Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title Page Title: TRA2A promoted paclitaxel resistance and tumor progression in triple negative breast cancers via regulating alternative splicing Tieju Liu1,2, Huizhi Sun1, Dongwang Zhu3, Xueyi Dong1,2, Fang Liu1,2, Xiaohui Liang1,2, Chen Chen1, Bing Shao1, Meili Wang1, Yi Wang1, Baocun Sun1,2* 1 Department of Pathology, Tianjin Medical University, Tianjin 300070, China 2 Department of Pathology, General Hospital of Tianjin Medical University, Tianjin 300052, China 3 Stomatology Hospital of Tianjin Medical University, Tianjin, China *Corresponding author: Prof. Baocun Sun, Department of Pathology and General Hospital of Tianjin Medical University, Tianjin, China; E-mail: [email protected]; [email protected] Tel:86-13602111192 Fax:86-22-83336813 Running title: TRA2A and paclitaxel resistance Keywords: triple-negative breast cancer; alternative splicing; TRA2A; paclitaxel resistance 1 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abbreviations AS, alternative splicing alt-exons, AS exons CE, cassette exon con-exons, constitutive exons DSS, disease-specific survival GO, Gene Ontology hnRNPs, heterogenous ribonucleoproteins IC50, inhibitory concentration 50 MTT, methylthiazol tetrazolium nt, nucleotide OS, overall survival pre-mRNAs, precursor messenger RNAs PTX, paclitaxel PTXR, PTX resistant ROC, receiver-operating characteristic SI, splicing index STRING, Search Tool for the Retrieval of Interacting Genes/Proteins TAC, Transcriptome Analysis Console TNBC, triple-negative breast cancer TRA2, Transformer2 Grant Support: This work was partly supported by a grant from The National Natural Science Foundation of China (No. 81672870 to T. Liu and No. 81572872 to X. Zhao), Key project of the National Natural Science Foundation of China (No. 81230050 to B. Sun), and National Undergraduate Training Program for Innovation and Entrepreneurship (No. 201510062001 to H. Sun). 2 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Treatment of triple-negative breast cancer (TNBC) has been challenging and paclitaxel (PTX) resistance is one of the major obstacles to the better prognosis. Deregulation of alternative splicing (AS) may contribute to tumor progression and chemotherapy resistance. Human AS factor TRA2 has two separate gene paralogs encoding TRA2A and TRA2B proteins. TRA2B is associated with cancer cell survival and therapeutic sensitivity. However the individual role of TRA2A in cancer progression has not been reported. Here we report that TRA2A facilitates proliferation and survival, migration and invasion of TNBC cells. In addition, TRA2A promotes PTX resistance of TNBC by specifically controlling cancer-related splicing, which is independent of other splicing factors. TRA2A overexpression could promote AS of CALU, RSRC2 and PALM during PTX treatment of TNBC cells. The isoform shift of RSRC2 from RSRC2s to RSRC2l leads to a decreased RSRC2 protein expression, which could contribute to TNBC PTX resistance. TRA2A can regulate RSRC2 AS by specifically binding upstream intronic sequence of exon4. Strikingly, TRA2A expression is increased dramatically in TNBC patients, and has close relationship with decreased RSRC2 expression; both of them are associated with poor survival of TNBC. Collectively, our findings suggest that PTX targets the TRA2A-RSRC2 splicing pathway and deregulated TRA2A and RSRC2 expression may confer PTX resistance. In addition to providing a novel molecular mechanism of cancer-related splicing dysregulation, our study demonstrates that expression of TRA2A in conjunction with RSRC2 may provide valuable molecular biomarker evidence for TNBC clinical treatment decisions and patient outcome. 3 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Triple-negative breast cancer (TNBC) patients are usually managed with chemotherapy including paclitaxel (PTX). PTX polymerizes tubulin and promotes microtubule assembly and stabilization to disrupt normal microtubule dynamics and arrest cells in mitosis. The ideal tumour responses are that cancer cells arrest in mitosis and die following PTX chemotherapy. However cancer cells can maintain viability by undergoing viable cellular responses and enhance the malignant phenotype after chemotherapy (1). Although, initially responsive to PTX, TNBC often recur and metastasize due to the development of chemoresistance (2). Alternative splicing (AS) is the process by which splice sites in precursor messenger RNAs (pre-mRNAs) are differentially selected and paired to produce multiple mature mRNAs and protein isoforms with distinct structural and functional properties. Regulation of AS is tightly controlled during normal tissue differentiation. Deregulation of AS can lead to production of aberrant protein isoforms, which may contribute to tumor establishment, progression and resistance to therapeutic treatments (3-7). In general, the regulation of AS patterns is achieved through complex interplay between cis regulatory elements within the pre-mRNAs and the trans protein factors that bind them (8, 9). The trans protein factors have been found to function in tumorigenesis and drug resistance (10). Transformer2 (TRA2) proteins that are first discovered in insects, could form an essential component of the splicing complex that controls fly sexual differentiation (11, 12), and play a role in the regulation of pre-mRNA splicing. Human TRA2 gene has two separate gene paralogs encoding TRA2A and TRA2B proteins. Both TRA2A and TRA2B contain RNA recognition motifs and extended regions of serine and arginine residues, resembling the well characterized trans protein factors (12-14). Current data implicate TRA2 proteins solely in AS rather than constitutive splicing (12, 15). There are studies that demonstrate TRA2B expression levels are upregulated in breast, cervical, ovarian, and lung cancer, and TRA2B is associated with cancer cell survival and drug sensitivity (16, 17). The known splicing targets of TRA2B identified in normal tissues are important for cancer cell biology and are particularly implicated in cell division, motility and invasion (11, 18). Recent study (12) found that simultaneous depletion of TRA2A and TRA2B induced substantial shifts in splicing 4 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. of endogenous TRA2B target exons, and that both constitutive and alternative target exons were under dual TRA2A-TRA2B control. Following depletion of TRA2B, upregulated TRA2A was able to functionally substitute for TRA2B and largely maintained TRA2B target exon inclusion, suggesting TRA2A has the same function as TRA2B and could modulate splicing events. However, the individual role of TRA2A in cancer related splicing has not been reported so far. Here we analyzed TRA2A-mediated changes of the transcriptome and assessed the role of TRA2A in PTX resistance and cancer progression of TNBC. Materials and Methods Cell culture and lentiviral transduction MDA-MB-231 and 293T cells were obtained from the American Type Culture Collection in 2012 and authenticated using short tandem repeat (STR) analysis by Genewiz Inc. in 2014. STR analysis showed that the submitted samples were in good agreement with the reference cell lines. Hs578T cells were provided by the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China in 2016. The pEZ-Lv201 vector was used for overexpressing TRA2A, hnRNPm and RSRC2, and the psi-LVRU6GP was used for hnRNPm or RSRC2 silencing (GeneCopoeia). The shRNA target sequence was (5′-ggtccgagcagacattcttga-3′) for hnRNPM and (5′-ggaagagagcgactaaattca-3′) for RSRC2. Lentiviruses were produced by transient transfection of 293T cells, and the virus suspension was used to infect the target cells. Paclitaxel (PTX) treatment PTX (Selleckchem) was prepared as a 10 μM stock solution in DMSO. Briefly, 1×106 cells were plated in 100-mm culture dishes for 24 hs and then treated with 10 nM PTX. After 3 days, fresh 10 nM PTX-containing media was added for another 2 days, totaling 5 days of PTX treatment. Cells were then rinsed with PBS and maintained in drug-free culture with media replacement every 48 hs until proliferative cell clones established. Cytotoxicity assay and inhibitory concentration 50 (IC50) measurement Cell growth determination kit (MTT based, Sigma) was used and the manufacturer’s instruction was followed. Brifely,