Molecular Homology and Difference Between Spontaneous Canine Mammary Cancer and Human Breast Cancer

Published OnlineFirst July 31, 2014; DOI: 10.1158/0008-5472.CAN-14-0392

Cancer Microenvironment and Immunology Research

Molecular Homology and Difference between Spontaneous Canine Mammary Cancer and Human Breast Cancer

Deli Liu1, Huan Xiong1, Angela E. Ellis2, Nicole C. Northrup2, Carlos O. Rodriguez Jr3, Ruth M. O'Regan4, Stephen Dalton1, and Shaying Zhao1

Abstract Spontaneously occurring canine mammary cancer represents an excellent model of human breast cancer, but is greatly understudied. To better use this valuable resource, we performed whole-genome sequencing, whole-exome sequencing, RNA-seq, and/or high-density arrays on twelve canine mammary cancer cases, including seven simple carcinomas and four complex carcinomas. Canine simple carcinomas, which histologically match human breast carcinomas, harbor extensive genomic aberrations, many of which faithfully recapitulate key features of human breast cancer. Canine complex carcinomas, which are characterized by proliferation of both luminal and myoepithelial cells and are rare in human breast cancer, seem to lack genomic abnormalities. Instead, these tumors have about 35 chromatin-modification genes downregulated and are abnormally enriched with active histone modification H4-acetylation, whereas aberrantly depleted with repressive histone modification H3K9me3. Our findings indicate the likelihood that canine simple carcinomas arise from genomic aberrations, whereas complex carcinomas originate from epigenomic alterations, reinforcing their unique value. Canine complex carcinomas offer an ideal system to study myoepithelial cells, the second major cell lineage of the mammary gland. Canine simple carcinomas, which faithfully represent human breast carcinomas at the molecular level, provide indispensable models for basic and translational breast cancer research. Cancer Res; 74(18); 1–12. 2014 AACR.

Introduction population of pet dogs (70 million estimated in the United Spontaneous cancers in pet dogs represent one of the best States) provides a valuable resource facilitating basic and cancer models (1–8). First, these cancers are naturally occur- clinical research. Importantly, the dog genome has been > ring and heterogeneous, capturing the essence of human sequenced to a 7.6X coverage (11), yielding a genome assem- cancer, unlike genetically modiﬁed or xenograft rodent models. bly nearly as accurate as the mouse or rat genome (11, 12), Second, as companion animals, dogs share the same environ- unlike another companion animal, the cat. This makes many ment as humans and are exposed to many of the same genomic analyses possible with the dog but not with the cat. carcinogens. Indeed, environmental toxins, advancing age, and As in women, mammary cancer is among the most frequent obesity are also risk factors for canine cancer (1). Third, dogs cancers in female dogs. The annual incidence rate is estimated better resemble humans in biology, for example, similar telo- at 198 per 100,000 (1), which is comparable with the rate of 125 mere and telomerase activities (9) and frequent spontaneous per 100,000 for breast cancer in women in the United States epithelial cancers (1), unlike mice (10). Fourth, numerous (13). Mammary cancer is especially common in dogs that are anatomic and clinical similarities are noted for the same not spayed or are spayed after the second estrus, with the risk types/subtypes of cancer between the two species, and similar for malignant tumor development expected at 26% (1). How- treatment schemes are used (2–4). Furthermore, the large ever, unlike human breast cancer, canine mammary cancer is poorly characterized at the genome-wide level. For example, only ﬁve canine mammary cancer cases have recently under- gone approximately 2X whole-genome sequencing (WGS; 1Department of Biochemistry and Molecular Biology, Institute of Bioinfor- matics, University of Georgia, Athens, Georgia. 2College of Veterinary ref. 14), and a limited number have been analyzed with gene Medicine, University of Georgia, Athens, Georgia. 3School of Veterinary expression microarray (15–17). This drastically differs from Medicine, University of California, Davis, California. 4The Winship Cancer Center, Emory School of Medicine, Atlanta, Georgia. their human counterparts, where thousands of breast cancer genomes and transcriptomes are characterized, with several Note: Supplementary data for this article are available at Cancer Research – Online (http://cancerres.aacrjournals.org/). studies cited here (18 23). Like other cancer types, many anatomic and clinical simi- Corresponding Author: Shaying Zhao, Department of Biochemistry and Molecular Biology, Institute of Bioinformatics, University of Georgia, B304B larities are documented between canine mammary cancer and Life Sciences Building, 120 Green Street, Athens, GA 30602-7229. Phone: human breast cancer (24). Various molecular homologies are 706-542-9147; Fax: 706-542-1738; E-mail: [email protected] also reported, for example, WNT signaling alteration (15, 17). doi: 10.1158/0008-5472.CAN-14-0392 Meanwhile, canine mammary cancer also differs from human 2014 American Association for Cancer Research. breast cancer in certain aspects. For example, dogs have only

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one or two estrous cycles a year followed by a prolonged luteal RNA were then extracted from the dissected tissues using the phase, which is 63 days for the dog compared with 14 days for DNeasy Blood & Tissue Kit (cat. no. 69504), RNeasy Plus Mini the human. During this unusually long luteal phase, the Kit (cat. no. 74134), or AllPrep DNA/RNA Mini Kit (cat. no. mammary gland is continuously exposed to high levels of 80204) from Qiagen. Only samples with a 260/280 ratio of progesterone (24). Another variation that may be related to approximately 1.8 (DNA) or approximately 2.0 (RNA) and this hormonal difference is described below. showing no degradation and other contaminations on the Mammary gland epithelium consists of an inner layer of agarose gels were subjected to further analyses. The synthesis luminal cells and an outer layer of myoepithelial cells that of cDNA, primer design, and PCR or qPCR with genomic DNA or border the basal lamina. Compared with luminal cells, myoe- cDNA samples were conducted as described (6). Primers used pithelial cells are significantly understudied and poorly char- are listed in the Supplementary Methods. acterized (25–30). Although their importance in mammary gland development and pathogenesis has been noted Array comparative genomic hybridization analyses (26, 29, 31), myoepithelial cells have traditionally received far Array comparative genomic hybridization (aCGH) experi- less attention than luminal cells. This is at least partially ments were conducted at the Florida State University Micro- because they rarely proliferate in human breast cancer (32, array Facility, with 385 K canine CGH array chips from Roche 33). However, in canine mammary cancer, myoepithelial cell NimbleGen Systems, Inc. Copy-number abnormalities (CNA) proliferation is much more common, occurring in >20% canine were identified as described (5). tumors compared with <0.1% human tumors (34). To more effectively use this unique feature of canine mammary cancer Paired-end WGS, whole-exome sequencing, and RNA-seq for a better understanding of myoepithelial cells, we set out to All three types of sequencing were conducted using the comprehensively compare spontaneous canine mammary can- Illumina platform, following the protocols from the manufac- cers with and without myoepithelial cell proliferation and to turer. Paired-end WGS of >12X sequence coverage was per- evaluate their molecular similarities to and differences from formed in collaboration with the Emory Genome Center (50 bp human breast cancers. or 100 bp paired-end sequencing of 200 bp fragments) or the BGI-America (90 bp paired-end sequencing of 500 bp frag- Materials and Methods ments). Whole-exome sequencing (WES) was conducted in Canine mammary tissue samples collaboration with the Hudsonalpha Institute for Biotechnol- Fresh-frozen (FF) and formalin-fixed paraffin-embedded ogy (Huntsville, AL). First, exome-capturing was achieved by (FFPE) normal and tumor tissue samples of spontaneous using a solution-based SureSelect Kit from Agilent, covering 50 canine mammary cancer were obtained from the University Mb canine exons and adjacent regions. Then, paired-end of California-Davis School of Veterinary Medicine (Davis, CA) sequences of 50 bp of approximately 200-bp fragments were and the Animal Cancer Tissue Repository of the Colorado State generated from the captured targets to reach the coverage of University (Fort Collins, CO). Samples were collected from 134X to 245X. RNA-seq was performed at Hudsonalpha, yield- client-owned dogs that develop the disease spontaneously, ing 42 to 94 million paired-end sequence reads of 50 bp per under the guidelines of the Institutional Animal Care and Use sample. Committee and with owner informed consent. The breed, age, Sequence data analyses histopathologic descriptions, and other information are pro- Sequence read alignment, mutation discovery, translocation vided in Supplementary Table S1. and chimeric fusion gene identification, clustering, and other fl Immunohistochemical analyses analyses are provided in the Supplementary Methods. Brie y, Immunohistochemical (IHC) experiments were performed WGS, WES, and RNA-seq sequence reads were aligned to the following standard protocols with 5-mm FFPE sections. Pri- dog reference genome (11). Then, uniquely mapped WES reads mary antibodies were used as described (35), including those were used to detect base substitutions and small indels, and fi fi against smooth muscle myosin heavy chain clone ID8 signi cantly mutated genes were identi ed as described (20). (MAB3568), acetyl-H4 (06-866), H3K9me3 (07-442), and H3- Uniquely mapped WGS read pairs were used to identify somatic K4/K9-me3 (06-866), all from Millipore; H3K4me3 (Abcam; translocations and chimeric fusion genes. Uniquely mapped ab8580), estrogen receptor alpha clone E115 (Abcam; ab32063); RNA-seq reads were used to quantify each gene's expression and E-cadherin (R&D Systems; AF648). Alexa Fluor488–, 647– level, as well as to detect chimeric fusion transcripts and or 594–conjugated secondary antibodies are from Jackson sequence mutations. ImmunoResearch. Images were taken with a Zeiss LSM 710 Results confocal microscope. Canine simple carcinomas have no myoepithelial cell Tissue dissection, DNA and RNA extraction, and PCR proliferation, whereas canine complex carcinomas have analyses luminal and myoepithelial cells, both proliferating Cryosectioning of FF tissues, hematoxylin and eosin (H&E) The 12 cases subjected to genome-wide characterization staining, and cryomicrodissection were performedas described represent two major histologic subtypes of canine mam- (5) to enrich tumor cells for tumor samples and mammary mary cancer (34), five with myoepithelial cell proliferation gland epithelial cells for normal samples. Genomic DNA and (complex carcinomas) and 7 without (simple carcinomas;

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Supplementary Table S1). Tumor cells in simple carcinomas mentary Tables S2A–S2C), with the extensiveness of CNA in express only luminal markers such as E-cadherin (Fig. 1A), correlation with the tumor progression stage. The only excep- and histologically match typical human breast carcinomas tion to this is an inflammatory carcinoma, in which no CNAs (Fig. 1A and C). Tumors with myoepithelial cell proliferation were detected. Second, CNAs also occurred in genomic sites include four complex carcinomas, a subtype that is rare recurrently altered in human breast cancer, for example, in humans (32), and one carcinoma with two distinct amplification of human 8q and dog chromosome 13 that histologic regions, one considered simple and the other encode genes including MYC (Fig. 2A). Third, although large considered complex. Complex carcinomas have prominent deletions were discovered, one resulting in PTEN loss (Fig. 2A; expression of both the luminal marker E-cadherin and the Supplementary Table S2G), amplifications prevailed over dele- myoepithelial marker smooth muscle myosin heavy chain tions in most tumors. Notably, two large amplicons of >4 Mb, (SMHC; Fig. 1B), indicating dual proliferation of luminal and harboring 54 and 43 genes, respectively, were uncovered (Fig. myoepithelial cells. This is also visible in H&E-stained sec- 2C). This led to amplification and overexpression of oncogenes tions (Fig. 1D). Besides this histologic difference, the tumors such as BRAF, PIM1, and CCND3 (Supplementary Tables S2E also vary in cancer progression stages (in situ,invasive,or and S2F). metastatic to the lung) and in estrogen receptor (ER) þ expression (five ER tumors and seven ER tumors; Sup- Translocations and a superamplicon were discovered in plementary Table S1). a canine simple carcinoma by paired-end WGS To further explore the two >4 Mb amplicons described CNAs are frequent in canine simple carcinomas above, we sequenced the tumor and normal genomes of case Reminiscent of human breast cancer, canine simple carci- 76 (Fig. 2A) to a >15X sequence coverage (Supplementary Fig. noma genomes harbor extensive CNAs. First, we observed both S1 and Supplementary Table S2D). For comparison purposes, focal and broad CNAs totaling from 10 Mb to >100 Mb and similar sequencing was performed on the case having the most affecting hundreds of genes per tumor (Fig. 2A and B; Supple- extensive CNAs (case 406434, with pulmonary metastasis) and

Merge DAPI Merge DAPI Merge DAPI Merge DAPI Merge DAPI Merge DAPI Merge DAPI Merge DAPI

SMHC E-cad SMHC E-cad SMHC E-cad SMHC E-cad SMHC E-cad SMHC E-cad SMHC E-cad SMHC E-cad

Merge Merge Merge Merge Merge Merge Merge Merge CD

Figure 1. Myoepithelial cell proliferation is absent in canine simple carcinomas but prominent in canine complex carcinomas. A and B, representative images of immunostaining with the myoepithelial marker SMHC and the luminal marker E-cadherin (E-cad) of normal (N) and tumor (T) tissues of two simple carcinoma cases (A), one in situ (ID 159) and the other invasive (ID 401188), and two complex carcinomas (B). Top, enlarged view of the areas indicated below. Red arrows, luminal cells; yellow arrows, myoepithelial cells; scale bar, 100 mm. C and D, H&E staining of the same tissues.

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ABComplex carcinomas 1 600 Amp Del 10 300 20 of No. 0 Amp/Del Genes 401130 402421 403802518 32510 115 30 115 Complex carcinomas Half simple/half 401130 402421 403802 518 32510 Inflammatory X complex carcinoma Inflammatory 600

MYC 300 BRAF No. of No.

0 PTEN Amp/Del Genes 159 401188 76 5 341400 406434 159 401188 76 5 341400 406434 Simple carcinomas

CDWGS Chr12 Chr16 Chr12: 9,098,400-14,228,400 Chr16: 9,683,000-13,988,600 Normal Normal Amplification 150 MPD 0 0 150 9,098,339 10,091,064 14,228,400 Tumor Tumor Chr12

MPD Chr16 0 0 9,683,709 10,055,887 13,988,600 Tumor vs. Normal aCGH Tumor vs. Normal

-T/N Amplification 2 –3 0 3 150 –3 0 3 150

Log 0 20M 40M 60M 0 20M 40M 60M EF Chr12 (+) ZFAND3 N C ZFAND3 A20-type AN1-type Chr16 (–) Transmembrane MGAM Gene fusion MGAM CN Glucoamylase P-type IIMaltase P-type I Lumenal Splicing Fusion N C gene A20-type Glucoamylase

Figure 2. Large-scale genomic aberrations are frequent in canine simple carcinomas but rare in canine complex carcinomas. A, CNAs found in four complex carcinomas (labeled), one half complex and half simple carcinoma (ID 32510), and seven simple carcinomas (which include the inflammatory tumor 115 and the six tumors at the second panel) by aCGH. The images were drawn as described (5), with each line representing a canine chromosome and vertical lines above/ below the chromosome indicating amplifications/deletions, respectively. Notable amplified/deleted genes are shown. B, the total numbers of amplified (shaded bars) and deleted (empty bars) genes of each carcinoma shown in A. C, two >4 Mb amplicons discovered in simple carcinoma 76 in A, by both WGS and aCGH. The x-axis indicates chromosomal coordinates in Mb, whereas the y-axis indicates the mapped read-pair density (MPD) values of WGS or the tumor against normal log2 ratios of aCGH. D, the proposed mechanism for superamplicon formation. Prior sequence amplifications led to two translocations (represented by the dashed lines), resulting in a circle, which was further amplified. The numbers indicate the chromosomal coordinates in bp. E, a fusion gene created by the second translocation shown in D. The translocation occurred in the intron of both genes as indicated (exons are represented by the vertical bars). An in-frame fusion transcript then emerged via splicing. F, the A20-type domain of ZFAND3 and the glucoamylase domain of MGAM are preserved in the fusion protein.

another case having hardly any CNAs (case 32510). WGS gene PIM1 and 17 other genes (Supplementary Tables S2E and revealed fewer translocations and inversions than CNAs in S2F), was then further amplified. these tumors. Furthermore, reminiscent of the human breast cancer MCF7 genome (36), some translocations are associated The superamplicon harbors a potentially oncogenic with amplification, creating a superamplicon with loci from fusion gene, ZFAND3–MGAM, created via a translocation different chromosomes colocalized and coamplified (Fig. 2D). The superamplicon also harbors a newly created fusion On the basis of chimeric sequence reads that span the gene. It consists of the first four exons of ZFAND3, a zinc translocation junctions (Supplementary Table S2H) and PCR finger gene located on chromosome 12, and the last 22 to 49 confirmation (Supplementary Fig. S1), we propose a mecha- exons of MGAM, which encodes maltase-glucoamylase and is nism for the superamplicon formation (Fig. 2D). First, a circle, located on chromosome 16 (Fig. 2E). The fusion gene, termed consisting of approximately 1-Mb sequences from chromo- ZFAND3-MGAM, arose from a translocation occurring in some 12 and approximately 0.4 Mb from chromosome 16, introns; transcription and splicing then yielded an in-frame emerged via two translocations that were likely facilitated by fusion transcript. This was confirmed by the detection of prior sequence amplification. The circle, which harbors onco- chimeric sequence fusion points via WGS, RNA-seq, and PCR

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analyses (Supplementary Fig. S1 and Supplementary Table of four simple carcinoma cases to 134X to 245X coverage S2H). (Supplementary Fig. S2 and Supplementary Table S3A). This As a result of in-frame fusion, the A20-type zinc finger analysis again revealed several dog–human homologies. First, domain of ZFAND3 and the glucoamylase domain of MGAM base transitions, particularly C ! T/G ! A changes, dominate are kept intact in the fusion product (Fig. 2F). This seems to be base transversions in most tumors (Fig. 3A), indicating similar significant. First, the A20 zinc finger protein has been reported mutation mechanisms (e.g., deamination of 5mC to T) in both to inhibit tumor necrosis factor–induced apoptosis (37). Sec- the species. The only exception (tumor 406434) has C ! A/G ! ond, MGAM, an integral membrane protein with its catalytic T transversions predominating, which is not an experimental domains facing the extracellular environment, is normally artifact of WES (39) based on our analyses (Supplementary expressed in the intestine to digest starch into glucose (38). Table S3F), and concurrently harbors an altered POLD1. This, Indeed, we did not detect MGAM expression in normal mam- likewise, is consistent with human cancer studies that link C ! mary tissues, unlike ZFAND3 (Supplementary Fig. S1C). How- A/G ! T changes to POLD1 mutations (40). Second, the ever, in carcinoma 76, both MGAM and ZFAND3–MGAM are mutation rate varies greatly among the carcinomas, with tumor amplified and overexpressed. ZFAND3–MGAM, which lacks 5 having 907 genes significantly mutated, compared with 0 to 31 the transmembrane domain and becomes intracellular, could genes for tumors of similar or more advanced stages (Fig. 3A). promote oncogenesis by accelerating carbohydrate metabo- This hypermutation is likely linked to defective DNA repair as lism via its glucoamylase domain and meanwhile inhibiting well, because tumor 5 has as many as 24 DNA repair-associated apoptosis via its A20-type domain. Of course, whether this is genes mutated (Supplementary Table S3D). Third, many true or not requires further studies. known human breast cancer genes are also mutated in these canine tumors (Supplementary Tables S3B and S3C), including Somatic sequence mutations are frequent in canine BRCA1, IGF2R, FOXC2, DLG2, and USH2A as described below. simple carcinomas as revealed by WES USH2A is one of the most significantly mutated genes in our To examine somatic base substitutions and small indels, we study (P ¼ 2.78E12), having one nonsense-, 12 missense-, and performed WES on the matching tumor and normal genomes three synonymous mutations (Fig. 3B; Supplementary Table

AD1 C T/G A C G/G C C A/G T GO:0016568~Chromatin modification 1.86×10–8 0.5 T C/A G • GO:0016570~Histone modification 2.15×10–4 POLD1 T G/A C GO:0045449~Regulation of transcription 1.17×10–8 alteration T A/A T GO:0006396~RNA processing 2.40×10–7 0 GO:0006281~DNA repair 2.62×10–5 ID 406434 76 159 5 GO:0007049~Cell cycle 0.006 Significantly 31 3 0 907 24 DNA repair genes mutated genes GO:0030198~Extracellular matrix organization 1.55×10–4 (BRCA1, etc.) GO:0019838~Growth factor binding 1.74×10–4 GO:0005976~Polysaccharide metabolic process 0.002 5

B 76 518 USH2A 115 159 GO:0007155~Cell adhesion 0.020 403802 402421 401130 406434 341400 401188 Laminin Laminin Transmembrane Complex Simple EGF-like G-like carcinomas carcinomas Laminin N- Fibronectin Fibronectin Cytoplasmic terminal type III type III Histone methyltransferases EHMT1, EHMT2, MLL2, MLL4, SETD1A, or associated factors SETDB1, SMYD5, SUZ12

Histone demethylases JMJD1C, PHF2 C P = 0.02 P = 0.03 Histone methylation readers CHD6, MSL3 300 P = 0.73 Histone acetyltransferases or CREBBP, CSRP2BP, ING3, KAT2A, MSL1, associated factors MSL2, NCOA3

Histone deacetylase SIRT1 150 Histone acetylation readers BRD3, BRD4

Number of Histone ubiquitination HUWE1, UBR5

Histone deubiquitination ATXN7L3, BAP1, UIMC1, USP16, USP21, USP36

coding mutatioins per Mb coding mutatioins 0 Simple Complex Normal Other chromatin remodelers ASF1A, ARID1B, BCORL1, DNMT3B, SUPT6H carcinomas carcinomas samples

Figure 3. Coding sequence mutations are frequent in canine simple carcinomas; chromatin-modification genes are downregulated in canine complex carcinomas. A, the fractions (the y-axis) of somatic base substitution types of simple carcinomas (IDs indicated by the x-axis) detected by WES. The total number of significantly mutated genes in each tumor is also shown, and tumor 5 has many DNA repair genes mutated. B, synonymous (green dots) and nonsynonymous substitutions (yellow dots), and a nonsense mutation (red star) uncovered in the USH2A gene in tumor 5. C, the base substitution (compared with the dog reference genome) rates of the three sample types in coding regions with 30X to 300X RNA-seq read coverage. The P values were calculated by t tests. D, the heatmap of 751 genes differentially expressed at FDR 0.2 between simple and complex carcinomas (red, upregulation; green, downregulation). Right, enriched functions of each gene cluster indicated; bottom, the 35 chromatin modifiers downregulated in complex carcinomas.

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S3G). Critically, USH2A is also prominently mutated in human downregulated in complex carcinomas. These include H3K9 breast cancer, ranking as the 21st most significantly mutated methyltransferase genes SETDB1, EHMT1, EHMT2, and SUZ12, gene in the Cancer Genome Atlas (TCGA) study (23). USH2A along with the demethylase genes JMJD1C and PHF2 (Supple- alterations may contribute to mammary cancer pathogenesis mentary Table S4E). Meanwhile, we also examined H4-acety- in both dogs and humans. lation because at least eight of the downregulated genes (CREBBP, CSRP2BP, ING3, KAT2A, MSL1, MSL2, NCOA3, and Canine complex carcinomas have hardly any genomic SIRT1) are involved in histone acetylation or deacetylation. CNAs and their sequence mutation rates also appear low Another active modification, H3K4me3, was studied as well Unlike simple carcinomas, we observed very few genomic because the H3K4 methyltransferase genes SETD1A, MLL2, and CNAs in complex carcinomas, making their genomes appear MLL4 are among those downregulated. normal (Fig. 2A; Supplementary Table S2A). Their sequence mutation rates are also low, based on our analysis with RNA- In canine normal mammary glands, both active and seq data. Briefly, to achieve a more accurate mutation finding, repressive histone modifications are significantly we used only regions with 30X to 300X RNA-seq read coverage, depleted in myoepithelial cells when compared with which distribute across the genome and amount to 4.5 to 9.4 luminal cells Mb sequence per sample (Supplementary Table S3I). The To understand the alteration in cancer, we first investigated analysis indicates that the mutation rates of complex carci- canine normal mammary glands where both luminal and nomas are significantly lower than those of simple carcinomas, myoepithelial cells are clearly visible. These include the normal but are comparable with those of normal mammary gland tissue from case 159, where myoepithelial cells form a nearly tissues (Fig. 3C). continuous layer surrounding the luminal cells (159N in Fig. 1A), and case 402421, where myoepithelial cells are not as Numerous chromatin-modification genes are prominent but are still noticeable (402421N in Fig. 1B). Inter- downregulated in canine complex carcinomas estingly, in these normal glands, active modifications, H4- RNA-seq analysis revealed 751 total genes differentially acetylation and H3K4me3, and repressive modification expressed at FDR 0.2 between simple and complex carci- H3K9me3 are both significantly depleted in the myoepithelial nomas (Fig. 3D). Strikingly, among these genes, chromatin cells (Fig. 4A; Supplementary Fig. S4), with the intensity modification and transcription regulation are the most signif- reduced by half in most cases (Fig. 4B), when compared with icantly enriched functions (Supplementary Tables S4A–S4C). the luminal cells. Indeed, a total of 35 known chromatin-modification genes were found to be downregulated in complex carcinomas (Fig. In canine complex carcinomas, active modification H4- 3D; Supplementary Table S4E), and more than 40% of them acetylation is abnormally enriched, whereas repressive remain so at P 0.05 when further compared with normal modification H3K9me3 is aberrantly depleted mammary gland tissues. Moreover, chromatin modification Compared with normal mammary glands and simple car- stays as the most significantly enriched function amid genes cinomas, complex carcinomas harbor significantly more (327 in total) differentially expressed among the three types of myoepithelial cells (Fig. 1). Yet, unlike normal mammary samples (Supplementary Fig. S3 and Supplementary Table S4H glands (Fig. 4), both myoepithelial and luminal cells in complex and S4I). The same overall conclusions were reached at FDR carcinomas were found to be equally enriched with active 0.1 (Supplementary Table S4F and S4G). modifications (Fig. 5A–H; Supplementary Fig. S5 and Supple- Amid the 35 chromatin genes downregulated in complex mentary Table S5). This is especially so for H4-acetylation, with carcinomas, 30 encode histone modifiers, covering methyla- the intensity being equal or stronger than luminal cells in tion and demethylation, acetylation and deacetylation, and normal mammary glands and in simple carcinomas. The ubiquitination and deubiquitination (Fig. 3D). Intriguingly, repressive modification H3K9me3, to the contrary, becomes both active and repressive modifiers were noted (see the significantly more depleted in both cell types in complex paragraph that follows). Furthermore, the identified histone carcinomas (Fig. 5I–L; Supplementary Fig. S5 and Supplemen- acetyltransferases and deacetylase modify histones H3, H4, and tary Table S5). H2A, influencing not only gene transcription (e.g., CREBBP), þ but also chromatin packing (e.g., MSL1 on H4K16 acetylation; Redox genes are upregulated in canine ER carcinomas, ref. 41). Besides histone-modification genes, other types of whereas cell cycle and DNA repair genes are upregulated chromatin-remodeling genes were also found downregulated in canine ER carcinomas in complex carcinomas (Fig. 3D), most of which (e.g., ARID1B, RNA-seq analyses also revealed a clear difference between þ þ ASF1A, and DNMT3B) are known to be mutated in human canine ER and ER tumors(Fig.6A),withmostER tumors cancers (42, 43). being complex carcinomas, whereas most ER tumors being To understand the significance of the observed change in simple carcinomas. Among the 1,350 differentially expressed chromatin-modification genes, many encoding histone modi- genes at FDR 0.2 (Supplementary Table S6A), approxi- þ fiers (Fig. 3D), we investigated histone modification. Specifi- mately half are upregulated in ER carcinomas and are cally, we performed IHC experiments to examine H3K9me3, a significantly enriched in redox functions (Fig. 6B; Supple- repressive modification that is associated with gene silencing mentary Table S6B, S6C, and S6E). These genes encode and heterochromatin and for which six relevant genes are approximately 25 dehydrogenases or oxidases, and 32 gene

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Figure 4. In canine normal mammary glands, active and repressive histone modifications are both depleted in myoepithelial cells when compared with luminal cells. A, representative IHC images of active modifications acetyl-H4 and H3K4me3, repressive modification H3K9me3, and modification H3-K4/K9-me3 (positive only when H3K4me3 and H3K9me3 are present simultaneously). Yellow arrows, myoepithelial cells; red arrows, luminal cells; scale bar, 100 mm. B, the intensity of each histone modification was measured from 10 individual cells of each type from different regions across the tissue section. The P values were calculated by Wilcoxon tests.

products are associated with mitochondria, including the 82 of 100 times, all canine simple carcinomas and the ER electron transport chain. Another half of the 1,350 differen- complex carcinoma (ID 518) group with the human basal-like þ tially expressed genes are upregulated in ER carcinomas, tumors. The remaining canine complex carcinomas (all ER ), among which approximately 118 genes are associated with however, fail to cluster with any specific human subtypes. One the cell cycle, for example, mitosis, spindle, microtubule clustering example is shown in Fig. 6C and Supplementary cytoskeleton, etc. (Fig. 6B; Supplementary Table S6D and Fig. S6B. S6F). Other significant functions comprise DNA repair (38 genes) and protein serine/threonine kinase activity (17 Discussion genes). The same overall conclusions were reached at FDR In this study, we performed an initial comprehensive char- 0.1 (Supplementary Fig. S6A). acterization of the genomes, transcriptomes, and epigenomes of two major canine mammary cancer histologic subtypes. Canine simple carcinomas and the ER complex Even with a small sample size (12 cases), the analysis reveals a carcinoma cluster closely with basal-like human breast remarkable molecular heterogeneity of spontaneous canine carcinomas in PAM50 classification mammary cancers. It also emphasizes their unique value and To directly compare the canine mammary cancers with raises a number of important questions that could profoundly human breast cancers, we randomly selected 20 human tumors affect human breast cancer research. for each subtype among a total of 195 luminal A, 111 luminal B, 53 HER2-enriched, and 87 basal-like tumors of the TCGA RNA- Canine simple carcinomas, without myoepithelial cell seq study (23). This, along with all seven normal-like tumors in proliferation, harbor extensive genomic aberrations and TCGA, amounts to 87 human carcinomas covering all five are molecularly homologous to human breast intrinsic subtypes. We then performed PAM50 clustering (44) carcinomas on these 87 human carcinomas together with our 12 canine Canine simple carcinomas investigated have no myoepithe- carcinomas. This analysis was repeated 100 times, ensuring lial cell proliferation and are histologically comparable with that each TCGA tumor was sampled at least once. Notably, in human in situ or invasive ductal or lobular carcinomas.

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Figure 5. In canine complex carcinomas, active histone modification H4-acetylation is enriched, whereas repressive modification H3K9me3 is depleted in both luminal and myoepithelial cells. A–C, representative IHC images of acetyl-H4 of simple carcinomas (A), complex carcinomas (B), and normal mammary glands (C). The merged (top) and split images (acetyl, acetyl-H4) are shown; scale bar, 100 mm. D, the immunofluorescence staining intensity of acetyl-H4 determined from at least three different areas of the first split image, labeled as "Acetyl" in A–C. The pairwise comparison P values were calculated by Wilcoxon tests. E–H, for H3K4me3 (K4) and I-L for H3K9me3 (K9) are presented in the same way as A–D. Unlike the 159N sample of Fig. 4A, the normal mammary glands in C, G, and K consist of mostly luminal cells, with myoepithelial cells either absent (401188N) or very few (402421N).

Significantly, these canine cancers faithfully recapitulate key human clinical trials, can significantly speed up new antican- molecular features of human breast cancer. First, analogous to cer drug development. For example, for drugs targeting the their human counterparts (18–23, 36), the genomes of these PI3K pathway (Fig. 7C; ref. 48), their efficacy, toxicity, dosage, canine carcinomas harbor extensive genetic lesions, including and schedule can be more accurately evaluated through clin- numerous CNAs, fusion gene-creating translocation, equally ical trials with canine patients, before entering human clinical complex superamplicon, and comparable sequence mutation trials. This will significantly reduce the cost and accelerate the types. The only exception is an inflammatory carcinoma, drug discovery process. whose human equivalent (inflammatory breast cancer) is also devoid of CNAs (21). Second, notable human breast cancer Can canine simple carcinomas serve as a much-needed genes (18–20, 23, 45, 46) are altered in these canine cancers as spontaneous cancer model of basal-like human breast well. Examples include (i) amplification and/or overexpression cancer? of the oncogenes BRAF, MYC, PIK3CA, PIK3R1, CCND3, and Canine simple carcinomas cluster with basal-like human TBX3; (ii) deletion and/or underexpression of tumor suppres- tumors with an 82% chance in our PAM50 classification, sors PTEN, PTPRD, and CDH1 (47); and (iii) mutations of indicating their closer resemblance to this subtype than other BRCA1, NF1, MAP3K1, and RUNX1 (Supplementary Table intrinsic subtypes. This may be explained by the observation S7B). Third, many of the altered pathways are shared between that none of the canine tumors carry HER2 amplification or the two species, for example, cell adhesion, Wnt signaling, PI3K overexpression. Furthermore, many harbor extensive CNAs signaling, and DNA repair (Fig. 7C; Supplementary Table S7; and are ER with genes related to DNA repair and cell cycle ref. 23), consistent with other canine mammary cancer studies significantly upregulated, consistent with the basal-like sub- (15, 17). type profile (23). This is especially so considering that the ER þ These strong molecular homologies make canine simple complex carcinoma clusters similarly as well. The only ER carcinomas valuable in human breast cancer research. For canine simple carcinoma has a prominent PTEN deletion, also example, for cancers with large genomic CNAs, we can apply a feature of basal-like tumors (23). the dog–human comparison strategy for effective driver-pas- Clearly, studies with a larger sample size are needed to senger discrimination as described (7). Critically, as elegantly determine if canine simple carcinomas as a whole or even just a discussed in several publications (2–4), these canine cancers, subset do indeed closely match the basal-like subtype. If which bridge a gap between traditional rodent models and confirmed, these canine cancers could serve as a much-needed

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A ER+ ER–

Figure 6. Subtype analysis reveals homology between canine B GO:0055114~Oxidation reduction 1.31×10–4 mammary cancers and human GO:0046395~Carboxylic acid catabolic process 3.11×10–4 breast cancers. A, examples þ –4 of ER (ID 401188) and ER (ID GO:0051188~Cofactor biosynthetic process 7.33×10 341400) canine carcinomas GO:0015031~Protein transport 0.003 determined by IHC; scale bar, GO:0005524~ATP binding 9.91×10–5 100 mm. B, genes (1,350 total) GO:0006260~DNA replication 0.002 differentially expressed between þ GO:0008610~Lipid biosynthetic process 0.002 ER and ER canine carcinomas at FDR 0.2. The tumor IDs are GO:0005524~ATP binding 8.16×10–4 indicated. Tumor 401188 (purple) GO:0042325~Regulation of phosphorylation 0.012 þ is ER but a simple carcinoma, GO:0005739~Mitochondrion 0.016 whereas tumor 518 (orange) is ER –45 but a complex carcinoma. Right, GO:0007049~Cell cycle 3.12×10 signiﬁcantly enriched functions of GO:0006260~DNA replication 4.88×10–15 each gene cluster indicated. C, GO:0006281~DNA repair 4.15×10–8 canine simple carcinomas and the 5 GO:0004672~Protein kinase activity 0.006

76 518 115 ER complex carcinoma cluster 159 GO:0030695~GTPase regulator activity 0.028 with the basal-like human breast 32510 402421 401188 403802 401130 406434 341400 carcinomas in PAM50 + – classiﬁcation. The heatmap ER ER represents a clustering example of C 12 canine tumors and 87 TCGA C1 C2 C3 C4 C5 human tumors (see text). The C1 Human: All basal-like (20*) composition of each cluster Dog: All simple carcinomas and ER– complex speciﬁed at the top of the heatmap carcinoma (ID 518) is explained at the right side. C2 Human: Luminal A (9) and luminal B (1)

Human: Luminal A (3), luminal B (2) and normal-like (2) C3 Dog: Half simple/half complex carcinoma and the rest complex carcinomas, all ER+

C4 Human: HER2-enriched (16) and luminal B (5)

C5 Human: HER2-enriched (4), luminal A (8), luminal B (12) and normal-like (5)

Simple Complex * Number in () represents the total number of tumors of subtype indicated.

spontaneous cancer model. Compared with other subtypes, sequence mutation rates as low as normal tissues. Thus, it is basal-like cancers are aggressive, have a poor prognosis, and unlikely that these tumors arise from genetic aberrations, currently lack effective treatments. Canine mammary cancer unlike simple carcinomas. Meanwhile, complex carcinomas could make significant contributions toward understanding could acquire genomic changes as they progress to later stages, and treating this worst subtype of human breast cancer. as shown by tumor 518 (Supplementary Table S2A) and another complex carcinoma with pulmonary metastasis Canine complex carcinomas, with myoepithelial cell (14). Second, chromatin modification and transcription regu- proliferation, appear to originate from epigenomic lation stand out as the most enriched functions among genes rather than genomic alterations differentially expressed between simple and complex carcino- Complex carcinomas, featuring dual proliferation of luminal mas, with numerous chromatin-modification genes downre- and myoepithelial cells, likely originate from epigenomic, gulated in complex carcinomas. Importantly, complex carci- rather than genomic, abnormalities (Fig. 7). First, their gen- nomas are aberrantly enriched with the active histone mod- omes appear normal without CNAs detected and with ification H4-acetylation, whereas abnormally depleted with the

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AB Altered stem cell/ Tumor ID Chromatin H3K9me3 depletion common progenitor Complex carcinoma – modification Aberrant epigenome Stem cell 518, ER carcinogenesis genes altered SOX2 Acetyl-H4 enrichment H3K4me3 401130, ER+ Epigenomic abnormalities? Common 402421, ER+ progenitor 403802, ER+ 32510, ER+ H3K9me3 down- H3K4me3 regulation Complex carcinoma Acetyl-H4 Myoepithelial Luminal progenitor progenitor C Ref-MEK-ERK 341400, ER– PI3K/AKT BRAF – 406434, ER PIK3CA 76 – 5, ER Genomic 76 DNA repair + gene mutations 401188, ER aberrations PTEN MEK 1/2 WNT Myoepithelial Alveolar Ductal – 76, ER 401188 CTNNB1 MYC BRCA1 – Simple carcinoma 159, ER AKT ERK 1/2 406434 406434 5

Simple carcinoma carcinogenesis

Figure 7. Canine complex carcinomas possibly arise from epigenomic alterations, whereas canine simple carcinomas likely originate from genomic aberrations. A, the proposed carcinogenic mechanism. The mammary gland development hierarchy is modiﬁed from a publication (50). B, epigenomic alterations in complex carcinomas, with histone modiﬁcations enriched (darker shading) or depleted (lighter shading). C, genomic alterations in simple carcinomas, with notable gene and pathway alterations (activation, darker shading; inactivation, lighter shading) indicated in the respective tumors (e.g., PTEN deletion in tumor 401188).

repressive modification H3K9me3. Thus, it is possible that the cates that proliferating luminal and myoepithelial cells in epigenomes of complex carcinomas are altered, turning on complex carcinomas may have derived from altered com- genes that normally should be silenced to promote tumori- mon progenitors. Third, compared with simple carcinomas genesis. Obviously, more studies are needed to confirm this and normal mammary tissues, glucose metabolic genes are possibility and to understand the underlying mechanisms. upregulated in complex carcinomas, consistent with this Myoepithelial cell proliferation is rare in human breast stem cell or progenitor origin theory. For simple carcinomas, cancer (32, 33). As a result, myoepithelial cells receive far less we hypothesize that they originate from either luminal attention than luminal cells and are poorly understood progenitors, because of their close resemblance to the (25, 27, 28, 30). However, the few laboratories that study basal-like subtype, which is reported to have a luminal myoepithelial cells have noted their importance. For exam- progenitor origin (49), or differentiated luminal cells because ple, myoepithelial cells are thought to be a part of the of luminal cell properties (see case 159 in Fig. 1). Of course, mammary stem cell niche, mediate the cross-talk between further studies with a larger sample size are needed to test luminal cells and extracellular matrix, contribute to the these hypotheses. maintenance of the apicobasal polarity of luminal cells, and In summary, we performed the first comprehensive char- serve as a tumor suppressor (26, 29, 31). Canine mammary acterization of the genomes, transcriptomes, and epigen- cancer, in which myoepithelial cell proliferation is much omes of canine simple carcinomas and complex carcinomas, more common, provides an ideal system to address such two major histologic subtypes of canine mammary cancer. functions and to better understand the second major cell The analysis reveals that canine simple carcinomas, which lineage of the mammary gland (e.g., by answering questions have no myoepithelial cell proliferation and histologically such as whether a prolonged luteal phase promotes myoe- match typical human breast carcinomas, faithfully recapit- pithelialcellproliferation). ulate many molecular features of human breast cancer. Notably, canine simple carcinomas closely cluster with Do canine complex carcinomas derive from mammary basal-like human breast tumors in PAM50 classification, gland stem cells or luminal/myoepithelial common and, thus, could serve as a much-needed spontaneous cancer progenitors? model for the basal-like subtype. Canine complex carcino- Several observations indicate the possibility that complex mas are characterized with dual proliferation of luminal and carcinomas arise from mammary gland stem cells or lumi- myoepithelial cells, which is rare in human breast cancer. nal/myoepithelial common progenitors (Fig. 7A). First, one Our analysis indicates that these canine cancers may arise of these tumors (ID 518) expresses the pluripotency marker from epigenomic rather genomic alterations. Canine com- SOX2, indicating stem cell property. Second, unlike normal plex carcinomas, hence, provide a unique system to inves- mammary glands that present a clearly different epigenomic tigate the roles of myoepithelial cells, the second major cell landscape between luminal and myoepithelial cells, no such lineage of the mammary gland, in normal developmental and difference was observed in complex carcinomas. This indi- pathogenic processes.

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Canine and Human Mammary Cancer Molecular Comparison

Data access Other (consultation on issues related to animal cancer, references, sample acquisition): N.C. Northrup Sequence data have been submitted to the NCBI SRA database with accession numbers SRP023115, SRP023472, and Acknowledgments SRP024250. aCGH data have been submitted to the GEO The authors thank Drs. Timothy D. Read, Shawn Levy, Steven H. Miller, and database with the accession number GSE54535. their respective teams, and the BGI, for their outstanding sequencing or aCGH work; Teri Guerrero, Irene Mok, and Dr. Susan E. Lana for their help on sample collection; Dr. John M. Rosenfeld of Millipore for providing antibodies; Drs. Nancy Disclosure of Potential Conﬂicts of Interest Manley and Jie Li for their help on IHC and H&E analyses; and Drs. Dong M. Shin, No potential conﬂicts of interest were disclosed. J. David Puett, Charles M. Perou, and Malcolm Hayes for their help on the study. Confocal imaging was performed at the UGA Biomedical Microscopy Core. Authors' Contributions Conception and design: D. Liu, S. Zhao Grant Support Development of methodology: D. Liu, H. Xiong, N.C. Northrup, S. Zhao This work was funded by the American Cancer Society, the Georgia Cancer Acquisition of data (provided animals, acquired and managed patients, Coalition, NCI R01 CA182093, and the AKC Canine Health Foundation (to provided facilities, etc.): D. Liu, A.E. Ellis, N.C. Northrup, C.O. Rodriguez Jr, S. Zhao), as well as by pilot project funds from NCI P50 CA128613 (PI, Dr. Dong S. Dalton, S. Zhao M. Shin) and GM085354 (PI, S. Dalton). The costs of publication of this article were defrayed in part by the payment of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, advertisement computational analysis): D. Liu, S. Zhao page charges. This article must therefore be hereby marked in Writing, review, and/or revision of the manuscript: D. Liu, A.E. Ellis, S. Zhao accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): D. Liu, H. Xiong, A.E. Ellis Received February 11, 2014; revised June 24, 2014; accepted June 29, 2014; Study supervision: R.M. O'Regan published OnlineFirst July 31, 2014.

References 1. Meuten DJ. Tumors in domestic animals. 4th ed. Ames, Iowa: Iowa that partly overlaps with human breast cancer profiles. BMC Cancer State University Press; 2002. 2010;10:618. 2. Paoloni M, Khanna C. Translation of new cancer treatments from pet 16. Paw Owski KM, Maciejewski H, Dolka I, Mol JA, Motyl T, Krol M. Gene dogs to humans. Nat Rev Cancer 2008;8:147–56. expression profiles in canine mammary carcinomas of various grades 3. Rowell JL, McCarthy DO, Alvarez CE. Dog models of naturally occur- of malignancy. BMC Vet Res 2013;9:78. ring cancer. Trends Mol Med 2011;17:380–8. 17. Rao NA, van Wolferen ME, Gracanin A, Bhatti SF, Krol M, Holstege FC, 4. Gordon I, Paoloni M, Mazcko C, Khanna C. The Comparative Oncology et al. Gene expression profiles of progestin-induced canine mammary Trials Consortium: using spontaneously occurring cancers in dogs to hyperplasia and spontaneous mammary tumors. J Physiol Pharmacol inform the cancer drug development pathway. PLoS Med 2009;6: 2009;60 Suppl 1:73–84. e1000161. 18. Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, Wedge 5. Tang J, Le S, Sun L, Yan X, Zhang M, Macleod J, et al. Copy number DC, et al. The landscape of cancer genes and mutational processes in abnormalities in sporadic canine colorectal cancers. Genome Res breast cancer. Nature 2012;486:400–4. 2010;20:341–50. 19. Banerji S, Cibulskis K, Rangel-Escareno C, Brown KK, Carter SL, 6. Youmans L, Taylor C, Shin E, Harrell A, Ellis AE, Seguin B, et al. Frederick AM, et al. Sequence analysis of mutations and translocations Frequent alteration of the tumor suppressor gene APC in sporadic across breast cancer subtypes. Nature 2012;486:405–9. canine colorectal tumors. PLoS ONE 2012;7:e50813. 20. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, et al. The 7. Tang J, Li Y, Lyon K, Camps J, Dalton S, Ried T, et al. Cancer driver- consensus coding sequences of human breast and colorectal cancers. passenger distinction via sporadic human and dog cancer compari- Science 2006;314:268–74. son: a proof-of-principle study with colorectal cancer. Oncogene 21. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. 2014;33:814–22. The genomic and transcriptomic architecture of 2,000 breast tumours 8. Parker HG, Shearin AL, Ostrander EA. Man's best friend becomes reveals novel subgroups. Nature 2012;486:346–52. biology's best in show: genome analyses in the domestic dog. Annu 22. Naylor TL, Greshock J, Wang Y, Colligon T, Yu QC, Clemmer V, et al. Rev Genet 2010;44:309–36. High resolution genomic analysis of sporadic breast cancer using 9. Nasir L, Devlin P, McKevitt T, Rutteman G, Argyle DJ. Telomere lengths array-based comparative genomic hybridization. Breast Cancer Res and telomerase activity in dog tissues: a potential model system to 2005;7:R1186–98. study human telomere and telomerase biology. Neoplasia 2001;3: 23. Cancer Genome Atlas N. Comprehensive molecular portraits of human 351–9. breast tumours. Nature 2012;490:61–70. 10. Rangarajan A, Weinberg RA. Opinion: Comparative biology of mouse 24. Sleeckx N, de Rooster H, Veldhuis Kroeze EJ, Van Ginneken C, Van versus human cells: modelling human cancer in mice. Nat Rev Cancer Brantegem L. Canine mammary tumours, an overview. Reprod 2003;3:952–9. Domest Anim 2011;46:1112–31. 11. Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, 25. Adriance MC, Inman JL, Petersen OW, Bissell MJ. Myoepithelial cells: Kamal M, et al. Genome sequence, comparative analysis and haplo- good fences make good neighbors. Breast Cancer Res 2005;7:190–7. type structure of the domestic dog. Nature 2005;438:803–19. 26. Gudjonsson T, Adriance MC, Sternlicht MD, Petersen OW, Bissell MJ. 12. Ji X, Zhao S. DA and Xiao-two giant and composite LTR-retrotran- Myoepithelial cells: their origin and function in breast morphogenesis sposon-like elements identified in the human genome. Genomics and neoplasia. J Mammary Gland Biol Neoplasia 2005;10:261–72. 2008;91:249–58. 27. Lakhani SR, O'Hare MJ. The mammary myoepithelial cell–Cinderella or 13. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer ugly sister? Breast Cancer Res 2001;3:1–4. J Clin 2012;62:10–29. 28. Moumen M, Chiche A, Cagnet S, Petit V, Raymond K, Faraldo MM, 14. Beck J, Hennecke S, Bornemann-Kolatzki K, Urnovitz HB, Neumann S, et al. The mammary myoepithelial cell. Int J Dev Biol 2011;55: Strobel P, et al. Genome aberrations in canine mammary carcinomas 763–71. and their detection in cell-free plasma DNA. PLoS ONE 2013;8: 29. Polyak K, Hu M. Do myoepithelial cells hold the key for breast tumor e75485. progression? J Mammary Gland Biol Neoplasia 2005;10:231–47. 15. Klopfleisch R, Lenze D, Hummel M, Gruber AD. Metastatic canine 30. Sopel M. The myoepithelial cell: its role in normal mammary glands and mammary carcinomas can be identified by a gene expression profile breast cancer. Folia Morphol 2010;69:1–14.

www.aacrjournals.org Cancer Res; 74(18) September 15, 2014 OF11

Liu et al.

31. Hu M, Yao J, Carroll DK, Weremowicz S, Chen H, Carrasco D, et al. 40. Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, Regulation of in situ to invasive breast carcinoma transition. Cancer et al. Germline mutations affecting the proofreading domains of POLE Cell 2008;13:394–406. and POLD1 predispose to colorectal adenomas and carcinomas. Nat 32. Tan PH, Ellis IO. Myoepithelial and epithelial-myoepithelial, mesen- Genet 2012;45:136–44. chymal and fibroepithelial breast lesions: updates from the WHO 41. Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Classification of Tumours of the Breast 2012. J Clin Pathol 2013;66: Schotta G, et al. Loss of acetylation at Lys16 and trimethylation at 465–70. Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 33. Hayes MM. Adenomyoepithelioma of the breast: a review stressing its 2005;37:391–400. propensity for malignant transformation. J Clin Pathol 2011;64: 42. Wilson BG, Roberts CW. SWI/SNF nucleosome remodellers and 477–84. cancer. Nat Rev Cancer 2011;11:481–92. 34. Goldschmidt M, Pena L, Rasotto R, Zappulli V. Classification and 43. Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to grading of canine mammary tumors. Vet Pathol 2011;48:117–31. therapy. Cell 2012;150:12–27. 35. Bryson JL, Griffith AV, Hughes B III, Saito F, Takahama Y, Richie ER, 44. Prat A, Perou CM. Deconstructing the molecular portraits of breast et al. Cell-autonomous defects in thymic epithelial cells disrupt endo- cancer. Mol Oncol 2011;5:5–23. thelial-perivascular cell interactions in the mouse thymus. PLoS ONE 45. Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, et al. 2013;8:e65196. A census of human cancer genes. Nat Rev Cancer 2004;4:177–83. 36. Volik S, Zhao S, Chin K, Brebner JH, Herndon DR, Tao Q, et al. End- 46. Samuels Y, Wang ZH, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High sequence profiling: sequence-based analysis of aberrant genomes. frequency of mutations of the PIK3CA gene in human cancers. Science Proc Natl Acad Sci U S A 2003;100:7696–701. 2004;304:554. 37. Lademann U, Kallunki T, Jaattela M. A20 zinc finger protein inhibits 47. Wendt MK, Taylor MA, Schiemann BJ, Schiemann WP. Down-regu- TNF-induced apoptosis and stress response early in the signaling lation of epithelial cadherin is required to initiate metastatic outgrowth cascades and independently of binding to TRAF2 or 14-3-3 proteins. of breast cancer. Mol Biol Cell 2011;22:2423–35. Cell Death Differ 2001;8:265–72. 48. Gordon V, Banerji S. Molecular pathways: PI3K pathway targets in 38. Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorods- triple-negative breast cancers. Clin Cancer Res 2013;19:3738–44. kaya E, Harrison M, et al. Glucoamylase: structure/function relation- 49. Molyneux G, Geyer FC, Magnay FA, McCarthy A, Kendrick H, Natrajan ships, and protein engineering. Biochim Biophys Acta 2000;1543: R, et al. BRCA1 basal-like breast cancers originate from luminal 275–93. epithelial progenitors and not from basal stem cells. Cell Stem Cell 39. Costello M, Pugh TJ, Fennell TJ, Stewart C, Lichtenstein L, Meldrim JC, 2010;7:403–17. et al. Discovery and characterization of artifactual mutations in deep 50. Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, et al. Aberrant coverage targeted capture sequencing data due to oxidative DNA luminal progenitors as the candidate target population for basal tumor damage during sample preparation. Nucleic Acids Res 2013;41:e67. development in BRCA1 mutation carriers. Nat Med 2009;15:907–13.

OF12 Cancer Res; 74(18) September 15, 2014 Cancer Research

Molecular Homology and Difference between Spontaneous Canine Mammary Cancer and Human Breast Cancer

Deli Liu, Huan Xiong, Angela E. Ellis, et al.

Cancer Res Published OnlineFirst July 31, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-0392

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