endocrine resistance in estrogen receptor-positive breast cancer. Modern Pathology, 28(5), 677–685. http://doi.org/10.1038/modpathol.2014.146 Gökmen-Polar, Y., Neelamraju, Goswami, C. P., Gu, X., Nallamothu, G., Janga, S. C., & Badve, (2015). Expression levels of SF3B3 correlate with prognosis and This is the author's manuscript of article published in final edited form as: 1 Expression levels of SF3B3 correlate with prognosis and endocrine resistance in estrogen 2 receptor positive breast cancer 3 4 Yesim Gökmen-Polar1*, Yaseswini Neelamraju3, Chirayu P. Goswami4, Xiaoping Gu1, 5 Gouthami Nallamothu1, Sarath Chandra Janga3,4,5, and Sunil Badve1,2,6* 6 Departments of 1Pathology and Laboratory Medicine, 2Medicine, Indiana University School of 7 Medicine, Indianapolis, IN, 3Department of Biohealth Informatics, School of Informatics and 8 Computing, IUPUI, Indianapolis, IN, 4Center for Computational Biology and Bioinformatics, 9 Indiana University School of Medicine; 5Department of Medical and Molecular Genetics, 10 Indiana University School of Medicine; 6Indiana University Melvin and Bren Simon Cancer 11 Center, Indianapolis, IN. 12 13 Grant Support 14 This work was supported by the Susan G Komen for the Cure® and 100 Voices of Hope to Sunil 15 Badve. 16 17 Disclosure of Potential Conflicts of Interest 18 The authors disclose no potential conflicts of interest. 19 20 *Address of corresponding: 21 Yesim Gökmen-Polar, PhD 22 Department of Pathology and Laboratory Medicine 23 Indiana University School of Medicine 24 635 Barnhill Dr., MS 0038 25 Indianapolis, IN 46202 26 [email protected] 27 28 Sunil Badve, MD, FRCPath 29 Department of Pathology and Laboratory Medicine 30 Indiana University School of Medicine 31 350 West 11th Street, IUHPL 4050 32 Indianapolis, IN 46202, USA 33 [email protected] 34 35 Running head: SF3B1 and SF3B3 expression in endocrine resistance 36 Keywords: Breast cancer, SF3B1, SF3B3, endocrine resistance, splicing factors 37 1 38 Abstract 39 De novo or acquired resistance to endocrine therapy limits its utility in a significant 40 number of estrogen receptor-positive (ER-positive) breast cancers. It is crucial to identify novel 41 targets for therapeutic intervention and improve the success of endocrine therapies. Splicing 42 factor 3b, subunit 1 (SF3B1) mutations are described in luminal breast cancer albeit in low 43 frequency. In this study, we evaluated the role of SF3B1 and SF3B3, critical parts of the SF3b 44 splicing complex, in ER-positive endocrine resistance. 45 To ascertain the role of SF3B1/SF3B3 in endocrine resistance, their expression levels 46 were evaluated in ER-positive/endocrine-resistant cell lines (MCF-7/LCC2 and MCF-7/LCC9) 47 using a real-time quantitative Reverse Transcription PCR (qRT-PCR). To further determine their 48 clinical relevance, expression analysis was performed in a cohort of 60 paraffin-embedded ER- 49 positive, node-negative breast carcinomas with low, intermediate, and high Oncotype DX 50 recurrence scores. Expression levels of SF3B1 and SF3B3 and their prognostic value were 51 validated in large cohorts using publicly available gene expression datasets including The Cancer 52 Genome Atlas. 53 SF3B1 and SF3B3 levels were significantly increased in ERα-positive cells with acquired 54 tamoxifen (MCF-7/LCC2; both P<0.0002) and fulvestrant/tamoxifen resistance (MCF-7/LCC9; 55 P=0.008 for SF3B1 and P=0.0006 for SF3B3). Expression levels of both MCF-7/LCC2 and 56 MCF-7/LCC9 were not affected by additional treatments with E2 and/or tamoxifen. 57 Furthermore, qRT-PCR analysis confirmed that SF3B3 expression is significantly upregulated in 58 Oncotype DX high risk groups when compared with low risk (P=0.019). Similarly, in publicly- 59 available breast cancer gene expression datasets, overexpression of SF3B3, but not SF3B1, was 2 60 significantly correlated with overall survival. Furthermore, the correlation was significant in ER- 61 positive, but not in ER-negative tumors. 62 This is the first study to document the role of splicing factor SF3B3 in endocrine 63 resistance and prognosis in ER-positive breast cancer. Potential strategies for therapeutic 64 targeting of the splicing mechanism(s) need to be evaluated. 65 66 3 67 INTRODUCTION 68 The process of alternative splicing results in synthesis of multiple mRNA variants from a 69 single gene. Earlier studies reported the importance of splice variants in a number of key genes 70 regulating signaling pathways such as apoptosis, metabolism, and angiogenesis. Alterations in 71 expression of some splicing factors, genes that regulate splicing process of mature messenger 72 RNA species from primary transcripts, have also been reported in various cancers including 73 breast cancer.1 The utilization of next-generation sequencing technologies have further revealed 74 that the presence of somatic mutations in splicing factors.2 Among them, the splicing factor 75 SF3B1 mutations occurred in several types of hematological malignancies as well as in solid 76 cancers such as breast, pancreatic cancers, and uveal melanomas.3-7 These mutations were shown 77 to be associated with alternative splicing in lymphocytic leukemia and uveal melanomas.8, 9 78 However, the impact of these factors in the clinical setting has not been well established in breast 79 cancer, and their clinical utility is not clear. 80 SF3B1 is one of the 35 most frequently mutated genes using next-generation sequencing 81 on 510 breast tumors.5 However, the frequency is low (2% of all tumors). Of the 15 nonsilent 82 mutations, the majority were missense mutations. Patients with ER-positive and HER2 subtypes 83 harbored the majority of these mutations. The SF3B1 was also amongst the 18 significantly 84 mutated genes in untreated ER-positive breast tumors from 77 patients accrued from two 85 neoadjuvant aromatase inhibitor clinical trials.10 Taken together, these studies indicate the 86 potential role of SF3B multiprotein complex in ER-positive breast cancers. 87 SF3B1 encodes the largest subunit of the SF3B protein complex, a key component of the 88 core spliceosome complex essential for the accurate excision of introns from premessenger RNA 89 to form mature mRNA. SF3B1 interacts with other SF3B subunits including SF3B3.11-14 Based 4 90 on the preclinical data, SF3B1 mutation (R1074H) confers resistance to spliceosome inhibitors, 91 such as pladienolide, and impairs its binding to SF3B complex.15 Furthermore, SF3B1 and 92 SF3B3 interactions are believed to be necessary to form a binding site for this class of 93 inhibitors.15, 16 Therefore, these genes may serve as potential new therapeutic targets in ER- 94 positive breast cancer. However, there is a relative lack of data regarding the expression and 95 prognostic value of these genes in ER-positive breast cancer and their clinical utility in endocrine 96 therapy. Despite the presence of SF3B1 mutations, no SF3B3 mutations were detected in either 97 The Cancer Genome Atlas or in neoadjuvant aromatase inhibitor clinical trials. Decreased SF3B3 98 expression has been reported in breast cancer, but attributed to loss of 16q, the location of the 99 gene.17 In this study, the expression of SF3B1 and SF3B3 was analyzed in breast cancer cell lines 100 with acquired resistance to endocrine therapy and in a cohort of Oncotype DX cases using qRT- 101 PCR. The findings were further confirmed in publicly available breast cancer datasets, including 102 The Cancer Genome Atlas, and correlated their prognostic relevance and clinical utility for 103 endocrine therapy. Here, we report the clinical relevance of SF3B1 and SF3B3 in development of 104 innate and acquired endocrine resistance in ER-positive breast cancers. 105 106 METHODS 107 Breast cancer cell lines 108 LCC2 (MCF-7/LCC2; resistant to tamoxifen), LCC9 (MCF-7/LCC9; resistant to 109 fulvestrant [Faslodex; ICI 182,780] and cross-resistant to tamoxifen), and AZ (MCF-7/AZ 110 control) cell lines were kind gifts from Dr. R. Clarke (Georgetown University Medical School, 111 Washington DC).18, 19 Cell lines have been carefully maintained in a humidified tissue culture o 112 incubator at 37 C in 5% CO2:95% air atmosphere and stocks of the earliest passage cells have 5 113 been stored. The cell lines were grown in phenol-red–free DMEM containing 5% charcoal- 114 stripped fetal calf serum (CCS) and 100 mg/mL penicillin as described previously.18, 19 115 Oncotype DX samples 116 All protocols were reviewed and approved by the Institutional Review Board of Indiana 117 University. Sixty archival formalin-fixed, paraffin-embedded tumor blocks were obtained from 118 patients with ER-positive (greater than 1% expression as per ASCO-CAP guidelines) node- 119 negative breast carcinomas at the Indiana University Simon Cancer Center based on their 120 Oncotype DX recurrence score (19 low score, 21 intermediate score, and 20 high score). Four of 121 the 60 cases had a lobular histology. Demographic and clinical characteristics of the patients 122 were acquired from medical charts (Suppl Table S1). 123 RNA isolation and qRT-PCR 124 RNA was extracted from 10μm-thick sections of archival paraffin blocks using 125 RecoverAll™ total nucleic acid isolation kit (Life Technologies, Grand Island, NY). For breast 126 cancer cell lines, RNAs were isolated (RNeasy isolation kit, Qiagen, VA) and treated with Turbo 127 DNase® (Ambion, Foster City, CA) to remove contaminating DNA. The quality of RNA was 128 assessed using the Nanodrop® ND-1000 spectrophotometer (ThermoScientific, Wilmington, 129 DE). Total RNAs were reverse-transcribed using the high capacity cDNA reverse transcription 130 kit (Life Technologies) according the manufacturer’s instructions. The mRNA levels of SF3B1 131 and SF3B3 were analyzed by qRT-PCR using TaqMan gene expression assays on an ABI Prism 132 7900 platform (Applied Biosystems, Foster City, CA) with ACTB and GUSB as endogenous 133 controls for normalization. All qRT-PCR reactions from tumor blocks and breast cancer cell 134 lines were performed in duplicates and triplicates, respectively.