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LOXL1 Confers Antiapoptosis and Promotes Gliomagenesis Through Stabilizing BAG2

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Cell Death & Differentiation (2020) 27:3021–3036 https://doi.org/10.1038/s41418-020-0558-4 ARTICLE LOXL1 confers antiapoptosis and promotes gliomagenesis through stabilizing BAG2 1,2 3 4 3 4 3 1 1 Hua Yu ● Jun Ding ● Hongwen Zhu ● Yao Jing ● Hu Zhou ● Hengli Tian ● Ke Tang ● Gang Wang ● Xiongjun Wang1,2 Received: 10 January 2020 / Revised: 30 April 2020 / Accepted: 5 May 2020 / Published online: 18 May 2020 © The Author(s) 2020. This article is published with open access Abstract The lysyl oxidase (LOX) family is closely related to the progression of glioma. To ensure the clinical significance of LOX family in glioma, The Cancer Genome Atlas (TCGA) database was mined and the analysis indicated that higher LOXL1 expression was correlated with more malignant glioma progression. The functions of LOXL1 in promoting glioma cell survival and inhibiting apoptosis were studied by gain- and loss-of-function experiments in cells and animals. LOXL1 was found to exhibit antiapoptotic activity by interacting with multiple antiapoptosis modulators, especially BAG family molecular chaperone regulator 2 (BAG2). LOXL1-D515 interacted with BAG2-K186 through a hydrogen bond, and its lysyl 1234567890();,: 1234567890();,: oxidase activity prevented BAG2 degradation by competing with K186 ubiquitylation. Then, we discovered that LOXL1 expression was specifically upregulated through the VEGFR-Src-CEBPA axis. Clinically, the patients with higher LOXL1 levels in their blood had much more abundant BAG2 protein levels in glioma tissues. Conclusively, LOXL1 functions as an important mediator that increases the antiapoptotic capacity of tumor cells, and approaches targeting LOXL1 represent a potential strategy for treating glioma. In addition, blood LOXL1 levels can be used as a biomarker to monitor glioma progression. Introduction Gliomas represent approximately 70% of malignant primary Edited by M. Bianchi brain tumors in adults and are characterized by high fi These authors contributed equally: Hua Yu, Jun Ding, Hongwen Zhu recurrence and low ve-year survival rate [1]. Due to the existence of the blood–brain barrier, current efficient clin- Supplementary information The online version of this article (https:// ical treatments for glioma are almost limited to temozolo- doi.org/10.1038/s41418-020-0558-4) contains supplementary material, which is available to authorized users. mide (TMZ) chemotherapy and ionizing radiation (IR) [2]. Glioma cells can also acquire resistance to apoptosis, * Xiongjun Wang making the current treatments ineffective. Thus, targeting [email protected] antiapoptotic factors might be a good solution to improve 1 Precise Genome Engineering Center, School of Life Sciences, patient survival. Guangzhou University, Guangzhou 510006, China LOXs are copper-dependent monoamine oxidases that 2 CAS Key Laboratory of Tissue Microenvironment and Tumor, are involved in the early stage of collagen and elastin Institute of Health Sciences, Chinese Academy of Sciences, polymerization in the extracellular matrix (ECM) and in University of Chinese Academy of Sciences, Shanghai 200031, later collagen-elastin crosslinking, thereby increasing the China stability of ECM [3]. The LOX family consists of LOX and 3 Department of Neurosurgery, Shanghai Jiao Tong University LOX-like (LOXL) proteins, including LOXL1, LOXL2, fi ’ Af liated Sixth People s Hospital, 600 Yishan Road, Xuhui LOXL3 and LOXL4 [4]. The carboxyl termini of LOX District, Shanghai 200233, China family proteins are highly conserved with a catalytic 4 Department of Analytical Chemistry and CAS Key Laboratory of domain of 205 amino acids shared by LOXL1-4, while the Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, amino termini of LOXL1-4 differ substantially, determin- China ing the various biological functions of these four LOXLs 3022 H. Yu et al. [5]. In general, LOXs have many important biological Materials and methods functions, including the regulation of cell differentiation, mobility, migration and gene expression [6]. Recent studies Cell lines, antibodies and reagents have shown that LOXs expressions are increased under hypoxic conditions in malignant tumors, including eso- U87, LN18 and 293FT cell lines were purchased from phageal cancer, colorectal tissue cancer, bladder cancer and American Type Culture Collection (ATCC, USA) or head and neck cancer, and that they function in promoting obtained from the Cell Bank of Type Culture Collection of tumor metastasis [7–9]. In other types of tumors, such as Chinese Academy of Sciences China (Shanghai, China). prostate, liver, lung, breast and stomach cancers, LOXs are GSC11 cell was a kind gift from Dr. Weiwei Yang involved in inhibiting tumor proliferation [10–12]. There laboratory (Shanghai Institute of Biochemistry and Cell are also a few reports about the role of LOX family in Biology, Chinese Academy of Sciences, University of glioma. For example, the polymorphisms in LOX gene, 22 Chinese Academy of Sciences, Shanghai 200031, China). G/C and 473 G/A, were associated with increased sus- Cells were tested for mycoplasma contamination and ceptibility to glioma [13]. LOX expression was regulated authenticated using the short tandem repeat (STR) method by IDH1 status in astrocytomas [14]. And active LOX in the previous report [23]. would modulate migration by association with FAK/pax- Antibodies against LOXL1 (H00004016-D01P), LOXL4 illin in invasive astrocytes [15]. More importantly, recent (NBP2-32692), BAG2 (AF800) were purchased from research discovered a symbiotic glioma-macrophage Novus Biologicals (USA) for western blot. Antibodies interplay, which could be considered as a novel target for against IRF-1(#8478), CEBPA (#8178), cMyc (ab56), PTEN-deficient glioma [16]. Also, LOXL1 could play an GATA1 (ab181544) and AP1 (ab31419) were purchased aggressive role in glioma through its antiapoptotic capacity from cell signaling technology (USA) or Abcam (UK) for via Wnt/beta-catenin signaling [17]. In addition, the chromatin IP. lncRNA, LOXL1-AS, was required for maintaining EGFRi, Afatinib (BIBW2992, S1011); VEGFRi, Axiti- mesenchymal characteristics of glioblastoma via NF-κB nib (S1005); Notchi, RO4929097(S1575); PI3Ki, pathway [18]. However, it is still elusive how LOXL1 is LY294002(S1105); SMOi, Smoothened Agonist (SAG) upregulated and exerts antiapoptotic function by directly HCl, (S7779); Srci, Bosutinib (SKI-606, S1014); PKCi, Go forming an axis with other proteins during glioma 6983(S2911); Akti, MK-2206 2HCl (S1078); ERKi, progression. LY3214996 (S8534); p38i, SB203580(S1076); DKK1 In the present study, we found that LOXL1 was (Millipore, GF170). involved in gliomagenesis and LOXL1 expression was specifically upregulated through the VEGFR-Src-CEBPA Plasmids, lentivirus packaging and infection axis. Growth factor receptor signaling stimulation results in the activation of transcriptional programs required for The LOXL1-overexpressing plasmid was constructed by survival, proliferation, invasion, and angiogenesis cloning LOXL1 from a LN18 cell line cDNA library using [19, 20]. Forced expression of LOXL1 significantly KOD FX Neo (TOYOBO) DNA polymerase and then increased the antiapoptotic capacity of U87 cells, while subcloned into the pCDH-3′SFB vector to generate Flag- LOXL1 depletion in LN18 or GSC11 cells heavily LOXL1. LOXL1 and LOXL4 knockdown plasmids were impaired the cell survival rate under suspended culture constructed by introducing annealing small hairpin RNA conditions. Protein-protein interaction network analysis (shRNA) into the pGIPZ vector. The pGIPZ control vector indicated that LOXL1 could interact with various impor- was generated with the control oligonucleotide 5′- tant proteins, especially BAG2. BAG2 is a cochaperone CTCGCTTGGGCGAGAGTAA-3′. The pGIPZ-LOXL1- and well-known protein that antagonizes apoptosis. It has shRNA plasmid was generated with shLOXL1#1: 5′- been reported that BAG2 expression is increased in pro- cctgggaactacatcctca-3′ oligonucleotide or shLOXL1#2: 5′- teasome inhibitor-induced apoptosis [21]. Additionally, gcattaaagcagcgtatc-3′ oligonucleotide targeting the coding BAG2 plays a pro-oncogenic role in triple-negative breast region of LOXL1. The target sequences used to construct cancer cells through its antiapoptotic activity [22]. In our pGIPZ-LOXL4-shRNA are described in the previous study, LOXL1 stabilized BAG2 by blocking K186 ubi- reports [24, 25]. The siRNAs targeting human BAG2, SRC quitination, rendering glioma cells resistant to apoptosis in and CEBPA were purchased from GenePharma (Shanghai, nonadherent conditions. Our data suggest that LOXL1 can China). be a potential biomarker for guiding the clinical treatment Lentiviruses were amplified using standard methods in of glioma, and the development of new drugs targeting sub-confluent HEK293FT cells. GBM cell lines were LOXL1 may improve the curative efficacy and prolong infected with the lentiviruses in the presence of polybrene survival in glioma patients. (Sigma) at a final concentration of 8 µg/ml. Cells were LOXL1 confers antiapoptosis and promotes gliomagenesis through stabilizing BAG2 3023 incubated with the lentivirus mixture for 72 h, digested with 3D suspension culture trypsin for passage into fresh growth medium, and then sorted based on green fluorescence to determine stable A total of 5 × 105 cells were suspended for 48 h in a 3D expression or knockdown. The constructed stable cell lines Insert PS scaffold for 6-well plates, which was purchased were
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