Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features Chad J. Creightona,1, Xiaoxian Lia,1, Melissa Landisa,1, J. Michael Dixonb, Veronique M. Neumeisterc, Ashley Sjolundc, David L. Rimmc, Helen Wonga, Angel Rodrigueza, Jason I. Herschkowitza, Cheng Fand, Xiaomei Zhanga, Xiaping Hec, Anne Pavlicka, M. Carolina Gutierreza, Lorna Renshawb, Alexey A. Larionovb, Dana Faratianb, Susan G. Hilsenbecka, Charles M. Peroud, Michael T. Lewisa,2, Jeffrey M. Rosena,2, and Jenny C. Changa,2,3 aDepartment of Molecular and Cellular Biology, Division of Biostatistics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030; bWestern General Hospital, Edinburgh EH4 2XU, United Kingdom; cYale University School of Medicine, New Haven, CT 06510; and dDepartments of Genetics and of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 Communicated by Robert A. Weinberg, Whitehead Institute for Biomedical Research, Cambridge, MA, May 26, 2009 (received for review December 19, 2008) Some breast cancers have been shown to contain a small fraction of Global gene expression analyses have identified at least four ,cells characterized by CD44؉/CD24؊/low cell-surface antigen profile major subtypes of human breast cancers (luminal A/B, basal-like that have high tumor-initiating potential. In addition, breast cancer and ERBB2-enriched) (4, 5). More recently Herschkowitz et al. (6) cells propagated in vitro as mammospheres (MSs) have also been described another subtype, involving relatively uncommon breast shown to be enriched for cells capable of self-renewal. In this study, cancers, termed ‘‘claudin-low,’’ which are characterized by com- we have defined a gene expression signature common to both paratively high expression of mesenchymal markers such as vimen- CD44؉/CD24؊/low and MS-forming cells. To examine its clinical sig- tin and low expression of epithelial markers, notably claudins and nificance, we determined whether tumor cells surviving after con- E-cadherin. During the course of normal development, the trans- differentiation of epithelial cells to a more mesenchymal state /ventional treatments were enriched for cells bearing this CD44؉ ؊/low ؉ ؊/low (7–13) known as epithelial-to-mesenchymal transition (EMT), is an CD24 -MS signature. The CD44 /CD24 -MS signature was essential process; this process has also been implicated in cancer CELL BIOLOGY found mainly in human breast tumors of the recently identified progression (14). Indeed, an increased representation after therapy ‘‘claudin-low’’ molecular subtype, which is characterized by expres- of cancer cells expressing mesenchymal markers, notably those sion of many epithelial-mesenchymal-transition (EMT)-associated described in claudin-low tumors, may account for treatment resis- genes. Both CD44؉/CD24؊/low-MS and claudin-low signatures were tance (15–17). It is not known directly at this time whether the more pronounced in tumor tissue remaining after either endocrine claudin-low tumors show elevated resistance to therapy. therapy (letrozole) or chemotherapy (docetaxel), consistent with the Defining the regulatory pathways in subpopulations of residual selective survival of tumor-initiating cells posttreatment. We con- tumor cells that survive conventional therapy could ultimately provide firmed an increased expression of mesenchymal markers, including important new therapeutic targets. With this objective in mind, we first ϩ Ϫ/low Ϫ vimentin (VIM) in cytokeratin-positive epithelial cells metal- derived a gene signature from CD44 /CD24 /lineage cells and loproteinase 2 (MMP2), in two separate sets of postletrozole vs. formed cancer MS cells, both isolated from human breast cancers. We ϩ Ϫ/low pretreatment specimens. Taken together, these data provide sup- then compared this CD44 /CD24 -MS signature to the previously porting evidence that the residual breast tumor cell populations defined intrinsic subtypes of breast cancer to determine whether there was any subtype that was highly enriched for this subpopulation. To surviving after conventional treatment may be enriched for subpopu- examine the signature’s clinical and therapeutic significance, we eval- lations of cells with both tumor-initiating and mesenchymal features. uated it in breast tumors before and after therapy (letrozole or Targeting proteins involved in EMT may provide a therapeutic strat- docetaxel), doing so with the hypothesis that the tumor cells surviving egy for eliminating surviving cells to prevent recurrence and improve after treatment would have an increased expression of the CD44ϩ/ long-term survival in breast cancer patients. CD24Ϫ/low-MS signature. Finally, relevant key markers from this sig- nature were confirmed in additional paired before and after treatment CD44ϩ/CD24Ϫ/low markers ͉ gene expression signature ͉ patient specimens. tumor-initiating cancer cells ͉ mesenchymal features ͉ treatment resistance Results espite recent improvements in breast cancer mortality, many Gene Transcription Patterns in Breast Cancer Cells with Tumor-Initi- Dpatients relapse after an initial response to conventional ating Potential. To define a gene expression signature, we used two endocrine therapy and chemotherapies. Several alternative but not methods to obtain breast cancer subpopulations that have been necessarily mutually exclusive hypotheses have been proposed to explain this treatment failure and recurrence. In particular, it has Author contributions: J.M.D., A.R., X.Z., A.P., M.C.G., A.A.L., J.M.R., and J.C.C. designed been suggested that a small subpopulation of cells within tumors, research; X.L., M.L., J.M.D., V.M.N., A.S., D.L.R., H.W., A.R., J.I.H., C.F., X.Z., A.P., M.C.G., L.R., often designated as ‘‘tumor-initiating cells’’ or ‘‘cancer stem cells,’’ C.M.P., M.T.L., and J.C.C. performed research; X.L., M.L., V.M.N., D.L.R., H.W., J.I.H., X.H., may be resistant to therapy and hence may reinitiate tumor growth A.P., D.F., S.G.H., M.T.L., and J.M.R. contributed new reagents/analytic tools; C.J.C., X.L., M.L., V.M.N., A.S., D.L.R., H.W., A.R., J.I.H., C.F., X.H., A.P., M.C.G., L.R., A.A.L., D.F., S.G.H., after treatment (1). The population containing these cells can be C.M.P., M.T.L., J.M.R., and J.C.C. analyzed data; and C.J.C., X.L., M.L., A.S., D.L.R., A.R., isolated by FACS using specific cell surface markers. For example, S.G.H., C.M.P., M.T.L., J.M.R., and J.C.C. wrote the paper. ϩ Ϫ/low in cells from metastatic pleural effusions, the CD44 /CD24 / The authors declare no conflict of interest. Ϫ lineage subpopulation was shown to be enriched for tumor- Freely available online through the PNAS open access option. ϩ initiating cells (2). Recently, we have reported that this CD44 / 1C.J.C., X.L., and M.L. contributed equally to this work. Ϫ/low CD24 subpopulation of cells appears to be more relatively 2M.T.L., J.M.R., and J.C.C. contributed equally to this work. resistant to chemotherapy in paired primary human breast cancer 3To whom correspondence should be addressed at: 1 Baylor Plaza, BCM 600, Houston, TX biopsies (3). This increase was associated with an enhanced mam- 77030. E-mail: [email protected]. mosphere (MS)-forming ability, an in vitro surrogate assay of This article contains supporting information online at www.pnas.org/cgi/content/full/ self-renewal capacity (3). 0905718106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905718106 PNAS Early Edition ͉ 1of6 Fig. 1. A gene transcription signature of breast cancer cells with putative tumor- initiating potential. (A) Venn diagram of the intersection between genes elevated in can- cer mammospheres (MSs, rich in CD44ϩ/ CD24Ϫ/low cells) compared with primary can- cers and genes elevated in CD44ϩ/CD24Ϫ/low cells obtained from FACS assay (P Ͻ 0.01, fold change Ͼ1.5 for each comparison). P value for the overlap between the two gene sets by one-sided Fisher’s exact test. (B) As with A but for genes lower in cancer MSs and genes lower in CD44ϩ/CD24Ϫ/low flow-sorted cells. (C) Heat map of genes in the CD44ϩ/CD24Ϫ/ low-MS signature (from parts A and B). Each row represents a transcript; each column, a sample (yellow: high expression). Associations of the genes with selected Gene Ontology (GO) annotation terms are indicated. Genes in the indicated region are listed by name. suggested previously to be enriched for putative tumor-initiating E-cadherin. We compared the CD44ϩ/CD24Ϫ/low-MS signature to each cells: CD44ϩ/CD24Ϫ/low markers and formed cancer MSs (2). These subtype to determine whether any group showed enrichment. For each analyses involved 36 tumors representing all subtypes of breast tumor in the Herschkowitz dataset, we derived an ‘‘R value’’ in relation cancer (18 luminal A/B, 13 basal-like, and 5 ERBB2-enriched) (Fig. to our CD44ϩ/CD24Ϫ/low-MS signature, with the R value being a S1). We performed comparative gene expression profiling in the measure for how much each individual Herschkowitz tumor manifested populations that have been shown by others to be enriched for our signature (see Methods). tumor-initiating cells (CD44ϩ/CD24Ϫ/low or formed cancer MSs) vs. Only the previously defined claudin-low group showed a clear non-tumor-initiating cells (all ‘‘other’’ flow-sorted fractions or bulk association with the CD44ϩ/CD24Ϫ/low-MS signature (Fig. 2A). We tumor, respectively), and then analyzed to look for significant obtained the same results using a number of other analytical overlap between the gene expression patterns of cells isolated by the approaches, including one-sided Fisher’s exact tests and GSEA (see two alternative enrichment methods. SI Text and Fig. S3).