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ONCOGENOMICS Mammary Luminal Epithelial Cells Oncogene (2003) 22, 2680–2688 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc cDNA microarray analysis of genes associated with ERBB2 (HER2/neu) overexpression in human mammary luminal epithelial cells Alan Mackay*,1,6, Chris Jones2,6, Tim Dexter2, Ricardo LA Silva3, Karen Bulmer2, Allison Jones2, Peter Simpson2, Robert A Harris1, Parmjit S Jat4, A Munro Neville1, Luiz FL Reis3, Sunil R Lakhani2,5 and Michael J O’Hare1 1LICR/UCL Breast Cancer Laboratory, University College London, London, UK; 2The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, London, UK; 3Ludwig Institute for Cancer Research, Sao Paolo, Brazil; 4Ludwig Institute for Cancer Research, University College London, London, UK; 5Royal Marsden Hospital, London, UK To investigate changes in gene expression associated with the disease within their lifetime. Overexpression of the ERBB2, expression profiling of immortalized human proto-oncogene ERBB2 (HER2/neu) is observed in 25– mammary luminal epithelial cells and variants expressing 30% of all such cancers, and is an established adverse a moderate and high level of ERBB2 has been carried out prognostic factor (Slamon et al., 1987, 1989; Ross and using cDNA microarrays corresponding to approximately Fletcher, 1999; Menard et al., 2001) yielding a median 6000 unique genes/ESTs. A total of 61 significantly up- or survival of 3years, compared with 6–7 years when downregulated (>2.0-fold) genes were identified and unassociated with ERBB2. Overexpression also corre- further validated by RT–PCR analysis as well as lates with tumor size, lymph node metastases, high microarray comparisons with a spontaneously ERBB2- nuclear grade, high percentage of S-phase cells, aneu- overexpressing breast cancer cell line and ERBB2-positive ploidy and estrogen receptor (ER) and progesterone primary breast tumors. The expression and clinical receptor (PR) negativity (Ross and Fletcher, 1998). relevance of proteins predicted to be associated with ERBB2 overexpression may also predict resistance to ERBB2 overexpression in breast cancers were analysed both chemotherapy and endocrine therapy, although together with their clinical relevance by antibody screen- this remains controversial (Houston et al., 1999; Miles ing using a tissue array. Differentially regulated genes et al., 1999; Yu and Hung, 2000). The ERBB2 receptor include those involved in cell–matrix interactions including has become the first oncogene product to be targeted for proline 4-hydroxylase (P4HA2), galectin 1 (LGALS1) breast cancer therapy, by the humanized anti-ERBB2 and galectin 3 (LGALS3), fibronectin 1 (FN1) and p- monoclonal antibody trastuzumab (Herceptin) (Baselga cadherin (CDH3), and cell proliferation (CRIP1, et al., 1996; Cobleigh et al., 1999; Slamon et al., 2001). IGFBP3) and transformation (S100P, S100A4). A There is much evidence to suggest that ERBB2 is not number of genes associated with MYC signalling were the only gene activated by amplification at 17q12–q21 in also differentially expressed, including NDRG1, USF2 breast cancer (Tomasetto et al., 1995; Bieche et al., 1996; and the epithelial membrane proteins 1 and 3 (EMP1, Kauraniemi et al., 2001). Owing to the potential EMP3). These data represent profiles of the transcrip- contribution of these other genes within the ERBB2 tional changes associated with ERBB2-related pathways amplicon (17q12–q21) to breast tumorigenesis in vivo,it in the breast, and identify novel and potentially useful has been difficult to determine the precise role played by targets for prognosis and therapy. ERBB2-mediated signalling, and to establish the me- Oncogene (2003) 22, 2680–2688. doi:10.1038/sj.onc.1206349 chanisms by which anti-ERBB2 therapeutic agents exert their antiproliferative effects. Keywords: ERBB2 (HER2/neu); breast cancer; cDNA We have previously established a model of ERBB2 microarray; expression profiling; tissue array overexpression in conditionally immortalized human ONCOGENOMICS mammary luminal epithelial cells. Transfection of the HB4a human mammary epithelial cell line with ERBB2 cDNA resulted in two cell lines, C3.6 and C5.2, which Introduction showed ‘moderate’ and ‘high’ overexpression of ERBB2, respectively (Harris et al., 1999). These cells do not Breast cancer is one of the most common malignancies exhibit amplification at 17q12–q21, and thereby provide in the Western world, with one in 12 women developing an opportunity to examine ERBB2-related signalling independent of the effects of other related coamplified *Correspondence: A Mackay, LICR/UCL Breast Cancer Laboratory, genes. Charles Bell House, Riding House Street, London W1W 7EJ, UK; Accordingly, we have investigated the transcriptional E-mail: [email protected] changes associated with ERBB2 overexpression in 6These authors contributed equally to this work Received 16 October 2002; revised 23December 2002; accepted 3 breast epithelial cells using cDNA microarrays employ- January 2003 ing 9930 cDNA clones representing approximately 6000 ERBB2-related gene expression using microarrays A Mackay et al 2681 unique genes/ESTs. A total of 61 statistically significant expressed genes, 132 (46.7%) were found to be differentially expressed genes (greater than 2.0-fold) significant in both C5.2 and C3.6, including LGALS1, associated with ERBB2 overexpression were identified. S100P, NDRG1, CDH3, EMP1, FN1, IGFBP3 and Their clinical relevance was demonstrated at the protein LGALS3. level by carrying out immunohistochemistry on a tissue We also examined a breast cancer cell line, BT474, array comprising 48 ERBB2-positive and 47 ERBB2- using our cDNA microarrays. BT474 contains the negative breast carcinomas. 17q12–q21 amplicon, and allows for comparison with our ERBB2-transfected cell lines which do not. BT474 mRNA was reverse transcribed and hybridized with HB4a for direct comparison with the C3.6 and C5.2 Results data, in two replicated reverse-labelled experiments. In cDNA microarray analysis total, 274 statistically significant (SAM, 1% false discovery), differentially expressed (greater than 1.7- Using the normalized data from our 9930 clone cDNA fold) genes are published in supplementary table S3. microarrays, gene lists were generated by SAM (version Primary breast tumors 731 and 732 were also analysed 1.12) for differentially expressed genes in the compar- by cDNA microarrays, hybridized against the normal isons between C3.6 (moderately overexpressing ERBB2) luminal epithelial HB4a cells. This analysis generated and HB4a, C5.2 (highly overexpressing ERBB2) and lists of 187 and 91 differentially expressed genes, HB4a, as well as between C3.6 and C5.2. Significance respectively, corresponding to a total of 152 unique was assigned using a False Discovery Rate threshold of genes (supplementary Table S3). 1% in conjunction with a ratio threshold of 1.7. A set of The Venn diagram in Figure 2 shows a summary of 143significantly upregulated and 140 significantly overlaps of differentially expressed genes between the downregulated genes from the three comparisons was ERBB2-transfected breast luminal epithelial cell line generated. In individual experiments, 61 genes were C5.2, the ERBB2-amplicon-containing breast cancer cell found to be differentially expressed in C3.6 compared to line BT474, and ERBB2-overexpressing invasive ductal HB4a, 180 between C5.2 and HB4a and 91 between breast carcinomas examined by cDNA microarrays. The C5.2 and C3.6 (mean array ratios, SAM scores and s.d.’s only gene to segregate with ERBB2 (differential in all for the list of 283are provided in supplementary Table S2). analyses) was IGFBP3. Four genes, including FN1, By performing the three-way comparisons, the C5.2 PTRF and TIP-1, overlapped between C3.6/C5.2 and versus C3.6 ratios can be predicted from the individual BT474. None of the differentially expressed genes in the variant versus parental experiments. The actual : pre- C5.2 and C3.6 experiments are found at the 17q12–q21 dicted plot is shown in Figure 1, and gives a correlation amplicon. Eight genes that were found to be differential coefficient of 0.968, allowing for an array-based control through analysis of C3.6/C5.2 were also up- or down- of the reproducibility of the data. For journal format regulated in the tumor samples, but were not discovered brevity, a ratio threshold of 2.0 in the C5.2 versus HB4a in the BT474 breast cancer cell line. These include the comparison was used to produce a list of 36 upregulated upregulated genes LGALS1, CRIP1, VIM and PRDX2, and 25 downregulated unique genes, ranked according and the downregulated genes RPL17, MYL9, ADORA3 to ratio value (Table 1). Of the 283differentially and GTF3A. Figure 1 Predicted versus actual array ratio plot for C5.2 versus C3.6 comparison, calculated from individual comparisons against parental cell line HB4a Oncogene ERBB2-related gene expression using microarrays A Mackay et al 2682 Table 1 List of differentially expressed genes by cDNA microarray analysis C5.2 versus C3.6 versus C5.2 versus Symbol Ensembl Name HB4a HB4a C3.6 ERBB2 ENSG00000141736 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 15.72 1.13 6.47 LGALS1 ENSG00000100097 Lectin, galactoside-binding, soluble, 1 (galectin1) 12.39 1.41 6.01 PRO2605 ENSG00000130600 Hypothetical protein PRO2605 10.87 0.97 8.18 CRIP1 ENSG00000133512 Cysteine-rich protein 1 (intestinal) 9.30 1.53 5.07 S100P ENSG00000163993 S100 calcium binding protein P 5.48 6.16 0.70 CPS1 ENSG00000021826 Carbamoyl-phosphate synthetase 1, mitochondrial
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