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Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and C-JUN to Bypass PUMILIO Posttranscriptional Regulation Wayne O

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Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and C-JUN to Bypass PUMILIO Posttranscriptional Regulation Wayne O Published OnlineFirst October 10, 2016; DOI: 10.1158/0008-5472.CAN-16-0844 Cancer Tumor and Stem Cell Biology Research Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation Wayne O. Miles1,2, Antonio Lembo3,4, Angela Volorio5, Elena Brachtel5, Bin Tian6, Dennis Sgroi5, Paolo Provero3,4, and Nicholas Dyson1 Abstract Alternative polyadenylation (APA) is a process that changes the targets. mRNA stability and luciferase assays demonstrated APA- posttranscriptional regulation and translation potential of dependent alterations in RNA and protein levels of affected mRNAs via addition or deletion of 30 untranslated region (30 candidate genes. Examination of clinical parameters of these UTR) sequences. To identify posttranscriptional-regulatory events tumors found those with APA of NRAS and c-JUN to be smaller affected by APA in breast tumors, tumor datasets were analyzed and less proliferative, but more invasive than non-APA for recurrent APA events. Motif mapping of the changed 30 UTR tumors. RT-PCR profiling identified elevated levels of poly- regions found that APA-mediated removal of Pumilio regulatory adenylation factor CSTF3 in tumors with APA. Overexpression elements (PRE) was unusually common. Breast tumor subtype– of CSTF3 was common in triple-negative breast cancer cell specific APA profiling identified triple-negative breast tumors as lines, and elevated CSTF3 levels were sufficient to induce APA having the highest levels of APA. To determine the frequency of of NRAS and c-JUN. Our results support the hypothesis that these events, an independent cohort of triple-negative breast PRE-containing mRNAs are disproportionately affected by tumors and normal breast tissue was analyzed for APA. APA- APA, primarily due to high sequence similarity in the motifs mediated shortening of NRAS and c-JUN was seen frequently, and utilized by polyadenylation machinery and the PUM complex. this correlated with changes in the expression of downstream Cancer Res; 76(24); 1–11. Ó2016 AACR. Introduction cific fluctuations in the levels of RBPs and miRNAs may be particularly important, as transcripts from oncogenes and tumor Cancer genome sequencing has identified driver mutations suppressor genes contain a disproportionately high number of associated with disease. In addition, nongenetic changes such as posttranscriptional regulatory motifs. epigenetic reprogramming and aberrant posttranscriptional reg- In line with these ideas, we recently discovered that the ulation also impact tumor phenotypes. Posttranscriptional con- expression of the human oncogene E2F3 is posttranscription- trols that affect mRNA processing and stability include miRNAs ally controlled by the Pumilio complex (4). The Pumilio and RNA-binding proteins (RBP; ref. 1). These factors bind to seed complex is comprised of two RBPs in humans, Pumilio (PUM) sequences within the 30 untranslated region (UTR) of transcripts andNANOS(NOS),whichbindtoaconservedmotifof (2) and are commonly misregulated in cancers (3). Tumor-spe- 0 UGUAXAUA (5–7) and a GU-rich element within the 3 UTR of their substrates (2, 8). The Pumilio repressor has evolution- arily conserved roles in the regulation of proliferation and 1Department of Molecular Oncology, Massachusetts General Hospital Cancer differentiation (6–9). PUM complexes function to inhibit Center and Harvard Medical School, Charlestown, Massachusetts. 2Department translation (10) or induce the degradation of their substrates of Molecular Genetics, The Ohio State University Comprehensive Cancer Center, (4, 11–14). The PUM repressor regulates the translation poten- The Ohio State University, Columbus, Ohio. 3Department of Molecular Biotech- 4 tial of hundreds of transcripts including a large number of nology and Health Sciences, University of Turin, Turin, Italy. Center for Trans- oncogenes and tumor suppressor genes (5). lational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, 5 While analyzing the role of PUM in modulating E2F3 levels in Italy. Department of Pathology, Massachusetts General Hospital Cancer Center 0 and Harvard Medical School, Charlestown, Massachusetts. 6Department of cancer cell lines, we found the 3 UTR of E2F3 transcripts were Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Med- often shortened. This change in mRNA length removed a number ical School, Newark, New Jersey. of regulatory elements, including the Pumilio regulatory elements Note: Supplementary data for this article are available at Cancer Research (PRE) motifs (4). The shortened E2F3 mRNA in these cells was Online (http://cancerres.aacrjournals.org/). polyadenylated, indicating that they were generated by alter- Corresponding Authors: Nicholas Dyson, Massachusetts General Hospital, Har- native polyadenylation (APA). APA is a poorly understood vard Medical School, Building 149, 13th Street, Charlestown, MA 02129. Phone: developmental process that leads to the use of noncanonical 617-726-7800; Fax: 617-726-7808; E-mail: [email protected]; and polyadenylation sites (15–17). APA affects the inclusion/exclu- Wayne O. Miles,The Ohio State University, 820 Biomedical Research Tower, sion of regulatory elements and can alter mRNA stability and 460 West 12th Street, Columbus, OH 43219. Phone: 614-366-2869; E-mail: translation potential (18). During normal development, APA is [email protected] utilized to regulate differentiation (15, 16, 19–21). In cultured doi: 10.1158/0008-5472.CAN-16-0844 human cells, APA has been linked with genes responsible for Ó2016 American Association for Cancer Research. cell proliferation (22, 23). www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst October 10, 2016; DOI: 10.1158/0008-5472.CAN-16-0844 Miles et al. APA is also a likely contributor to oncogenesis (24), as APA has tories/A300-202A), PABPN1 (Abcam/AB75855), FOXO1 (Cell been correlated with poor patient prognosis in a variety of Signaling Technology/2880), JUN (Cell Signaling Technology/ malignancies (25–27). Despite this, little is known about APA 9165), PTEN (Cell Signaling Technology/9552), and NRAS in tumor development. Our finding that the E2F3 mRNA is altered (Abcam/ab77392). by APA in cancer cell lines, prompted us to investigate this phenomenon further. To do this, we looked for mRNAs that are Luciferase assays altered by APA in human breast tumor samples compared with Cells were transfected and luciferase levels were measured 48 Æ ¼ normal tissue. This analysis found that APA is widespread in hours after transfection (data are expressed as mean SD, n 3). breast tumors and that these APA events disproportionately Luciferase readings were taken using the Dual-Luciferase Reporter remove putative PUM-regulatory elements. Assay System (Promega). Breast tumor subtype–specific APA profiles were then generated and unexpectedly, this revealed that triple-negative breast tumors Lentiviral shRNA have a distinct APA profile from other subtypes. Four candidate The DNA preparation, transfections, and virus preparation APA events were then selected and their frequency assayed in an methods have been published elsewhere (4, 35). independent cohort of triple-negative breast tumors. These results siRNA transfection led us to focus on two recurrent APA-altered mRNAs, c-JUN and Cells were transfected with siRNAs targeting PABPN1 and a NRAS, for detailed analysis. These genes were selected as they scrambled control (Dharmacon) using Lipofectamine RNAiMAX. displayed high levels of APA in tumors. Here, we show that these Cells were analyzed using RT-PCR and Western blotting 2 days mRNAs are targets of PUM-mediated repression and demonstrate after transfection. Individual siRNAs targeting PUM1, PUM2, and that APA circumvents this regulation. Strikingly, both c-JUN and CSTF3 were from Life Technologies (Sequences in Supplementary NRAS were known to be deregulated in triple-negative breast Table S2). tumors (28, 29), but the underlying mechanism was unclear. In addition, APA of NRAS and c-JUN altered the expression of downstream target genes. Intriguingly, unlike previous reports, RNA stability assays m APA in this tumor cohort correlated with smaller, slower growing Cells were treated with actinomycin D (5 mol/L) and collected but more invasive node-positive tumors. Collectively, these find- every hour. Transcript levels were assayed using RT-PCR. Cells fi ings indicated that APA of PUM substrates is a common feature of were depleted using shRNA speci c to PUM1, PUM2, and scram- triple-negative breast tumors. These results show that recurrent bled controls and placed under puromycin selection for 4 days. APA of PRE-containing mRNAs allows transcripts to circumvent Knockdown was validated by Western blot analysis. PUM-mediated repression and provide a mechanistic explanation 0 for the increased activity of NRAS and c-JUN in tumors. 3 RACE RT-PCR Cells were lysed after selection for lentiviral infection and RNA Materials and Methods was extracted using the Qiagen RNeasy kit. cDNA was generated using an extended oligo-dT primer (P7 oligo-dT). This cDNA was Bioinformatics used in a standard RT-PCR reaction using a gene and poly-A site– We used the data from ref. 30 [Gene Expression Omnibus (31) specific primer as a reverse P7 primer (36). (GSE3744)] to compare 38 tumor samples with 7 normal breast samples. Analysis of differential APA between tumors and normal Patients
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