Arch Toxicol 1992;66: 496–502. Cullen KJ, Smith HS, Hill S, et al. Growth factor messenger RNA expression by human breast fibroblasts from benign and malignant lesions. Cancer Res 1991;51:4978–85. Diaz NM, Palmer JO, Mcdivitt RW. Carcinoma arising within fibroadenomas of the breast. A clinicopathologic study of 105 patients. Am J Clin Pathol 1991;95:614–22. Docheva D, Hunziker EB, Fassler R, et al. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol Cell Biol 2005;25:699–705. Dupont WD, Page DL, Parl FF, et al. Long-term risk of breast cancer in women with fibroadenoma. N Engl J Med 1994;331:10–5. Garin-Chesa P, Old LJ, Rettig WJ. Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc Natl Acad Sci USA 1990;87:7235–9. Goruppi S, Chiaruttini C, Ruaro ME, et al. Gas6 induces growth, beta-catenin stabilization, and T-cell factor transcriptional activation in contact-inhibited C57 mammary cells. Mol Cell Biol 2001;21:902–15. Hardy K, Mansfield L, Mackay A, et al. Transcriptional networks and cellular senescence in human mammary fibroblasts. Mol Biol Cell 2005;16:943–53 [Epub Dec 1 2004]. Jones C, Mackay A, Grigoriadis A, et al. Expression profiling of purified normal human luminal and myoepithelial breast cells: identification of novel prognostic markers for breast cancer. Cancer Res 2004;64:3037–45. Joseph LJ, Le Beau MM, Jamieson GA JR, et al. Molecular cloning, sequencing, and mapping of EGR2, a human early growth response gene encoding a protein with ‘‘zincbinding finger’’ structure. Proc Natl Acad Sci USA 1988a;85:7164–8 [Erratum in: Proc Natl Acad Sci USA 1989;86(2):515]. Joseph LJ, Chang LC, Stamenkovich D, et al. Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. An abundant transcript induced by transformation of fibroblasts. J Clin Invest 1988b;81:1621–9. Kuijper A, Preisler-Adams SS, Rahusen FD, et al. Multiple fibroadenomas harbouring carcinoma in situ in a woman with a family history of breast/ovarian cancer. J Clin Pathol 2002a;55:795–7.
Kuijper A, Buerger H, Simon R, et al. Analysis of the progression of fibroepithelial tumours of the breast by PCR-based clonality assay. J Pathol 2002b;197:575–81. Lester SC. Chapter 23: The breast. In: Kumar V, Abbas AK, Fausto N, editors. Robbins and Cotran pathologic basis of disease. 7th ed. Elsevier Saunders; 2004. p. 1150. Marxfeld H, Grenet O, Bringel J, Staedtler F, Harleman JH. Differentiation of spontaneous and induced mammary adenocarcinomas of the rat by gene expression profiling. Exp Toxic Pathol 2006;58:151–61. McMartin DN, Sahota PS, Gunson DE, et al. Neoplasms and related proliferative lesions in control Sprague–Dawley rats from carcinogenicity studies. Historical data and diagnostic considerations. Toxicol Pathol 1992;20:212–25. Nesland JM, Holm R, Johannessen JV. A study of different markers for neuroendocrine differentiation in breast carcinomas. Pathol Res Pract 1986;181:524–30. Rosen J, He M, Umbricht C, Alexander HR, Dackiw AP, Zeiger MA, et al. A six-gene model for differentiating benign from malignant thyroid tumors on the basis of gene expression. Surgery 2005;138:1050–6. Rudland PS, Warburton MJ, Monaghan P, et al. Thy-1 antigen on normal and neoplastic rat mammary tissues: changes in location and amount of antigen during differentiation of cultured stem cells. J Natl Cancer Inst 1982;68:799–811. Sawyer EJ, Hanby AM, Poulsom R, et al. Beta-catenin abnormalities and associated insulin-like growth factor overexpression are important in phyllodes tumours and fibroadenomas of the breast. J Pathol 2003;200:627–32. Skubitz KM, Skubitz AP. Gene expression in aggressive fibromatosis. J Lab Clin Med 2004;143:89–98. Stafyla V, Kotsifopoulos N, Grigoriades K, et al. Lobular carcinoma in situ of the breast within a fibroadenoma. A case report. Gynecol Oncol 2004;94:572–4. Velazquez-Fernandez D, Laurell C, Geli J, Hoog A, Odeberg J, Kjellman M, et al. Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy. Surgery 2005;138:1087–94. Wiseman BS, Werb Z. Stromal effects on mammary gland development and breast cancer. Science 2002;296: 1046–9. Xie WF, Zhang X, Sandell LJ. The 2.2-kb promoter of cartilage-derived retinoic acid-sensitive protein controls gene expression in cartilage and embryonic mammary buds of transgenic mice. Matrix Biol 2000;19:501–9. Zhou X, Tan FK, Guo X, et al. Small interfering RNA inhibition of SPARC attenuates the profibrotic effect of transforming growth factor beta1 in cultured normal human fibroblasts. Arthritis Rheum 2005;52:257–61. Zumbrunn J, Trueb B. Primary structure of a putative serine protease specific for IGF-binding proteins. FEBS Lett 1996;398:187–92.
Experimental and Toxicologic Pathology 58 (2006) 145–150
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Gene expression in fibroadenomas of the rat mammary gland in contrast to spontaneous adenocarcinomas and normal mammary gland Heike Marxfelda,b,, Frank Staedtlerc, Johannes H. Harlemana a
Preclinical Safety, Novartis Pharma AG, Basel, Switzerland Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany c Biomarker Development, Novartis Pharma AG, Basel, Switzerland b
Received 17 March 2006; accepted 14 June 2006
Abstract Fibroadenomas are considered a benign lesion in rodent carcinogenicity studies. However, the entity adenocarcinoma arising in fibroadenoma does exist and in humans there is evidence of certain forms of fibroadenomas to confer greater risk of subsequent breast cancer. In this study, we aim to elucidate the molecular features of both spontaneous fibroadenomas and adenocarcinomas. The gene expression of the two tumour types is examined and compared to mammary gland in the same developmental state and examined for similarities which might indicate common molecular pathways. In the present study no similarities were discovered. We conclude that in the tumours examined here, no progression to adenocarcinoma is likely. Further studies are needed, examining a greater number of tumours and including cases of adenocarcinoma arising in fibroadenoma. r 2006 Elsevier GmbH. All rights reserved. Keywords: Gene expression profiling; Rat; Spontaneous mammary tumours; Adenocarcinoma; Fibroadenoma
Introduction Fibroadenomas are the most common neoplasm of the human breast and the rat mammary gland (Lester, 2004; Chandra et al., 1992; McMartin et al., 1992). It has been noted in both humans and rats that carcinoma can develop in the area of a fibroadenoma (e.g., Stafyla et al., 2004; Diaz et al., 1991; Bader et al., 1993). In humans, the risk factors are not yet clearly defined; it seems possible that only a certain form of fibroadenoma (complex fibroadenoma) confers greater risk (Dupont et al., 1994). In rats, no specification for Corresponding author. Present address: Charles River Laboratories, Tranent, Edinburgh, EH33 2NE, Scotland, UK. Tel.: +44 1875 618512; fax: +44 1875 614555. E-mail address: [email protected] (H. Marxfeld).
0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.06.007
certain histologic types conferring greater risk are known. So far, fibroadenomas are counted among benign lesions in toxicology studies, while adenocarcinoma and adenocarcinoma arising in fibroadenoma are recorded as malignant. The aberrations in the gene expression of fibroadenomas are not well characterized in humans or rats. Recently, gene expression profiling has been successfully applied to distinguish benign from malignant tumours (e.g., thyroid tumours: Rosen et al., 2005, adrenocortical neoplasms: Velazquez-Fernandez et al., 2005). Thus, we hypothesized that gene expression profiling could be used to elucidate the relationship of adenocarcinoma and fibroadenoma of the rat mammary gland on a molecular level and whether the gene expression profile of the fibroadenomas contained any of the genes characterizing adenocarcinomas.
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Materials and methods
Microarray experiment
Female Sprague–Dawley rats were obtained from the following suppliers:
Total RNA was obtained by acid guanidinium isothiocyanate–phenol–chloroform extraction (Trizol; Invitrogen Life Technologies, San Diego, CA, USA) and purified on an affinity resin (RNeasy; Qiagen, Hilden, Germany) according to manufacturer’s instructions. DNA microarray experiments were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Inc. 2002). Rat-specific RAE230A gene expression probe arrays (Affymetrix, Inc., Santa Clara, CA, USA) were used containing 15,923 probe sets interrogating primarily annotated genes. Per tissue and per animal, one chip was used. The resulting image files (.dat files) were processed using the Microarray Analysis Suite 5 (MAS5) software (Affymetrix, Inc.). Tab delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).
Sixty retired breeding animals from Hsd, Harlan (Netherlands), at the age of 8 months (spontaneous fibroadenoma and adenocarcinoma) were used. Three 12–14-week-old post lactating Sprague–Dawley rats (normal mammary gland) were obtained from Taconic (Borup, Denmark). Animals were housed in MUT-2881, Muttenz, Switzerland. Room temperature was 20–24 1C with a humidity of 40–70%. For 12 h a day, fluorescent light was turned on in the animal quarters. Animals were kept in groups of three in a type IV Macrolon cage on sterilized softwood particle bedding (Rettenmaier & So¨hne, Ellwangen-Holzmu¨hle, Germany) under optimal hygenic conditions. KLIBA-NAFAG no. 3893.0.25 pelleted standard diet was fed ad libitum (Provimi Kliba AG, Kaiseraugst, Switzerland). Tap water from the local supply was available ad libitum from polyethylene bottles. Animals were identified by electronic transducers (DATAMARS SA RF-identification technology, Bedano-Lugano, Switzerland).
Permission for the animal experiment was obtained from Kantonales Veterina¨ramt, Basel (Permission no.: Tierversuchsbewilligung 5087). Animals were palpated weekly for occurence of mammary tumours and were daily observed for signs of illness. If tumours reached a size of 1 cm3 or the animal seemed moribund, it was euthanized. Tumours were cut into two halves, one half was immediately snap-frozen in liquid nitrogen and stored at 80 1C until RNA extraction. The other half was fixed in phosphate-buffered 4% formaldehyde. After embedding in paraffin, sections were cut at nominally 3 mm and stained with haematoxylin and eosin for histological evaluation. Three samples from each normal mammary gland, fibroadenoma and adenocarcinoma were chosen for microarray analysis.
Table 1.
Data analysis To determine genes which were diffentially expressed between the tumour groups and their control group, a one-way analysis of variance (ANOVA) was applied to genes that had a present call in all of the samples. Genes with a p-valueo0.05 were considered statistically significant. Group average 3-fold changes were calculated by using the average of the tumour groups compared with the control group. A filter on raw data (raw data 4100) was applied to the genes 3-fold upregulated, and this filter was omitted in the 3-fold downregulated genes (Silicongenetics, Redwood City, CA 94063, USA). Immunohistochemistry was performed with the following antibodies. Buffer: Ci6: 10 mM citrate buffer pH 6.0 (Milan analytica AG, La Roche, Switzerland). Borat: 5 ml stock solution added to 250 ml aqua dest. Stock solution: 31 g boric acid (Sigma B 0396) in 500 ml aqua dest, calibrated to pH 7 by adding NaOH. 5 ml of stock solution.
Primary antibodies, source of antibodies, pre-treatments, dilutions and secondary antibody
Antibody
Source
Pre-treatment
Dilution
Secondary antibody
Pan-cytokeratin Vimentin Actin Thy-1
Sigma C-1801 DAKO DAKO Research diagnostics
1:10000 1:100 1:800 1:20
Goat Goat Goat Goat
NSE
DAKO
Borat 20 Ci6 MW 100 98 1C Ci6 MW 100 98 1C Ci6 MW 450 98 1C ampli EDTA MW 100 98 1C
1:50
Goat anti mouse
anti anti anti anti
mouse mouse mouse mouse
Ci6: citrate buffer pH 6, Borat: borate buffer, EDTA: ethylenediaminetetraacetic acid TEC: Tris–EDTA–citrate buffer, MW: microwave
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EDTA: 25 ml stock solution in 250 ml aqua dest, adjusted to pH 8. Stock solution:14.6 g EDTA (Sigma) in 500 ml aqua dest. MW: microwave with temperature sensor (H2500 microwave processor, Energy Beam Sciences, Inc.). Borat 2: slides cooked in a pressure cooker for 2 min, then left to cool for 20 min in buffer. The secondary antibody goat anti mouse (JacksonImmunoResearch, West Grove, PA, USA) was used in a dilution of 1: 1000 (diluted in Ventana antibody diluent (Hauptman–Woodward Medical Research Inst., Inc., Buffalo, NY, USA). Slides were dewaxed in graded alcohol, pre-treated according to the information given in Table 1 and stained with an automated staining system (Ventana Medical Systems, 6 Buffalo, NY, USA).
Table 2.
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Results In fibroadenomas, genes related to fibroblasts were found to be overexpressed compared to both normal post-lactating mammary gland and adenocarcinomas. These could be divided into genes directly involved in collagen synthesis, fibrosis and growth factors/ transcription factors (Table 2).
Collagen synthesis Extracellular steps in collagen synthesis require the presence of procollagen C-proteinase and procollagen Nproteinase as well as lysyl oxidase. Procollagen C proteinase, its enhancer protein Pcolce and lysyl oxidase were up-regulated. The collagens found are col1a1,
Genes related to fibroblasts, genes also reported on myoepithelial cells or myofibroblasts marked with*
Affymetrix identifier
Short name
Gene name
Post-lactating control mean (range)
Fibroadenoma mean (range)
Extracellular matrix Collagen synthesis 1370864_at 1387854_at 1369955_at 1368172_a_at 1368171_at
Col1a1 Col1a2 Col5a1 Lox Lox
Collagen alpha1
0.833 0.822 0.892 0.568 0.354
3.269 3.427 4.915 2.934 1.793
1367749_at 1367563_at 1386912_at
Lum Sparc* Pcolce
1368237_at
Tnmd
Fibroblast-related genes 1369422_at 1369651_at 1368860_at 1369415_at
Fap* Thy1* Tdag Bhlhb2
1387306_a_at 1370341_at
Egr2 NSE*
Extracellular matrix homeostasis 1367571_a_at Igf2 1387180_at 1369947_at
Il1r2 Ctsk
Growth factors 1367571_a_at
Igf2
1387306_a_at 1386884_at
Egr2 Prss11*
Collagen, type V, alpha 1 Lysyl oxidase (an H-rev gene with its expression downregulated in HRAStransformed rat 208F fibroblasts) Lumican Osteonectin Procollagen C-proteinase enhancer protein Myodulin Fibroblast activation protein Thymus cell surface antigen T-cell death-associated gene Basic helix-loop-helix domain containing, class B2 Early growth response 2 Neuron-specific enolase Insulin-like growth factor II (somatomedin A) Interleukin 1 receptor, type II Cathepsin K
Insulin-like growth factor II (somatomedin A) Early growth response 2 Protease, serine, 11
(0.431–1.343) (0.475–1.169) (0.411–1.788) (0.393–0.76) (0.226–0.452)
(3.081–3.628) (3–4.033) (3.733–6.194) (1.522–5.015) (1.245–2.187)
0.802 (0.559–1.427) 0.673 (0.387–1.071) 0.814 (0.471–1.627)
2.726 (2.257–3.268) 2.456 (2.164–2.758) 4.613 (4.039–5.645)
1.152 (1.034–1.383)
15.75 (9.527–22.69)
1.447 (0.853–2.206) 0.825 (0.496–1.304) 0.5 (0.344–0.68) 0.271 (0.219–0.317)
7.648 5.579 2.331 1.056
0.977 (0.778–1.198) 0.8 (0.7–0.9)
6.4 (5.848–6.816) 2.98 (1.89–4.01)
1.968 (1.513–2.368)
8.044 (5.841–12.58)
0.557 (0.346–1) 1.062 (0.607–1.645)
2.869 (2.571–3.237) 5.522 (4.616–7.61)
1.968 (1.513–2.368)
8.044 (5.841–12.58)
0.977 (0.778–1.198) 0.867 (0.564–1.205)
6.4 (5.848–6.816) 2.702 (2.193–3.361)
(6.041–8.772) (3.554–7.338) (1.639–3.496) (0.912–1.152)
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col1a2, col5a1 which occur in skin and form fibrils with each other (Alberts et al., 2002). Lumican is a major component of dermal connective tissues and necessary for the development of orderly collagen fibrils (Chakravarti et al., 1998). Osteonectin is a downstream target of TGFbeta1 and can induce the expression of collagen1a2 (Zhou et al., 2005). TNMD has been found in dense connective tissue of tendons and is involved in the maturation of collagen fibrils (Docheva et al., 2005). In adenocarcinoma, only coll2a1 was found.
Fibroblasts Expression of fibroblast activation protein is highly specific for tumour fibroblasts in reactive stroma of cancer (Garin-Chesa et al., 1990). Thymus cell surface antigen 1 expression on fibroblasts promotes focal adhesion and actin stress fibre formation and inhibits migration. These fibroblasts maintain continuity with the surrounding layer and spread rather than migrate (Barker et al., 2004a, b). Tdag51 is implicated in the senescence of mammary fibroblasts (Hardy et al., 2005). DEC1 (BHLHB2)/Stra13/Sharp2, a basic helix-loophelix (bHLH) transcription factor, has been suggested to be involved in the control of proliferation and/or differentiation of several cells including fibroblasts. (OMIM). EGR-2 is regulated by fibroblast mitogens (Joseph et al., 1988a, b). IGF-2 has been described as overexpressed in the stroma of human fibroadenomas (Sawyer et al., 2003). PRSS11 is a serine protease, which can cleave insulin growth factor binding proteins, and thus causes the release of IGFs (Zumbrunn and Trueb, 1996). Another set of genes which was overexpressed in fibroadenomas were genes related to a putative myoepithelial or myofibroblast differentiation of tumour cells. Neuron-specific enolase has been described as a marker for neuroendocrine differentiation in breast cancer. The occurrence of neurocrine markers in fibroadenomas is under debate, in normal breast, only myoepithelial cells are positive for NSE (Nesland et al., 1986). Osteonectin has been described as a marker for myoepithelial cells as well as on fibroblasts (Jones et al., 2004). THY-1 has been described on myoepithelial-like cells in the rat mammary gland (Barraclough et al., 1987). Rudland and colleagues described THY-1 on
Table 3.
both myoepithelial cells and fibroblasts (Rudland et al., 1982). Fibroblast activation protein and PRSS11 have been found in myofibroblasts of aggressive fibromatosis (Skubitz and Skubitz, 2004). Only two genes related to epithelial cells have been found: GAS6 and CDRAP. CDRAP and the transcription factor BHLHB2 were the only genes which were expressed in both adenocarcinoma and fibroadenoma where it was expressed in lower levels than in adenocarcinomas (Table 3). GAS6 has anti-apoptotic functions and signals via PI3K and Src as well as inducing beta-catenin stabilization, T-cell factor activation and growth in C57 mammary cells (Goruppi et al., 2001). A detailed report about the gene expression in spontaneous adenocarcinoma will be published elsewhere (Marxfeld et al., 2006). Immunohistochemistry was performed to locate the signal of NSE and THY-1 as both antigens have been described on fibroblasts and myoepithelial cells. Vimentin and pan-cytokeratin staining was performed to determine the nature of the stromal tumour cells. Tumour cells stained positive with vimentin and were negative for pan-cytokeratin, consistent with fibroblasts (data not shown). These cells also showed positive staining with both NSE (Fig. 1) and THY-1 (Fig. 2). No staining was observed in stromal cells of adenocarcinoma or in densely fibrotic areas of fibroadenomas.
Fig. 1. Immunohistochemical staining of NSE antigen (Dako) in cytoplasm of fibroblasts in fibroadenoma (DAB method) surrounding glandular elements.
Genes found in both fibroadenoma and adenocarcinoma
Genes in both adenocarcinoma and fibroadenoma 1369320_at 1369415_at
Cdrap Bhlhb2
Cartilage-derived retinoic acid sensitive protein Basic helix-loop-helix domain containing, class B2
6.346 (4.834–9.108) 0.271 (0.219–0.317)
19.32 (12.3–28.29) 1.056 (0.912–1.152)
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Fig. 2. Immunohistochemical staining of Thy-1 antigen (Research diagnostics) in cytoplasm of fibroblasts in fibroadenoma (DAB method) surrounding glandular elements.
Discussion In our study, we detected only CDRAP in both fibroadenoma and adenocarcinoma. This gene is detected in breast cancer and transient endogenous expression of CDRAP in the mammary bud is found (Xie et al., 2000). The rest of the gene expression profile differed as most genes in fibroadenoma seemed to be expressed by fibroblasts. This led to the question if the stromal component might be the neoplastic part of the tumour on the one hand and whether it is possible that this stroma still could provide a permissible milieu for the adjacent epithelium to form cancerous lesions on the other. It has become increasingly noticed that only an interplay of stroma and epithelium lead to neoplastic conversion (Wiseman and Werb, 2002; Barcellos-Hoff and Ravani, 2000). A factor which could contribute to the growth stimulation of epithelial cells we detected is IGF-2. IGF-2 was described in the stroma of breast cancer and was suspected to have a growth promoting effect on adjacent epithelial tumour cells (Cullen et al., 1991). But, as already menitioned, IGF-2 is also expressed in the stroma of human fibroadenomas without known association with epithelial conversion (Sawyer et al., 2003). Furthermore, lumican, FAP and osteonectin have been reported in breast cancer stroma. The role of these genes should be investigated in adenocarcinomas, fibroadenomas and adenocarcinomas arising in fibroadenomas for their contribution to tumourigenesis before fibroadenoma can be unequivocally be termed a benign tumour without risk for cancer development. It has, however, to be noted that in the case of the adenocarcinomas and fibroadenomas we investigated, the stroma of the two tumour types seemed to be
149
differently composed: in fibroadenoma, we found dermal collagens and an activated type of fibroblast, which was able to express NSE and THY-1, while the adenocarcinomas expressed RNA for a collagen found in cartilage and vitreous body and showed in their stromal cells no staining for THY-1 or NSE. It is possible that the stroma represents the neoplastic part of the tumour. In human fibroadenomas, it has been reported that the stromal part of the tumour was clonal in origin, while the epithelial part was polyclonal. It has also been proposed that fibroadenoma could progress to both phyllodes tumour and adenocarcinoma (Kuijper et al., 2002a, b). Phyllodes tumour is a biphasic tumour of the human breast, which contains a malignant stromal component. This entity, however, is not known in the rat. In the course of time most rat fibroadenomas seem rather to develop into a fibrous mass with no or atrophic epithelium. In our analysis, we found TDAG51 a gene participating in senescence of fibroblasts, which might participate in this process. In human cases, this has also been associated with cessation of hormonal stimulation after menopause. Further studies are needed to characterize the stroma of fibroadenoma in a time course during tumour development. In this context, the roles of NSE and THY-1 could be investigated as well, as staining with both antibodies was lost in areas of fibroadenomas containing dense fibrotic tissue. Concluding, there is up to now little evidence that fibroadenoma plays a role as precursor lesion for adenocarcinoma, although further studies should be initiated to investigate the rarely occuring adenocarcinoma arising in fibroadenoma.
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