Transgelin 2 Promotes Paclitaxel Resistance, Migration, and Invasion

Published OnlineFirst September 5, 2019; DOI: 10.1158/1535-7163.MCT-19-0261

Cancer Biology and Translational Studies Molecular Cancer Therapeutics Transgelin 2 Promotes Paclitaxel Resistance, Migration, and Invasion of Breast Cancer by Directly Interacting with PTEN and Activating PI3K/Akt/GSK-3b Pathway Leichao Liu1, Ti Meng1, Xiaowei Zheng2, Yang Liu1, Ruifang Hao1, Yan Yan1,3, Siying Chen1, Haisheng You1, Jianfeng Xing3, and Yalin Dong1

Abstract

MDR and tumor migration and invasion are still the main breast cancer cells were also enhanced by Transgelin 2 over- obstacles to effective breast cancer chemotherapies. Transgelin expression in vivo. Moreover, Transgelin 2 overexpression 2 has recently been shown to induce drug resistance, tumor activated the PI3K/Akt/GSK-3b pathway by increasing the migration, and invasion. The aim of this study was to deter- phosphorylation levels of Akt and GSK-3b and decreasing mine the biological functions of Transgelin 2 and the mech- the expression of PTEN. We also found that Transgelin 2 anism underlying how Transgelin 2 induces paclitaxel (PTX) could directly interact with PTEN and was located upstream resistance and the migration and invasion of breast cancer. We of PTEN. Furthermore, the PI3K/Akt pathway inhibitor detected that the protein level of Transgelin 2 was significantly MK-2206 reversed the resistance to paclitaxel and inhibited upregulated in breast cancer tissues compared with adjacent the migration and invasion of breast cancer cells. These nontumor tissues. A bioinformatics analysis indicated that findings indicate that Transgelin 2 promotes paclitaxel Transgelin 2 was significantly related to clinicopathologic resistance and the migration and invasion of breast cancer parameters and patient prognosis. Overexpression of Trans- by directly interacting with PTEN and activating the gelin 2 enhanced the migration and invasion of human breast PI3K/Akt/GSK-3b pathway. Transgelin 2 may therefore be cancer cells and decreased the sensitivity of breast cancer cells useful as a novel biomarker and therapeutic target for to paclitaxel. Meanwhile, the tumorigenesis and metastasis of breast cancer.

Introduction molecular mechanisms of MDR and tumor metastasis is significant for patients with breast cancer to develop new effective Breast cancer is the most common malignancy among women therapeutic strategies. worldwide (1). Although treatment with a combination of che- Paclitaxel (PTX) is widely applied in first-line chemotherapies motherapy and surgery has made remarkable progress in patients for treating breast cancer (4, 5). However, the efficacy of PTX is with breast cancer during the past few years, the recurrence and often hampered to acquired resistance in patients. Various cancer death remain very high due to high frequency of metastasis mechanisms have been proposed to explain PTX resistance, and recurrence. Even though additional drugs were used, breast including alterations in the level of expressed tubulin, overexpres- cancer cells generally gained MDR, finally causing tumor migra- sion of ATP-binding cassette (ABC) transporter proteins, and tion and invasion over time (2, 3). Therefore, exploring the apoptotic modulation (6–8). Several studies have found that the epithelial–mesenchymal transition (EMT) is related to acquisition of the MDR phenotype (9–11). The EMT is known to be involved in cancer 1 fi Department of Pharmacy, The First Af liated Hospital of Xi'an Jiaotong Uni- metastasis and is a process in which epithelial cancer cells lose versity, Xi';an, Shaanxi, P.R. China. 2Department of Pharmacy, The First Hospital of Xi'an, Xi'an, Shaanxi, P.R. China. 3School of pharmacy, Xi'an Jiaotong Uni- their typical epithelial characteristics and acquire mesenchymal versity, Xi'an, Shaanxi, P.R. China. traits. Cancer cells undergoing the EMT become migratory and invasive, enabling metastasis and chemotherapy resistance. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Therefore, identifying the molecular mechanism underlying tumor progression may ultimately lead to innovative therapeutic L. Liu and T. Meng contributed equally to this article. strategies against breast cancer. Corresponding Authors: Yalin Dong, The First Affiliated Hospital of Xi'an Transgelin 2, which is encoded by TAGLN2, belongs to the Jiaotong University, Xi'an, Shaanxi 710061, P.R. China. Phone: 8629-8532- family of actin-binding proteins and is a 22,391-Da protein 3241; Fax: 8629-8532-3240; E-mail: [email protected]; Jianfeng Xing, containing 199 amino acids. Deregulation of Transgelin 2 is School of Pharmacy, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China. Phone: 8629-8265-5139; E-mail: [email protected] considered to be associated with tumorigenesis and tumor development in various human malignancies (12–14). We previously Mol Cancer Ther 2019;18:2457–68 used proteomic technologies to reveal the expression of Transge- doi: 10.1158/1535-7163.MCT-19-0261 lin 2 to be 15.48-fold higher in PTX-resistant human breast cancer 2019 American Association for Cancer Research. cells (MCF-7/PTX) established by our laboratory than in breast

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cancer drug-sensitive cells (MCF-7/S; ref. 15). Furthermore, our Biochemistry and Cell Biology, Chinese Academy of Sciences, data demonstrated that Transgelin 2 can modulate the expression and MCF-7/PTX cell line was established as previously levels of MDR1, MRP1, and BCRP, suggesting that Transgelin 2 is described (19). All cell lines were grown in DMEM supple- critical for the occurrence of MDR in breast cancer cells (16). mented with 10% FBS, 1% penicillin, and streptomycin at 37C However, whether Transgelin 2 mediates the migration and in a humidified atmosphere of 5% CO2. The other culture invasion of breast cancer cells remains unclear. conditions of MCF-7/PTX cells werethesameastheMCF-7/S The PI3K/Akt/GSK-3b pathway is known to participate in the cells except for maintaining in 30 nmol/L paclitaxel (19). Cells regulation of various biological processes, such as the inhibition in exponential phase growth were observed under the inverted of apoptosis, induction of cell proliferation, and promotion of light microscope (Olympus). cancer metastasis (17). Our previous study demonstrated that Transgelin 2 activated the PI3K/Akt pathway in MCF-7/PTX cells, siRNA transfection, plasmid transient transfection, and thereby inhibiting mitochondrial apoptosis and inducing breast lentiviral infection cancer resistance (18). Nevertheless, the role of Transgelin 2 in the MCF-7/PTX cells were seeded in 6-well plate (5 105 cells/ PI3K/Akt/GSK-3b pathway is still unknown and needs to be well). For siRNA transfection, cells were incubated in RPMI1640 explored further. without antibiotics for 24 hours, after which 0.1 nmol/L double- In this study, we found that Transgelin 2 was upregulated in stranded siRNA against TAGLN2 or nonspecific control siRNA breast cancer tissues, and that a strong expression of Transgelin 2 (GenePharma) was transfected with Lipofectamine 2000 (Invi- indicated a poor prognosis in patients with breast cancer. More- trogen). For plasmid transient transfection, 2 mg eukaryotic over, Transgelin 2 has a strong effect on the PTX resistance and the expression vector alone (pcDNA3.1) or pcDNA3.1-TAGLN2 invasion and metastasis of breast cancer cells both in vitro and (GenePharma) was transfected to MCF-7/S cells with the same in vivo. The molecular mechanism assays demonstrated that method. All the processes above were conducted according to the Transgelin 2 could suppress the expression of PTEN and activate manufacturer's instruction. the PI3K/Akt/GSK-3b pathway. MDA-MB-231 cells (2 106 cells) were prepared and infected at a multiplicity of infection of 10 with negative control (Lenti-EV) or Transgelin 2-overexpressing lentiviruses (Lenti-TAGLN2) for Materials and Methods 10 hours at 37C in the presence of 5 mg/mL of polybrene. Chemicals and antibodies Selecting process was performed in 3 mg/mL puromycin 96 hours Paclitaxel and MK-2206 were purchased from Sike Pharma- after transfection. The lentivirus was purchased from GeneChem ceutical and Selleck Chemicals, respectively. 3-(4,5-Dimethylthia- and labeled with GFP. zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was from The expression level of Transgelin 2 and transfection efficiency Sigma. Annexin-V FLUOS staining kit was obtained from Invitro- were detected by qPCR and Western blot assays, supplemented by gen. The human monoclonal anti-N-cadherin, anti-P-gp, and fluorescent inverted microscope (ZEISS) to observe GFP expres- anti-BCRP were purchased from Abcam. The human monoclonal sion after lentiviral infection. anti-vimentin, anti-MRP1, and anti-Transgelin 2 antibodies were purchased from GeneTex. The human anti-Bcl-2 and anti-Bcl-2– MTT assay associated X (Bax) were from Epitomics. The human monoclonal Cells (5 105/mL) were seeded in 96-well plates with different anti-E-cadherin, anti-PTEN, anti-Akt, anti-phospho-Akt (p-Akt), treatments for indicated duration. A solution of 20 mL of MTT anti-caspase-3, anti-caspase-9, anti-PARP, and anti-Snail were (5 mg/mL) was added into each well and incubated for 4 hours. purchased from Cell Signaling Technology. The human polyclon- Then 150 mL of dimethyl sulfoxide was added into each well to al anti-b-actin antibody was obtained from Biosynthesis Biotech- solubilize the formazan for 15 minutes. The absorbance was read nology and horseradish peroxidase–conjugated secondary anti- at 490 nm on a microplate reader (BioTek). Each experiment was body was obtained from Cwbiotech. repeated three times. The following equation was used to calcu- late the reversal index (RI): RI ¼ IC50 of paclitaxel/IC50 of Clinical samples and databases paclitaxel plus MK-2206. Fifty-five tissue samples of formalin-fixed, paraffin-embedded breast cancer and adjacent nontumor tissue specimens from Flow cytometry assay female patients who had been diagnosed with breast cancer and Cells with different treatments after 48 hours were harvested received primary surgical treatment between 2008 and 2013 were and washed and resuspended with cold PBS. Then staining obtained from the First Affiliated Hospital of Xi'an Jiaotong process was conducted using the Annexin V-FITC/PI Apoptosis University (Xi'an, China), whose ethics committees approved the Detection Kit (KeyGen Biotech) or Annexin V-APC/7-AAD Apo- protocol. The mRNA expression microarray data and correspond- ptosis Detection Kit (KeyGen Biotech) according to manufac- ing overall survival information of 1,904 breast cancer samples turer's instructions. The stained cells were analyzed using FACS- were downloaded from the METABRIC (Molecular Taxonomy of Canto II flow cytometry (Becton Dickinson Company). Breast Cancer International Consortium) databases to analyze the association between Transgelin 2 expression and clinicopatho- qPCR logic features as well as the prognostic value of Transgelin 2 in Total mRNA of cells was extracted using RNAfast2000 kit breast cancer. (Fastagen). All PCR reactions were performed using the Prime Script RT Master Mix Perfect Real Time kit (DRR036A, TaKaRa) Cell lines and cell culture and SYBR Premix Ex Taq II (TaKaRa) according to manufacturer's MCF-7/S, MDA-MB-231, and T-47D human breast cancer cell instructions. Each sample was run independently in triplicate. The lines were purchased from the Cell Bank of Shanghai, Institute of primer sequences and product length are listed in Supplementary

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Transgelin 2 Confers Resistance, Migration, and Invasion

Table S1. The experiments were run on the Bio-Rad CFX96 Real- overnight. Then cells were maintained in serum-free culture at time system (Bio-Rad). b-actin was used as an internal control. 37 C in a humidified atmosphere of 5% CO2. Migration photo- graphs were captured at 0, 12, 24, and 48 hours after scratching. Western blotting assay Experiments were repeated in triplicate independently. The per- Cells with different treatments were lysed in RIPA buffer con- cent wound closure were calculated using the following equation: taining protease inhibitor on ice. Then equal amount of protein percent wound closure (%) ¼ [1 (Lt/L0)] 100%. lysates were electrophoretically separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). Transwell invasion assay After blocking with 5% nonfat dried milk for 2 hours, the The invasiveness of cells was evaluated by Boyden chamber membranes were incubated with primary antibodies overnight method. Coated with Matrigel Matrix (BD Biosciences), the at 4C. Another incubation with a horseradish peroxidase– polycarbonate filters (8-mm pore size, Corning) were incubated conjugated secondary antibody was performed in the following at 37C for 5 hours. Next, 5 105 cells suspended in 200-mL day for 2 hours at 37C, after when the protein bands were serum-free RPMI1640 were added into the top chamber, whereas detected using the Super Signal West Pico kit (Thermo Fisher 800 mL of complete media was added to the bottom chamber. Scientific). All Western blot experiments were repeated at least After 48 hours, cells migrated through Matrigel and adhered onto 3 times. the bottom chamber were fixed in 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet, and counted under IHC upright microscope (five fields per chamber). Each invasion assay IHC staining was applied to 3-mm thick sections of paraffin- was repeated in three independent experiments. embedded tissue specimens using the PV-9001 Detection Kit (ZSGB-BIO) according to the manufacturer's instructions. Briefly, In vivo xenograft tumor model formalin-fixed paraffin-embedded tissues were baked at 60C for BALB/C nude mice aged 4 weeks were acquired from 2 hours and then deparaffinized in xylene for 10 minutes and Shanghai SLAC Laboratory Animal Center of the Chinese rehydrated in a graded series of ethanol solutions. The tissues were Academy of Sciences. All animal experiments were approved immersed in 0.01 M citric acid buffer at 121C for 5 minutes and by the ethical committee for animal care of Xi'an Jiaotong then cooled and washed with 0.1 M PBS 3 times. By being treated University. For in vivo tumor growth, 14 female BALB/C nude with 3% hydrogen peroxide for 10 minutes, endogenous perox- mice were used to establish a subcutaneous tumor model. idase was blocked. Then the tissues were incubated with primary MDA-MB-231 cells (1 106) infected with Lenti-TAGLN2 (or antibodies overnight at 4C, followed by the secondary antibody Lenti-EV as a control) were injected subcutaneously into the for 30 minutes at room temperature. Sections were stained with flank of mice (n ¼ 10 per group). Tumor growth was monitored 3,30-diaminobenzidine and counterstained using hematoxylin by estimating the tumor volume as length width2 0.5. For for 5 seconds, dehydrated in a graded series of ethanol solutions, the metastasis model, MDA-MB-231 cells (1 106)were immersed in xylene, and examined with the aid of a microscope injected into the tail vein (n ¼ 5pergroup).Themicewere (Axio Image M2, Zeiss). killed 6 weeks later, whose lung tissues were removed and fixed Transgelin 2 expression was evaluated by a pathologist in a in formalin. The presence of lung metastases was then assessed blinded manner on the basis of the staining intensity and the by hematoxylin and eosin staining. percentage of positive cells. Staining intensity was scored on the following 4-point scale: negative, 0; weak, 1; intermediate, 2; and Coimmunoprecipitation assay strong, 3. The percentage of positive cells was classified as follows: Protein lysates from cells were extracted and incubated with 0%–5%, 0; 6%–25%, 1; 26%–50%, 2; 51%–75%, 3; and >75%, 4. 20-mL protein A/G-agarose beads at 4C; 0.5 hours later, beads The final result was calculated by multiplying the percentage of were removed and the residue was incubated with 8-mL antibody positive cells by the staining intensity to obtain a total score against PTEN or Transgelin 2 or normal IgG and 20-mL proteinA/G ranging from 0 to 12. Total scores of 0, 1–4, 5–8, and 9–12 were beads with gentle rocking at 4C overnight. The next day, isolated regarded as indicating negative expression and low, moderate, beads were washed 3 times with PBS buffer. The supernatant (30 and high expression levels, respectively. mL) was examined by Western blot assay using relative antibodies. Each experiment was repeated at least 3 times. Mammosphere formation assay Mammosphere culture was done in a serum-free DMEM/F12 The measurement for the affinity of Transgelin 2 and PTEN (Invitrogen) supplemented with 2% B27 (Invitrogen), 20-mg/L The equilibrium-binding constant (KD) of Transgelin 2 and human epidermal growth factor (Invitrogen), 10-mg/L human PTEN was determined by OpenSPR. Briefly, the PTEN (40 mg/mL) basic fibroblast growth factor (Invitrogen), and 5-mg/L insulin. was covalently immobilized on COOH-sensor chips by the Single cells prepared from mechanical and enzymatic dissociation N-3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride were plated in 6-well ultralow attachment plates at a density of and N-hydroxysuccinimide chemistry. Then, Transgelin 2 was 104 cells/mL in culture. Single cell status was confirmed under continuously diluted into several different concentrations using microscope. After 14 days, the number of mammospheres the running buffer and injected into the chip from low to high (420 mm) was counted under an upright microscope. Experiments concentrations. BSA was used as a negative control. In each cycle, a were repeated in triplicate, independently. 250-mLsamplewasflowed through the chip for 5 minutes at a constant flow rate of 20 mL/minute. After detection, 0.02% SDS was Scratch wound healing assay added to dissociate the peptides from target protein. The kinetic Cells were grown in 6-well plate until confluent. An artificial parameters of the binding reactions were calculated and analyzed scratch wound was created, followed by being serum-starved by TraceDrawer software (Ridgeview Instruments AB).

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Statistical analysis sitivity of breast cancer cells to paclitaxel (Fig. 1D). Consistently, Statistical analysis was performed using SPSS (version 19.0) flow cytometry analysis indicated that after treatment with software. Data obtained in triplicate experiments performed in a 20 nmol/L paclitaxel, the percentage of apoptotic cells in parallel manner were expressed as mean standard deviation Transgelin 2-overexpressing cells (23.82 %) was less than that values. One-way ANOVA was used for comparison tests. The in control cells (41.35 %; Fig. 1E). In addition, qPCR and Western relationships between Transgelin 2 expression levels and clinico- blotting revealed that the overexpression of Transgelin 2 in MCF-7 pathologic parameters were tested using the x2 test. The prog- and MDA-MB-231 cells significantly increased the mRNA and nostic value of Transgelin 2 expression in the prognosis of breast protein levels of P-gp, MRP1, and BCRP (Fig. 1F and G). Together cancers was analyzed by Kaplan–Meier curves and compared by these findings strongly indicate that strong expression of the log-rank test using GraphPad prism software. The cut-off value Transgelin 2 contributed to PTX resistance, which leads us to for the Transgelin 2 expression level was set using X-tile (version propose that targeting Transgelin 2 could be a useful strategy for 3.6.1) software. Probability values of P < 0.05 were considered improving the sensitivity of breast cancer cells to PTX. statistically significant. Overexpression of Transgelin 2 enhanced the migration and invasion of human breast cancer cells Results Mammosphere formation, qPCR, Western blotting, scratch Transgelin 2 expression was significantly upregulated in breast wound healing, and Transwell invasion assays were performed cancer to clarify whether overexpression of Transgelin 2 could alter the To explore the clinical significance of Transgelin 2, we applied migration and invasion abilities of MCF-7/S cells. Transgelin 2 IHC to the 54 collected pairs of breast cancer tissues and adjacent overexpression led to a distinct increase in the mammosphere- nontumor tissues. The results showed that Transgelin 2 was forming ability of breast cancer cells (Fig. 2A). In accordance with mainly expressed in breast cancer tissues in the cell cytoplasm, these observations, the expression of epithelial marker E-cadherin and the expression level was significantly higher than that in the was dramatically decreased, whereas the levels of mesenchymal adjacent nontumor tissues (Fig. 1A and B). We then obtained markers N-cadherin and vimentin were increased (Fig. 2B and C). 1,904 breast cancer cases with detailed clinical information and In addition, cells that overexpressed Transgelin 2 exhibited sig- RNA-seq results from the METABRIC databases. We analyzed the nificantly enhanced migration and invasion characteristics associations between Transgelin 2 mRNA expression and clini- (Fig. 2D and E). Our results imply that upregulation of Transgelin copathologic features in the patients with breast cancer (Supple- 2 induces the EMT process, leading to enhancement of the mentary Table S2). As Supplementary Figure S1 shows, the migratory and invasive ability of breast cancer cells. Transgelin 2 mRNA expression in breast cancer was not associated with age or menopausal status; however, a high Transgelin 2 Overexpression of Transgelin 2 enhanced the xenograft growth mRNA expression was significantly associated with larger tumors, and metastasis poor histologic grade, high tumor stage, negative estrogen recep- We next explored the effect of Transgelin 2 in vivo. To investigate tor (ER) expression, progesterone receptor (PR), positive Her-2 the effect of Transgelin 2 on cancer progression, MDA-MB-231 expression, and invasive ductal tumors. A subsequent multivar- cells infected with Lenti-TAGLN2 (or Lenti-EV as a control) were iate logistic regression analysis found that the cancer type, ER subcutaneously injected into nude mice. The tumor volumes were status, and histologic grade were independently associated with significantly higher in the Lenti-TAGLN2 group than in the control Transgelin 2 mRNA expression in breast cancers (Supplementary group (Fig. 3A). Moreover, Kaplan–Meier survival analysis indi- Table S3). Furthermore, Kaplan–Meier survival analysis was used cated that survival was worse in the Transgelin 2-overexpressing to reveal the prognostic value of Transgelin 2. Breast cancer group than in the control group (Fig. 3B). A metastasis model was patients with a high Transgelin 2 mRNA expression had poorer established to further validate the role of Transgelin 2 in tumor prognosis than those with low Transgelin 2 expression in breast metastasis, with the results showing that overexpression of Trans- cancers (184.8 vs. 159.0 months, P ¼ 0.0111; Fig. 1C). gelin 2 increased lung metastases of MDA-MB-231 cells (Fig. 3C These results indicate that Transgelin 2 can be considered a and D). diagnostic marker for breast cancer. Transgelin 2 could regulate the expression of phospho-Akt, Overexpression of Transgelin 2 decreased the sensitivity of phospho-GSK-3b, and the expression of PTEN breast cancer cells to paclitaxel We previously revealed that knockdown of Transgelin 2 To investigate the biological functions of overexpressed Trans- reversed the resistance to PTX in MCF-7/PTX cells by inactivating gelin 2 in breast cancer, we detected the expression levels of the PI3K/Akt pathway and then promoting activation of the Transgelin 2 in three breast cancer cell lines. We found that the mitochondrial apoptotic pathway (18). Akt activation plays a expression level was low in MCF-7 and MDA-MB-231 cells pivotal role in inducing the EMT by inhibiting GSK-3b, (Supplementary Fig. S2A). We then overexpressed Transgelin 2 leading to the stabilization and nuclear localization of Snail, in these two cell lines using plasmid transient transfection and thereby triggering tumor migration (20). To confirm whether lentivirus stable transfection, respectively. As shown in Supple- the PI3K/Akt/GSK-3b pathway can regulate the EMT and the mentary Figure S2B, mRNA and protein expression levels of migration of breast cancer cells, we used the Western blot Transgelin 2 were both substantially increased compared with assay to measure the expression of several proteins involved in the control group in both two cell lines. the PI3K/Akt/GSK-3b pathway. Remarkably, downregulated To identify the effect of Transgelin 2 in paclitaxel resistance, we PTEN and upregulated phospho-Akt, phospho-GSK-3b (inactive measured the sensitivity to paclitaxel in breast cancer cells. Sur- form), and Snail were observed in MCF-7/PTX cells, along prisingly, Transgelin 2 overexpression decreased the chemosen- with the elevation of Transgelin 2, demonstrating that the

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Transgelin 2 Confers Resistance, Migration, and Invasion

ABCAdjacent nontumor tissue Breast cancer tissue

1.0 15 Low transgelin 2 (n = 947) 0.8 High transgelin 2 (n = 957) Log-rank test P = 0.0111 10 0.6 Anti-Transgelin 2 Anti-Transgelin 0.4 5

Fraction survival 0.2

Immunohistochemical score Immunohistochemical 0 0.0 0 100 200 300 400 Months Anti-Transgelin 2 Anti-Transgelin Breast cancer tissue

Adjacent nontumor tissue

D 100 MCF-7/S Control E MCF-7/S pcDNA3.1

4 Control 4 PTX 4 pcDNA3.1+PTX 4 pcDNA3.1+PTX 80

pcDNA3.1-T 10 10 10 10

60 3 3 3 3 10 10 10 10

40 2 2 2 2 10 10 10 10 PI PI PI PI

20 1 1 1 1 10 10 10 10 Relative cell viability (%) 0 0 0 0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 1010 0 101 102 103 104 1010 0 101 102 103 104 10 0 101 102 103 104 1010 0 101 102 103 104 Paclitaxel (log nmol/L) Annexin V FITC Annexin V FITC Annexin V FITC Annexin V FITC

150 MDA-MB-231 WT MDA-MB-231 Lenti-EV Control WT+PTX Lenti-EV+PTX Lenti-T+PTX 5

Lenti-T 5 100 5 5 10 10 10 10 4 4 4 4 10 10 10 10 3 3 50 3 3 7-AAD 7-AAD 7-AAD 7-AAD 10 10 10 10 2 2 2 2 10 10 10 10 Relative cell viability (%) 0 2 3 4 5 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 10 10 10 2 3 4 5 10 10 10 10 102 103 104 105 102 103 104 105 Paclitaxel (log nmol/L) Annexin V APC Annexin V APC Annexin V APC Annexin V APC

MCF-7 MDA-MB-231 F Control G MCF-7 pcDNA3.1 1.5 pcDNA3.1-T Control pcDNA3.1 pcDNA3.1-T WT Lenti-EV Lenti-T P-gp P-gp 1.0 MRP1 MRP1 0.5 BCRP BCRP

0.0 b-Actin b-Actin normalized to b -Actin

Quality of mRNA expression MDR1 MRP1 BCRP

Control MDA-MB-231 Control pcDNA3.1 pcDNA3.1-T 2.0 Lenti-EV 1.6 2.0 Lenti-T WT 1.5 1.2 Lenti-EV 1.5 Lenti-T 1.0 0.8 1.0

0.5 0.4 0.5

0.0 0.0 0.0 Quality of protein normalized to b -Actin normalized to b -Actin normalized to b -Actin P-gp P-gp Quality of mRNA expression

Quality of protein expression Quality of protein MRP1 BCRP MRP1 BCRP MDR1 MRP1 BCRP

Figure 1. Transgelin 2 expression was significantly upregulated in breast cancer and overexpression of Transgelin 2 decreased the sensitivity of breast cancer cells to paclitaxel. A, IHC staining of Transgelin 2 in breast cancer tissues and adjacent nontumor tissues. Serial sections of breast cancer tissues were stained with antibody to TAGLN2 and viewed at (a, b) 200 magnification and (c, d) 400 magnification. B, The protein level of Transgelin 2 in breast cancer tissues and adjacent nontumor tissues. C, Kaplan–Meier survival analysis was used to compare overall survival of the patients with breast cancer with low and high scores for Transgelin 2. D, MCF-7 and MDA-MB-231 cells transfected with plasmid or lentivirus were treated with various concentrations of paclitaxel and cell viability was examined by MTT assay. E, Flow cytometry assay detected the percentage of apoptotic cells in MCF-7 and MDA-MB-231 cells transfected with plasmid or lentivirus. F and G, The mRNA and protein expressions of P-gp, MRP1 and BCRP were assessed in MCF-7 and MDA-MB-231 cells transfected with plasmid or lentivirus. Results were expressed as mean SD from three experiments. , P < 0.05; , P < 0.01; , P < 0.001 vs. control.

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MCF-7 MDA-MB-231 WT Lenti-EV Lenti-T A Control pcDNA3.1 pcDNA3.1-T

60 80

40 60

40 20 20 0

Number of mammosphere of Number Control pcDNA3.1 pcDNA3.1-T 0 Number of mammosphere of Number

WT Lenti-T Lenti-EV

MCF-7 Control 1.2 Control MCF-7 pcDNA3.1 pcDNA3.1-T pcDNA3.1 B 0.6 C pcDNA3.1-T Control pcDNA3.1 pcDNA3.1-T E-cadherin 0.8 0.4 N-cadherin 0.4 0.2

Vimentin

-actin β to normalized

Quality of protein expression protein of Quality 0.0

Quality of mRNA expression mRNA of Quality 0.0 β-Actin

Vimentin E-cadherin Vimentin N-cadherin E-cadherin N-cadherin

MDA-MB-231 MDA-MB-231 WT 2.0 Lenti-EV WT 1.0 Lenti-T WT Lenti-EV Lenti-T Lenti-EV 1.5

0.8 E-cadherin Lenti-T

-actin β 0.6 1.0 N-cadherin 0.4 0.5 Vimentin

0.2 protein of Quality

normalized to to normalized

-actin β normalized to to normalized 0.0

0.0 β-Actin Quality of mRNA expression mRNA of Quality

Vimentin Vimentin E-cadherin N-cadherin E-cadherin N-cadherin

MDA-MB-231 D MCF-7 WT Lenti-EV Lenti-T Control pcDNA3.1 pcDNA3.1-T Control WT pcDNA3.1 0 h Lenti-EV 0 h 100 pcDNA3.1-T 80 Lenti-T 80 12 h 60 12 h 60 40 40 24 h 24 h 20

20 Percent wound closure (%) closure wound Percent

Percent wound closure (%) closure wound Percent 0 48 h 0 48 h 12 h 24 h 48 h

12 h 24 h 48 h

MCF-7 MDA-MB-231 E Control pcDNA3.1 pcDNA3.1-T WT Lenti-EV Lenti-T

300 Control 200 pcDNA3.1 pcDNA3.1-T 150 200

100 100

Cell numbers of invation of numbers Cell Cell numbers of invasion of numbers Cell 0 0 Control pcDNA3.1 pcDNA3.1-T WT Lenti-T Lenti-EV

Figure 2. Overexpression of Transgelin 2 enhanced the migration and invasion in human breast cancer cells. A, MCF-7 and MDA-MB-231 cells were transfected with plasmid or lentivirus, and their mammosphere forming abilities were examined (original magnification 100). B and C, The mRNA and protein expressions levels of E-cadherin, N-cadherin, and vimentin were assessed in MCF-7 and MDA-MB-231 cells transfected with plasmid or lentivirus by qRT-PCR and Western blot methods. D, Migration of MCF-7 and MDA-MB-231 cells transfected with plasmid or lentivirus was measured by wound healing assay (original magnification 100). E, Invasiveness of MCF-7 and MDA-MB-231 cells transfected with plasmid or lentivirus was detected by Transwell invasion assay (original magnification 100). Data were presented as mean SD from three experiments. , P < 0.05; , P < 0.01 vs. control.

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Transgelin 2 Confers Resistance, Migration, and Invasion

A B Lenti-EV 1.5 100 Lenti-T

Lenti-T ) 3 Lenti-EV 80 1.0 60

40 0.5

20 P < 0.0001 Tumor volume (cm volume Tumor 0.0 (%) probability Survival 0 5 10 15 20 25 0 10 20 30 40 Days Days

C D Lung metastasis

15 Lenti-EV

in lungs in 5

Metastasic nodules Metastasic Lenti-T 0 Lenti-EV Lenti-T

Figure 3. Overexpression of Transgelin 2 enhanced the xenograft growth and metastasis. A, Tumor growth curves for MDA-MB-231 tumors transfected with Lenti-T or Lenti-EV (n ¼ 10 each group, P < 0.05). B, Mice survival of Transgelin 2-overexpressed MDA-MB-231 and control group (n ¼ 10 each group; P < 0.0001). C, Lung metastases were detected by hematoxylin and eosin staining (n ¼ 5 each group). D, The number of lung metastatic nodules was determined (n ¼ 5 each group, P < 0.05).

PI3K/Akt/GSK-3b pathway was activated in MCF-7/PTX cells. Transgelin 2 directly interacted with PTEN and was located However, no significant change was found in the expression levels upstream of PTEN of Akt and GSK-3b (Fig. 4A). PTEN is another very important tumor suppressor factor In view of the above findings, we hypothesized that the PI3K/ following p53, which functions in many cellular process- Akt/GSK-3b pathway is involved in the Transgelin 2-induced es (21). Our results indicate that Transgelin 2 can negatively migration and invasion of breast cancer cells. To verify this regulate PTEN expression, suggesting relevance between PTEN hypothesis, Transgelin 2 was knocked down by siRNA in MCF- and Transgelin 2. A coimmunoprecipitation assay was carried 7/PTX cells. As expected, compared with the control, TAGLN2 out to confirm the interaction between PTEN and Transgelin 2 knockdown resulted in distinct decreases in the expression levels protein in cells. Anti-PTEN and anti-Transgelin 2 antibodies of Transgelin 2, phospho-Akt, phospho-GSK-3b, and Snail, pulled down Transgelin 2 protein and PTEN protein, whereas that of tumor suppressor PTEN protein was significantly respectively (Fig. 4E). However, the regulatory relationship increased (Fig. 4B). In contrast, overexpression of Transgelin 2 in between Transgelin 2 and PTEN remains unclear. We there- MCF-7/S cells decreased the PTEN protein level and upregulated fore simultaneously upregulated or knocked out the expres- the expression levels of phospho-Akt and phospho-GSK-3b sion of Transgelin 2 and PTEN in MCF-7/S or MCF-7/PTX (Fig. 4C). Meanwhile, activation of the Akt signaling pathway cells, respectively, and then detected the expression of the subsequently elevated the expression of prosurvival factor Bcl-2 PI3K/Akt signaling pathway with the aim of defining the but suppressed proapoptotic factor Bax and the cleavage of relative positions of Transgelin 2 and PTEN. We found that caspase-3, caspase-9, and PARP. In contrast, the downstream when Transgelin 2 was overexpressed in MCF-7/S cells, the molecule Snail was strongly upregulated (Fig. 4D). expression of PTEN was inhibited and the PI3K/Akt signaling In brief, these observations provide strong evidence that the pathway was activated; however, when Transgelin 2 and PTEN PI3K/Akt/GSK-3b pathway was activated in MCF-7/PTX cells, were both upregulated, the PI3K/Akt pathway was compara- which exhibit a high expression level of Transgelin 2, and that tively inhibited (Fig. 4F). Similarly, knocking out Transgelin 2 the level of tumor suppressor PTEN protein was regulated by led to the restoration of PTEN and the inhibition of the Transgelin 2. PI3K/Akt pathway, whereas the simultaneous knockout of

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A B None Control siRNA MCF-7/S MCF-7/PTX MCF-7/S NoneCcontrolTAGLN2 siRNATAGLN2 siRNA1TAGLN2 siRNA2 siRNA3 1.5 TAGLN2 siRNA1 Transgelin 2 MCF-7/PTX TAGLN2 siRNA2 1.2 Transgelin 2 TAGLN2 siRNA3 PTEN PTEN

Akt 1.0

-Actin b -Actin b 0.8 Akt p-Akt p-Akt GSK-3b 0.4 0.5

GSK-3b Quality of protein of Quality

p-GSK-3b protein of Quality normalized to to normalized normalized to to normalized 0.0 p-GSK-3b Snail b 0.0 Akt Snail Snail b-Actin PTEN p-Akt Akt GSK-3 PTEN p-Akt Snail p-GSK-3b b-Actin GSK-3b Transgelin 2 p-GSK-3b Transgelin 2

C Control D 2.4 Control Control pcDNA3.1pcDNA3.1-T pcDNA3.1 pcDNA3.1 pcDNA3.1-T pcDNA3.1 Control 1.5 pcDNA3.1-T Bcl-2 pcDNA3.1-T PTEN

1.6 -Actin Bax b

Akt -Actin b 1.0 Cleaved-caspase-3 p-Akt Cleaved-caspase-9 0.8 GSK-3b 0.5 Cleaved-PARP b to normalized

p-GSK-3 Snail 0.0

Quality of protein expression protein of Quality normalized to to normalized b 0.0 Bax -Actin b-Actin Bcl-2 Snail b Quality of protein expression protein of Quality Akt PTEN p-Akt GSK-3 Cleaved-PARP p-GSK-3b Cleaved-caspase-3Cleaved-caspase-9

NC - + -- E F TAGLN2 - - ++ MCF-7/S MCF-7/PTX PTEN - - - + 1.5 None 2.0 TAGLN2 Transgelin 2 NC PTEN TAGLN2 1.5

PTEN -Actin) 1.0

-Actin) b b TAGLN2+PTEN

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IP:IgG

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Bcl-2 (normalized to to (normalized WB:PTEN to (normalized

Relative mRNA expression mRNA Relative 0.0

Relative protein expression protein Relative 0.0 Bax NC - + - - Akt Bax - - b-Actin PTEN p-Akt Bcl-2 TAGLN2 + + PTEN - - - + Transgelin 2

G siRNA H PTEN-TAGLN2 NC - + - - Transgelin 2_1000 nmol/L fitted Transgelin 2_800 nmol/L fitted Transgelin 2_500 nmol/L fitted Transgelin 2_400 nmol/L fitted TAGLN2 - - + + Transgelin 2_200 nmol/L fitted Transgelin 2_1000 nmol/L Transgelin 2_800 nmol/L Transgelin 2_500 nmol/L ---+ Transgelin 2_400 nmol/L Transgelin 2_200 nmol/L PTEN 1.5 None Transgelin 2 1.0 TAGLN2 140 siNC PTEN PTEN 120

1.0 siTAGLN2 0.8 -Actin)

siTAGLN2 + siPTEN b Akt 0.6 100

p-Akt 0.5 0.4 80

Bcl-2 0.2 60

Signal

-Actin) to (normalized b

0.0 to (normalized Bax expression protein Relative 0.0 40 Relative mRNA expression mRNA Relative NC – + – – b Akt Bax 20 -Actin PTEN p-Akt Bcl-2 TAGLN2 – – ++ PTEN ––– + 0 Transgelin 2 siRNA -20 0 50 100 150 200 250 300 350 400 Time (sec)

Figure 4. The PI3K/Akt/GSK-3b pathway was activated in MCF-7/PTX cells and Transgelin 2 directly interacted with PTEN and was located upstream of PTEN. A, The expressions of Transgelin 2 and PI3K/Akt/GSK-3b pathway signal factors PTEN, Akt, p-Akt, GSK-3b, p-GSK-3b, and Snail of MCF-7/S and MCF-7/PTX cells were determined by Western blot assay. B, MCF-7/PTX cells were transfected with control siRNA (0.1 nmol/L) or TAGLN2 siRNA (0.1 nmol/L) for 48 hours, and the protein expressions of Transgelin 2, PTEN, Akt, p-Akt, GSK-3b, p-GSK-3b, and Snail were examined. Data were represented as mean SD from three experiments. , P < 0.01 vs. MCF-7/S cells or control. C, PI3K/Akt/GSK-3b pathway signal factors PTEN, Akt, p-Akt, GSK-3b, and p-GSK-3b expression levels were determined in MCF-7/S cells transfected with pcDNA3.1 or pcDNA3.1-T. D, PI3K/Akt/GSK-3b pathway downstream apoptosis molecules Bcl-2, Bax, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, and Snail expression levels were compared in MCF-7/S cells transfected with pcDNA3.1 or high-TAGLN2. Data were presented as mean SD from three experiments. , P < 0.05; , P < 0.01 vs. control. E, The interaction between PTEN and Transgelin 2 protein was detected by coimmunoprecipitation assay. F, The expression of Transgelin 2 and PTEN was upregulated by plasmids transfection. The efﬁciency of plasmids transfection was determined by qRT-PCR. The protein level of Transgelin 2, PTEN and PI3K/Akt pathway signal factors Akt, p-Akt and its downstream apoptosis molecules Bcl-2, Bax were detected by Western blot. G, The expression of Transgelin 2 and PTEN was downregulated by RNAi. The efﬁciency of RNAi was determined by qRT-PCR. The protein level of Transgelin 2, PTEN and PI3K/Akt pathway signal factors Akt, p-Akt and its downstream apoptosis molecules Bcl-2, Bax were detected by Western blot. H, Kinetic analysis of Transgelin 2 by localized surface plasmon resonances (LSPR) with PTEN immobilized on COOH-sensor chips.

Transgelin 2 and PTEN reactivated the PI3K/Akt pathway mentary Table S4 and Supplementary Table S5). Then the (Fig. 4G). These results indicate that Transgelin 2 is upstream afﬁnity of Transgelin 2 and PTEN was studied at the protein of PTEN. Furthermore, to understand the regulation of structure level. Their afﬁnity was detected by localized surface Transgelin2onPTEN,weconductedChIP-seqtodetectthe plasmon resonances (LSPR) and the result showed a strong transcriptional regulatory sites of Transgelin 2 on PTEN. The interaction between Transgelin 2 and PTEN with a KD of result showed that Transgelin 2 might be a transcriptional 2,610 nmol/L (Fig. 4H). These results showed that Transgelin regulatory factor of PTEN; furthermore, it could regulate 2 could directly interact with PTEN at the level of transcrip- PTEN expression through direct binding of PTEN (Supple- tion and protein.

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A m B 100 MCF-7/S IC50 = 3.11 ± 3.43 mol/L 100 MCF-7/PTX Control m MCF-7/PTX IC50 = 3.81 ± 3.18 mol/L 20 nmol/L 80 80 40 nmol/L 80 nmol/L 60 60

40 40

20 20

Relative cell viability (%) viability cell Relative Relative cell viability (%) viability cell Relative

0 0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 1.5 2.0 2.5 3.0 3.5 4.0 MK-2206 (Log nmol/L) Paclitaxel (Log nmol/L)

C MCF-7/PTX Control PTX MK-2206 MK-2206+PTX 0.36% 0.54% 0.86% 10.32% 0.27% 2.04% 1.18% 10.03%

1.88%

D 1.5 Control 20 nmol/L 40 nmol/L 80 nmol/L

1.0

-Actin b

0.5 normalized to to normalized

Quality of mRNA expression mRNA of Quality 0.0

MDR1 MRP1 BCRP E +MK-2206 (nmol/L) 2.4 Control 20 nmol/L Control 20 40 80 40 nmol/L 80 nmol/L

P-gp 1.6

-Actin b

MRP1 0.8

BCRP to normalized

Quality of protein expression protein of Quality 0.0 b-Actin P-gp MRP1 BCRP

Figure 5. PI3K/Akt pathway inhibitor MK-2206 reversed resistance to paclitaxel of breast cancer cells. A, The cytotoxicity of MK-2206 to MCF-7/S and MCF-7/PTX cells was represented. B, The effect of MK-2206 (20, 40, and 80 nmol/L) on the sensitivity of MCF-7/PTX cells to paclitaxel was assessed by MTT assay. C, MCF-7/PTX cells treated with 1,000 nmol/L paclitaxel minus or plus SAA (80 nmol/L) for 48 hours and apoptotic cells were detected using Annexin V-FITC/PI-double staining analyzed by ﬂow cytometry. D and E, MCF-7/PTX cells were treated with MK-2206 for 48 hours, and the expressions of P-gp, MRP1 and BCRP were measured by qRT-PCR and Western blot assays. Data were presented as mean SD from three experiments. , P < 0.05; , P < 0.01 vs. control.

PI3K/Akt pathway inhibitor MK-2206 reversed resistance to To conﬁrm the role of the PI3K/Akt/GSK-3b pathway, MK-2206 paclitaxel and inhibited migration and invasion of breast was used to investigate the correlation between activation of Akt cancer cells and increased PTX resistance and cell motility induced by Trans- MK-2206 is a potent AKT inhibitor that has been proved to have gelin 2 in breast cancer cells. First of all, the cytotoxicity and effect good tolerance at the bioactive dose of inhibiting AKT signal (22). of resistance reversal of MK-2206 toward cells were evaluated

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+MK-2206 (nmo/L) A 150 Control D Control 20 40 80 +MK-2206 (nmol/L) 20 nmol/L Control 20 40 80 40 nmol/L 80 nmol/L 100 0 h 80 Control 20 nmol/L 50 40 nmol/L 60 80 nmol/L 12 h Number of mammosphere 0 Control 20 nmol/L 40 nmol/L 80 nmol/L 40

B Control 24 h 2.0 20 nmol/L 20 40 nmol/L Percent wound closure (%) 1.5 80 nmol/L 0 b -Actin 48 h 12 h 24 h 48 h 1.0

0.5 normalized to Quality of mRNA expression Quality of mRNA 0.0

Vimentin E-cadherin N-cadherin E 300 Control +MK-2206 (nmol/L) +MK-2206 (nmo/L) 20 nmol/L 1.5 Control C Control 20 40 80 40 nmol/L Control 20 40 80 20 nmol/L 80 nmol/L 40 nmol/L 200 E-cadherin 80 nmol/L 1.0 b -Actin 100 N-cadherin

0.5 Cell numbers of invation 0 Vimentin normalized to Control 20 nmol/L 40 nmol/L 80 nmol/L Quality of protein expression 0.0 b-Actin

Vimentin E-cadherin N-cadherin F +MK-2206 (nmol/L) G +MK-2206 (nmol/L) Control 20 40 80 2.0 Control Control 20 40 80 20 nmol/L Transgelin 2 1.8 Control 40 nmol/L Bcl-2 20 nmol/L 1.5 80 nmol/L PTEN 40 nmol/L b -Actin Bax 80 nmol/L 1.2 b -Actin Akt 1.0 Cleaved-Caspase-3

p-Akt Cleaved-Caspase-9 0.5 0.6 normalized to b

GSK-3 normalized to

Quality of protein expression Cleaved-PARP

0.0 Quality of protein expression p-GSK-3b 0.0 b Snail Akt PTEN p-Akt Bax b-Actin GSK-3b b Bcl-2 Snail p-GSK-3 -Actin Transgelin 2 Cleaved-PARP

Cleaved-Caspase-3Cleaved-Caspase-9

Figure 6. MK-2206 inhibited the migration and invasion and suppressed the PI3K/Akt/GSK-3b pathway in MCF-7/PTX cells. A, After treated with MK-2206, mammosphere-forming ability of MCF-7/PTX cells was examined (original magnification 100). B and C, The mRNA and protein expressions of E-cadherin, N-cadherin, and vimentin were assessed in MCF-7/PTX cells treated with MK-2206 by qRT-PCR and Western blot methods. D, Migration of MCF-7/PTX cells treated with MK-2206 was measured by wound-healing assay (original magnification 100). E, Invasiveness of MCF-7/PTX cells treated with MK-2206 was detected by Transwell invasion assay (original magnification 100). Data were shown as mean SD from three experiments. , P < 0.05; , P < 0.01 vs. control. F, The expressions of Transgelin 2, PTEN, Akt, p-Akt, GSK-3b, and p-GSK-3b were determined in MCF-7/PTX cells treated with MK-2206 for 48 hours. G, Bcl-2, Bax, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, and Snail expression levels were examined in MCF-7/PTX cells treated with MK-2206.

using the MTT assay. As shown in Figure 5A, MK-2206 inhibited compared with the control group (Fig. 6A). Meanwhile, the growth of both MCF-7/S and MCF-7/PTX cells in a dose- E-cadherin expression increased sharply, whereas N-cadherin dependent manner. The IC50 values were 3.11 3.43 and 3.81 and vimentin were both clearly reduced (Fig. 6B and C). Similarly, 5.18 mmol/L (Fig. 5A) respectively, indicating that MCF-7/PTX the migration and invasion abilities of MCF-7/PTX cells treated cells did not produce resistance to MK-2206. Consequently, with MK-2206 alone for 48 hours were both reduced (Fig. 6D MK-2206 was used at three nontoxic concentrations (20, 40, and and E), indicating that MK-2206 can attenuate the Transgelin 80 nmol/L, which produced inhibitions <10%) in the subsequent 2-mediated EMT and the motility and invasiveness of MCF-7/PTX experiments. The growth curves showed that MK-2206 at these cells. three concentrations augmented the sensitivity of MCF-7/PTX cells to PTX by 2.02-, 3.14-, and 4.67-fold, respectively MK-2206 suppressed Transgelin 2 expression and activation of (Fig. 5B; Supplementary Table S6). We also found that the PI3K/Akt/GSK-3b pathway in MCF-7/PTX cells apoptosis rate (69.67%) was effectively increased in MCF-7/PTX Western blotting was used to detect the effects of MK-2206 on cells following treatment with PTX (1,000 nmol/L) combined the PI3K/Akt/GSK-3b pathway and Transgelin 2. After treating with MK-2206 (80 nmol/L; Fig. 5C). Moreover, the mRNA and MCF-7/PTX cells with MK-2206 for 48 hours, the levels of Trans- protein levels of P-gp, MRP1, and BCRP were consistently reduced gelin 2, phospho-Akt, and phospho-GSK-3b were downregulated (Fig. 5D and E). Our data supported that MK-2206 has a strong with a concomitant increased expression of PTEN, whereas the ability to reverse PTX resistance in MCF-7/PTX cells. levels of Akt and GSK-3b were unaffected (Fig. 6F). Meanwhile, qPCR, Western blotting, scratch wound healing, and Transwell the expression levels of its downstream factor Bcl-2 and Snail were invasion assays were performed to elucidate if MK-2206 can reduced. A particularly interesting ﬁnding was that the expression inhibit the migration and invasion of breast cancer. The number levels of Bax, cleaved-caspase-3, cleaved-caspase-9, and cleaved- of mammospheres of these cells decreased by about 2-fold PARP were increased notably after MK-2206 treatment (Fig. 6G).

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Discussion knocked down in MCF-7/PTX cells, indicating that the mechanism underlying Transgelin 2-mediated PTX resistance, Transgelin 2, located on chromosome 1q21-q25, is an migration, and invasion in breast cancer involves the important cytoskeletal actin–binding protein that influences PI3K/Akt/GSK-3b pathway. It is particularly interesting that the dynamics of the actin cytoskeleton. Some recent studies we also found that Transgelin 2 directly interacted with have described Transgelin 2 as a potential oncogenic factor in PTEN and was located upstream of PTEN, thus activating the many human malignancies (14). Studies have also shown that PI3K/Akt pathway. We also detected the effect of Transgelin 2 overexpression of Transgelin 2 was associated with tumor on PTEN stability, but the results showed that the overexpres- migration and invasion (23). Moreover, Transgelin 2 was sion of Transgelin 2 did not have significant effect on the half- reported to be correlated with lymph node metastasis, distant life or the proteasome-mediated degradation of PTEN (Sup- metastasis, and the TNM classification in colorectal cancer, plementary Fig. S3). We therefore speculate that Transgelin 2 and it may be useful as a new biomarker for predicting the might inhibit the expression of PTEN at the translation level progression and prognosis of colorectal cancer (24). On the via the non–proteasome–ubiquitin pathway; this mechanism other hand, there is accumulating evidence that a high level of requires further investigation. To confirmtheroleofTransge- Transgelin 2 in cancer cells could contribute to drug lin 2 in the PI3K/Akt/GSK-3b pathway, we treated MCF-7/PTX resistance (25). cells with the Akt inhibitor MK-2206 and found that inacti- This study is the first to find a high expression level of vation of the PI3K/Akt/GSK-3b pathway exerted a feedback Transgelin 2 in MCF-7/PTX cells and its association with cell effect on Transgelin 2 expression, which was essential to PTX resistance, migration, and invasion. In this study, we reverse the PTX resistance and suppress migration and inva- were planning to collect clinical samples from patients who sion (Supplementary Fig. S4). had undergone PTX chemotherapy treatment prior to breast In summary, this study found that Transgelin 2 plays an surgery to explore the effect of chemotherapy on Transgelin 2 important role in the PTX resistance, migration, and invasion of expression. However, the difficulty of collecting such speci- breast cancer cells. A high level of Transgelin 2 could activate mens meant that we collected only three specimens in which the PI3K/Akt/GSK-3b pathway by interacting with PTEN and the Transgelin 2 expression level was significantly higher than thus participate in the PTX resistance, migration, and invasion that in the adjacent nontumor tissue. Besides, considering of breast cancer. These results provide insights into the hormone levels, we found that the expression of Transgelin 2 mechanism of drug resistance mediated by Transgelin 2 and was inversely correlated with ER and PR status. We also indicate that Transgelin 2 is a potential therapeutic target in found that ER status was independently associated with breast cancer. Transgelin 2 mRNA expression in breast cancers. What is more, we had detected the expression of ER in drug- Disclosure of Potential Conflicts of Interest resistant cell lines and found that ER expression was low in No potential conflicts of interest were disclosed. drug resistant cells while the expression of human epidermal growth factor receptor 2 (Her-2) was upregulated (19). These Authors' Contributions suggest that Transgelin 2 might be a possible candidate as Conception and design: L. Liu, J. Xing, Y. Dong therapeutic target for ER-negative breast cancer. However, Development of methodology: L. Liu, T. Meng, X. Zheng, Y. Liu, Y. Yan whether hormone status has effect on the expression and Acquisition of data (provided animals, acquired and managed patients, function of Transgelin 2 and the therapeutic effect of Trans- provided facilities, etc.): T. Meng, R. Hao gelin 2 in ER-negative breast cancer remain unclear. There- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Liu, X. Zheng, R. Hao, S. Chen fore, ER status should be taken into account in the follow-up Writing, review, and/or revision of the manuscript: L. Liu, T. Meng, S. Chen study of Transgelin 2. Administrative, technical, or material support (i.e., reporting or organizing It is widely believed that the PI3K/Akt/GSK-3b pathway data, constructing databases): T. Meng plays an important role in physiologic processes in cancer Study supervision: H. You, J. Xing, Y. Dong cells, such as the cell cycle, migration, differentiation, and apoptosis (26). Multiple investigations have discovered hyper- Acknowledgments b activity of Akt/GSK-3 /Snail signaling to be necessary for cell We thank Hongying Wang (Xi'an Jiaotong University, Shaanxi, proliferation, the EMT process, and also migration and China) for her support in the establishment of xenograft tumor invasion (27–32). PTEN, a negative regulator of the PI3K/Akt model. The ChIP-Seq high throughput sequencing and subsequent pathway, can dephosphorylate phosphatidylinositol (3,4,5)- bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, triphosphate to phosphatidylinositol (4,5)-bisphosphate, China). and it therefore decreases the activation of the PI3K/Akt The work is supported by National Natural Science Foundation of China (no. 81473177, 81672954). pathway (33). In this study, we observed that the PI3K/Akt/ GSK-3b pathway was activated when the negative regulator PTEN was downregulated in MCF-7/PTX cells. Overexpression The costs of publication of this article were defrayed in part by the of Transgelin 2 in MCF-7/S cells via plasmid transfection payment of page charges. This article must therefore be hereby marked immensely greatly increased ABC transporters and decreased advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate PTEN expression, resulting in the activation of the PI3K/Akt/ this fact. GSK-3b pathway and upregulation of Snail expression, thereby facilitating cell proliferation, migration, and invasion. Received March 11, 2019; revised July 3, 2019; accepted August 27, 2019; However, the opposite results were found when TAGLN2 was published first September 5, 2019.

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2468 Mol Cancer Ther; 18(12) December 2019 Molecular Cancer Therapeutics

Transgelin 2 Promotes Paclitaxel Resistance, Migration, and Invasion of Breast Cancer by Directly Interacting with PTEN and Activating PI3K/Akt/GSK-3β Pathway

Leichao Liu, Ti Meng, Xiaowei Zheng, et al.

Mol Cancer Ther 2019;18:2457-2468. Published OnlineFirst September 5, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-19-0261

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