Comparative oncogenomics identifies breast tumors SPECIAL FEATURE enriched in functional tumor-initiating cells Jason I. Herschkowitza, Wei Zhaob,c, Mei Zhanga, Jerry Usaryc,d, George Murrowc, David Edwardsa, Jana Knezevica, Stephanie B. Greenea, David Darrc,d, Melissa A. Troesterc, Susan G. Hilsenbecke, Daniel Medinaa, Charles M. Perouc,d,f, and Jeffrey M. Rosena,1 FEATURE aDepartment of Molecular and Cellular Biology and eLester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030; and bCurriculum in Bioinformatics and Computational Biology, cLineberger Comprehensive Cancer Center, dDepartment of Genetics, and fDepartment of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599 BREAST CANCER SPECIAL Edited by Kornelia Polyak, Dana-Farber Cancer Institute, Boston, MA, and accepted by the Editorial Board April 5, 2011 (received for review January 26, 2011) The claudin-low subtype is a recently identified rare molecular model exhibits histological heterogeneity reminiscent of human subtype of human breast cancer that expresses low levels of BCs, including a subset of the tumors expressing the estrogen tight and adherens junction genes and shows high expression of receptor (ER). In addition antiestrogens are able to significantly epithelial-to-mesenchymal transition (EMT) genes. These tumors are delay tumor formation in this model (8). Last, these p53-deficient enriched in gene expression signatures derived from human tumor- tumors exhibit genetic instability and/or aneuploidy, which likely initiating cells (TICs) and human mammary stem cells. Through cross- play a critical role in progression (6). fi fi species analysis, we discovered mouse mammary tumors that have Using gene expression pro ling for classi cation we show that, similar gene expression characteristics as human claudin-low tumors like human tumors, p53 null mouse mammary tumors fall into mul- and were also enriched for the human TIC signature. Such claudin- tiple molecular groups, including basal-like, luminal, and claudin- low tumors were similarly rare but came from a number of distinct low subtypes. The claudin-low tumors contain a majority of spindle- mouse models, including the p53 null transplant model. Here we shaped cells, a histology originally described for carcinosarcomas, present a molecular characterization of 50 p53 null mammary which are now called epithelial-to-mesenchymal transition (EMT) tumors (9). Like their human counterparts, the p53 null claudin-low tumors compared with other mouse models and human breast GENETICS tumor subtypes. Similar to human tumors, the murine p53 null tumors exhibit high expression of EMT inducers and a core EMT signature (10). Unlike many other mouse tumor models, p53 null tumors fell into multiple molecular subtypes, including two basal- tumors show extensive genomic instability. Accordingly, we deter- like, a luminal, a claudin-low, and a subtype unique to this model. mined that these different p53-deficient murine subtypes contain The claudin-low tumors also showed high gene expression of EMT distinct genomic DNA copy number changes, as assessed by array inducers, low expression of the miR-200 family, and low to absent comparative genomic hybridization (aCGH). We also show, by lim- expression of both claudin 3 and E-cadherin. These murine subtypes iting dilution transplantation, that p53 null claudin-low tumors have also contained distinct genomic DNA copy number changes, some of a marked enrichment of functional tumor-initiating cells (TICs). which are similarly altered in their cognate human subtype coun- These data show the utility of this heterogeneous tumor model and terpart. Finally, limiting dilution transplantation revealed that p53 provide functional data further demonstrating the stem cell char- null claudin-low tumors are highly enriched for TICs compared with acteristics of the claudin-low subtype. the more common adenocarcinomas arising in the same model, thus providing a unique preclinical mouse model to investigate the Results therapeutic response of TICs. p53 Null Tumors Show Variable Histology. Previously, we hypothe- sized that the heterogeneity observed in human BC might arise genetically engineered mouse model | gene profiling | array comparative not only because of activation of different oncogenes or loss of genomic hybridization specific tumor suppressor genes, but might also be dependent on the cell of origin in which these genetic changes occur (11). Ini- reast cancer (BC) is the second leading cause of cancer- tially transplanted p53 null mammary epithelial cells gave rise to Brelated deaths among women in the United States (1). The phenotypically normal ductal outgrowths, which then stochasti- large compendium of underlying genetic alterations and the re- cally developed mammary tumors. We therefore hypothesized sulting histological and molecular subtypes illustrate the hetero- that the deletion of this single tumor suppressor gene might give geneous nature of this disease. Both this intertumor heterogeneity rise to a spectrum of heterogeneous tumors, depending on the cell and the cellular heterogeneity found within a breast tumor (intra- of origin in which additional stochastic changes occurred. To test tumor heterogeneity) are major obstacles to effective treatments. this hypothesis, we collected 50 p53 null tumors that arose in wild- One common feature of BC (and most cancers) is the loss of the type BALB/c mice after transplantation of p53 null BALB/c tumor suppressor p53 function. p53 has been shown to be mutated mammary tissue into the cleared fat pads of 3-wk-old mice (6). in ≈40% of BCs, associated with poor clinical outcomes, and a higher frequency of mutations occurs in more-aggressive molec- ular subtypes, including the basal-like subtype of human BC (2). Author contributions: J.I.H., D.M., C.M.P., and J.M.R. designed research; J.I.H., W.Z., M.Z., Mice homozygous for p53 loss have been shown to develop J.U., G.M., D.E., J.K., S.B.G., D.D., M.A.T., S.G.H., and D.M. performed research; J.I.H., W.Z., M.Z., D.E., S.G.H., D.M., C.M.P., and J.M.R. contributed new reagents/analytic tools; J.I.H., lymphomas and sarcomas with a short latency (3, 4). When W.Z., M.Z., J.U., J.K., S.B.G., M.A.T., S.G.H., D.M., C.M.P., and J.M.R. analyzed data; and crossed into the BALB/c background, mammary tumors were J.I.H., W.Z., C.M.P., and J.M.R. wrote the paper. +/− observed in p53 mice (5). To circumvent the appearance of Conflict of interest statement: C.M.P. has an equity interest in University Genomics and other tumor types that occurred with short latency, the model was BioClassifier LLC. fi −/− further modi ed (6); namely, 6-wk-old p53 glands were re- This article is a PNAS Direct Submission. K.P. is a guest editor invited by the Editorial moved and transplanted into 3-wk-old wild-type BALB/c recipi- Board. ents. These mice develop mammary tumors stochastically with an Data deposition: The data reported in this paper have been deposited in the Gene Ex- average latency of approximately 12 mo. Interestingly, the p53 pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE27101). null epithelium initially forms normal ductal growths, which ex- 1To whom correspondence should be addressed: E-mail: [email protected]. hibit few genetic changes compared with wild-type outgrowths This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (7). Unlike many transgenic mouse models, the p53 null tumor 1073/pnas.1018862108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1018862108 PNAS Early Edition | 1of6 Like some other genetically engineered mouse model (GEMM) MMTV-Wnt1 tumors. Basal 2 tumors (8 of 50, 16%) clustered next mammary tumor models, the p53 null model gave rise to tumors to the Basal 1 tumors but showed a higher expression of the murine with a diversity of histological phenotypes (Fig. S1 and Dataset luminal cluster than did Basal 1 (Fig. 1C). Basal 1 p53 null tumors S1) (9, 12). Approximately 10% of the tumors contained a ma- showed an increased proliferation signature separating them from jority of spindle-shaped cells, a histology originally described for Basal 2 and the other p53 null subtypes (Fig. 1G and Fig. S2), and carcinosarcomas, now called EMT tumors (9). they also showed high p16 expression, which is a hallmark of im- paired RB1 function (15). Basal tumors (eight of nine) tested stain- p53 Null Tumors Cluster into Distinct Tumor Subtypes. In a previous ed positively for K5, as expected (Fig. S3 and Dataset S1); however, study we profiled 13 distinct mouse models, including the p53 null paradoxically, five of eight tested stained positively for the ER, of model (13). However, with only five p53 null tumor samples, we which four of five were of the Basal 2 subtype. were not able to appreciate the full spectrum of molecular het- Luminal. Eight of 50 p53 null tumors (16%) clustered close to- erogeneity represented in this mouse model. Now, with a total of gether and with the mouse luminal models MMTV-Neu and 50 tumors from the p53 null model, we see that these tumors MMTV-PyMT. As we have seen for other luminal models, these cluster into five distinct tumor subtypes when performing hier- tumors express luminal-specific genes like XBP1 but are missing archical clustering analysis using our previously defined mouse ER and estrogen responsive genes; accordingly, only one of eight intrinsic gene list (13) (Fig. 1); furthermore, we used SigClust of the luminal tumors stained positively for ER. Interestingly, like (14) to assess the significance of this clustering and objectively human luminal tumors, p53 null luminal tumors showed lower determined that the p53 null model did populate multiple sta- levels of p18INK4C, and p18 null mice develop predominantly tistically significant groups/subtypes, as follows.