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Inherited Variation in Mir-290 Expression Suppresses Breast Cancer Progression by Targeting the Metastasis Susceptibility Gene Arid4b

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Inherited Variation in Mir-290 Expression Suppresses Breast Cancer Progression by Targeting the Metastasis Susceptibility Gene Arid4b Published OnlineFirst February 27, 2013; DOI: 10.1158/0008-5472.CAN-12-3513 Cancer Tumor and Stem Cell Biology Research Inherited Variation in miR-290 Expression Suppresses Breast Cancer Progression by Targeting the Metastasis Susceptibility Gene Arid4b Natalie Goldberger1, Renard C. Walker1, Chang Hee Kim2, Scott Winter1, and Kent W. Hunter1 Abstract The metastatic cascade is a complex and extremely inefficient process with many potential barriers. Understanding this process is of critical importance because the majority of cancer mortality is associated with metastatic disease. Recently, it has become increasingly clear that microRNAs (miRNA) play important roles in tumorigenesis and metastasis, yet few studies have examined how germline variations may dysregulate miRNAs, in turn affecting metastatic potential. To explore this possibility, the highly metastatic MMTV-PyMT mice were crossed with 25 AKXD (AKR/J Â DBA/2J) recombinant inbred strains to produce F1 progeny with varying metastatic indices. When mammary tumors from the F1 progeny were analyzed by miRNA microarray, miR-290 (containing miR-290-3p and miR-290-5p) was identified as a top candidate progression-associated miRNA. The microarray results were validated in vivo when miR-290 upregulation in two independent breast cancer cell lines suppressed both primary tumor and metastatic growth. Computational analysis identified breast cancer progression gene Arid4b as a top target of miR-290-3p, which was confirmed by luciferase reporter assay. Surprisingly, pathway analysis identified estrogen receptor (ER) signaling as the top canonical pathway affected by miR-290 upregulation. Further analysis showed that ER levels were elevated in miR-290–expressing tumors and positively correlated with apoptosis. Taken together, our results suggest miR-290 targets Arid4b while simul- taneously enhancing ER signaling and increasing apoptosis, thereby suppressing breast cancer progression. This, to the best of our knowledge, is the first example of inherited differences in miRNA expression playing a role in breast cancer progression. Cancer Res; 73(8); 1–11. Ó2013 AACR. Introduction influence metastatic potential (2). These efforts have resulted fi The metastatic cascade is a complex and extremely ineffi- in the identi cation of a number of genes associated with – cient process with many potential barriers. Because as much as metastatic potential in both mouse and humans (2 5). As these 90% of cancer-associated mortality is associated with meta- genes do not appear to be frequently somatically mutated in fi static disease rather than primary tumor burden, understand- breast cancer, identi cation and characterization of inherited ing the molecular mechanisms and pathways exploited by metastasis susceptibility genes provide novel insights into the cancer cells during the metastatic process is of critical impor- mechanisms underlying the terminal stages of breast cancer tance. Decades of metastasis research have led to a variety of progression. fi different models of the metastatic process (1). Although the More recently, there has been a signi cant interest in metastatic process likely involves a combination of models, our characterizing the role of microRNAs in tumor progression. — laboratory has focused on inherited genetic variants that The discovery of microRNAs (miRNA small, noncoding RNAs) in 1993 sparked a paradigm shift in our understanding of gene regulation. Initially, miRNAs were classified as func- tionless stretches within "junk DNA" but are currently viewed fi 1 Authors' Af liations: Laboratory of Cancer Biology and Genetics, and as powerful regulators of gene expression as it relates to 2Laboratory of Molecular Technology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland cellular development, death, and proliferation (6), as well as disease (7). Because miRNAs negatively regulate gene expres- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). sion posttranscriptionally, they can act as either tumor sup- pressors or oncogenes (8), depending on their gene targets and Current address for C.H. Kim: DxTerity Diagnostics, Rancho Dominguez, California. cellular context (9). Recently, much attention has been focused on the role dysregulated miRNAs have on tumorigenesis (10) Corresponding Author: Kent W. Hunter, Metastasis Susceptibility Sec- fi tion, Laboratory of Cancer Biology & Genetics, CCR/NCI/NIH, Building 37 and metastasis (11) and whether miRNA pro les can be used Room 5046D, 37 Convent Drive, Bethesda, MD 20892. Phone: 301-435- for diagnosis and prognosis in cancer (12). Most interesting, 8957; Fax: 301-480-2772; E-mail: [email protected] germline mutations in microRNAs have been associated with doi: 10.1158/0008-5472.CAN-12-3513 skeletal and growth defects (13), chronic lymphocytic leukemia Ó2013 American Association for Cancer Research. (CLL; ref. 14), and breast cancer (15), thereby supporting the www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst February 27, 2013; DOI: 10.1158/0008-5472.CAN-12-3513 Goldberger et al. notion that germline mutations in certain miRNAs may create Controls Kit. Four replicates of each sample were hybridized to a predisposition towards tumorigenesis. GeneChip Mouse Gene 1.0 ST Array in the GeneChip Hybrid- In this study, we analyzed miRNA expression levels in the ization Oven 645 while shaking at 60 rpm at 45C for 16 hours. mammary tumors of the (PyMT Â AKXD) F1 progeny Washing and staining were conducted on the GeneChip Flu- described previously (16) to determine whether a link exists idics Station 450 and scanned on the GeneChip Scanner 3000. between inherited differences in miRNA expression and mam- Data were collected using the GeneChip Command Console mary tumor progression. We provide evidence that miR-290- Software (AGCC). All reagents, software, and instruments used, 3p, a member of the pluripotency miR-290-295 cluster (17–19), except for the Agilent 2100 Bioanalyzer, were from Affymetrix. an ortholog of the human miR-371-373 cluster (19), is differ- entially expressed between the highly metastatic AKR/J and RNA isolation poorly metastatic DBA2/J strains of mice. Increased expression Tumors were snap-frozen upon harvesting and stored at of miR-290 suppresses tumor growth and progression in a À80C. All tumors were homogenized on dry ice in an RNase- tumor autonomous manner, in part, by targeting the recently free environment. The RNA was isolated using the mirVana described tumor progression gene, Arid4b (20). To the best of miRNA Isolation Kit (Ambion). The RNA for all remaining our knowledge, this is the first example of an inherited samples, including cell lines, was isolated using the RNAeasy miRNA expression difference being associated with tumor Kit (Qiagen). progression. Quantitative real-time PCR and Western blot Materials and Methods Total RNA was reverse-transcribed with iScript cDNA Syn- Cell lines thesis Kit (Bio-Rad) and PCR-amplified using QuantiTect SYBR All cells were cultured in Dulbecco's Modified Eagle's Media Green PCR Kit (Qiagen) on the Applied Biosystems 7900HT (DMEM) with 10% FBS and antibiotics. Puromycin was used Fast Real-Time PCR System (Applied Biosytems). The standard for selection. curve method was used for quantitation and normalized to endogenous control Ppib levels. TaqMan MicroRNA Assays Mouse strains (Applied Biosystems) were used to measure miRNA expression. The AKXD RI mice were generated as described (21). The Expression of miRNA was defined from the threshold cycle, and 634Mul PyMT mouse strain FVB/N-TgN(MMTV-PyVT) (22) was relative expression levels were calculated using the 2 À DDCt then bred to 18 of the AKXD RI strains to generate 18 (PyMT Â method (23) after normalization with reference snoRNA135. AKXD) F1 sublines (16). Primers for reverse transcription (RT)-PCR: Ppib and Arid4b. miRNA microarray analysis of AKXD RI tumors Mouse PPIB: 50-GGAGATGGCACAGGAGGAAAGAG-30 The LMT_miRNA_v2 microarray was designed using the (forward) Sanger miR9.0 database (http://microrna.sanger.ac.uk) and Mouse PPIB: 50-TGTGAGCCATTGGTGTCTTTGC-30 (reverse) manufactured as custom-synthesized 8 Â 15K microarrays 0 0 Mouse ARID4B: 5 -AACAAAGGTGCAGGTGAAGC-3 (Agilent Technologies). Each mature miRNA was represented (forward) by þ and À (reverse complement) strand sequences. Each Mouse ARID4B: 50-ACATCAGTGCCCACTGTCAA-30 (reverse) probe has 4 replicates within each microarray, providing Mouse ESR1: 50-TCTCTGGGCGACATTCTTCT-30 (forward) technical replicates for consistency and performance of the 0 0 microarray. Each unique mature miRNA was represented by 8 Mouse ESR1: 5 -CATGGTCATGGTAAGTGGCA-3 (reverse) 0 0 probes (4 þ strand and 4 À strand). A total of 3,556 unique LMT Mouse EGFR: 5 -GGCGTTGGAGGAAAAGAAAG-3 (forward) 0 0 seq IDs (miRNA, positive and negative controls, Æstrand) were Mouse EGFR: 5 -ATCCTCTGCAGGCTCAGAAA-3 (reverse) on the microarray, each with 4 replicates. Mouse C3: 50-GGCCTTCTCTCTAACAGCCA-30 (forward) Total RNA (1 mg) was labeled using the miRCURY LNA Mouse C3: 50-TGCAGGTGACTTTGCTTTTG-30 (reverse) 0 microRNA Array Power Labeling Kit (Exiqon Inc.). The 3 -end Mouse DLC1: 50-CCTGGCTGGAATAGCATCAT-30 (forward) of the RNA was enzymatically labeled with Hy3 for the sample Mouse DLC1: 50-ATGCATGGGTCAAGGAAGAG-30 (reverse) fl RNA and Hy5 uorescent dye (Exiqon) for the reference RNA Mouse IL6ST: 50-CTGAGGGACCGGTGGTGT-30 (forward) (Ambion reference
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