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Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [D. A. P., I. E. K., K. P.]; Harvard Medical School, Boston, Massachusetts 02115 [D. A. P., I. E. K., S. N., D. S., C. M. K., K. P.]; Department of Pathology, Massachusetts General Hospital, Charlestown, Massachusetts 02129 [S. N., D. S.]; Department of Surgery, Brigham and Women’s Hospital, Boston, Massachusetts 02115 [C. M. K.]; and Departments of Surgery [J. R. M.] and Pathology [G. R.], Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Introduction |
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To identify such alterations we generated SAGE4 libraries from normal mammary epithelial cells and in situ, invasive, and metastatic carcinomas. SAGE analyzes 14-bp tags derived from a defined position of the cDNAs without a priori knowledge of the sequence of the genes expressed (3) . The SAGE tag numbers directly reflect the abundance of the mRNAs; therefore, SAGE data are highly accurate and quantitative. Although several previous studies analyzed the gene expression profiles of breast carcinomas, no study used an unbiased comprehensive gene expression profiling approach and highly purified, uncultured, patient-derived tissues as starting material (4, 5, 6) . Therefore, we believe that our study is a faithful description of the in vivo gene expression profiles of normal and cancerous mammary epithelial cells.
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Materials and Methods |
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Generation and Analysis of SAGE Libraries.
For immunomagnetic purification, minced breast tissue was digested in DMEM/F12 medium (Life Technologies, Inc., Rockville, MD) supplemented with 1% fetal bovine serum, 2 mg/ml collagenase I (C0130; Sigma Chemical Co.), and 2 mg/ml hyaluronidase (H3506; Sigma Chemical Co.) at 37°C for 2 h. Cells were collected by centrifugation; trypsinized; and resuspended in PBS, 1% BSA, and 2 mM EDTA and purified using the Epithelial Enrich kit (Dynal, Oslo, Norway) following the manufacturer’s recommendations. In the case of libraries derived from frozen sections, mRNA was prepared from OCT-embedded tissue using µMACS kit (Miltenyi Biotec). SAGE libraries were generated following a modified micro-SAGE protocol, including a 1% SDS washing/heating step after each enzymatic reaction to ensure complete inactivation of the enzymes (7)
. As part of the Cancer Gene Anatomy Project SAGE consortium, SAGE libraries were arrayed at the Lawrence Livermore National Laboratories and sequenced at the Washington University Human Genome Center or at the National Institute Sequencing Center (NIH, Bethesda, MD). The data have been posted on the Cancer Gene Anatomy Project web-site as part of the SAGEmap database (8
, 9)
. Approximately 50,000 SAGE tags were obtained from each library. The exact numbers were: N1 (49, 351), N2 (38, 371), D1 (42, 556), D2 (29, 768), IDC1 (39, 996), IDC2 (67, 703), MET1 (45, 539), and MET2 (60, 975). SAGE libraries were analyzed using the SAGE 2000 software (derivation of tags, library comparisons, and Monte Carlo analysis for statistical significance); the data were subsequently transferred into Microsoft Access (link to Unigene database) and Excel (to sort data and convert it into tab-delimited files). For comparisons, only SAGE tags that occurred at least twice/library/50,000 tags were included. To compensate for the unequal tag numbers in the eight different libraries, tag numbers were normalized to 50,000-tags/library before comparisons. Hierarchical clustering was applied to data using the Cluster program developed by Eisen et al. (10)
. Data were log-transformed and filtered for at least one observation abs Val 5 and Maxval-Minval>2. Using these settings, 3,987 genes (of 16,808 total) were included in the analysis. Results in Fig. 1
were displayed with the TreeView program (10)
.
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. In addition, we also identified genes with an expression pattern that correlated with histological grade or the presence of the ER (Table 2, A and B)
. Data were also analyzed using a clustering algorithm to delineate patterns of gene expression among all eight libraries (Fig. 1)
. Clusters of coexpressed genes suggested that the two normal mammary epithelial libraries, despite being derived from two different patients, were the most similar to each other. Only 43 tags were expressed differentially between the two normal libraries. The four invasive and metastatic libraries appeared to be quite similar to each other. Interestingly the two DCIS libraries were very dissimilar (225 tags showed an at-least 2-fold statistically significant difference) and clustered according to their predicted clinical behavior. High-grade DCIS have high recurrence rates and worse overall prognoses when compared with intermediate or low-grade DCIS. In agreement with this, the high-grade comedo DCIS (DCIS-1) was more similar to invasive tumors, whereas the intermediate DCIS was more similar to normal mammary epithelium. Although the two normal and the DCIS-2 libraries were generated from cells purified using anti-Ber-Ep4-coated magnetic beads, whereas all other libraries were derived from macroscopically dissected (>95% pure) tumors, it is unlikely that the presence of contaminating cells (<5%; mostly fibroblasts) in the dissected samples would be the only reason for this intriguing clustering pattern. However, this possibility cannot be excluded.
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encode secreted proteins, chemokines (IL-8, GRO
, GROß, and MIP3), and cytokines (LIF, IL-6, and HIN-1) that may play autocrine and/or paracrine roles in the regulation of normal mammary epithelial cell growth, differentiation, and morphogenesis. HIN-1, for example, is a novel growth-inhibitory cytokine hypermethylated in a large fraction of breast cancers that may play a role in epithelial cell proliferation and branching morphogenesis.5
Several other chemokines (SCYA2, SCYA7, SCYB5, SCYB6, and SYD1) and chemokine receptors (IL-4R, IL-6R, and IL-15R) were also more abundant (5–10-fold) in normal mammary epithelial cells, but because of their low tag numbers, the P of this difference was above P = 0.001. It is unlikely that these chemokines and cytokines were derived from contaminating leukocytes, because SAGE libraries were generated from immunomagnetic purified normal luminal mammary epithelial cells, and the purity of the cells was confirmed by reverse transcription-PCR analysis using known luminal (HIN-1 and EMA) and myoepithelial (calponin and CALLA/CD10) cell-specific transcripts (data not shown). Preliminary mRNA in situ and immunohistochemical experiments also confirmed that chemokines and cytokines are expressed in normal luminal mammary epithelial cells.6
Similarly, a recently identified novel chemokine, mammary enriched chemokine, or MEC, was also shown to be expressed in normal mammary epithelial cells and found to be down-regulated in breast carcinomas (12)
.
Another gene expressed at high levels in normal mammary epithelium and lost in tumors is the mitochondrial superoxide dismutase, SOD-2. A decreased level of SOD-2 in cancer cells is well documented and it may contribute to the high level of reactive oxygen species and subsequent oxidative stress characteristic of breast cancers (13
, 14)
. The expression levels of two transcriptional regulators, IK and C/EBP
, show a somewhat gradual decline from normal to in situ and then to invasive carcinomas. IK sequesters NFkß in the cytoplasm in an inactive form; therefore, loss of IK expression may lead to activation of the NFkß pathway. C/EBPs are transcription factors that regulate cell growth and differentiation in a cell type-specific manner. C/EBP has been shown to be required for growth arrest in mammary epithelial cells (15)
; thus, the gradual decline of C/EBP mRNA levels during breast tumor progression may correlate with increased proliferation rates.
Genes Differentially Expressed Among Various Tumors and Their Correlation with Histological Parameters.
In contrast to the relatively high number of normal mammary epithelium-specific genes, only three genes appeared to be highly expressed in all breast carcinomas. These included trefoil factor 3, X-box binding protein 1, and fatty acid synthase (Table 1B)
. Both trefoil factor 3 and fatty acid synthase previously have been demonstrated to be up-regulated in breast carcinomas (16, 17, 18)
. The IFN- inducible protein, IFI-6–16, is also highly represented in most of these breast cancer libraries and is likely to reflect the activation of the IFN/signal transducers and activators of transcription-signaling pathway observed in several previous studies (5
, 6)
. Interestingly, glutamine synthase and desmoplakin were the only two genes that were specifically up-regulated in both DCIS tumors. Both genes have been shown to be aberrantly expressed in various cancers. Using immunohistochemical analysis, the levels of desmoplakin protein were found to be decreased in poorly differentiated, advanced-stage breast carcinomas (19)
. Glutamine synthase, on the other hand, may be induced because of hypoxia and/or decreased intracellular glutamine levels (20)
. Another indication of metabolic alterations in cancer cells was the up-regulation of several metabolic enzymes involved in glycolytic (3-phosphoglycerate dehydrogenase and glyceraldehyde dehydrogenase) and mitochondrial (NADH:uniquinone dehydrogenase and NADH dehydrogenase 1) function in invasive carcinomas. There are numerous reports on metabolic differences between normal and cancer cells, the most dramatic being a highly active glycolytic pathway in cancers (21)
. Many transcripts encoding ribosomal proteins and immunoglobulins were also included in the "invasive" cluster. Their presence in invasive tumors is likely attributable to increased protein synthesis and the possible presence of contaminating lymphocytes in the dissected samples. Interestingly, the expression of the chemokine receptor 4 was also significantly higher in invasive carcinomas, correlating with recent data implicating chemokine receptors in invasive/metastatic behavior of breast carcinomas (22)
. Two SAGE tags seemed to be particularly interesting because of their restricted expression in invasive breast carcinomas. One corresponds to calmodulin-like skin protein, a calcium-binding protein implicated in keratinocyte differentiation, whereas the other, IBC-1, has no expressed sequence tag matches and corresponds to a novel gene.7
Both of these genes may potentially be used for breast cancer diagnosis and for differential diagnosis of in situ and invasive carcinomas.
The expression of certain genes appeared to correlate with the histological grade of the tumor and/or the presence of the ER. All four invasive/metastatic and one of the DCIS tumors were high nuclear grade, and most of the genes highly expressed in these tumors encode extracellular matrix proteins, such as various collagens, osteonectin, and BIGH3, a TGFß-induced gene. Although some of these genes could be expressed in contaminating fibroblasts, previous studies demonstrated that osteonectin is produced by most ER-negative breast carcinomas and may contribute to the aggressive phenotype and the presence of microcalcifications characteristic for these tumors. To demonstrate that these secreted factors can be produced by normal and/or cancerous epithelial cells themselves, we performed Northern blot analysis of normal organoids (purified breast ducts) and various breast cancer cell lines (Fig. 2)
. Correlating with previous studies, breast cancer cell lines do not seem to reproduce the in vivo expression pattern of these differentially expressed genes. However, some cells clearly express high levels of genes encoding extracellular matrix components and other secreted proteins, indicating that these proteins can be produced by the tumor cells themselves.
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inducible protein 27, appeared to be significantly higher in DCIS2 (Table 2B)
. Despite the fact that we analyzed three ER-negative (DCIS1, IDC1, and MET1) and three ER-positive (DCIS2, IDC2, and MET2) tumors, the only ER target that clearly correlated with the ER status of the tumors was cyclin D1. This could be attributable to the fact that both IDC2 and MET2 were of high nuclear grade, whereas most ER-positive tumors are of low-intermediate grade. All other known ER target genes were either present in all (cathepsin D and bcl-2) or in a subset of libraries (LIV-1-DCIS2 only; Trefoil factor 1, both normal and both DCIS). |
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. This is likely attributable to the restricted expression pattern of these genes, which makes them excellent candidate tumor markers. Searching against the completed human genome sequence, these SAGE tags will likely lead to the identification of novel transcripts. These transcripts could then be included in DNA arrays for high throughput analysis of breast and other tumors to determine their usefulness for cancer diagnosis and/or prognostication.
The high levels of SOD-2, IK, and C/EBP in normal mammary epithelial cells and the loss of expression in breast cancers correlates with previous studies (13
, 14
, 23)
. One of the novel findings reported here was that several of the genes highly expressed in normal mammary epithelial cells and down-regulated in tumors encode secreted proteins, chemokines, and cytokines. Chemokines are mediators of immune cell trafficking, and, although they are also involved in regulating cell movements during morphogenesis, their principal targets are bone marrow-derived cells (24
, 25) . Recent data indicate that in addition to fibroblasts and adipocytes, macrophages, eosinophils, and endothelial cells are also required for normal mammary gland development (26
, 27) . Although the function of leukocytes and endothelial cells in this process has not been fully elucidated, they may be necessary for the formation of terminal end buds and for branching morphogenesis. The high abundance of GRO, GROß, MIP3, IL-8, and, to a lesser degree, SCYA2, SCYA7, SCYB5, SCYB6, and SYD1 chemokines in normal mammary epithelial cells suggests that epithelial cells may actively recruit bone marrow-derived and endothelial cells. Alternatively, chemokines may play an as yet uncharacterized role in interepithelial cell communication, a hypothesis that deserves additional investigation.
There were very few genes that were universally up-regulated in all of the tumors examined, indicating a high degree of heterogeneity of breast carcinomas at the molecular level. Even based on the limited number of tumors analyzed, it is clear that tumors of different histological stage (invasive) or nuclear grade (high or intermediate) clearly show distinct gene expression patterns that are likely to reflect their clinical behavior. The same is true for ER-positive and -negative tumors. However, we found no genes that were up-regulated in both lymph node metastases that were not already expressed in invasive carcinomas. Similarly, there were very few genes that were up-regulated in both DCIS but not in invasive carcinomas. This could be attributable to the limited number of samples analyzed, or it may indicate that preinvasive and metastatic lesions may be even more heterogeneous than invasive ones. Alternatively, genes responsible for invasive and metastatic behavior may already be expressed in preinvasive and invasive lesions, respectively. Analysis of large numbers of in situ carcinomas without adjacent invasive lesions and analysis of many invasive carcinomas with and without lymph node metastasis would be required to answer this question.
Some of the cancer-specific genes we identified could be used for cancer diagnosis and/or molecular-based anticancer therapy. For example, breast and other cancer cells have very high levels of fatty acid synthase (Table 1B
; Ref. 18
), and its inhibition leads to apoptosis in in vitro cell cultures and in xenograft models (28)
. Similarly, two of the transcripts highly expressed in invasive breast cancers, calmodulin-like skin protein, and IBC-1, a novel gene, have restricted expression patterns that make them good candidate invasive breast cancer tumor markers.
There were several similarities and differences between our findings and previously published data on the molecular profiling of breast tumors (4, 5, 6) . Correlating with our data, trefoil factor, cytokeratin 18, X-box binding protein, cyclin D1, and transcriptional targets of the STAT/IFN pathway are highly up-regulated in a subset of breast cancers (4, 5, 6) . In contrast, our "normal cluster" is very different from that of previous studies. There are several possible explanations of this difference: (a) in contrast with this study, no previous study used purified, uncultured, normal luminal mammary epithelial cells; (b) in previous cDNA array experiments the reference RNA was derived from a mix of cancer cell lines, potentially masking differences between normal and cancerous mammary epithelial cells; and (c) some of the genes we identified, especially the ones with no EST matches, may not be present on the arrays. Future studies analyzing the same samples on different platforms (SAGE versus arrays) are required to resolve the observed differences.
In summary, using comprehensive gene expression profiling, we identified several genes and pathways that have not previously been implicated in breast cancer and which may play important roles in the initiation and progression of breast carcinomas. Because we analyzed a limited number of specimens, additional experiments using high-throughput techniques, such as mRNA in situ hybridization or immunohistochemical analysis on tissue-microarrays, are required to determine how commonly these genes are differentially expressed. Additional analysis of these genes and the biochemical pathways in which they are involved will not only further our understanding of breast oncogenesis, but will also provide new and valuable targets for translational research.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute-Cancer Gene Anatomy Project Contract S98-146A from the National Cancer Institute Cancer Gene Anatomy Project (to G. J. R. and K. P.), an American Society of Clinical Oncology Career Development Award (to K. P.), the National Cancer Institute Specialized Programs of Research Excellence in Breast Cancer at Dana-Farber Cancer Institute (to K. P.); and by the Dana-Farber Cancer Institute (to K. P., D. A. P., and I. E. K.). 
2 These two authors contributed equally to this study.
3 To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, Department of Adult Oncology, 44 Binney Street, D740C, Boston, MA 02115. Phone: (617) 632-2106; Fax: (617) 632-4005; E-mail: Kornelia_Polyak{at}dfci.harvard.edu
4 The abbreviations used are: SAGE, serial analysis of gene expression; DCIS, ductal carcinoma(s) in situ; IDC, invasive ductal carcinoma; MET, metastasis; HIN-1, high in normal-1; IBC-1, invasive breast carcinoma-1; C/EBP, CCAAT/enhancer binding protein; ER, estrogen receptor.
5 I. Krop, D. Sgroi, D. Porter, K. Lunetta, R. LeVangie, P. Seth, C. Kaelin, E. Rhei, M. Bosenberg, S. Schnitt, J. Marks, Z. Pagon, D. Belina, J. Razumovic, and K. Polyak. HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells. PNAS, in press.
6 D. Porter, J. Lahti-Domenici, S. Schmitt, and K. Polyak, unpublished data.
7 P. Seth, D. Porter, J. Lahti-Domenici, J. Marks, A. Richardson, and K. Polyak, unpublished data.
Received 5/ 8/01. Accepted 6/15/01.
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