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Aurora-B Knockdown Inhibits Osteosarcoma Metastasis By Wu et al. Cancer Cell Int (2020) 20:575 https://doi.org/10.1186/s12935-020-01674-1 Cancer Cell International PRIMARY RESEARCH Open Access Aurora-B knockdown inhibits osteosarcoma metastasis by inducing autophagy via the mTOR/ULK1 pathway Xin Wu1,2†, Jia‑ming Liu1,2†, Hong‑hai Song1,2, Qi‑kun Yang1, Hui Ying1, Wei‑lai Tong1,2, Yang Zhou1 and Zhi‑li Liu1,2* Abstract Background: Autophagy plays an essential role in metastasis of malignancies. Although our studies showed that Aurora‑B facilitate pulmonary metastasis in OS, the mechanism of Aurora‑B kinase on autophagy and metastasis in OS has not been explored. Methods: Clinical‑pathological parameters and follow‑up information was collected in OS patients. Immunohisto‑ chemical staining was performed to detect Aurora‑B and LC3 protein in OS tissues. Short hairpin RNA transfection was used to silence Aurora‑B in OS cells. Real‑time quantitative PCR (RT‑qPCR) was performed to detect Aurora‑B mRNA expression in OS cells. Aurora‑B and autophagy related protein were measured by Western blot. Transmission electron microscopy and laser scanning confocal microscopy were performed to observe the formation of autophagosomes and autolysosomes. Migratory and invasive ability of OS cells were measured by Wound healing and transwell assays. Orthotopic xenograft model was used to evaluate the efect of autophagy mediated by Aurora‑B inhibition on pul‑ monary metastasis of OS. Results: The elevated expression of Aurora‑B protein in OS tissues negatively associated with the overall survival of OS patients. Further investigation has found that Aurora‑B expression was negatively correlative with autophagy related protein LC3 in OS patient tissues. Knockdown Aurora‑B stimulates autophagy and inhibits migratory and invasive ability of OS cells. Mechanistically, Aurora‑B knockdown suppressed the mTOR/ULK1 signaling pathway and reactivation of the mTOR/ULK1 pathway decreased autophagy level. Furthermore, the inhibition efect of silencing Aurora‑B on migration and invasion of OS was reversed by chloroquine and mTOR activator in vitro and vivo. Conclusions: Our results suggest that silencing of Aurora‑B stimulate autophagy via decreasing mTOR/ULK1 and result in inhibiting OS metastasis. Targeted Aurora‑B/mTOR/ULK1 pathway may be a promising treatment strategy for OS patients. Keywords: Osteosarcoma, Aurora‑B, The mTOR/ULK1 pathway, Autophagy, Metastasis Background Osteosarcoma (OS), a highly aggressive tumor with a tendency to metastasize to the lung, is the most com- *Correspondence: [email protected] †Xin Wu and Jia‑ming Liu contributed equally to this work mon malignant bone tumor in children and adolescents 1 Department of Orthopedic Surgery, The First Afliated Hospital [1, 2], and metastasis to the lung is the leading cause of of Nanchang University, No.17 Yong Wai Zheng Street, Donghu District, death in patients with OS [3]. Despite the combination Nanchang, Jiangxi 330006, People’s Republic of China Full list of author information is available at the end of the article of surgery resection with neoadjuvant chemotherapy © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wu et al. Cancer Cell Int (2020) 20:575 Page 2 of 14 strategies for OS, the 5 year overall survival rate has invasion of OS cells by increasing the levels of autophagy remained unchanged, at 65–70%, over the past few mediated by mTOR inhibition. Our results identifed decades [4, 5]. In addition, the 5 year survival for met- a novel as a potential therapeutic target and prognostic astatic disease is around 20%, highlighting the need biomarker in OS patients. for novel therapeutic targets. Terefore, elucidation of the molecular and cellular drivers involved in the Material and methods pathogenesis of OS should facilitate the development Tissues specimens and patients of efective new strategies for the management of this Sixty-nine OS tissue samples were obtained by surgical malignancy. biopsy from the First Afliated Hospital of Nanchang Autophagy is an intracellular degradation process that University, China. Patients who had received radiother- removes and recycles damaged proteins and organelles to apy or chemotherapy before undergoing biopsy were extend cell longevity. Numerous studies have shown that excluded. Donors included 29 females and 40 males, with autophagy is exploited by tumor cells as a highly dynamic a mean age of 26 years (range 5–72 years). All OS tissues process to suppress initial stage of tumor in carcinogen- were subject to pathological examination, and the expres- esis by limiting chromosomal instability, restricting oxi- sion of Aurora-B and LC3 was evaluated through IHC dative stress, and preventing intratumoral necrosis and analysis. Te clinical parameters are shown in Table 1; local infammation [6, 7]. Furthermore, in the early stages follow-up information was missing for 10 of these of cancer metastasis, autophagy may restrict neoplasm patients. Informed study protocols were completed in metastasis by suppressing tumor necrosis and infam- accordance with the declaration of Helsinki and “Guiding matory cell infltration, and by reducing tumor-induced Opinions on the Treatment of Animals” in China. Te senescence. Studies have indicated that the induction of medical ethics committee of the First Afliated Hospital autophagy inhibits proliferation, invasion, and migra- of Nanchang University has approved this experimental tion in bladder cancer and OS cells [8–10]. However, the protocol (Jiangxi, China; NO. Y2019-126). underlying molecular regulatory mechanisms require further elucidation. Histology and IHC Aurora-B kinase, a member of the aurora kinase family, OS tissue samples were fxed in 4% paraformaldehyde is a ubiquitously expressed serine/threonine kinase that for 20 min, embedded in parafn, and sectioned to a phosphorylates histone H3 on Ser10 and variant centro- some protein A on Ser7 in early G2, resulting in the con- densation of chromatin 18. Aurora-B participates in the Table 1 Correlation of Aurora-B protein expression levels regulation of the spindle assembly complex, chromosome in OS tissues with clinical pathologic parameters segregation, and cytokinesis [11]. Terefore, silencing or Variables All cases Aurora-B expression P value loss of Aurora-B leads to defective chromosome segrega- Low High tion and polyploidy. Abundant evidence has shown that Aurora-B is expressed at high levels in various malig- Gender nant tumors, and represents an important antitumor Male 40 19 (47.5%) 21 (52.5%) 0.328 target [12–14]. Amplifcation or increased expression Female 29 18 (62.1%) 11 (37.9%) of Aurora-B has been shown to be associated with poor Age 20 33 19 (57.6%) 14 (42.4%) 0.631 prognosis in various human malignant tumors [15–17]. ≤ Further, Aurora-B is a therapeutic target in non-small > 20 36 18 (50%) 18 (50%) cell lung cancer refractory to anti-EGFR therapy [18].Our Location previous study indicated that the expression of Aurora-B Femur/Tibia 53 27 (50.9%) 26 (49.1%) 0.569 is elevated in OS tissues and cell lines, and that silenc- Elsewhere 16 10 (62.5%) 6 (37.5%) ing of Aurora-B inhibited the malignant phenotype of OS Tumor size (cm) 5 23 16 (69.6%) 7 (30.4%) 0.076 cells in vitro [19]. However, the mechanisms by which ≤ Aurora-B promotes OS pulmonary metastasis have not > 5 46 21 (45.7%) 25 (54.3%) been fully elucidated to date. Enneking staging I IIA 40 26 (65%) 14 (35%) 0.031* In this study, we investigated the role, and potential + IIB III 29 11 (37.9%) 18 (62.1%) underlying mechanisms, of Aurora-B in the pathogen- + esis of OS, and found that the expression of this kinase LC3B expression is negatively correlated with prognosis and autophagy Low 34 13 (38.2%) 21 (61.8%) 0.016* in OS, which had not been shown before. Furthermore, High 35 24 (68.6%) 11 (31.4%) Aurora-B silencing was shown to inhibit migration and P value was calculated by Pearson’s Chi-Square test. *P < 0.05 Wu et al. Cancer Cell Int (2020) 20:575 Page 3 of 14 thickness of ~ 3 µm. Tese slides were deparafnized and signaling technology 2775), anti-p-mTOR(ser2448)(Cell rehydrated, and then treated with 0.2% Triton X-100PBS signaling technology 2971), anti-mTOR(Cell signal- for 10 min and blocked with 3% hydrogen peroxide at ing technology 2972), anti-AMPK (Cell Signaling Tech- room temperature for 20 min. Tese slides were auto- nology, 2532), anti-pAMPKα (Tr172) (Cell Signaling claved in 10 mM citric acid solution to enhance the anti- Technology, 2535), anti-ULK1 (Cell Signaling Tech- gen retrieval for 2 min. Te samples were incubated with nology, 8054) and anti-Pulk1 (Ser555) (Cell Signaling anti-Aurora-B (Abcam ab45145) and anti-LC3 (Cell sign- Technology, 5869), mouse anti-human GAPDH (Ori- aling technology 2775) overnight at 4 °C.
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