LYN Is a Mediator of Epithelial-Mesenchymal Transition and a Target of Dasatinib in Breast Cancer

Published OnlineFirst March 9, 2010; DOI: 10.1158/0008-5472.CAN-09-3141

Molecular and Cellular Pathobiology Cancer Research LYN Is a Mediator of Epithelial-Mesenchymal Transition and a Target of Dasatinib in Breast Cancer

Yoon-La Choi1,4, Melanie Bocanegra4, Mi Jeong Kwon3, Young Kee Shin3, Seok Jin Nam2, Jung-Hyun Yang2, Jessica Kao4, Andrew K. Godwin5, and Jonathan R. Pollack4

Abstract Epithelial-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is considered a key process driving tumor cell invasiveness and metastasis. Using breast cancer cell lines as a model system, we sought to discover gene expression signatures of EMT with clinical and mechanistic relevance. A supervised comparison of epithelial and mesenchymal breast cancer lines defined a 200-gene EMT signature that was prognostic across multiple breast cancer cohorts. The immunostaining of LYN, a top-ranked EMT signature gene and Src-family tyrosine kinase, was associated with significantly shorter overall survival (P = 0.02) and correlated with the basal-like (“triple-negative”) phenotype. In mesenchymal breast cancer lines, RNAi-mediated knockdown of LYN inhibited cell migration and invasion, but not proliferation. Dasatinib, a dual-specificity tyrosine kinase inhibitor, also blocked invasion (but not proliferation) at nanomolar concentrations that inhibit LYN kinase activity, suggesting that LYN is a likely target and that invasion is a relevant end point for dasatinib therapy. Our findings define a prognostically relevant EMT signature in breast cancer and identify LYN as a mediator of invasion and a possible new therapeutic target (and theranostic marker for dasatinib response), with particular relevance to clinically aggressive basal-like breast cancer. Cancer Res; 70(6); 2296–306. ©2010 AACR.

Introduction acquire a spindly or stellate morphology, and increase motility to carry out orchestrated migrations. EMT is required for In breast cancer, mortality results not from tumor growth normal gastrulation and the formation of the three-layered per se but from the tumor invading through normal tissue embryo, and later for the formation of normal tissues and boundaries and metastasizing to distant sites. To invade organs, including the heart, musculoskeletal system, and pe- and metastasize, breast cancer cells must first dissociate from ripheral nervous system (2). one another and become motile. These events are reminiscent Increasing evidence suggests that in breast cancer, malig- of epithelial-mesenchymal transition (EMT), a process nant cells co-opt the EMT program (3). EMT provides a path- that occurs during tissue patterning in normal embryonic way by which cancerous layers of epithelial cells (carcinoma development. in situ) can dissociate and become motile, leading to invasion EMT is a coordinated cellular program whereby epithelial through the basement membrane into blood vessels or lym- cells in layers reversibly or irreversibly convert to mesenchy- phatics, and metastatic spread. As such, targeting EMT re- mal cells—fibroblast-like cells loosely embedded in extracel- presents an important new therapeutic strategy for the lular matrix (1). During EMT, epithelial cells dissociate, prevention or treatment of breast cancer. A framework of molecular and cellular events underlying EMT has been elucidated (4, 5). In different cell contexts, Authors' Affiliations: 1Department of Pathology and 2Division of Breast ligands such as hepatocyte growth factor (scatter factor), and Endocrine Surgery, Samsung Medical Center, Sungkyunkwan epidermal growth factor (and related growth factors), and University School of Medicine; 3Laboratory of Molecular Pathology, β College of Pharmacy, Seoul National University, Seoul, Korea; transforming growth factor can stimulate EMT, acting 4Department of Pathology, Stanford University, Stanford, California; through signal transduction pathways (including SRC, and 5Women's Cancer Program, Department of Medical Oncology, Fox RAS, and phosphoinositide 3-kinase) to alter cell adhesion Chase Cancer Center, Philadelphia, Pennsylvania (through adherens junctions and desmosomes) and cell Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). motility (through cytoskeletal reorganization). Downstream transcriptional regulators, such as Snail, Slug, and Twist, Y-L. Choi and M. Bocanegra contributed equally to this work. repress the expression of E-cadherin (a key mediator of ep- Corresponding Author: Jonathan R. Pollack, Department of Patholo- gy, Stanford University, 269 Campus Drive, CCSR-3245A, Stanford, ithelial cell-cell adhesion), while activating expression of CA 94305-5176. Phone: 650-736-1987; Fax: 650-736-0073; E-mail: mesenchymal markers, e.g., vimentin, N-cadherin, and [email protected]. smooth muscle actin. However, the pace of recent ad- doi: 10.1158/0008-5472.CAN-09-3141 vances in understanding EMT suggests that much yet re- ©2010 American Association for Cancer Research. mains unknown.

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LYN: EMT Mediator and Dasatinib Target in Breast Cancer

The molecular pathways of EMT have been studied largely ple RNA and 40 μgof“universal” reference RNA were dif- in the context of embryogenesis in model organisms, such as ferentially labeled with Cy5 and Cy3, respectively, then Drosophila, Xenopus, and mice, and in mammalian cell cul- cohybridized onto the microarray in a high-volume mixing ture systems (1). The latter include Madin-Darby canine kid- hybridization at 65°C for 40 h. Details of the array proces- ney cells, NBII rat bladder carcinoma cells, and NMuMG sing and sample labeling and hybridization methods have mouse mammary cancer cells, each of which can be stimu- been described in ref. (8). The complete microarray data lated in culture to undergo EMT. However, these canine and are available at the Stanford Microarray Database (9) and rodent cell culture model systems may not faithfully replicate Gene Expression Omnibus (accession GSE13915). Some of EMT events in human breast cancer. these microarray data were included in a recent study in- Breast cancer cell lines display varied morphologies when tegrating genomic and transcriptional profiles of breast grown in culture. Some appear epithelial-like, forming cell cancer lines (10). clusters, whereas others appear more fibroblast-like (mesen- Microarray data analysis. Background-subtracted fluo- chymal), with dispersed and spindle-shaped cells. The latter rescence log 2 ratios were globally normalized for each array, cell lines tend to express vimentin and to be more invasive and then mean centered for each gene (i.e., reporting relative in vitro and metastatic in vivo (6, 7), suggesting that they have to the average log 2 ratio across all samples). Subsequent undergone stable EMT conversion. These cell lines provide a analysis included only the 6,947 well-measured and variably useful model for studying the underpinnings of EMT in expressed genes, defined as those with intensities in the Cy5 breast cancer. Here, we set out to explore gene expression or Cy3 channel at least 1.5-fold above background in at least patterns associated with EMT in breast cancer cells in cul- 80% of samples, and with at least 3-fold ratio variation from ture and, in particular, to discover molecular signatures the mean in at least three samples. Differentially expressed and biomarkers of EMT with possible prognostic, mechanis- genes were identified by two-class significance analysis of mi- tic, and therapeutic relevance. croarrays (false discovery rate <5%; ref. 11). An EMT signature was defined by combining the top-ranked 100 genes Materials and Methods overexpressed in mesenchymal breast cancer lines compared with both epithelial breast cancer lines and normal breast Specimens. Breast cancer cell lines were obtained directly fibroblasts, and in epithelial breast cancer lines compared from the American Type Culture Collection or DSMZ, and with both mesenchymal breast cancer lines and normal grown in RPMI 1640 with 10% fetal bovine serum (FBS) breast fibroblasts. Gene Ontology (GO) term enrichment and 1% penicillin/streptomycin. Early-passage primary was done using FatiGO (12). The clinical relevance of the breast fibroblasts were prepared from reduction mammo- EMT signature was evaluated using publicly available micro- plasties or prophylactic mastectomies. In brief, surgical array data for primary breast tumor cohorts (13–15). Data breast tissue specimens were obtained following informed sets were mean centered and log transformed, and the consent by the Biosample Repository staff and surgery at corresponding EMT signature genes were identified by En- the Fox Chase Cancer Center. Deidentified tissue specimens trez Gene ID. Breast tumors were then clustered in the space were finely minced and incubated in a collagenase solution of the EMT signature genes, and the resultant two main sam- (DMEM/medium 199, 10% horse serum + collagenase, hyal- ple branches were evaluated by Kaplan-Meier analysis. uronidase, antibiotic/antimycotic, insulin, and hydrocorti- Western blot, immunohistochemistry, and DNA sequenc- sone) overnight at 37°C in a rotating water bath and then ing. Cells were lysed in 1× radioimmunoprecipitation assay centrifuged at 2,500 rpm for 10 min. The supernatant was buffer. Forty micrograms of total protein lysate were elec- decanted to a sterile tube and the residual tissue was rinsed trophoresed on a 4% to 15% polyacrylamide gel, transferred several times, resuspended in culture medium, combined to polyvinylidene difluoride membrane, and blocked in with the supernatant, and centrifuged as before. The result- TBST-T with 5% dry milk. Anti-LYN, p-LYN (Tyr507), SRC, ing tissue pellet was resuspended in fibroblast medium p-SRC (Tyr527), and p-p130cas (Tyr410) antibodies (Cell Sig- (DMEM, 15% FBS, penicillin/streptomycin, Cipro, fungizone, naling Technology) were used at 1:1,000 dilution with over- and gentamicin) and was plated in a swine skin gelatin and night incubation at 4°C in TBS-T with 5% bovine serum FBS–coated flask. The tissue was permitted to attach to albumin. Anti-vimentin, E-cadherin, p130cas, and glyceralde- flasks for 24 to 48 h. Once this occurred, cells were fed twice hyde-3-phosphate dehydrogenase (GAPDH) antibodies (San- weekly, increasing medium amounts incrementally. Cells ta Cruz Biotechnology) were used at 1:1,000 dilution. After were passaged (at a 1:2 split ratio) until characterized. A tis- incubation with a horseradish peroxidase–conjugated sec- sue microarray (TMA) was constructed from 970 clinically ondary antibody, detection was done using an enhanced annotated breast cancer cases (each represented by dupli- chemiluminescence kit (GE Healthcare). Band intensities cate 2-mm cores) archived at the Samsung Medical Center were quantified by densitometry using ImageJ software (Seoul, Korea). All research was conducted with institutional (16). Immunohistochemistry was done using 4-μm sections review board approval. of the breast cancer TMA. Following heat-induced antigen Expression profiling. Gene expression profiling was done retrieval, anti-LYN antibody (Santa Cruz Biotechnology) was using human exonic evidence-based oligonucleotide arrays used at 1:20 dilution, with chromogenic detection by perox- obtained from the Stanford Functional Genomics Facility idase-conjugated secondary antibody and 3,3′-diaminoben- and representing 24,207 human genes. Briefly, 40 μg of sam- zidine reagents (Envision detection kit; DAKO). Parallel

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TMA sections were stained with anti-CK5/6 (1:100; D5/16 B4, “contamination” in subsequently evaluated clinical samples). DAKO), epidermal growth factor receptor (EGFR; 1:30; E30, We defined a 200-gene “EMT signature” by combining the DAKO), HER2 (1:200; CB11, DAKO), estrogen receptor (ER; top 100 genes significantly overexpressed (false discovery 1:100; 6F11; Novocastra), and progesterone receptor (PR; rate <0.05) in mesenchymal breast cancer lines compared 1:50; 1A6; Novocastra) antibodies. DNA sequencing of LYN with both epithelial breast cancer lines and fibroblasts, and (exons 8–13, including intron-exon junctions) was done from the top 100 genes significantly overexpressed in epithelial PCR-amplified genomic DNAs (PCR primers and conditions in breast cancer lines compared with both mesenchymal breast Supplementary Table S1), with Sanger sequencing by Geneway cancer lines and fibroblasts (Supplementary Fig. S2; Fig. 1A). Research. Assuringly, the top 100 genes overexpressed in epithelial lines Small inhibitory RNA knockdown and dasatinib treat- were enriched for the GO term “intercellular junction” (dis- ment. Synthetic 21-nucleotide small inhibitory RNAs (siRNA) rupted in EMT; including genes CLDN3, CLDN4, CLDN7, directed against LYN and SRC,andanontargetingSMART pool, EVPL, MARVELD2, OCLN,andPKP3; corrected P = 0.001), were obtained from Dharmacon (sequences in Supplementary and related GO terms, although no GO term enrichment Table S2). Briefly, 200,000 cells were seeded in triplicate in six- was identified among the top 100 genes overexpressed in well plates and transfected with a final concentration of 50 nmol mesenchymal lines. siRNA for 16 h using Lipofectamine 2000 (Invitrogen). Dasatinib EMT is thought to underlie aggressive tumor behavior (1). (LC Laboratories) was reconstituted in DMSO at 200 mmol/L To determine a possible clinical relevance of our EMT signa- and used at the indicated concentrations. ture, we evaluated the signature genes in three different pri- Cell proliferation, migration, and invasion assays. The mary breast cancer cohorts using publicly available effect of gene knockdown or drug treatment on cell prolifer- microarray data. The first cohort, Sotiriou and colleagues ation was measured by quantifying the metabolic cleavage of (13), from the John Radcliffe Hospital (United Kingdom) the tetrazolium salt WST-1 (Roche Applied Science) in viable and Uppsala University Hospital (Sweden), comprised 169 cells. Motility and invasion were quantified by the Boyden cases of invasive ductal carcinoma, profiled with the Affyme- chamber assay (BD Biosciences). Briefly, 10,000 (migration) trix U133A GeneChips. To evaluate the EMT signature, we or 20,000 (invasion) cells were plated into 24-well inserts us- clustered those samples in the space of the EMT signature ing a 0.5% to 5% FBS gradient. Cells were fixed and stained genes (Supplementary Fig. S3) and compared clinical out- with crystal violet; cells traversing the membrane were comes between the two major sample clusters by Kaplan- counted. All assays were performed in triplicate, and mean Meier analysis. Notably, the sample cluster associated with values and SDs were reported. IC50 values were determined EMT genes overexpressed in mesenchymal lines (compared by fitting sigmoidal (four-parameter logistic) curves with with the converse pattern of genes overexpressed in epithe- Prism 4.0 software (GraphPad). lial lines) showed a strong trend toward decreased relapse- free survival (P = 0.058) and significantly decreased distant Results metastasis-free survival (P = 0.014; Fig. 1B). A similar analysis of a second cohort, van de Vijver and colleagues (295 cases Prognostic EMT signature from breast cancer cell lines. from the Netherlands Cancer Institute, profiled with Agilent Breast cancer cell lines grown in culture display distinct oligonucleotide microarrays; ref. 14), revealed significant as- morphologies, with a small subset of lines appearing more sociation with both metastasis-free (P < 0.001) and overall “fibroblast-like” (mesenchymal) and being more invasive survival (P = 0.017; Supplementary Fig. S3). Likewise, analysis (refs. 6, 7 and our observations), features suggestive of having of a third cohort, Bild and colleagues (171 cases profiled with undergone EMT. To explore the molecular variation associ- the Affymetrix U133 2.0 GeneChips; ref. 15), showed a signif- ated with this phenotype, we used whole-genome oligonucle- icant association with overall survival (P = 0.015; Supplemen- otide microarrays to profile the gene expression of 5 tary Fig. S3). mesenchymal breast cancer cell lines (BT549, Hs578T, Where sufficient clinical annotations were available, we al- MDA157, MDA231, and MDA436), compared with a diverse so evaluated the EMT signature in multivariate analysis. In set of 10 epithelial-like breast cancer cell lines (BT20, the Sotiriou and colleagues cohort (13), the EMT signature BT474, EFM19, MCF7, MDA-361, MDA-453, MDA-468, SKBR3, was a significant independent predictor of relapse-free and T47D, and UACC893) representing both luminal and basal- distant metastasis-free survival (Table 1). In the Netherlands like subtypes (10, 17). Molecular pathologic features of cell Cancer Institute data set, the signature was significant only lines and representative morphologies are shown in Fig. 1A. when ER status was omitted from the model (data not We also profiled five early-passage primary breast fibroblast shown). cultures to help identify characteristic profiles of EMT dis- Signature gene LYN functions in invasion. LYN is among tinct from fibroblasts. the highly ranked EMT signature genes overexpressed in Unsupervised cluster analysis of gene expression readily mesenchymal lines (Fig. 1A). LYN is a member of the Src- differentiated the three groups (epithelial, mesenchymal, nor- family kinases, a family of nonreceptor tyrosine kinases with mal fibroblasts; Supplementary Fig. S1), indicating robust roles in signal transduction, often deregulated in cancers and expression differences. We therefore sought to build a signa- linked to neoplastic transformation (18). LYN was of partic- ture that distinguished epithelial from mesenchymal breast ular interest because, as a kinase, it is “druggable” and might cancer cells, distinct from fibroblasts (anticipating stromal provide a therapeutic opportunity for targeting EMT. We first

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Figure 1. Cell line–derived EMT signature shows prognostic relevance. A, expression profiling of morphologically distinct breast cancer cell lines defines signature of EMT. Expression profiles were compared among breast cancer cell lines exhibiting epithelial-like and mesenchymal morphologies, and normal breast fibroblasts. Cell line characteristics (7, 10) are indicated (black box means yes), along with representative photos showing cell culture morphologies (equal cell numbers plated). The EMT signature (heat map shown) comprises the top-ranked 100 genes overexpressed in mesenchymal breast cancer cells compared with epithelial breast cancer cells and normal breast fibroblasts, and in epithelial breast cancer cells compared with mesenchymal breast cancer cells and normal breast fibroblasts. The top 20 genes, including the LYN tyrosine kinase (arrow), are shown. Expression ratio (log 2) scale is shown. B, EMT signature is predictive of clinical outcome. Primary breast tumors from a publicly available microarray data set (Sotiriou and colleagues; ref. 13) were clustered in the space of the EMT signature genes, and the two major sample clusters were then compared by Kaplan-Meier analysis (P values are shown). The “EMT+” group, associated with EMT genes overexpressed in mesenchymal lines (compared with the converse pattern of genes overexpressed in epithelial lines), showed increased risk of relapse (top) and distant metastasis (below); P values are shown.

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Table 1. Multivariate analysis

Variable Hazard ratio (95% CI) P

Sotiriou and colleagues (13), EMT signature 2.02 (1.07–3.81) 0.029 relapse-free survival ER negativity 1.62 (0.77–3.39) 0.20 Grade (2) 2.00 (0.92–4.36) 0.082 Grade (3) 1.13 (0.53–2.42) 0.76 Tumor size (2–5 cm) 2.66 (1.38–5.13) 0.004 Tumor size (>5 cm) 5.69 (1.59–20.4) 0.007 Lymph node positivity 0.73 (0.30–1.78) 0.48

Sotiriou and colleagues (13), EMT signature 2.72 (1.16–6.34) 0.021 distant metastasis-free survival ER negativity 1.48 (0.55–3.98) 0.44 Histologic grade (2) 2.20 (0.79–6.11) 0.13 Histologic grade (3) 1.24 (0.49–3.14) 0.65 Tumor size (2–5 cm) 5.13 (1.90–13.8) 0.001 Tumor size (>5 cm) 5.91 (0.98–35.8) 0.053 Lymph node positivity 1.09 (0.40–2.94) 0.87

TMA overall survival LYN positivity 2.29 (1.18–4.42) 0.014 ER negativity 1.28 (0.65–2.49) 0.48 Tumor size (2–5 cm) 2.36 (0.83–6.72) 0.108 Tumor size (>5 cm) 7.45 (2.47–22.5) <0.001 Lymph node positivity 2.06 (1.12–3.80) 0.020

evaluated LYN expression in a large cohort of primary breast morphologic changes (Supplementary Fig. S4). In cell lines, cancer cases (archived at the Samsung Medical Center, Seoul, knockdown of LYN did not alter cell proliferation levels but Korea) by imunohistochemistry on TMAs. LYN expression led to significantly decreased cell migration and invasion was found in 133 of 939 scorable breast cancer cases (Fig. 3C). Transfection of individual siRNAs from the pool (14.2%; representative images shown in Fig. 2A) and was sig- similarly inhibited invasion (Supplementary Fig. S5), effec- nificantly associated with shorter overall survival (P =0.02; tively excluding possible RNAi off-target effects. with a trend for relapse-free survival), most evident between To determine whether LYN overexpression/activation 2 and 6 years after surgery (Fig. 2B). In multivariate analysis, might be driven by DNA amplification, we analyzed our ar- LYN expression was a significant prognostic factor indepen- ray-based comparative genomic hybridization data for 49 dent of other clinical variables (Table 1). Notably, LYN ex- breast cancer cell lines (10) and 172 breast tumors (8). pression was associated with the triple-negative phenotype LYN (residing at cytoband 8q12.1) was not found to be fo- (P < 0.001; Fisher's exact test), as defined by imunohisto- cally amplified, although it resided within broad gains span- chemistry (HER2 and ER/PR negative). Forty-six percent of ning some or all of 8q in 20 of 49 (41%) breast cancer lines triple-negative cases were LYN positive, versus 4% of others, (including BT549 and Hs578T) and in 51 of 172 (30%) breast and 79% of LYN-positive cases were triple negative, versus tumors. To determine whether LYN activity might be asso- 15% for LYN-negative cases (Supplementary Table S3). LYN ciated with activating mutations, we sequenced exons 8 to expression was also associated with basal marker (CK5/6 13 (corresponding to the P-loop and activation segment of and/or EGFR) positivity (P < 0.001; Supplementary Table S3). thekinasedomain)ofLYN from PCR-amplified genomic To evaluate a functional connection between LYN and DNA of the five mesenchymal breast cancer cell lines and EMT, we first assayed protein levels in breast cancer cell 50 breast tumors (LYN positive from the TMA). No muta- lines by Western blot. Both total LYN and phospho-LYN tions were identified. (Tyr507; indicative of LYN activation; ref. 19) were elevated LYN is a likely target of dasatinib. We also sought to de- in mesenchymal compared with epithelial breast cancer termine whether we could inhibit LYN pharmacologically. lines (Fig. 3A). We next used a siRNA pool to knock down Dasatinib is a dual-specificity tyrosine kinase inhibitor, active LYN expression in two mesenchymal breast cancer lines against both ABL and the Src-family tyrosine kinases (of (BT549 and Hs578T). Knockdown of LYN (and p-LYN), con- which LYN is a member). Of note, dasatinib was also recently firmed by Western blot (75–90% knockdown), led to the de- reported to show selective growth inhibition of basal-like creased expression of the mesenchymal markers vimentin breast cancer cell lines (20, 21). In our studies, dasatinib (65–75% reduction) and N-cadherin (50–70% reduction), treatment of the mesenchymal (and also basal like) breast but not to increased E-cadherin (Fig. 3B) nor to observed cancer lines BT549 and Hs578T resulted in decreased cell

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Figure 2. LYN expression is associated with unfavorable outcome. A, LYN immunostaining on TMA. Representative breast cancer cases negative (arrow, region with tumor cells) or positive for expression of the LYN tyrosine kinase in tumor cells. Note that LYN is expressed in stromal cells in some breast cancer cases (also see Supplementary Fig. S7). B, LYN expression in tumor cells predicts unfavorable outcome. Kaplan-Meier analysis comparing LYN-positive (LYN+) and LYN-negative (LYN−) cases for relapse-free survival (top) and overall survival (bottom); P values are shown.

μ growth/viability (Fig. 4A), with an IC50 of 1.6 and 0.30 mol/ mediated LYN knockdown and dasatinib treatment did not L, respectively. Notably, these growth-inhibitory concentra- show additive effects in inhibiting BT549 and Hs578 cell inva- tions were, respectively, 188- and 35-fold higher than the re- sion (Fig. 5D), suggesting that LYN siRNA and dasatinib are in vitro ported dasatinib IC50 of LYN tyrosine kinase activity acting through the same target (i.e., LYN). (8.5 nmol/L; ref. 22), suggesting that the effect on growth The finding that LYN but not SRC promotes invasion in was likely not mediated through LYN. We also measured mesenchymal breast cancer lines suggests the possibility that the effect of dasatinib on cell invasion. In BT549 and LYN and SRC tyrosine kinases are phosphorylating distinct Hs578T cells, the IC50 for invasion was 0.028 and 0.026 target proteins. Although there are many known targets of μmol/L (Fig. 4B), or respectively, 57- and 12-fold lower Src-family kinases (24), as a starting point, we chose to focus CAS than the IC50 for cell growth, and more comparable (only on p130 (Crk-associated substrate), previously linked to 3-fold higher) to the reported IC50 for LYN kinase activity. cell motility/invasion (25, 26). In BT549 (mesenchymal) cells, Western blot confirmed dasatinib inhibition of LYN activity knockdown of LYN but not SRC led to decreased phospho- (i.e., p-LYN) at nanomolar concentrations (Fig. 4C). p130CAS (Tyr410) levels (65% reduction; Fig. 5F). Although a These data are consistent with dasatinib targeting LYN to similar reduction was not observed in Hs578T cells (Fig. 5F), inhibit cell invasion. However, it remained possible that dasa- this finding nevertheless supports a likelihood that the differ- tinib was acting instead through a different Src-family kinase, ential effects of LYN and SRC on cell invasion are manifested in particular SRC itself, which has been linked previously to through the phosphorylation/activation of distinct down- cell invasion (23). Several additional findings argue against stream targets. Future studies should clarify the mechanisms this possibility. First, in examining transcript levels of all linking LYN to EMT phenotypes. Src-family kinases, we found that LYN but not SRC was relatively overexpressed in invasive, mesenchymal breast cancer Discussion lines (P = 0.006; Mann-Whitney U test; Fig. 5A) and, more broadly, in basal-like breast cancer cell lines (P < 0.001; Sup- The broad goal of our study is to explore expression pat- plementary Fig. S6). Consistent with this finding, SRC was also terns of EMT, using breast cancer cell lines as a model sys- not expressed at higher protein levels (by Western blot) in tem. Comparing mesenchymal and epithelial breast cancer mesenchymal lines (Fig. 5B). Second, siRNA-mediated knock- lines (and distinct from normal breast fibroblasts), we de- down of SRC, confirmed by Western blot (Fig. 5C), did not fined a 200-gene EMT signature that was robustly prognostic inhibit BT549 and Hs578 cell invasion (Fig. 5D), suggesting across three breast cancer microarray data sets representing that LYN and not SRC mediates invasiveness. Last, siRNA- independent cohorts and different microarray platforms. The

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EMT signature was a significant predictor independent of (“Mammaprint”; ref. 29), a 21-gene “OncotypeDx” signature clinically used prognostic factors (tumor size, grade, lymph (30), “intrinsic” subtypes (31), wound signature (32), hypoxia − node, and ER status) in the Sotiriou and colleagues cohort, signature (33), stem cell (CD44+/CD24 /low) signature (34), and although not in the Netherlands Cancer Institute data set, stroma signature (35), among several others. Many of these in which there was some relation with ER status. Nonethe- signatures identify the same poor outcome cases and are less, the EMT signature does not seem to represent only an likely capturing the same underlying biology (36). Indeed, re- ER-negative/ER-positive or basal/luminal signature (despite cent studies support a connection between breast cancer having fewer basal-like lines in the epithelial group due to stem cells, the basal-like phenotype, and EMT (37, 38). Irre- repository availability at the start of our study). Notably ab- spective of clinical utility, our finding that the EMT signature sent among EMT signature genes was ESR1 (ER) itself, as well identifies aggressive tumors supports a clinical relevance as any of 89 empirically defined ER target genes (27) or key of EMT. basal-luminal discriminatory genes (CAV1, CD44, EGFR, MET, Among the top signature genes, we carried out additional ETS1, GATA3, KRT19, MME, and MSN; ref. 28). studies of LYN, a Src-family kinase. Members of this family of Although the EMT signature was prognostic, eventual clin- nonreceptor tyrosine kinases function in signal transduction, ical utility is less certain. The “prognostic space” for breast regulating diverse cellular activities, including growth, surviv- cancer is becoming increasingly crowded. Microarray-de- al, motility, and invasion (23). Src-family kinases, foremost rived prognosticators include a 70-gene outcome signature SRC, are also frequently deregulated in cancer, in which they

Figure 3. LYN overexpression contributes to invasiveness. A, LYN exhibits relative overexpression and activation in mesenchymal breast cancer lines. Western blot probed with anti-LYN, phosphorylated (activated) LYN (Tyr507), vimentin and N-cadherin (markers of EMT), E-cadherin (a marker of epithelial morphology; overnight exposure; positive control was not shown), and GAPDH (loading control). B, validation of LYN knockdown by siRNA, and effect on vimentin, N-cadherin, and E-cadherin levels. Transfected siRNA pools (LYN or control nontargeting siRNA) are indicated. C, LYN knockdown (compared with nontargeting control) does not alter cell proliferation (left; measured by WST-1 assay) but leads to significantly decreased cell migration (center) and invasion (right; measured by Boyden chamber assay). Columns, mean; bars, SD. *, P < 0.05; **, P < 0.01; Student's t test. OD, absorbance.

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Figure 4. Dasatinib inhibits cell invasion at concentrations that inhibit LYN. Dasatinib effect (dose-response curve) on (A) cell viability and (B) cell invasion for

BT549 cells (left) and Hs578T cells (right). IC50 values (indicated) were determined from sigmoidal (four-parameter logistic) curves. C, verification in BT549 cells that dasatinib treatment leads to decreased phosphorylated (activated) LYN, assayed by Western blot. have been linked to tumor development and progression growth, survival). The knockdown of LYN also led to reduced (18). LYN itself has been studied mainly in hematopoietic vimentin and N-cadherin (EMT markers) but not increased cells but was recently linked to prostate cancer (in which ex- E-cadherin, suggesting that LYN directs only a portion of pression was associated with growth, invasion, and metasta- the mesenchymal phenotype. Although a single potential sis; refs. 22, 39, 40), glioblastoma (41), and Ewing's sarcoma LYN activating mutation (D385Y, in activation segment) (42). In our studies, we found LYN to be expressed in 14% of was reported in a breast cancer case (among 80 samples breast cancers, in which immunostaining was prognostic, screened; ref. 44), we did not identify any mutations in 55 and associated with (although not equivalent to) the triple- samples. We also did not find focal DNA amplification in negative/basal-like subtype (about half of triple-negatives breast cancer lines or tumors. Therefore, LYN overexpres- were LYN positive). Of note, in breast cancer cases with pos- sion/activity is more likely controlled mainly by upstream itive immunostaining, most (or all) cancer cells expressed regulators that remain to be defined. LYN rather than only those cells at the leading edge of inva- Dasatinib is a dual-specificity tyrosine kinase inhibitor, sion. This finding is consistent with the idea that tumor phe- with activity against both ABL and the Src-family tyrosine notypes, like EMT and metastatic potential, might be kinases (45). It is currently used as a second-line therapy encoded in the bulk tumor (rather than in a select subpopu- for imatinib (Gleevec)–resistant chronic myeloid leukemia, lation; ref. 43). and efficacy is being explored in solid tumors (46). Indeed, In examining cell lines, we confirmed LYN overexpression recent studies indicate the selective growth inhibition of bas- and increased activity (p-LYN) in mesenchymal breast cancer al-like breast cancer lines (20, 21), and clinical trials are un- lines. Knockdown experiments revealed a function of LYN in der way (47). In our study, we found that dasatinib treatment cell motility and invasion but not in cell proliferation (i.e., also inhibited cell invasion and at levels comparable with

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LYN kinase inhibition and up to ∼60-fold lower than re- was expressed at higher levels in the invasive, mesenchymal quired to inhibit cell growth. These findings suggest that breast cancer lines and indeed generally at higher levels in LYN is a target of dasatinib and that invasion is a relevant basal-like breast cancer lines in which dasatinib was previ- end point for measuring drug response. ously shown to be selectively inhibitory (21). Second, knock- Although dasatinib is active against other Src-family ki- down of LYN (but not SRC) in mesenchymal breast cancer nases, in particular SRC, multiple lines of evidence support lines inhibited invasion (which was also inhibited by dasati- LYN as the presumptive target. First, LYN (but not SRC) nib). Third, the effects of LYN knockdown and dasatinib

Figure 5. Relevant dasatinib target is likely LYN and not SRC. A, heat map showing microarray expression levels (mean-centered log 2 ratios; fold change indicated) of ABL and Src-family kinase genes. LYN (but not SRC) is significantly overexpressed in mesenchymal (compared with epithelial) breast cancer lines (P = 0.006; Mann-Whitney U test). Note, however, that LYN seems to be expressed in the epithelial-like (and not highly invasive) lines BT20 and MCF7, suggesting that its role in EMT may be context specific. B, Western blot shows that SRC is not relatively overexpressed or phosphorylated (Tyr527) in mesenchymal breast cancer cell lines. GAPDH serves as loading control. C, validation of SRC knockdown by siRNA. Transfected siRNA pools (SRC, LYN, or control nontargeting siRNA) are indicated; SRC levels assayed by Western blot (GAPDH serves as loading control). D, knockdown of SRC (in contrast to LYN) does not inhibit cell invasion in mesenchymal lines BT549 and Hs578T. Note that the apparent augmentation of invasion observed with SRC knockdown in Hs578T cells is not reproducible in replicate experiments. E, LYN knockdown and dasatinib treatment each significantly inhibits invasion in mesenchymal lines BT549 and Hs578T; however, the effect is not additive. Dasatinib was used at 0.03 and 0.015 μmol/L for BT549 and Hs578T, respectively. *, P < 0.05; **, P < 0.01; n.s., not significant; Student's t test. F, knockdown of LYN (but not SRC) leads to reduced phospho-p130CAS (Tyr410) levels in BT549 cells, assayed by Western blot (GAPDH serves as loading control).

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LYN: EMT Mediator and Dasatinib Target in Breast Cancer

treatment were not additive, consistent with their sharing BT549 cell invasion. However, tumor invasion and metasta- the same target. Taken together, our data support LYN as sis, not growth per se, are the real drivers of cancer mortality. a relevant target of dasatinib in invasive breast cancer cells. Inhibiting tumor cell invasion and metastasis may represent Nonetheless, SRC may still function in other aspects of breast a distinct approach to control (rather than cure) cancer. Our cancer pathogenesis. Indeed, SRC was recently shown to sup- findings highlight the importance of incorporating pheno- port survival of breast cancer cells in the bone marrow (48). types, like cell invasion, into drug screening, and designing Our studies of LYN have important clinical implications. clinical trials that use such drugs earlier, to block tumor Foremost, our findings identify LYN as a novel target for metastasis, thereby effectively managing cancer. therapy in breast cancer, with particular relevance to clinically aggressive basal-like breast cancers. These typically Disclosure of Potential Conflicts of Interest triple-negative tumors are not treatable by standard therapies such as ER modulators or HER2 antagonists. Recent No potential conflicts of interest were disclosed. studies suggest a promise of poly(ADP-ribose) polymerase inhibitors (49), leveraging probable tumor defects in DNA Acknowledgments repair. Dasatinib, targeting Src-family kinases, represents We thank the Stanford Functional Genomics Facility for the microarray, an additional possible treatment (47). Importantly, our SMD for the database support, and the members of the Pollack Laboratory findings suggest that LYN immunostaining might be a (in particular Keyan Salari) for the helpful discussion. good “theranostic” biomarker for dasatinib (or similar inhibitor) response, and incorporating LYN immunostaining Grant Support into clinical trials is worthwhile. Given its role in invasion (which precedes metastasis), the inhibitory effects on LYN NIH grants CA97139 (J.R. Pollack), CA113916 (A.K. Godwin), CA09302 (M. Bocanegra), and CA130172 (M. Bocanegra); NIH contract N01-CN-43309 (A.K. might be best observed in adjuvant studies, which can be Godwin); Department of Defense (BC073467); California Breast Cancer Re- lengthy and challenging. search Program, 8KB-0135 (J.R. Pollack); and Basic Science Research Program Finally, our studies also underscore the relevance of EMT/ through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0071010; Y-L.Choi). invasion (rather than or in addition to cell growth) as a The costs of publication of this article were defrayed in part by the payment meaningful biological and clinical end point. Currently, most of page charges. This article must therefore be hereby marked advertisement in drug screening programs rely on assaying cell growth/cyto- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. “ toxicity. Indeed, BT549 cells were thus classified as resis- Received 08/25/2009; revised 12/01/2009; accepted 12/21/2009; published tant” to dasatinib (21), although clearly dasatinib inhibits OnlineFirst 03/09/2010.

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2306 Cancer Res; 70(6) March 15, 2010 Cancer Research

LYN Is a Mediator of Epithelial-Mesenchymal Transition and a Target of Dasatinib in Breast Cancer

Yoon-La Choi, Melanie Bocanegra, Mi Jeong Kwon, et al.

Cancer Res 2010;70:2296-2306. Published OnlineFirst March 9, 2010.

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