tra2a promoted paclitaxel resistance and tumor progression ... · tra2a promoted paclitaxel...

Cancer Biology and Signal Transduction

TRA2A Promoted Paclitaxel Resistance andTumor Progression in Triple-Negative BreastCancers via Regulating Alternative SplicingTieju Liu1,2, Huizhi Sun1, Dongwang Zhu3, Xueyi Dong1,2, Fang Liu1,2, Xiaohui Liang1,2,Chen Chen1, Bing Shao1, Meili Wang1, Yi Wang1, and Baocun Sun1,2

Abstract

Treatment of triple-negative breast cancer (TNBC) has beenchallenging, and paclitaxel resistance is one of themajor obstaclesto the better prognosis. Deregulation of alternative splicing (AS)may contribute to tumor progression and chemotherapy resis-tance. Human AS factor TRA2 has two separate gene paralogsencoding TRA2A and TRA2B proteins. TRA2B is associated withcancer cell survival and therapeutic sensitivity. However, theindividual role of TRA2A in cancer progression has not beenreported. Here we report that TRA2A facilitates proliferation andsurvival and migration and invasion of TNBC cells. In addition,TRA2A promotes paclitaxel resistance of TNBC by specificallycontrolling cancer-related splicing, which is independent of othersplicing factors. TRA2A overexpression could promote AS ofCALU, RSRC2, and PALM during paclitaxel treatment of TNBCcells. The isoform shift of RSRC2 from RSRC2s to RSRC2l leads to

a decreased RSRC2 protein expression, which could contributeto TNBC paclitaxel resistance. TRA2A can regulate RSRC2 ASby specifically binding upstream intronic sequence of exon4.Strikingly, TRA2A expression is increased dramatically inpatients with TNBC, and has a close relationship with decreasedRSRC2 expression; both are associated with poor survival ofTNBC. Collectively, our findings suggest that paclitaxel targetsthe TRA2A–RSRC2 splicing pathway, and deregulated TRA2Aand RSRC2 expression may confer paclitaxel resistance. Inaddition to providing a novel molecular mechanism of can-cer-related splicing dysregulation, our study demonstrates thatexpression of TRA2A in conjunction with RSRC2 may providevaluable molecular biomarker evidence for TNBC clinical treat-ment decisions and patient outcome. Mol Cancer Ther; 16(7);1377–88. �2017 AACR.

IntroductionTriple-negative breast cancer (TNBC) patients are usually man-

aged with chemotherapy including paclitaxel. Paclitaxel poly-merizes tubulin and promotes microtubule assembly and stabi-lization to disrupt normal microtubule dynamics and arrest cellsinmitosis. The ideal tumor responses are that cancer cells arrest inmitosis and die following paclitaxel chemotherapy. However,cancer cells can maintain viability by undergoing viable cellularresponses and enhance the malignant phenotype after chemo-therapy (1). Although, initially responsive to paclitaxel, TNBCoften recur and metastasize due to the development of chemore-sistance (2).

Alternative splicing (AS) is the process by which splice sites inprecursor messenger RNAs (pre-mRNA) are differentially selectedand paired to produce multiple mature mRNAs and proteinisoforms with distinct structural and functional properties. Reg-ulation of AS is tightly controlled during normal tissue differen-

tiation. Deregulation of AS can lead to production of aberrantprotein isoforms, which may contribute to tumor establishment,progression and resistance to therapeutic treatments (3–7). Ingeneral, the regulation of AS patterns is achieved through complexinterplay between cis regulatory elements within the pre-mRNAsand the trans protein factors that bind them (8, 9). The transprotein factors have been found to function in tumorigenesis anddrug resistance (10).

Transformer2 (TRA2) proteins that are first discovered ininsects, could form an essential component of the splicing com-plex that controls fly sexual differentiation (11, 12), and play arole in the regulation of pre-mRNA splicing. Human TRA2 genehas two separate gene paralogs encoding TRA2A and TRA2Bproteins. Both TRA2A and TRA2B contain RNA recognitionmotifsand extended regions of serine and arginine residues, resemblingthe well characterized trans protein factors (12–14). Current dataimplicate TRA2 proteins solely in AS rather than constitutivesplicing (12, 15).

There are studies that demonstrate TRA2B expression levels areupregulated in breast, cervical, ovarian, and lung cancer, andTRA2B is associated with cancer cell survival and drug sensitivity(16, 17). The known splicing targets of TRA2B identified innormal tissues are important for cancer cell biology and areparticularly implicated in cell division, motility, and invasion(11, 18). Recent study (12) found that simultaneous depletion ofTRA2A and TRA2B induced substantial shifts in splicing ofendogenous TRA2B target exons, and that both constitutive andalternative target exons were under dual TRA2A-TRA2B control.Following depletion of TRA2B, upregulated TRA2A was able tofunctionally substitute for TRA2B and largely maintained TRA2B

1Department of Pathology, Tianjin Medical University, Tianjin, China. 2Depart-ment of Pathology, General Hospital of Tianjin Medical University, Tianjin, China.3Stomatology Hospital of Tianjin Medical University, Tianjin, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthor: Baocun Sun, Tianjin Medical University, Qixiangtai RoadNo. 22, Heping District, Tianjin 300070, China. Phone/Fax: 86-22-8333-6813;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0026

�2017 American Association for Cancer Research.

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target exon inclusion, suggesting TRA2A has the same function asTRA2B and could modulate splicing events. However, the indi-vidual role of TRA2A in cancer related splicing has not beenreported so far. Here we analyzed TRA2A-mediated changes ofthe transcriptome and assessed the role of TRA2A in paclitaxelresistance and cancer progression of TNBC.

Materials and MethodsCell culture and lentiviral transduction

MDA-MB-231 and 293T cells were obtained from the ATCC in2012 and authenticated using short tandem repeat (STR) analysisby Genewiz Inc. in 2014. STR analysis showed that the submittedsamples were in good agreement with the reference cell lines.Hs578T cells were provided by the Cell Bank of Type CultureCollection of the Chinese Academy of Sciences, Shanghai, Chinain 2016. The pEZ-Lv201 vector was used for overexpressingTRA2A, hnRNPm and RSRC2, and the psi-LVRU6GP was usedfor hnRNPm or RSRC2 silencing (GeneCopoeia). The shRNAtarget sequencewas (50-ggtccgagcagacattcttga-30) for hnRNPM and(50-ggaagagagcgactaaattca-30) for RSRC2. Lentiviruses were pro-duced by transient transfection of 293T cells, and the virussuspension was used to infect the target cells.

Paclitaxel treatmentPaclitaxel (Selleckchem) was prepared as a 10 mmol/L stock

solution in DMSO. Briefly, 1 � 106 cells were plated in 100-mmculture dishes for 24 hours and then treated with 10 nmol/Lpaclitaxel. After 3 days, fresh 10 nmol/L paclitaxel-containingmedia was added for another 2 days, totaling 5 days of paclitaxeltreatment. Cells were then rinsed with PBS and maintained indrug-free culture with media replacement every 48 hours untilproliferative cell clones established.

Cytotoxicity assay and inhibitory concentration 50measurement

The Cell Growth Determination Kit (MTT based, Sigma) wasused and the manufacturer's instruction was followed. Briefly,cells were separately seeded into 96-well plates at 1 � 103 cells/well andwere treatedwith paclitaxel at different concentrations (aseries of dilutions, each a doubling dilution of the previous one)in 100 mL culture medium. After 48-hour culture, 10 mL MTT(methylthiazol tetrazolium) solution was added and incubatedfor 4 hours at 37�C. Subsequently the supernatant was removed,and 100 mL MTT solvent was added. Spectrophotometricallymeasure absorbance at a wavelength of 570 nm was performedby using a BioTek ELx800. Dose–response curves were plotted todetermine half maximal IC50 for paclitaxel using the GraphPadPrism6 (GraphPad Software). The assays were performed inde-pendently and repeated at least three times.

Plate clonogenic assayCells (5 � 104 cells/well) were cultured in 6-well plates over-

night and then exposed to 10 nmol/L paclitaxel for 5 days. Thecells were further cultured for 10 days in 6-well plates containingdrug-free medium. Clonogenic cells were determined as thoseable to form a colony consisting of at least 50 cells. The colonieswere fixed with methanol and stained with 0.5% crystal violet.

Wound-healing assayCells were implanted into 6-well plates for 90% confluence,

and then a sterilized tip was used to draw a line with the same

width on the bottom of the dishes. Images were captured at 72hours after the wounding. Data shown were representative ofthree independent repeats.

Cell migration and invasion assaysTranswell 24-well plates containing permeable polyethylene

terephthalatemembrane inserts with 8 mmol/L pores (Invitrogen)were used. A total of 1 � 105 cells in 100 mL DMEM without FBSwere placed on the top layer of Transwell inserts. Matrigel (1 mg/mL, BD Biosciences) was additionally coated on the inserts forinvasion assay. The bottom chamber was filled with 10% FBS-containing medium. The cells were incubated for 48 hours, andsubsequently nonmigratory or noninvading cells were removedfrom the top surface of the membrane. The cells that passedthrough the membrane were fixed with methanol and stainedwith 0.5% crystal violet. The number of migratory or invadingcells was counted using an inverted light microscope (Nikon).

RNA extraction and microarray analysisTotal RNA was extracted using TRIzol reagent (Tiangen Bio-

tech), and sent to Oebiotech for Affymetrix GeneChip HumanTranscriptome Array 2.0 analysis. The microarray data have beendeposited in NCBI's Gene Expression Omnibus (Liu and collea-gues, 2016) and are accessible through GEO Series accessionnumber GSE90145 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE90145).

Quantitative real-time polymerase chain reactionQuantitative real-time polymerase chain reaction (qRT-PCR)

was performed as previously described (19), using the primerslisted in Supplementary Table S3. Briefly, 2 mg of total RNA wasreverse-transcribed into cDNA using a Reverse Transcription Kit(Takara, RR037A). qRT-PCR analyses were performed withPower SYBR Green (Takara, RR820A) in 7500HT Real-TimePCR System (Applied Biosystems). GAPDH was used as anendogenous control. The qRT-PCR data of 47 cases of breastcancer tissue were presented by using the 2–DCt method (20). Thegene expression of interest was normalized to an internalcontrol (GAPDH) [DCt ¼ (Ct gene of interest � Ct GAPDH)],and three PCR replicates per cDNA sample were performed.Two–DCt values were calculated and compared between differ-ent groups by using independent-samples t test. The qRT-PCRdata of breast cancer cells were expressed as 2�DDCt using theequation [DDCt ¼ (Ct gene of interest � Ct GAPDH) treatedsample � (Ct gene of interest � Ct GAPDH) control sample]to calculate and were presented as fold change in expression.The fold change >2 or <�2 was considered as significant.

Semiquantitative RT-PCRThe assay was performed according to the recommended

thermal profile: 95�C for 5 minutes (preincubation), followedby 30 cycles at 95�C for 30 seconds (denaturation), 60�C for1 minute (annealing), and 72�C for 30 seconds (elongation).The amplified products were subjected to electrophoresis ina 2% agarose gel containing ethidium bromide (Bio-Rad).The expression of GAPDH was used to examine the integrityof the RNA in each sample and to standardize the amount ofcDNA added to each PCR tube. The following primers wereused: RSRC2: forward: 50-AGAAAACACAGGAGCCGGAG-30;reverse: 50-TGAGTGACTTCTGCCTCTTGA-30; GAPDH: forward:50-CCTGGCCAAGGTCATCCATGAC-30; reverse: 50-TGTCATAC-CAGGAAAT4-GAGCTTG-30.

Liu et al.

Mol Cancer Ther; 16(7) July 2017 Molecular Cancer Therapeutics1378



http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE90145




RSRC2 splicing reporter minigene assaysThe RSRC2 minigenes' construct was referred to RSRC2

PubMed ID NR_036434's sequence. The RSRC2 minigene 1contains exon3 (44 nucleotide, nt), 237 nt of flanking intronicsequences upstream from exon4, exon4 (78 nt), and exon5 (191nt). The RSRC2minigene 2 contains exon3 (44 nt), exon4 (78 nt),237 nt of flanking intronic sequences downstream from exon4and exon5 (191 nt). RSRC2 minigene 3 was constructed byaltering minigene 1's motif AGAAG into AGACG, AGAA intoATAA,GAAG intoGACG,AGAAG intoAGACG,AGAA intoAGTA,AGAAG into AGACG, and GAAG into GATG. These minigeneswere assembled into the pEZ-Lv206 vector and were customizedfrom GeneCopoeia, Inc. The sequences of RSRC2 minigenes hadbeen provided in Supplementary Data.

RSRC2 splicing reporter minigene assays were performed in293T cells. Briefly, 2 � 105 cells were seeded in 6-well plate 24hours before transfection using polyethylenimine. Differentamounts of TRA2A were cotransfected with the RSRC2minigenes

construct. Cells were collected 48 hours after transfection, andRNA was isolated for subsequent RT-PCR analysis.

Tissue specimensThe written informed consents from the patients for all the

breast cancer specimens were obtained. The study was conductedin accordance with Declaration of Helsinki and was approved bythe review board of Tianjin Medical University, China.

Immunohistochemistry and evaluation of IHC stainingInformation on these staining methods may be referenced to

the literatures (21–24). Scoring system was modified and usedaccording to published evaluation standard (25).

AntibodiesAntibodies used for Western blotting and IHC were as follows:

TRA2A (GeneTex), hnRNPm (Novus Biologicals), RSRC2 (NovusBiologicals).

UntreatedA

C

F G

D

E

B

GO Biological process

Fo

ld c

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mR

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exp

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RNA splicing

Nuclear mRNA splicing, via spliceosome

Cell-cycle checkpoint

Angiogenesis

Response to drug

DNA Replication

Cell adhesion

Apoptosis

Mitotic cell cycle

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TRA2A

0

TRA2AhnRNPm

hnRNPu

hnRNPA2B1 DHX9TRA2B

SRSF6

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3PTXControl

4

5

6

SMC1A

TRA2AHNRNPU

HNRNPM

HNRNPA2B1

SRSF6

DHX9

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Mean ± SD

hnRNPm

b-Actin

TRA2A

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b-Actin

PTXsi h

nRNPm

si hnRNPm

+PTXExogenous

hnRNPm

7%

7%

7.9%

7.9%

8.8%

8.8%

10.5%

11.4%

11.4%

5d PTX + 1wk 5d PTX + 2wk 5d PTX + 3wk 5d PTX + 4wk

Figure 1.

Paclitaxel (PTX) treatment induced a higher TRA2A expression in MDA-MB-231 cells.A,After paclitaxel treatment, the majority of these cells died within 2 weeks; blackarrowhead indicates few survived cells. B, The survived cells resumed proliferation and established characteristic clones within 3 to 4 weeks (black arrow).C, The RNA splicing pathway was activated after paclitaxel treatment. D, STRING analysis showed there were stronger interactions among hnRNPM, hnRNPu,hnRNPA2B1, SRSF6, and DHX9, whereas a weaker interaction between hnRNPm and TRA2A or hnRNPu and TRA2A. E, qPCR validated that the expression ofTRA2A and hnRNPm was higher than other splicing factors. The product of TRA2A gene paralogs, that is, TRA2B, did not show significant increase in contrast withhigher TRA2A expression after paclitaxel treatment. (� means fold changewas>2).F,Western blotting showed an increase in TRA2A and hnRNPmprotein levels underpaclitaxel treatment compared with normal conditions. TRA2A protein level did not increase following exogenous hnRNPm expression. G, TRA2A protein level did notdecrease following hnRNPm knockdown. TRA2A protein level showed a dramatic increase in hnRNP knockdown MDA-MB-231 cells with paclitaxel treatment.

TRA2A and Paclitaxel Resistance

www.aacrjournals.org Mol Cancer Ther; 16(7) July 2017 1379




Statistical analysisData analysis was performed with the SPSS16.0 software pack-

age (IBM). All P values were two-sided, and statistical significancewas measured at the 0.05 level.

ResultsPaclitaxel treatment induced a higher TRA2A expression inMDA-MB-231 cells

Themajority ofMDA-MB-231 died in 2weeks after treatedwithpaclitaxel 10 nmol/L for 5 days (Fig. 1A). A small number of cellssurvived (Fig. 1A, black arrowhead), resumed proliferation, andestablished characteristic clones within 3–4 weeks (Fig. 1B, blackarrow), and such cells were considered to be paclitaxel-resistant(PTXR) cells. Next these cells were sent for microarray analysis.Gene Ontology (GO) biological process revealed that the mostrepresented category wasmitotic cell cycle, apoptosis, response todrug, angiogenesis, and RNA splicing (Fig. 1C), etc. There were 8splicing factors identified by GO analysis, including DHX9,TRA2A, heterogeneous nuclear ribonucleoprotein (hnRNP)m,hnRNPu, hnRNPA2B1, SRSF6, SMC1A, RBM8A (SupplementaryTable S1). By Search Tool for the Retrieval of Interacting Genes/Proteins (STRING)-Known and Predicted Protein-Protein Inter-actions analysis (Fig. 1D), there were stronger interactions amonghnRNPM, hnRNPu, hnRNPA2B1, SRSF6, and DHX9, whereas a

weaker interaction between hnRNPm and TRA2A or hnRNPu andTRA2A. To validate these findings qRT-PCR was performed forhnRNPm, hnRNPu, hnRNPA2B1, SRSF6, DHX9, and TRA2A.Remarkably TRA2A and hnRNPm significantly showed higherexpression than other splicing factors (Fig. 1E). Consistently,Western blotting confirmed an increase in relative TRA2A andhnRNPmprotein levels under paclitaxel treatment comparedwithnormal conditions (Fig. 1F).

HnRNPm is a splicing factor that potentiates TGFb signaling,drives cells to undergo EMT, and promotes breast cancermetastasis in animals (26). Interestingly, TRA2A protein expres-sion neither increased following hnRNPm overexpression nordecreased with hnRNPm knockdown (Fig. 1F and G). In addi-tion, when MDA-MB-231 cells with hnRNPm knockdown weregiven paclitaxel treatment, TRA2A protein level still showed adramatic increase (Fig. 1G). These results suggested that TRA2Amight play an important role in PTXR, which was independentof hnRNPm.

TRA2 has two separated gene paralogs encoding TRA2A andTRA2B proteins. It has been demonstrated that TRA2B regulatessplicing patterns which are important to cancer cells splicing (15).Interestingly, TRA2B did not show significant increase in contrastwith higher TRA2A expression after paclitaxel treatment in ourstudy, suggesting TRA2A, rather than TRA2B, was contributed tocancer cells survival under paclitaxel treatment.

Control

TRA2A

A

D

G H

E F

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b-Actin

b-Actin

TRA2A

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MDA-MB-231 MDA-MB-231

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Paclitaxel (nmol/L)

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100 1,000

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100 1,000 10,000

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0 h

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MDA-MB-231

Hs578T

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0204060

80

0204060

80

MDA-MB-231

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Control TRA2A Control TRA2AHs578TMDA-MB-231

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Control TRA2A Control TRA2A Control TRA2A Control TRA2A

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Control TRA2A

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100200300400

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Hs578T ControlControl + PTX TRA2A + PTX

Hs578TControl + PTX TRA2A + PTX

TRA2AP < 0.001

P < 0.001

P = 0.005

P < 0.001 P = 0.006 P = 0.001 P = 0.002

P = 0.005

P < 0.001

P < 0.001

IC50=174.5

IC50=208.4

IC50=33.7 IC50=20.8

IC50=80.9

Hs578T

Figure 2.

TRA2A expression promoted PTXR, cancer cells survival and invasion. A,MDA-MB-231 and Hs578T cells transfected with TRA2A overexpression plasmid expressedhigher levels of TRA2A than control cells. B, The IC50 value for paclitaxel was higher in TRA2A-overexpressing cells. C, TRA2A-overexpressing cells formedmore colonies than control cells when given paclitaxel treatment.D, TRA2A silencing in PTXR cells resulted in fewer colonies than control cells when given paclitaxeltreatment. E, TRA2A silencing in PTXR cells induced IC50 decreased from 208.4 to 59.6 nmol/L. F, The TRA2A transfected cells displayed the faster speedof wound healing. G–H, The increased migration (G) and invasion (H) ability was observed in TRA2A-overexpressing cells. (�, P < 0.05).

Liu et al.





TRA2A expression was essential for the survival of PTXR tumorcells and promoted cancer cells migration and invasion

MDA-MB-231 and Hs578T cells were transfected with TRA2Aoverexpression plasmid and they expressed higher levels ofTRA2A than the empty vector transfected control cells as ana-lyzed by Western blotting (Fig. 2A). The MDA-MB-231 orHs578T cells were sensitive to the cytotoxicity of paclitaxelwith an IC50 of 33.7 or 20.8 nmol/L. In contrast, the over-expressing TRA2A cells were highly resistant to paclitaxel withan IC50 of 174.5 or 80.9 nmol/L (Fig. 2B). In line with the MTTdata, plate clonogenic assay showed that the TRA2A-overex-pressing cells had stronger survival ability after paclitaxel treat-ment and the numbers of cell colony formation were signifi-cantly higher in TRA2A overexpressing cells than that of controlcells (Fig. 2C). Remarkably TRA2A knockdown in PTXR cellsimpaired the clonogenic survival ability of the resistant cells(Fig. 2D) when given paclitaxel treatment, inducing the IC50

value decreased from 208.4 to 59.6 nmol/L (Fig. 2E).Moreover, quantitative analyses of wound-healing assay sug-

gested a significant difference in the speed of wound healing

between the TRA2A overexpression and the control cells. TheTRA2A-transfected cells displayed the faster speed of woundhealing (Fig. 2F).Meantime, the increasedmigration and invasionability was observed in TRA2A-overexpressing cells by Transwellassays (Fig. 2G and H).

Global regulation of the transcriptome by TRA2A incancer-related genes

The microarray data of MDA-MB-231–overexpressing TRA2Aand control cells were analyzed by Transcriptome AnalysisConsole (TAC) software to obtain a list of AS events. Figure3A showed the gene structure view of one example (MELKgene), there was an exon skipping event in control, but not inthe TRA2A-overexpressing cells. We identified 3290 TRA2A-regulated AS events, including cassette exon (CE), alternative5' Donor Site (A5D), alternative 3' Acceptor Site (A3A), intronretention (IR), mutually exclusive exons (MEE; Fig. 3B; Sup-plementary Table S2). Subsequent analysis indicated that theAS events could be negatively or positively regulated by TRA2A[decreased or increased splicing index (SI) value by TRA2A

Cassette Exon5

MELK

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329

700702

1,524

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TRA2aControl

Figure 3.

Global splicing and transcriptional regulation by TRA2A. A, Example of alternative exon affected by TRA2A. B, Quantification of the different AS eventsaffected by TRA2A. C, AS events could be negatively or positively regulated by TRA2A. D, Gene ontology of TRA2A-regulated AS targets. E–F, Functionalassociation network of TRA2A-regulated AS targets. The genes in (D) were analyzed using the STRING database, and subgroups were marked accordingto their functions. G, Gene ontology analyses of TRA2A-regulated gene expression events. H, Validation of PCNA, CDC25A, and CDC6 expression changesby qRT-PCR. I, hnRNPm and SRSF6 showed an increased expression following TRA2A upregulation. (� means fold change was >2 or <�2).






5MELKA

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CALU RSRC2 CEACAM1 LMCD1 PALM RFWD2

4

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1.0Overall survival

Overall survivalnon-TNBC Patients received PTX chemotherapy (n = 44)

Log rank (Mantel–Cox)

P = 0.023

Log rank (Mantel–Cox)P = 0.600




P = 0.003

TRA2A–

TRA2A+

TRA2A+-censored

TRA2A–-censored

TRA2A–

TRA2A+

TRA2A+-censored

TRA2A–-censored

TRA2A–

TRA2A+

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Log rank (Mantel–Cox)

Disease-specific survival

Disease-specific survivalOverall survival

TNBC non-TNBC

TNBC Patients received PTX chemotherapy (n = 37)Disease-specific survival

All patients (n = 100)

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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-PCRusing 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 fromqRT-PCRwere highly correlated to those observed bymicroarray data and TAC analysis. C–K, CALU, RSRC2, and PALM isoform shift occurred under paclitaxel (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 longisoformcontaining variable exons. The ratio of CALUl toCALUs (D) andRSRC2l to RSRC2s (G)was increased,whereas the ratio of PALMl to PALMs (J)was decreased.The total levels of CALU (E), RSRC2 (H), and PALM (K) were not significantly changed. L, TRA2A-positive signal was located in the nucleus in TNBC tissue. Thenegative expression of TRA2A presented in non-TNBC tissue (black arrow indicates a normal breast duct). M, TRA2A expression was higher in TNBC thanin non-TNBC. N, TRA2A expression significantly associated with poorer overall survival and disease-specific survival. O, For TNBC patients with paclitaxelchemotherapy, elevated TRA2A expression was significantly associated with poor survival. P, There was no significant difference in the survival between TRA2Apositive and TRA2A negative of non-TNBC patients with paclitaxel chemotherapy.

Liu et al.





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RSRC2s isoform expression played roles in PTXR of TNBC. A–B, Both TRA2A protein and mRNA expression was higher in PTXR than innon-PTXR of TNBC. C and D, RSRC2l (C) showed dramatically increased expression, whereas RSRC2s (D) displayed decreased expression in PTXRthan 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, nosignificant differences were found in CALUl, CALUs, PALMl, and PALMs levels. J and K, ROC curve analysis of TRA2A protein, TRA2A mRNA,and RSRCl for PTXR prediction. L and 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 paclitaxel (PTX)treatment. Forced RSRC2 expression in MDA-MB-231TRA2A cells led to fewer colonies formation (N) and lower IC50 (O) when given paclitaxeltreatment. (� , P < 0.05).






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The decreased RSRC2 protein expression could be induced by the shift from RSRC2s to RSRC2l mRNA, which was controlled by TRA2A. A, Normal breast tissueshowed strong RSRC2 protein expression and in most of low-grade TNBC, a moderate to strong staining of RSRC2 could be observed. In contrast, a decreasedstaining was observed in high-grade TNBC. B, RSRC2 protein expression was related to TNBC grade. C, RSRC2 protein expression showed negativelycorrelation with TRA2A protein expression. D, The mean expression of RSRC2l mRNA was lower in RSRC2þ cases than in RSRC2� cases. E, In contrast, themean expression of RSRC2s mRNA was higher in RSRC2þ cases than in RSRC2� cases. F, Survival analysis showed that the decreased RSRC2 proteinexpression was significantly associated with poor survival. (Continued on the following page.)

Liu et al.





expression; Fig. 3C], demonstrating that TRA2A was a generalsplicing factor that controls different types of AS when specif-ically binding to pre-mRNAs.

When analyzing cellular functions of TRA2A-regulated ASevents using GO, we found that TRA2A affected genes in the RNAprocessing pathway, including translational initiation, elongationand termination, as well as RNA splicing (Fig. 3D). Such func-tional enrichment is not surprising because TRA2A is RNA-bind-ing factor known to regulate splicing and translation. Intriguingly,TRA2A targets were also enriched with the cancer-related func-tions such as regulation of cell proliferation, includingmitotic cellcycle, cell division, and programmed cell death and apoptoticprocess. The TRA2A-regulated splicing targets involved in trans-lation control (Fig. 3E) and cell division (Fig. 3F) could befunctionally connected into well linked interaction networks.Taken together, these results suggested that the biological pro-cesses affected by TRA2A were related to proliferation, apoptosis,and tumorigenesis.

We also analyzed how TRA2A affected global gene expression.The genes regulated by TRA2A were associated significantly withcancer-related functions, as judged by GO (Fig. 3G). The expres-sion of cell proliferationmarker, PCNA,CDC25A, andCDC6,waselevated following TRA2A overexpression (Fig. 3H). Interestingly,theAS factor hnRNPmandSRSF6 showed an increased expressionfollowing TRA2A upregulation (Fig. 3I).

The AS regulated by TRA2A played roles in PTXR ofMDA-MB-231 cells

MELK, CALU, RSRC2, CEACAM1, LMCD1, PALM, andRFWD2 were selected for CE validation (Fig. 3A, Supplemen-tary Fig. S1). All the selected genes function as oncogene ortumor suppressor in cell proliferation and migration in tumorprogression (27–33). Subsequently, we designed qPCR primersto interrogate constitutive exons (con-exons) and AS exons (alt-exons) for each gene (Supplementary Table S3) and confirmedthat TRA2A either positively or negatively controlled all endog-enous AS events tested. The fold changes of con-exons and alt-exons were presented in Fig. 4A, and the relative changes of SIsobtained from qRT-PCR were highly correlated to thoseobserved by microarray data and TAC analysis (Fig. 4B; P ¼0.019; Supplementary Table S4).

We next examined the AS events of the selected 7 genesresponse to paclitaxel. A striking observation was that CALU,RSRC2, PALM AS events occurred during paclitaxel treatment(Fig. 4C–K) whereas MELK, CEACAM1, LMCD1, and RFWD2AS events were not shown (Supplementary Fig. S2). Uponpaclitaxel treatment, CALU or RSRC2 short isoform (CALUsor RSRC2s) was gradually converted to long isoform (CALUl orRSRC2l) resulting from exon inclusion. The expression ofCALUl or RSRC2l was increased 13-fold or 8-fold, whereasexpression of CALUs or RSRC2s was decreased 10-fold or 9-fold, respectively (Fig. 4C and F). The switch in CALU or RSRC2

isoform was also evidenced by the ratio of CALUl to CALUs orRSRC2l to RSRC2s, which increased approximately 143-fold or74-fold at day 14, respectively (Fig. 4D and G). Importantly,total levels of CALU (CALUt) or RSRC2 (RSRC2t) transcriptionvaried by less than 2-fold throughout the paclitaxel treatment(Fig. 4E and H). However, for PALM, exon8 skipping occurredin paclitaxel treatment, thus inducing a strong shift from thelong isoform (PALMl) to the short isoform (PALMs) emergedwith extended phases of paclitaxel treatment. The PALMlexpression was decreased 12-fold and the PALMs expressionwas increased 11-fold at day 14 (Fig. 4I). Correspondingly, theratio of PALMl to PALMs was decreased 130-fold (Fig. 4J).However, the total level of PALM (PALMt) was not significantlychanged (Fig. 4K).

TRA2A expression was specifically associatedwith PTXR of TNBC

TRA2A protein expression was detected in a cohort of 100breast cancer patients, including 37 TNBC and 63 non-TNBCpatients. TRA2A-positive signal was located in the nucleus andits expression was higher in TNBC than in non-TNBC (Fig. 4Land M, P ¼ 0.035). Moreover, survival analysis showed thatTRA2A expression was significantly associated with poorersurvival (Fig. 4N; Supplementary Table S5). On multivariateanalysis, TRA2A-positive expression remained associated withpoor survival after correcting for tumor stage [P ¼ 0.000, rela-tive risk ¼ 5.355 for overall survival (OS) and P ¼ 0.000,relative risk ¼ 5.058 for disease specific survival (DSS), respec-tively; Supplementary Table S5], supporting that TRA2A pos-itive expression was a prognostic marker independent of theclinicopathological parameters examined. In this cohort, allthe TNBC patients (n ¼ 37) and 69.8% (44/63) non-TNBCpatients received paclitaxel chemotherapy. For TNBC patients,elevated TRA2A expression was significantly associated withpoor survival (Fig. 4O; Supplementary Table S6) and wasstrong risk marker (P ¼ 0.001, relative risk ¼ 7.748 for OSand P ¼ 0.001, relative risk ¼ 7.061 for DSS, respectively;Supplementary Table S6). However, there was no significantdifference in the OS and DSS between TRA2A positive andTRA2A negative of non-TNBC patients with paclitaxel chemo-therapy (Fig. 4P). These results indicated that the TRA2A ex-pression was specifically associated with paclitaxel resistanceof TNBC but not with that of non-TNBC patients.

RSRC2 isoform shift related to TRA2A expressionoccurred in PTXR of TNBC

Forty-seven fresh and paraffin specimen of operable TNBCpatients with paclitaxel chemotherapy after surgery wereobtained. PTXR was defined as tumor recurrence and metastasis,invasive contralateral breast cancer, or death during the periodfrom the date of paclitaxel assignment to date of follow-up.Remarkably, the expression of both TRA2A protein and mRNA

(Continued.) G, MDA-MB-231 cells exhibited a significant shift in RSRC2 AS toward the RSRC2 exon4 inclusion isoform under paclitaxel (PTX) treatment or TRA2Aupregulation. H, RSRC2 protein expression was decreased under paclitaxel treatment or TRA2A upregulation. I, Schematic of the RSRC2 minigene construct. Theexon4 and its flanking introns were inserted between two constitutive exons. J, RT-PCR analysis of RNA harvested from 293T cells cotransfected with theRSRC2 minigene containing upstream sequence, and the indicated amounts of TRA2A showed that TRA2A promoted RSRC2 exon4 inclusion in a dose-dependentmanner. In contrast, cells transfected with theminigene 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 ofcells transfected with 2 mg of TRA2A and RSRC2 minigene constructs that contained upstream sequence (minegene1) displayed exon4 inclusion, whereasreplacing RAAG or AGAA in minegene1 sequence (minigene3) reduced exon4 inclusion. (� , P < 0.05).






was higher in PTXR (n ¼ 25) than in non-PTXR (n ¼ 22; Fig. 5Aand B; Supplementary Table S7). Moreover, RSRC2l showeddramatically increased expression and RSRC2s showed decreasedexpression in PTXR than in non-PTXR (Fig. 5C and D; Supple-mentary Table S7). However, the level of CALUl, CALUs, PALMl,and PALMs did not differ significantly between PTXR and non-PTXR (Fig. 5E–H; Supplementary Table S7).

Then TRA2A, CALUl, CALUs, RSRC2l, RSRC2s, PALMl, andPALMs mRNA levels were compared between TRA2Aþ andTRA2A� cases (Fig. 5I, Supplementary Table S8). The level ofTRA2A mRNA was significantly higher in TRA2Aþ than inTRA2A� as expected. In addition, we found that RSRC2lshowed increased expression while RSRC2s showed decreasedexpression in TRA2Aþ compared with TRA2A�. However, nosignificant differences were found in CALUl, CALUs, PALMl,and PALMs levels. Next, we obtained receiver-operating char-acteristic (ROC) curves with an AUC of 0.826 [95% confidenceinterval (CI), 0.699–0.954) for TRA2A protein, 0.956 (95% CI,0.906–1.006) for TRA2A mRNA, and 0.904 (95% CI, 0.819–0.989) for RSRC2l expression, whose increased expressionshowed strong capacity for PTXR prediction. The combinationof TRA2A and RSRC2l was superior to individual marker inPTXR prediction (Fig. 5J and K).

To further demonstrate the role of RSRC2s in PTXR of TNBCcells, RSRC2s mRNA and RSRC2 protein expression was steadi-ly silenced by using lentiviral shRNA RSRC2s (Fig. 5L). Asshown in Fig. 5L and M, the number of colonies and cellsurvival of MDA-MB-231 cells were significantly increased inshRSRC2s with paclitaxel treatment compared with the control.The downregulation of RSRC2s was also significantly associatedto a worse IC50 (33.7 nmol/L in control vs 168.9 nmol/L inshRSRC2s; Fig. 5M).

Now that knockdownofRSRC2s conferred PTXR to cancer cells,we askedwhether upregulation of RSRC2smay increase paclitaxelsensitivity. The rescue experiments for PTXR of TRA2A-overex-pressing cells had been performed by upregulation of RSRC2s inMDA-MB-231TRA2A cells. RSRC2s upregulation obviously inhib-ited cell proliferation and survival of MDA-MB-231TRA2A cells byclonogenic assay when given paclitaxel treatment (Fig. 5N). Inaddition, IC50 dropped from174.5 nmol/L inMDA-MB-231TRA2A

cells to 51.9 nmol/L in cells with RSRC2s forcedly expressed inthem (Fig. 5O).

The decreased RSRC2 protein expression could be a marker forpoor survival of TNBC

RSRC2 protein expression observed in the nuclei was alsoexamined in the 47 cases of TNBC specimen by IHC. RSRC2protein was strongly expressed in benign breast epithelium ofparacancerous tissues, with a pattern of patchy staining; that is,positive-stained normal luminal/ductal cells were surrounded byfewer unstained myoepithelial cells (Fig. 6A). Moreover, a mod-erate to strong staining of RSRC2 couldbeobserved inmost of lowgrade TNBC (Fig. 6A) with the positive rate of 81.8% (9/11; Fig.6B). In contrast, a decreased staining was observed in high-gradeTNBC (Fig. 6A) and the positive rate of RSRC2was 44.4% (16/36;c2 ¼ 4.727, P ¼ 0.030; Fig. 6B).

Importantly, RSRC2 protein expression showed negativelycorrelation with TRA2A protein expression by Spearman analysis(P¼ 0.009; Fig. 6C). In addition, the mean expression of RSRC2lmRNA was bottom and RSRC2s mRNA was higher in RSRC2þ

cases than in RSRC2� cases (Fig. 6D and E), suggesting RSRC2

protein expression was related to RSRC2s. Survival analysisshowed that the decreased RSRC2 protein expression was signif-icantly associated with poor survival (Fig. 6F).

A shift from RSRC2s to RSRC2l mRNA regulated by TRA2Ainduced the decreased RSRC2 protein expression

By searching PubMed, we found that RSRC2s's sequence wasin accord with the shortest transcript (NM_023012.5) of RSRC2gene which could encode a functional protein. Furthermore,RSRC2l's sequence was in accord with transcripts holdingexon4 that included transcript variant 4 (NR_036435) andvariant 5 (NR_036434) of RSRC2 gene. The additional exon4includes a premature stop codon, so RSRC2l is a nonsense-mediated mRNA decay candidate and does not make a func-tional protein. As shown in Fig. 6G, MDA-MB-231 cells withpaclitaxel treatment exhibited a significant shift in RSRC2 AStoward the RSRC2 exon4 inclusion isoform. RT-PCR analysis ofRSRC2 mRNA using specific primers showed that expression ofRSRC2l-containing exon4 was increased, whereas expression ofRSRC2s was decreased. Meantime, we found the forced TRA2Aoverexpression resulted in an analogous RSRC2 AS pattern asobserved with paclitaxel treatment (Fig. 6G). Next, RSRC2protein expression was determined by Western blot analysesin cancer cells with paclitaxel treatment or exogenous TRA2Aoverexpression (Fig. 6H). The result showed the decline of abiological active RSRC2 protein following increased RSRC2land decreased RSRC2s mRNA expression induced by paclitaxeltreatment or TRA2A upregulation. Taken together, we hypoth-esize that TRA2A is a specific regulator of RSRC2 AS by pro-moting the expression of the RSRC2 exon4 retaining mRNAisoform, and thereby inhibits the generation of biologicalactive RSRC2 protein.

Next, we intended to establish a direct link between exon4inclusion of RSRC2l and TRA2A. We constructed minigenesplicing reporters (Fig. 6I) according to the literature (34) andcotransfected 293T cells with TRA2A and the RSRC2 minigenes.Cells transfected with the minigene containing upstream intro-nic sequence (minigene1) showed a remarkable shift in RSRC2AS pattern toward the RSRC2 exon4-inclusion isoform in aTRA2A dose-dependent manner, resulting in an increase inexon4 inclusion from 8% in control cells to 95% in cellstransfected with the highest dosage of TRA2A (Fig. 6J). Incontrast, cells transfected with the minigene containing down-stream intronic sequence (minigene2) displayed no differencein RSRC2 AS pattern compared with control cells (Fig. 6J). qRT-PCR analysis of exon-included and -skipped products con-firmed these results (Fig. 6K).

It is well demonstrated that upregulated TRA2A is able tomaintain TRA2B target exon inclusion following depletion ofTRA2B (12), suggesting TRA2A could control the splicing targetsbound by TRA2B. Furthermore, it is reported that the RAAG orAGAA tetranucleotide is specifically recognized by TRA2B (16).An overview of the 237 bp upstream intronic sequence surround-ing exon4 in RSRC2 minigene1 is observed to be particularly richin these kinds of motifs (Supplementary Data). Therefore, wesupposed AGAA or RAAG sequence as the potential bindingmotifs for TRA2A. To better demonstrate the interaction, wedecided to replace the suspected sequences within this region byconstructing mingene3, and remarkably replacing these motifsreduced TRA2A's splicing inclusion (Fig. 6L). Thus, TRA2A isnecessary and sufficient to stimulate RSRC2 exon4 inclusion via

Liu et al.





its interaction with AGAA or RAAG motifs located in intronsupstream from exon4.

DiscussionTNBC commonly acquires resistance to the first line che-

motherapy, paclitaxel, and this is one of major obstacles to thebetter prognosis. AS and the related splicing factors are deregu-lated in cancer. Each of the "hallmarks of cancer," includingchemotherapy resistance is associated with a switch in splicing,toward a more aggressive invasive cancer phenotype (35, 36).In this study, we analyzed microarray profiles and identified 8splicing factors related to PTXR, in which the expression levelof TRA2A was the highest. Conformably, we had also foundthat ectopic expression of TRA2A promoted stronger resistanceto paclitaxel in TNBC cells whereas TRA2A knockdown inthe PTXR cells induced the reversion of paclitaxel sensitivity.However, the paralogs product of TRA2A, that is, TRA2B, didnot show significant increase after paclitaxel treatment inour study. In addition, STRING analysis show there is inter-action between TRA2A and hnRNPm, and hnRNPm showedthe second higher level following paclitaxel treatment in ourstudy. However, we found that the elevated TRA2A levelwas independent of hnRNPm expression. Interestingly,hnRNPm and the other AS factor SRSF6 showed an increasedexpression following TRA2A upregulation. All of these resultssuggest that individual TRA2A expression has a role in medi-ating PTXR of TNBC.

Functionally, we provided evidence that ectopic expressionof TRA2A in TNBC cells was associated with a highly aggres-sive phenotype, as constitutive expression of TRA2A stimu-lated breast cancer cell proliferation and survival, migration,and invasion. Microarray analysis demonstrated that TRA2Atargets were enriched with the cancer-related functions,and proliferative markers of tumor cells such as PCNA,CDC25A, and CDC6 showed elevated expression followingTRA2A overexpression, suggesting TRA2A's function in tumorprogression.

We further demonstrated that TRA2A predominantly func-tioned as a splicing factor when directly bound to pre-mRNAsand the AS events could be negatively or positively regulated byTRA2A. By arbitrarily examining the AS events, we found thatTRA2A control AS of MELK, CALU, RSRC2, CEACAM1, LMCD1,PALM, and RFWD2, which might contribute to aggressive phe-notype of TRA2A-overexpressing cells. Therefore, it is possiblethat TRA2A may affect cell growth and survival through control-ling AS. Importantly, we found that following paclitaxel treat-ment there was significantly isoform shift of CALU, RSRC2, andPALM in survived cancer cells, hence demonstrating that cellsused AS controlled by TRA2A to orchestrate a switch in isoformexpression of cancer-related genes, which in turn promoted PTXRand cancer progression.

Our data of breast cancer patient samples showed that TRA2Aexpression could be a marker for poor survival of breast cancer.Specifically, TRA2A expression was associated with PTXR ofTNBC, but not with that of non-TNBC patients. Further study infresh TNBC tissues not only confirmed TRA2A's role in PTXR, butalso demonstrated isoform shift of RSRC2 occurred in PTXRdevelopment of TNBC. RSRC2 has been identified as tumorsuppressor and it might be associated with chemotherapy sensi-

tivity (37). In our study, in vitro experiment showed that RSRC2expression obliteration resulted in PTXR in TNBC cells and theforced RSRC2 expression in TRA2A overexpressing cells recoveredcancer cells' paclitaxel sensitivity. Moreover, RSRC2 proteinexpression was decreased in resected tumor tissues when com-pared with normal breast acini. The decreased RSRC2 expressionlevels have been reported to be correlated with tumor metastasisand invasion, as well as shorter post-operative survival in esoph-ageal cancer (29).

However, the mechanism of RSRC2 downregulation intumor aggressiveness was unclear until now. Here, we dem-onstrated that RSRC2's function could be controlled by TRA2Athrough splicing. RSRC2 is located at 12q24 and has differentisoforms in which only the shortest isoform makes functionalprotein. At the molecular level, we found that TRA2A directlybound to RSRC2 pre-mRNA at AGAA or RAAG rich sequencesand stimulated inclusion of RSRC2 variable exons, which ledto RSRC2 isoform switching from RSRC2s to RSRC2l andcaused the decreased RSRC2 protein levels during PTXR devel-opment of TNBC. Consistently, there was significantly negativecorrelation between RSRC2 and TRA2A expression in TNBCtissues, further confirming the regulation of RSRC2 by TRA2Ain vivo. Crucially, like TRA2A overexpression, the decreasedexpression of RSRC2 was associated with poor prognosis inTNBC.

In summary, this study represents an important example ofhow a splicing factor can control critical AS events in chemother-apy resistance and cancer progression of TNBC. These resultssuggest a causal role for the TRA2A in TNBC progression andmodulating RSRC2 splicing might be a potential therapeuticintervention for TNBC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Liu, B. SunDevelopment of methodology: T. Liu, H. Sun, X. Dong, F. Liu, M. Wang,Y. Wang, B. SunAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Liu, H. Sun, D. Zhu, X. Dong, F. Liu, X. Liang, B. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Liu, D. Zhu, X. Dong, C. Chen, B. Shao, M. Wang,Y. Wang, B. SunWriting, review, and/or revision of the manuscript: T. Liu, B. SunAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Liu, X. Dong, F. Liu, X. Liang, C. Chen, B. ShaoStudy supervision: T. Liu

Grant SupportThis 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 ScienceFoundation of China (no. 81230050; to B. Sun), and National Under-graduate Training Program for Innovation and Entrepreneurship (no.201510062001; to H. Sun).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 7, 2017; revised December 7, 2016; accepted April 7, 2017;published OnlineFirst April 17, 2017.






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2017;16:1377-1388. Published OnlineFirst April 17, 2017.Mol Cancer Ther Tieju Liu, Huizhi Sun, Dongwang Zhu, et al. Triple-Negative Breast Cancers via Regulating Alternative SplicingTRA2A Promoted Paclitaxel Resistance and Tumor Progression in

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