Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells

OncoTargets and Therapy, Jun 2016

Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells Ying Liu,1,2,* Baocun Sun,1–3,* Tieju Liu,1,2,* Xiulan Zhao,1,2 Xudong Wang,3 Yanlei Li,1,2 Jie Meng,2 Qiang Gu,1,2 Fang Liu,1,2 Xueyi Dong,1,2 Peimei Liu,2 Ran Sun,2 Nan Zhao1 1Department of Pathology, General Hospital of Tianjin Medical University, 2Department of Pathology, Tianjin Medical University, 3Department of Pathology, Cancer Hospital of Tianjin Medical University, Tianjin, People’s Republic of China *These authors contributed equally to this work Abstract: Tumor cell vasculogenic mimicry (VM), a newly defined pattern of tumor blood supply, signifies the functional plasticity of aggressive cancer cells forming vascular networks. VM and cancer stem cells (CSCs) have been shown to be associated with tumor growth, local invasion, and distant metastasis. In our previous study, CSCs in triple-negative breast cancer were potential to participate in VM formation. In this study, breast CSCs were isolated from the triple-negative breast cancer cell line MDA-MB-231 by using mammosphere culture. Western blotting and reverse transcription polymerase chain reaction showed that mammosphere cells displayed an increased expression of AURKA protein kinase and stem cell marker c-myc and sox2. The VM formation by mammosphere cells was inhibited by AURKA knockdown or the addition of AURKA inhibitor MLN8237. In the meantime, MLN8237 induced the increased E-cadherin and decreased c-myc, sox2, and β-catenin expressions. The function of AURKA in VM formation was further confirmed using a xenograft-murine model. The results suggested that AURKA protein kinase is involved in VM formation of CSCs and may become a new treatment target in suppressing VM and metastasis of breast cancer. Keywords: AURKA, cancer stem cells, vasculogenic mimicry, breast cancer

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Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells

OncoTargets and Therapy Function of a Ur Ka protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells Ying liu 1 2 Baocun sun 2 Tieju liu 1 2 Xiulan Zhao 1 2 Xudong Wang 0 Yanlei li 1 2 Jie Meng 1 Qiang gu 1 2 Fang liu 1 2 Xueyi Dong 1 2 Peimei liu 1 ran sun 1 n an Zhao 2 0 Department of Pathology, cancer hospital of Tianjin Medical University , Tianjin, People's republic of china 1 Department of Pathology, Tianjin Medical University 2 Department of Pathology, g eneral hospital of Tianjin Medical University Tumor cell vasculogenic mimicry (VM), a newly defined pattern of tumor blood supply, signifies the functional plasticity of aggressive cancer cells forming vascular networks. VM and cancer stem cells (CSCs) have been shown to be associated with tumor growth, local invasion, and distant metastasis. In our previous study, CSCs in triple-negative breast cancer were potential to participate in VM formation. In this study, breast CSCs were isolated from the triple-negative breast cancer cell line MDA-MB-231 by using mammosphere culture. Western blotting and reverse transcription polymerase chain reaction showed that mammosphere cells displayed an increased expression of AURKA protein kinase and stem cell marker c-myc and sox2. The VM formation by mammosphere cells was inhibited by AURKA knockdown or the addition of AURKA inhibitor MLN8237. In the meantime, MLN8237 induced the increased E-cadherin and decreased c-myc, sox2, and β-catenin expressions. The function of AURKA in VM formation was further confirmed using a xenograftmurine model. The results suggested that AURKA protein kinase is involved in VM formation of CSCs and may become a new treatment target in suppressing VM and metastasis of breast cancer. - 8 1 0 2 l u J 3 1 n o 3 5 1 . 4 5 2 . 5 3 1 . 5 y b / m o c . s s rvpee l.yno behavior and increased metastatic ability.7 Our previous study8 showed that CSCs were able to participate in VM formation and that CSC subpopulation inside TN breast cancer was able to organize VM for which transdifferentiative capacity of CSCs might be needed. The AURKA gene is located at chromosome 20q13.2 and encodes serine–threonine kinase, which is composed of 403 amino acids and has vital cellular functions in mitosis. AURKA is considered as an oncogene and plays important roles in the development of breast CSCs by inducing epithelial– mesenchymal transition (EMT).9 AURKA gene amplification is a common genetic aberration in breast cancer, especially in TN tumors.10 Given that both AURKA and VM formation could promote breast cancer invasion and metastasis, the relationship of AURKA and VM in TN breast cancer remains unknown. This study aimed to demonstrate the potential contribution of AURKA to VM in TN breast cancer. Materials and methods .dow lsue cell culture and isolation of breast cscs /ww ano The human breast cancer cell lines MDA-MB-231, Hs578T, / ttsp rspe and MCF-7 were obtained from the American Type Culture : h ro Collection (Manassas, VA, USA). This study did not use human from F tissues or the primary cultured tumor cells, only cell lines were ddea used, thus, such permission was not required, according to lon General Hospital of Tianjin Medical University review board. odw These cells were cultured in Dulbecco’s Modified Eagle’s rypa Medium (Sigma-Aldrich Co., St Louis, MO, USA) suppleehT mented with 10% fetal bovine serum, 100 units/mL penicilnad lin, and 100 mg/mL streptomycin (Thermo Fisher Scientific, tsge Waltham, MA, USA) in a humidified 5% CO2 incubator at raT 37°C. At the logarithmic growth phase, the MDA-MB-231 cno or Hs578T cells were digested with 0.25% trypsin and then O seeded at 1×105 into six-well, ultralow adherent plates covered with poly 2-hydroxyethyl methacrylate (Sigma-Aldrich). Each well also contained 2 mL of serum-free suspension medium or Dulbecco’s Modified Eagle’s Medium/F12 (1:1; Thermo Fisher Scientific) supplemented with 2% B27 (Thermo Fisher Scientific), 0.5% epidermal growth factor (Pepro Tech; Rocky Hill, NJ, USA), and 0.5% basic fibroblast growth factor (Pepro Tech). Cell growth was daily observed under an inverted microscope (Nikon USA, Garden City, NY, USA). reverse transcription polymerase chain reaction analysis To assess the expression levels of c-myc, sox2, E-cadherin, and β-catenin, we extracted total RNA from the cell lines by using Trizol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Polymerase chain 3474 reaction was designed to amplify specific mRNAs by using published sequences. The primer sequences were listed as follows: c-myc sense 5′-TACCCTCTCAACGACAGCAG-3′, antisense 5′-TCTTGACATTCTCCTCGGTG-3′, Sox2 sense 5′-GGGAAATGGAGGGGTGCAAAAGAGG-3′, antisense 5′-TTGCGTGAGTGTGGATGGGATTGGTG-3′, β-catenin sense 5′-AAGGTCTGAGGAGCAGCTTC-3′, antisense 5′-TGGACCATAACTGCAGCCTT-3′, E-cadherin sense 5′-GTCACTGACACCAACGATAATCCT-3′, antisense 5′TTTCAGTGTGGTGATTACGACGTTA-3′, α-catenin sense 5 ′ - G C (...truncated)


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Ying Liu, Baocun Sun, Tieju Liu, Xiulan Zhao, Xudong Wang, Yanlei Li, Jie Meng, Qiang Gu, Fang Liu, Xueyi Dong, Peimei Liu, Ran Sun, Nan Zhao. Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells, OncoTargets and Therapy, 2016, pp. 3473-3484, DOI: 10.2147/OTT.S93015