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 signif- icant 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 devel- opment 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 Ó2019 American Association for Cancer Research. cells (MCF-7/PTX) established by our laboratory than in breast www.aacrjournals.org 2457 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 5, 2019; DOI: 10.1158/1535-7163.MCT-19-0261 Liu et al. 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 Â 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 Â 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 Â 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