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Title Page Title: TRA2A promoted paclitaxel resistance and tumor progression in triple negative breast cancers via regulating alternative splicing
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026
TRA2A overexpression, the decreased expression of RSRC2 was associated with
poor prognosis in TNBC.
In summary, this study represents an important example of how a splicing factor can
control critical AS events in chemotherapy resistance and cancer progression of
TNBC. These results sugguest a causal role for the TRA2A in TNBC progression and
modulating RSRC2 splicing might be a potential therapeutic intervention for TNBC.
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Figure Legends Figure 1 Paclitaxel (PTX) treatment induced a higher TRA2A expression in MDA-MB-231 cells. (A) After PTX treatment, the majority of these cells died within 2 weeks, black arrowhead indicated few survived cells. (B) The survived cells resumed proliferation and established characteristic clones within 3-4 weeks (black arrow). (C) RNA splicing pathway was activated after PTX treatment. (D) STRING analysis showed there were stronger interactions among hnRNPM, hnRNPu, hnRNPA2B1, SRSF6 and DHX9 while a weaker interaction between hnRNPm and TRA2A or hnRNPu and TRA2A. (E) qPCR validated that the expression TRA2A and hnRNPm was higher than other splicing factors. The product of TRA2A gene paralogs, i.e. TRA2B, didn’t show significant increase in contrast to higher TRA2A expression after PTX treatment. (F) Western blotting showed an increase in TRA2A and hnRNPm protein levels under PTX treatment compared to normal conditions. TRA2A protein level didn’t increase following exogenous hnRNPm expression. (G) TRA2A protein level didn’t decrease following hnRNPm knockdown. TRA2A protein level showed a dramatic increase in hnRNP knockdown MDA-MB-231 cells with PTX treatment. Figure 2 TRA2A expression promoted PTXR, cancer cells survival and invasion. (A) MDA-MB-231 and Hs578T cells transfected with TRA2A overexpression plasmid expressed higher levels of TRA2A than control cells. (B) The IC50 value for PTX was higher in TRA2A overexpressing cells. (C) TRA2A overexpressing cells formed more colonies than control cells when given PTX treatment. (D) TRA2A silencing in PTXR cells resulted in fewer colonies than control cells when given PTX treatment. (E) TRA2A silencing in PTXR cells induced IC50 decreased from 208.4 nM to 59.6 nM. (F) The TRA2A transfected cells displayed the faster speed of wound healing. (G-H) The increased migration (G) and invasion (H) ability was observed in TRA2A overexpressing cells. Figure 3 Global splicing and transcriptional regulation by TRA2A. (A) Example of alternative exon affected by TRA2A. (B) Quantification of the different AS events affected by TRA2A. (C) AS events could be negatively or positively regulated by TRA2A. (D) Gene ontology of TRA2A-regulated AS targets. (E-F) Functional association network of TRA2A-regulated AS targets. The genes in (D) were analyzed using the STRING database, and subgroups were marked according to their functions. (G) Gene ontology analyses of TRA2A-regulated gene expression events. (H) Validation of PCNA, CDC25A and CDC6 expression changes by qRT-PCR. (I) hnRNPm and SRSF6 showed an increased expression following TRA2A upregulation.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026
Figure 4 TRA2A expression and the AS events regulated by TRA2A played key roles in PTXR of TNBC. (A) Validation of TRA2A-regulated cassette exon events by qRT-PCR using MDA-MB-231 cells transfected with TRA2A or control vectors (* mean fold change was >2 or <-2). (B) The relative changes of splicing indexes obtained from qRT-PCR were highly correlated to those observed by microarray data and TAC analysis. (C-K) CALU, RSRC2 and PALM isoform shift occurred under PTX treatment. qRT-PCR analysis of levels of CALU (C), RSRC2 (F) and PALM (I) isoforms using primers that specifically detected either short isoform or long isoform containing variable exons. The ratio of CALUl to CALUs (D) and RSRC2l to RSRC2s (G) was increased while the ratio of PALMl to PALMs (J) was decreased. The total levels of CALU (E), RSRC2 (H) and PALM (K) weren’t significantly changed. (L) TRA2A positive signal was located in the nucleus in TNBC tissue. The negative expression of TRA2A presented in non-TNBC tissue (black arrow indicated a normal breast duct). (M) TRA2A expression was higher in TNBC than in non-TNBC. (N) TRA2A expression significantly associated with poorer overall survival and disease-specific survival. (O) For TNBC patients with PTX chemotherapy, elevated TRA2A expression was significantly associated with poor survival. (P) There was no significant difference in the survival between TRA2A positive and TRA2A negative of non-TNBC patients with PTX chemotherapy. Figure 5 RSRC2s isoform expression played roles in PTXR of TNBC. (A-B) Both TRA2A protein and mRNA expression was higher in PTXR than in non-PTXR of TNBC. (C-D) RSRC2l (C) showed dramatically increased expression while RSRC2s (D) displayed decreased expression in PTXR than in non-PTXR. (E-H) The level of CALUl (E), CALUs (F), PALMl (G) and PALMs (H) did not differ significantly between PTXR and non-PTXR. (I) The level of TRA2A, RSRC2l and RSRC2s mRNA was significantly different between TRA2A protein positive and negative cases. However no significant differences were found in CALUl, CALUs, PALMl and PALMs levels. (J-K) ROC curve analysis of TRA2A protein, TRA2A mRNA and RSRCl for PTXR prediction. (L-M) ShRNA mediated RSRC2 knockdown (mRNA and protein) resulted in PTXR in MDA-MB-231 cells. Compared with control cells, cells with RSRC2 knockdown formed more colonies (L) and showed a worse IC50 (M) when given PTX treatment. (N-O) Forced RSRC2 expression in MDA-MB-231TRA2A cells led to fewer colonies formation (N) and lower IC50 (O) when given PTX treatment. Figure 6 The decreased RSRC2 protein expression could be induced by the shift from RSRC2s to RSRC2l mRNA which controlled by TRA2A. (A) Normal breast tissue showed strong RSRC2 protein expression and in most of low grade TNBC, a moderate to strong staining of RSRC2 could be observed. In contrast, a decreased staining was observed in high grade TNBC. (B) RSRC2 protein expression was related to TNBC grade. (C) RSRC2 protein expression showed negatively correlation with TRA2A protein expression. (D) The mean expression of RSRC2l mRNA was lower in RSRC2+ cases than in RSRC2- cases. (E) In contrast, the mean expression of RSRC2s mRNA was higher in RSRC2+ cases than in RSRC2- cases. (F) Survival analysis showed that the decreased RSRC2 protein expression was significantly associated with poor survival. (G) MDA-MB-231 cells exhibited a significant shift in RSRC2 AS toward the RSRC2 exon4 inclusion isoform under PTX treatment or TRA2A upregulation. (H) RSRC2 protein expression was decreased under PTX
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026
treatment or TRA2A upregulation. (I) Schematic of the RSRC2 minigene construct. The exon4 and its flanking introns were inserted between two constitutive exons. (J) RT-PCR analysis of RNA harvested from 293T cells cotransfected with the RSRC2 minigene containing upstream sequence and the indicated amounts of TRA2A showed that TRA2A promoted RSRC2 exon4 inclusion in a dose-dependent manner. In contrast, cells transfected with the minigene containing downstream sequence displayed no difference in RSRC2 AS pattern compared with control cells. (K) qRT-PCR analysis of RSRC2 exon4 inclusion using RNA samples shown in C. Ratios of exon4 inclusion and skipping were plotted. (L) RT-PCR analysis of cells transfected with 2 μg of TRA2A and RSRC2 minigene constructs that contained upstream sequence (minegene1) displayed exon4 inclusion, while replacing RAAG or AGAA in minegene1 sequence (minigene3) reduced exon4 inclusion.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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. 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. 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. 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. 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. 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. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026
Published OnlineFirst April 17, 2017.Mol Cancer Ther Tieju Liu, Huizhi Sun, Dongwang Zhu, et al. splicingin triple negative breast cancers via regulating alternative TRA2A promoted paclitaxel resistance and tumor progression
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 17, 2017; DOI: 10.1158/1535-7163.MCT-17-0026