Recent studies suggest that thousands of genes may contribute to breast cancer pathophysiologies when deregulated by genomic or epigenomic events. Here, we describe a model "system" to appraise the functional contributions of these genes to breast cancer subsets. In general, the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors, although some significant differences are documented. The cell lines that comprise the system also exhibit the substantial genomic, transcriptional, and biological heterogeneity found in primary tumors. We show, using Trastuzumab (Herceptin) monotherapy as an example, that the system can be used to identify molecular features that predict or indicate response to targeted therapies or other physiological perturbations.

SUMMARY A handful of tumor-derived cell lines form the mainstay of cancer therapeutic development, yielding drugs with impact typically measured as months to disease progression. To develop more effective breast cancer therapeutics and more readily understand their clinical impact, we constructed a functional metabolic portrait of 46 independently-derived breast cell lines. Our analysis of glutamine uptake and dependence identified a subset of triple negative samples that are glutamine auxotrophs. Ambient glutamine indirectly supports environmental cystine acquisition via the xCT antiporter, which is expressed on 1/3 of triple negative tumors in vivo. xCT inhibition with the clinically approved anti-inflammatory Sulfasalazine decreases tumor growth revealing a therapeutic target in breast tumors of poorest prognosis, and a lead compound for rapid, effective drug development.

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis [1][2][3] . Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype [3][4][5] ; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent Correspondence should be addressed to R.P. (rep15@columbia.edu). 14 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS L.H.S., S.K.G.-S., R.P. and Å.B conceived and designed the study; L.H.S., S.K.G.-S., J.S., G.J., K.H., S.K., J.V.-C., H.O., T.S., L.M., S.P.E., H.H., R.P. and Å.B. collected the samples; L.H.S., K.L., M.J., L.M., T.L., M.S., J.I. and H.H. performed and analyzed immunohistochemistry experiments; L.H.S. and C.P. designed and performed methylation analyses; L.H.S., C.P., M.M. and K.H. performed nucleotide sequencing experiments; L.H.S., J.S., G.J. and K.H. performed and analyzed aCGH experiments; L.H.S., M.M.P., S.S. and V.V.V.S.M. designed and performed FISH experiments; L.H.S., S.K.G.-S. and M.K. performed statistical analyses; R.P. and Å.B. supervised the study; and L.H.S. wrote the paper with assistance from R.P. and Å.B. and input from all coauthors.Note: Supplementary information is available on the Nature Genetics website. [1][2][3][4][5] . Of these, basal-like breast cancer (BBC) comprises 10-20% of all breast cancer and is one of the subtypes with the worst prognosis 2-5 . The term BBC was coined because these tumors express cytokeratin markers typical of basally oriented epithelial cells of the normal mammary gland, such as CK5, CK14 and CK17 (refs. 1,3,5 ). In addition to having characteristic cytokeratin expression, BBCs are highly proliferative, poorly differentiated and genomically unstable, and they pose clinical challenges because they rarely express the three most common therapeutically targeted 'Achilles' heels' of breast cancer: the estrogen receptor (ER), progesterone receptor and HER2 receptor (refs. 1,3,5,7 ).Intriguingly, breast tumors initiated by an inherited mutation of BRCA1 are nearly always basal-like 3,5 . BRCA1 dysfunction is thought to be tumorigenic primarily owing to defective BRCA1-dependent DSB repair, which precipitates an accumulation of secondary mutations 10 ; however, only general genomic patterns at relatively low resolution have been described (reviewed in ref. 5 ). Despite these advances in delineating BBC, the molecu...

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Germ line mutations of the BRCA1 gene confer a high risk of breast cancer and ovarian cancer to female mutation carriers. The BRCA1 protein is involved in the regulation of DNA repair. How specific tumor-associated mutations affect the molecular function of BRCA1, however, awaits further elucidation. Cell lines that harbor BRCA1 gene mutations are invaluable tools for such functional studies. Up to now, the HCC1937 cell line was the only human breast cancer cell line with an identified BRCA1 mutation. In this study, we identified three other BRCA1 mutants from among 41 human breast cancer cell lines by sequencing of the complete coding sequence of BRCA1. Cell line MDA-MB-436 had the 5396 + 1G>A mutation in the splice donor site of exon 20. Cell line SUM149PT carried the 2288delT mutation and SUM1315MO2 carried the 185delAG mutation. All three mutations were accompanied by loss of the other BRCA1 allele. The 185delAG and 5396 + 1G>A mutations are both classified as pathogenic mutations. In contrast with wildtype cell lines, none of the BRCA1 mutants expressed nuclear BRCA1 proteins as detected with Ab-1 and Ab-2 anti-BRCA1 monoclonal antibodies. These three new human BRCA1 mutant cell lines thus seem to be representative breast cancer models that could aid in further unraveling of the function of

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer. (Mol Cancer Res 2005;3(12):655 -67)

Epithelial-mesenchymal transition (EMT) promotes cancer invasion and metastasis, but the integrative mechanisms that coordinate these processes are incompletely understood. In this study, we used a cross-species expression profiling strategy in metastatic cell lines of human and mouse origin to identify 22 up-regulated and 12 down-regulated genes that are part of an essential genetic program in metastasis. In particular, we identified a novel function in metastasis that was not previously known for the transcription factor Forkhead Box Q1 (Foxq1). Ectopic expression of Foxq1 increased cell migration and invasion in vitro, enhanced the lung metastatic capabilities of mammary epithelial cells in vivo, and triggered a marked EMT. In contrast, Foxq1 knockdown elicited converse effects on these phenotypes in vitro and in vivo. Neither ectopic expression nor knockdown of Foxq1 significantly affected cell proliferation or colony formation in vitro. Notably, Foxq1 repressed expression of the core EMT regulator E-cadherin by binding to the E-box in its promoter region. Further mechanistic investigation revealed that Foxq1 expression is regulated by TGF-β1, and that Foxq1 knockdown blocked TGF-β1-induced EMT at both morphological and molecular levels. Our findings highlight the feasibility of cross-species expression profiling as a strategy to identify metastasis-related genes, and they reveal that EMT induction is a likely mechanism underlying a novel metastasis-promoting function of Foxq1 defined here in breast cancer.

We describe a survey of genetic changes by comparative genomic hybridization (CGH) in 11 human breast cancer cell lines recently established in our laboratory. The most common gains took place at 8q (73%), 1q (64%), 7q (64%), 3q (45%) and 7p (45%), whereas losses were most frequent at Xp (54%), 8p (45%), 18q (45%) and Xq (45%). Many of the cell lines displayed prominent, localized DNA amplifications by CGH. One-third of these loci affected breast cancer oncogenes, whose amplifications were validated with specific probes: 17q12 (two cell lines with ERBB2 amplifications), 11q13 (two with cyclin-D1), 8p11–p12 (two with FGFR1) and 10q25 (one with FGFR2). Gains and amplifications affecting 8q were the most common genetic alterations in these cell lines with the minimal, common region of involvement at 8q22–q23. No high-level MYC (at 8q24) amplifications were found in any of the cell lines. Two-thirds of the amplification sites took place at loci not associated with established oncogenes, such as 1q41–q43, 7q21–q22, 7q31, 8q23, 9p21–p23, 11p12–p14, 15q12–q14, 16q13–q21, 17q23, 20p11–p12 and 20q13. Several of these locations have not been previously reported and may harbour important genes whose amplification is selected for during cancer development. In summary, this set of breast cancer cell lines displaying prominent DNA amplifications should facilitate discovery and functional analysis of genes and signal transduction pathways contributing to breast cancer development. © 1999 Cancer Research Campaign