TROAP regulates cell cycle and promotes tumor progression through Wnt/β‐Catenin signaling pathway in glioma cells

Abstract Aims Experimental evidence demonstrated a crucial role of TROAP (Trophinin‐associated protein) in regulating the cell proliferation of multiple tumors, while TROAP expression and function were largely unknown in glioma. We aimed to investigate the oncogenic role of TROAP and its potential mechanisms in gliomagenesis. Methods Four gene expression databases (GEO, TCGA, GTEx and CCLE) were enrolled in our study and used for TROAP expression and survival analysis. TROAP expression was quantified by qRT‐PCR, western blot and immunohistochemistry assays in glioma tissues and cell lines. TROAP knockdown and overexpression vector were constructed and transfected into glioma cells. CCK‐8, colony formation, transwell, and wound healing assays were used to evaluate cell viability, migration and invasion, flow cytometry to determine cell cycle arrest. Gene set enrichment analysis (GSEA) was conducted to screen the pathway involved in TROAP‐high phenotype. The expression of cell cycle and Wnt/β‐Catenin signaling proteins were analyzed by immunofluorescence and western blot. Results Based on the bioinformatic analysis and a series of functional assays, we found the TROAP was enriched in glioma tissues and cell lines, its overexpression was correlated with the clinicopathologic characteristics and poor prognosis. TROAP knockdown inhibited cell proliferation, migration, invasion, and G1/S cell cycle arrest compared with control group in glioma. Mechanism analysis revealed that TROAP activated Wnt/β‐Catenin pathway and upregulated its downstream targets expression, while silencing β‐Catenin or Axin2 could reverse the tumor‐promoting effects caused by TROAP, confirming that TROAP‐induced malignant phenotype and tumorigenesis via Wnt/β‐Catenin signaling pathway. Conclusion The present study found that TROAP accelerated the progression of gliomagenesis through Wnt/β‐Catenin pathway, and TROAP might be considered as a novel target for glioma therapy.


| INTRODUC TI ON
Glioma, the most common malignant primary intracranial tumors due to carcinogenesis of brain glial cells, shows a gloomy prognosis. 1,2 Glioma, especially glioblastoma (GBM), is characterized by multifocal infiltration around the adjacent brain parenchyma.
According to pathological characteristics, glioma is classified into four grade (Grade Ⅰ-Ⅳ), depending on which the overall survival time (OS) varies strikingly. For instance, patients with GBM (Grade Ⅳ) had a 5-year survival rate of 5.6%, much shorter than 94.1%, the 5-year survival rate of pilocytic astrocytoma (PA, GradeⅠ) patients. 3 Although surgical intervention and adjuvant therapy have evolved over decades, the prognosis of patients with glioma still faces significant challenges. 4,5 Therefore, an in-depth underlying molecular mechanisms associated with the malignant phenotype of glioma is of great value in the development of potential gene therapies.
Trophinin-associated protein(TROAP, formally known as Tastin), a proline-rich protein characterized by 778 amino acids, was first identified as a soluble cytoplasmic protein that participated in early embryo implantation. 6,7 A previous study has shown that TROAP was required for microtubular cytoskeleton regulation, centrosome integrity, and spindle assembly during the mitosis and cell cycle progression. [8][9][10] Expect for TROAP-induced physiological processes, several studies proved the dysregulated expression of TROAP in tumorigenesis. Results of a genomics microarray analysis indicated that TROAP presented aberrantly elevated expression in human cancer cell lines such as Jurkat and Hela cells. 11 In recent years, aberrant expression of TROAP has been found to be responsible for the invasive behavior of various malignancies, including breast cancer, colorectal cancer, prostate cancer, gastric cancer, and hepatocellular carcinoma. [12][13][14][15][16] However, the oncogenic functions of TROAP in astrocytoma were still unclear.
Wnt/β-Catenin signaling pathway exerted crucial roles in many aspects of cell behavior such as proliferation, stem cell maintenance, tissue homeostasis, and cell fate decisions, imbalance in the signaling properties of Wnt/β-Catenin could induce deregulated cell growth related to tumorigenesis. 17, 18 Ye et al found that knocking down TROAP could inhibit tumor progression via activating Wnt3/survivin signaling, meanwhile induces cell cycle arrest at S phase through cyclinA2/cyclinB1 caspase pathway in prostate cancer. 14 Several studies have reported that abnormal activation and mutation of Wnt pathway were linked to the initiation and progression of glioma. 19,20 Yet, the biological functions of TROAP and its detailed relationship with Wnt/β-Catenin signaling were still elusive.
In our study, we aimed to ascertain TROAP expression in glioma and provide evidence for the oncogenic role of TROAP in the malignant phenotype of glioma and the underlying potential molecular mechanisms.

| RNA extraction and quantitative real-time PCR(qRT-PCR)
RNA was harvested using the Trizol reagent (Invitrogen), according to the manufacturer's guidelines. The RNA was transcribed into cDNA by miscriptreverse transcription kit (Takara).

| Protein preparation and western blot analysis
Tissues and cells were lysed in RIPA buffer (Beyotime) containing phosphatase inhibitors and protease inhibitors. Then, cell lysates were separated by SDS-PAGE and the protein blots were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore).
Western blot analysis was performed by the following antibodies:

| Wound healing and transwell assays
For wound healing assay, cells were seeded in 6-well plates and wounds were made with yellow pipette tips, then the cell monolayers were washed twice with PBS. Images of the width of wound were photographed at 0, 12, 24, 36, and 48 h later.
For transwell assay, the experiment was performed with the 8μm-pore chamber inserted into 24-well plates, 5 × 10 5 transfected cells were placed into upper chamber coated with Matrigel (Dilution 1:7). DMEM containing 10% FBS was added to the lower chamber.
After 24 h incubation, cells that had invaded through the membrane were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet for 15 min, counted and photographed with an inverted microscope.

| Cell cycle analysis
Cells were seeded in 6-well plates at a destiny of 5 × 10 5 cells / well and cultured to 70%-80% confluence. Cells were harvested and washed twice with ice-cold PBS, then fixed with 70% cold ethanol at 4% overnight and incubated with RNase and propidium iodide (PI). Cell cycle was detected by EPICS XL Flow Cytometer (Beckman, Coulter) and Modfit software was used for data analysis.
nih.gov/geo/). The TCGA, CCLE, GTEx RNA-seq data of glioma were obtained from UCSC Cancer Browser (https://xenab rowser. net/). performed to identify differentially enriched biological pathways between the high and low TROAP groups. The expression of TROAP was used as a phenotype label. The normalized enrichment score (NES) and false discovery rate (FDR) were performed to sort enriched biological pathways in each phenotype. Moreover, other R packages including "ggpubr", "ggplot2", "limma" etc, were applied for visualizing the results of bioinformatic analysis.

| Statistical analysis
Statistical analysis was performed using Origin 2018 and Sigmaplot version 14.0 for Windows. Data were expressed as the Means ± SD.
Student's t-test was used to analyze differences between two groups. Comparisons between more than three groups were determined using one-way ANOVA analysis of variance followed by the Turkey post hoc test. The χ 2 test examines the relationship and GBM cells compared with neuro stem cells (NSCs) and normal human astrocyte (NHA), suggesting that TROAP had the potential to promote the malignant phenotype of glioma (GSE67089, Figure 1F).
Then, the results of qRT-PCR and western blot assays revealed that in comparison with control group, TROAP expression was markedly upregulated with increasing WHO grade in glioma samples (p < 0.05, Figure 1G,H). In addition, we also measured TROAP mRNA and protein level in a normal astrocyte cell line (HA1800) and 5 glioma cell lines (U251, SF295, TJ905, A172, PT2). Findings showed that TROAP expression was higher in glioma cell lines (especially U251 and SF295) compared to normal astrocyte cells (p < 0.05, Figure 1I,J).

| TROAP overexpression was associated with clinical features and poor prognosis
We reviewed the clinical characteristics of 70 patients with glioma and explore the association between TROAP expression and the clinicopathological features via immunohistochemistry assay (

Pleomorphic xanthoastrocytoma and Oligodendroglioma) and
high-grade glioma (such as Anaplastic Oligodendroglioma and Glioblastoma Multiforme) tissues (Figure 2A,B). Overexpression of TROAP protein was significantly correlated with WHO grade, Ki67 and P53mut ( Figure 2C, Table 2). To further investigate the potential role of TROAP in glioma, we performed Kaplan-Meier analysis of overall survival time (OS) in the TCGA glioma database and found that patients stratified by a media cutoff of TROAP level with lower TROAP expression had longer OS than those with higher levels of TROAP ( Figure 2D). Similarly, we also analyzed the TROAP expression profiles of patients (LGG and HGG) involved in our study for whom OS data were available, and the results were consistent with the conclusion from TCGA database, suggesting the high level of TROAP indicated poor prognosis of patients with glioma ( Figure 2E).

| TROAP knockdown inhibited glioma cell proliferation by inducing G1/S cell cycle arrest
To

| Upregulated TROAP promoted glioma cells invasion and migration in vitro
The role of TROAP in cell invasion and migration was explored by

| TROAP facilitated gliomagenesis via activating Wnt/β-Catenin signaling pathway
The molecular mechanisms in the process of TROAP-induced oncogenic phenotype were evaluated. Firstly, the results of GSEA enrichment analysis identified 23 TROAP-associated significantly enriched pathways (adj.p.value < 0.05, Figure 5A-B), including 19 activated pathways (normalized enrichment score, NES > 0) and 4 inactivated pathways (NES < 0). As shown in Figure 5C, the Wnt/ β-Catenin belonged to the activated signaling pathway. To our knowledge, Axin2 and β-Catenin were two crucial regulatory molecular involved in Wnt/β-Catenin pathway. Immunofluorescence with these evidence, silencing TROAP could induce significantly attenuated proliferation of Hela cell lines. 11 While, studies on its potential functional mechanism in glioma were elusive. In the present study, mRNA and protein expression of TROAP was significantly increased in glioma tissues and cell lines, TROAP overexpression was correlated with lower survival rates, which also proved the results of bioinformatic analysis. Therefore, we speculated that the downregulation of TROAP might be a potential strategy for treating glioma. Reorganization of cytoskeleton in the essential mechanism underlied the cell motility. TROAP has been validated as a MT-associated protein that maintained the dynamic feature of centrosome, contributing to normal structural function. 22 Canonical Wnt signaling pathway was controlled by β-Catenin, whose degradation was mediated by Axin2. 30 Hence, our immunofluorescence assay showed that TROAP overexpression resulted in the elevated Axin2 and β-Catenin level, while these genes were downregulated once TROAP silenced, suggesting that TROAP might be an upstream positive regulator in Wnt/β-Catenin signaling pathway. We hypothesized that aberrantly upregulated TROAP might prevent β-Catenin from being degraded by Axin2, leading to the substantial accumulation of β-Catenin in cytoplasm and then transportation into nucleus, where it activated the downstream target genes to activate Wnt/β-Catenin signaling, inducing tumorigenesis. [31][32][33] Furthermore, gene set enrichment analysis (GSEA) analysis uncovered similar results. To get more accurate molecular mechanism, accordingly, our study reported that TROAP overexpression increased the level of two key pathway regulatory factors, including β-Catenin and Axin2, and a series of downstream targets proteins (C-myc, CyclinD1, MMP7 and TCF4). Conversely, the phenomenon would be partly reversed by silencing Axin2 and β-Catenin.
In conclusion, our study revealed that TROAP could promote malignant phenotype of glioma cells in vitro, at least in part, activating Wnt/β-Catenin signaling pathway, thus proving the role of TROAP and underlying molecular mechanism of TROAP in gliomagenesis for the first time. We hope our findings on TROAP-induced glioma progress will provide new clues for guiding therapeutic strategies in glioma.

ACK N OWLED G EM ENT
The authors would thank all the member of the Department of Neurosurgery and the institute of Brain Science and Brain-Like Intelligence. This study was supported by the Nature Science Foundation of Shandong Province (ZR2014HM077).
F I G U R E 6 TROAP-induced malignant phenotype via activating the Wnt/β-Catenin signaling in glioma cells. (A, B) Western blot analysis showing the expression of Wnt/β-Catenin signaling-related proteins after silencing Axin2 or β-Catenin in TJ905 cells ectopically overexpressing TROAP. The statistics were shown in the histogram C and D. The effect of TROAP overexpression on cell proliferation(E-H), migration and invasion (I, J) was partly eliminated by silencing Axin2 or β-Catenin (*p < 0.05, **p < 0.01, ***p < 0.001, respectively)

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest were disclosed.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original contribution presented in the study are included in the article/supplement material. Future inquiries can be directed to the corresponding authors.