Published OnlineFirst April 11, 2016; DOI: 10.1158/0008-5472.CAN-15-2654 Cancer Tumor and Stem Cell Biology Research Ly6E/K Signaling to TGFb Promotes Breast Cancer Progression, Immune Escape, and Drug Resistance Midrar AlHossiny1, Linlin Luo2, William R. Frazier1, Noriko Steiner1, Yuriy Gusev1,2, Bhaskar Kallakury3, Eric Glasgow1, Karen Creswell1, Subha Madhavan1,2, Rakesh Kumar4, and Geeta Upadhyay1,2 Abstract Stem cell antigen Sca-1 is implicated in murine cancer stem cell Ly6K/E was required for TGFb signaling and proliferation in biology and breast cancer models, but the role of its human breast cancer cells, where they contributed to phosphorylation homologs Ly6K and Ly6E in breast cancer are not established. of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cyto- Here we report increased expression of Ly6K/E in human breast kine-induced PDL1 expression and activation and binding of NK cancer specimens correlates with poor overall survival, with an cells to cancer cells. Finally, we found that Ly6K/E promoted drug additional specific role for Ly6E in poor therapeutic outcomes. resistance and facilitated immune escape in this setting. Overall, Increased expression of Ly6K/E also correlated with increased our results establish a pivotal role for a Ly6K/E signaling axis expression of the immune checkpoint molecules PDL1 and involving TGFb in breast cancer pathophysiology and drug CTLA4, increased tumor-infiltrating T regulatory cells, and response, and highlight this signaling axis as a compelling realm decreased natural killer (NK) cell activation. Mechanistically, for therapeutic invention. Cancer Res; 76(11); 3376–86. Ó2016 AACR. Introduction GDF10, a novel tumor suppressive cytokine (6). Recently, Ly6K and Ly6E, another members of the Ly6 gene family, have been In recent years, it has become increasingly clear that the shown to be involved in human malignancies and have been process of breast cancer oncogenesis and therapeutic sensitivity suggested as potential therapeutic targets for cancer immuno- is profoundly affected by pathways that also infringe on cancer therapy (9–14). Here we set out to investigate their role in stem cell biology (1, 2). One of the gene families at the interface breast cancer progression and the underlying cellular signaling of cancer stem cell biology and cancer biology is the human Ly- mechanisms and relate these findings to clinical cases of breast 6 gene family, which is related to the murine stem cell antigen-1 cancer using clinical informatics. gene (Sca-1) or mouse (m) Ly6A. It encodes a set of glycosy- lated cell surface proteins that have been recognized as stem cell markers (1, 3, 4). The mLy6A confers resistance to radiotherapy Materials and Methods and promotes metastatic behavior of mammary tumors in Bioinformatics analysis animal models (5). Earlier work from our laboratory has Oncomine (www.oncomine.org) was used to visualize gene demonstrated that mLy6A regulates TGFb,PTEN,andERK/AKT expression microarray dataset. ProgeneV2 online tool (http:// cell signaling pathways (6–8). Mechanistically, mLy6A binds to www.abren.net/PrognoScan/) was used to study survival out- TGFb receptor-1 (TbR1), disrupts the TbR1 ligand complex, and come. The Cancer Genome Atlas (TCGA) Breast Dataset is avail- inhibits Smad2/3 signaling. In addition, increased mLy6A able from http://tcga-data.nci.nih.gov/tcga/. expression in tumor cells correlates with a reduced level of Reagents and reporter plasmids 1Department of Oncology, Georgetown University Medical Center, Washington, DC. 2Innovation Center for Biomedical Informatics (ICBI), TGFb1, IFNg, and IL4 was obtained from R&D Systems. The Georgetown University Medical Center,Washington, DC. 3Department Smad-responsive TGFb reporter plasmids were used as described of Pathology, Georgetown University Medical Center,Washington, DC. previously (6). 4Department of Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC. shRNAs and cell lines Note: Supplementary data for this article are available at Cancer Research Ly6K sh1 (cat. no. TRCN0000117952), Ly6K sh2 (cat. no. Online (http://cancerres.aacrjournals.org/). TRCN0000117953), Ly6E sh1 (cat. no. TRCN0000154460), and Corresponding Author: Geeta Upadhyay, Georgetown University Medical Cen- Ly6E sh2 (cat. no. TRCN0000155331) shRNAs cloned into ter, 3970 Reservoir Road NW, Washington, DC 20007. Phone: 202-687-2156; pLKO.1 were obtained from Sigma Inc. Lentivirus was produced Fax: 202-687-7505; E-mail: [email protected] in 293T cells by cotransfection of the pMD2.g and VSVG vectors. doi: 10.1158/0008-5472.CAN-15-2654 At 24 hours after transfection, the medium was replaced and virus Ó2016 American Association for Cancer Research. was collected. Cells were infected with lentivirus for 24 hours in 3376 Cancer Res; 76(11) June 1, 2016 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst April 11, 2016; DOI: 10.1158/0008-5472.CAN-15-2654 Ly6K/E–TGFb Axis in Breast Cancer Tumorigenesis the presence of 4 mg/mL of polybrene and selection carried out 100 pg/mL TGFb1 for 30 minutes, 10 ng/mL IFNg, or 50 ng/mL with 1 mg/mL of puromycin. IL4 for overnight. For overexpression, the open reading frame containing clones (Ly6K, Ly6E) in G418 selection markers were obtained by Origene Western blot analysis MD. The cells were transfected using Fugene HD and selected in Cells were lysed in a buffer containing 60 mmol/L octylglu- 1 mg/mL G41. coside, 0.5% Nonidet P-40, 0.1% SDS, 0.25% sodium deox- The KHYG-1 natural killer (NK) cell line stably expressing à ycholate, 50 mmol/L Tris-HCl (pH 7.5), 125 mmol/L NaCl, KIR2DS1 002 have been generated and used as described 1 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, phosphatase, previously (37). Prior to each assay, cells were screened for and a protease inhibitor mixture (Roche Molecular Biochem- the surface expression of KIR2DS1 by immunolabeling icals) at 4C. A total of 20 to 50 mg of lysate was separated in a – using 2DS1 MoAB CD158a/h (clone EB6B or 11PB6) and 4% to 12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to fl ow assay. Zeocin was removed prior to NK-cell coculture nitrocellulose membranes. Primary antibody was incubated for assays. either 1.5 hours at room temperature or overnight at 4C. The cells were obtained by ATCC, which provided cells Secondary antibody was incubated for 30 minutes at room authenticated by short random repeat DNA sequencing. Upon temperature, and proteins were visualized with West Pico arrival, cells were propagated and stored in multiple vials as Stable (Pierce). For Western blot analysis, the primary anti- recommended. bodies anti-Ly6K (cat no. AF6648; R&D Systems), Ly6E (cat no. NBP1 68553; Novus Biologicals), pS423/425 Smad3 (cat no. Colony assay 9520), pS463/465 Smad1/5 (cat no. 9516), Smad2/3 (cat no. Cells (5,000/dish) were seeded into 100-mm dishes in 10 mL of 9523), and Smad5 (cat no. 12534) were purchased from Cell DMEM containing 5% FBS. After 7 to 10 days, colonies were fixed Signaling Technology. in 50% trichloroacetic acid, stained with sulforhodamine B, and solubilized in 10 mmol/L Tris-HCl (pH 10), and absorbance was Semiquantitative IHC measured at 560 nm (6). IHC was performed using tissue staining HRP-DAB System kits (R&D Systems). Antigen retrieval was performed using Nude mice xenograft assay citrate buffer (10 mmol/L citric acid, 0.05% Tween 20, pH 500,000 cells were injected subcutaneously into opposite 6.0). The primary antibodies were used for 1 hour at room flanks in five-week-old athymic nude mice. For the mice temperature using anti-Ly6K antibody (AF6648; R&D Systems) receiving T47D cells, mice were transplanted with controlled at 1:100 and anti-Ly6E antibody (NBP1 68553; Novus Biolo- release (60 days release time, 0.18 mg/pellet total dose) 17b- gicals) at 1:400 dilution on clinical samples of breast cancer estradiol pellet (Innovative Research of America). Tumor vol- (tissue microarrays; USBiomax, Histopathology and Tissue ume was determined by caliper measurements at weekly inter- Shared Resources at Georgetown University). Slides were semi- vals. All animal studies were performed under a protocol quantitatively scored in a blind fashion using both intensity approved by Georgetown University's Animal Care and Use and percent positive cells. Intensity was scored on a scale of 0 to ¼ ¼ ¼ ¼ Committee. 3asfollows:0 negative, 1 weak, 2 moderate, and 3 intense. Percent positive labeling was scored on a scale of 0 to 3asfollows:0¼ negative, 1 ¼ <10%, 2 ¼ 11%–50%, and 3 ¼ Quantitative real-time PCR >50%. Both these values were added for final numerical score. A Total RNA was extracted using the Qiagen RNAeasy Mini Kit score of 2 and below was considered negative and a score of 3 andcDNAwaspreparedaccordingtothemanufacturer'spro- and above was considered positive. tocol (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed in triplicate in an ABI 7900 instrument using SYBR- NK-cell activation assay Green detection (Applied Biosystems) according to the manu- NK-cell activation was measured by the release of granzyme B facturer's protocol. All primer sequences are described in Sup- and the proinflammatory cytokine MIP1a from the plementary Table S1. The expression of each target gene was KIR2DS1Ã002 NK (KHYG-1) cell line (37,49). Briefly, 2DS1 normalized to the expression of GAPDH using SDS2.4 software NK cells were cocultured with indicated cells at a ratio of 1:2 for (Applied Biosystems). 48 hours at 37 Cina5%CO2 environment. Each assay condition consisted of 1  105 effectors (2DS1 NK cells) and Reporter assays 0.5  105 targets in 200-mL culture media in one well of a Cells were seeded in triplicate into 24-well plates at a density of 48-well plate.