Background
Human gliomas are the most common and deadly type of primary intracranial tumors and account for approximately 80% of all primary brain malignancies. Gliomas are histopathologically classified into four tumor grades (I–IV) according to the World Health Organization (WHO). WHO grade IV or glioblastoma multiforme (GBM) confers the worst prognosis, with a median survival time of merely 12 to 15 months following primary diagnosis [
1,
2].
With advancement of gene technology, molecular signatures have become prominent in the classification of gliomas in recent years. Whole-genome analysis of patient cases through The Cancer Genome Atlas (TCGA), revealed a molecular classification scheme for GBM which includes four molecular subtypes: proneural, neural, classical, and mesenchymal. Among these four subtypes, the mesenchymal subtype was distinguished from the others as being particularly aggressive [
3‐
5]. Thus, there is an urgent need for the exploration of novel biomarkers and therapeutic targets for GBM molecularly classified as the mesenchymal subtype.
Transgelin-2 (TAGLN2) is an actin-cross-linking protein containing a calponin homolog (CH) domain, with a molecular weight of 24 kDa. It has been reported that TAGLN2 is involved in the regulation of cell transformation and cell morphology [
6,
7]. More recently, the dysregulation of TAGLN2 in a variety of malignant tumor types, including colorectal cancer [
8], bladder cancer [
9], lung cancer [
10], uterine cervical squamous cell carcinoma [
11], and breast cancer [
12], has been discovered through proteomic analysis, and thus reveals an important role for TAGLN2 in tumor progression. The expression pattern and clinical significance of TAGLN2 in human gliomas, however, have not been determined. Furthermore, it remains unknown as to whether TAGLN2, because of its role in cell transformation and cell morphology, is involved in the regulation of the epithelial-mesenchymal transition (EMT).
Here, we used publicly available datasets to determine the pattern of TAGLN2 expression in human gliomas, and its relationship with tumor grade, and other clinicopathological indicators and molecular features of gliomas. TAGLN2 function was investigated both in vitro and in vivo as well as potential pathways regulating it. Our findings indicate that TAGLN2 might be a significant prognostic indicator and a potential therapeutic target for human gliomas.
Methods
Clinical specimens and databases
Archived paraffin embedded glioma tissues (WHO grades II-IV) were collected from patients (
n = 46) who underwent surgery in the Department of Neurosurgery, Qilu Hospital of Shandong University. Normal brain tissue samples (
n = 5) were taken from trauma patients who underwent partial resection of normal brain as decompression treatment for severe head injuries. mRNA expression microarray data and accompanying clinical information for samples in The Cancer Genome Atlas Research Network (
n = 667; TCGA,
http://cancergenome.nih.gov) were used for analysis. Four external independent glioma databases (Rembrandt, CGGA, Gravendeel, and GSE4271) were also mined.
Immunohistochemistry (IHC)
Sections (4 μm) were obtained from formalin-fixed, paraffin-embedded tissues of different grades of human gliomas. Sections were boiled in sodium citrate buffer (pH 6.0) for antigen retrieval, and endogenous HRP activity was blocked with 3% H2O2. Slides were blocked with 10% normal goat serum and incubated with primary antibody (mouse anti-TAGLN2 monoclonal antibody, 1:25; Santa Cruz; Dallas, TX, USA) at 4 °C overnight. Signal was visualized using standard protocols with horse radish peroxidase conjugated secondary antibody and 3, 3′-diaminobenzidine (DAB) as the substrate. For negative controls, sections were incubated with normal mouse serum rather than primary antibody. Slides were counterstained with hematoxylin, and representative images were obtained using an Olympus inverted microscope.
Cell culture
U87MG and U251 human GBM cell lines were purchased from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). The patient-derived primary GBM cells (GBM#P3, mesenchymal subtype) were kindly provided by Professor Rolf Bjerkvig, Department of Biomedicine, University of Bergen, Norway. GBM#P3 cells were cultured in Neurobasal Medium (Thermo Fisher Scientific; Waltham, MA, USA) containing B27 supplement (20 μL/mL), FGF (20 ng/mL) and EGF (20 ng/mL). Cells were maintained at 37 °C in a humidified chamber containing 5% CO2.
Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis
Correlation analysis of
TAGLN2 was performed in gene expression profiles available in the TCGA dataset with Matlab software (
https://cn.mathworks.com). To identify biological processes and the KEGG signaling pathways associated with
TAGLN2 expression in gliomas, genes positively and negatively correlated with
TAGLN2 (
P < 0.01) were analyzed using the DAVID web tool (
http://david.abcc.ncifcrf.gov/home.jsp). Association between
TAGLN2 expression and hallmark gene sets from the Molecular Signatures Database (MSigDB) were analyzed using gene set enrichment analysis (GSEA) software (
http://software.broadinstitute.org/).
TAGLN2 silencing
Small interfering RNA (siRNA) targeting TAGLN2 were synthesized (GenePharma; Shanghai, China). siRNAs were transfected with Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific; Waltham, MA, USA) according to the manufacturer’s protocol. Stable knockdown of TAGLN2 in cells was generated using lentiviral transduction of sh-TAGLN2 (Genepharm). Knockdown efficiency was evaluated 48 h after transfection by Western blotting. siRNA sequences (n = 2) that generated efficient knockdown are the following: si-TAGLN2#1: 5′-GCAAGAACGUGAUCGGGUU-3′; and si-TAGLN2#2: 5′-UAUGUGAGCUCAUUAAUGC-3′. The second sequence of siRNA was used for the functional assays in vitro.
Western blotting
Harvested cells were lysed with heat denaturation in RIPA cell lysis buffer. Protein lysates (20 μg) were run on SDS-PAGE, and proteins were transferred to polyvinylidene difluoride (PVDF) membrane. Blots were incubated primary antibodies against TAGLN2 (Santa Cruz); N-cadherin, E-cadherin, β-catenin, Snail, Slug, Twist, p21, p27, CDK2, Survivin, c-Myc, Cyclin D1, CD44, GAPDH (Cell Signaling Technology; Danvers, MA, USA); and FoxM1, Cyclin B1, Smad, p-Smad, CHI3L1 (Abcam; Cambridge, UK). Specific proteins were detected with enhanced chemiluminescence (ECL, Millipore, Bredford, USA). Band density was measured (ImageJ software) and normalized to GAPDH.
3D tumor spheroid invasion assay
Glioma spheroids were generated by incubating cells in the spheroid formation matrix for 72 h in a 3D culture qualified 96-well spheroid formation plate. Spheroids with a diameter of >200 mm were embedded into the invasion matrix (Trevigen, Gaithersburg, USA) composed of basement membrane proteins in the 96-well plate. Glioma spheroids were photographed every 24 h under Nikon microscopy. The spheroid at 0 h was used as a reference point for measurement of the area invaded by sprouting cells.
Immunofluorescence
Transfected cells were fixed with 4% paraformaldehyde for 15 min at room temperature, rinsed with phosphate buffered saline (PBS), permeabilized with 0.4% Triton X-100 for 10 min, and blocked with 10% goat serum for 60 min at room temperature. Coverslips were incubated at 4 °C overnight with anti-N-cadherin and anti-E-cadherin antibody, followed by incubation for 1 h with an Alexa-conjugated secondary antibody. Alexa Fluor® 594 labeled phalloidin (Life Technologies, USA) was used to visualize F-actin in the cytoskeleton, and nuclei were stained with DAPI. Representative images were obtained with a Nikon inverted fluorescence microscope.
Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). U87MG or U251 (at 2 × 103 cells/well) were incubated in 96-well plates for 24, 48, and 72 h. CCK-8 solution (10 μL) was added to each well, the plates were incubated for 1 h at 37 °C, and absorbance at 450 nm wavelength (OD450) was measured in a Microplate Reader (Bio-Rad). For the EdU assay, cells were incubated with 200 μL of 5-ethynyl-20-deoxyuridine (Ribo-Bio; Guangzhou, China) for 2 h at 37 °C. Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.4% Triton X-100 for 10 min, and incubated with Apollo® reagent (100 μL) for 30 min. Nuclei were stained with DAPI, and representative images obtained with a Nikon inverted fluorescence microscope.
Flow cytometry
Cell cycle analysis was performed by determining DNA content with propidium iodide (PI) staining (BD Biosciences; San Jose, CA, USA). Briefly, U87MG and U251 glioma cells were harvested, re-suspended and stained with propidium iodide (PI; BD Biosciences) in the presence of RNase A for 20 min. Apoptosis was evaluated in U87MG and U251 cells with Annexin V-FITC and PI staining (20 min; BD Biosciences). Cells were analyzed using a flow cytometer (BD Biosciences) according to the manufacturer’s instructions.
Implantations in nude mice
To establish intracranial gliomas, U87MG and U251 cells (1 × 106) were infected with Lenti-si-TAGLN2 or Lenti-Control virus and then implanted stereotactically into the brain of 4-week-old nude mice (SLAC laboratory animal Center; Shanghai, China). Tumor tissues were harvested, formalin-fixed and paraffin-embedded, sectioned (4 μm) and incubated with antibodies against TAGLN2 (Santa Cruz, USA), N-cadherin and Ki-67 (Abcam, UK).
Statistical analysis
Survival curves were estimated by the Kaplan-Meier method and compared using the log-rank test. The cut-off level was set at the median value of TAGLN2 expression levels. The expression pattern of TAGLN2 in different glioma subtypes and the associations of TAGLN2 with isocitrate dehydrogenase 1 (IDH1) mutation, methylation of O-methylguanine-DNA methyltransferase (MGMT) promoter, codeletion of 1p/19q, telomerase reverse transcriptase (TERT) loss, and alpha thalassemia/mental retardation syndrome X-linked (ATRX) mutation were performed using the TCGA dataset. A two-tailed χ2 test was used to determine the association between TAGLN2 expression and clinicopathological characteristics. Pearson correlation was used to evaluate the linear relationship between the expression of different genes. The one-way ANOVA test or Student’s t test were used for all other data comparisons using GraphPad Prism 6 software. All data are presented as the mean ± standard error. All tests were two-sided, and P-values <0.05 were considered to be statistically significant.
Discussion
Human gliomas have been well characterized molecularly. Yet the significance of many of the changes in gene expression have not been attributed to any underlying biological function. Here, we investigated the function of TAGLN2, a gene that was found to be highly expressed in GBMs compared to LGGs. High TAGLN2 expression was associated with the mesenchymal molecular phenotype in human gliomas and thus poor prognosis in glioma patients. In contrast, low TAGLN2 mRNA levels were linked to other positive prognostic markers, including IDH1 and ATRX mutations, methylated MGMT, 1p/19q codeletion, and loss of TERT. Finally, functional studies using RNA knockdown implicated a role for TAGLN2 in promoting cell invasion and proliferation in human gliomas.
Glioma progression is a dynamic process in which EMT is a key event driving invasion of tumor cells [
26,
27]. Several EMT-related factors have been previously associated with increased invasion and poor prognosis in gliomas [
19]. Here, we observed that TAGLN2 depletion significantly decreased glioma cell invasiveness and reversed EMT features, including changes in epithelial (E-cadherin) and mesenchymal markers (N-cadherin, Snail, Slug, Twist) in glioma cells. Using immunofluorescence staining, we observed F-actin cytoskeletal changes induced by TAGLN2 depletion in gliomas.
TAGLN2 silencing appeared to specifically suppress the F-actin-rich leading edge in glioma cells, thus reducing the formation of invadopodia during cell invasion. According to the study by Na et al., TAGLN2 blocks actin depolymerization and competes with cofilin to protect F-actin during the formation of immunological synapse in T cells, and knockout of TAGLN2 significantly destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading [
28,
29]. Therefore, we hypothesize that TAGLN2 may promote invadopodia formation of glioma cells via competing with cofilin and suppressing actin depolymerization. Altogether, these results indicated that TAGLN2 may serve as a crucial regulator of invasion and aggressiveness by inducing mesenchymal-like properties in gliomas.
Abnormal cell proliferation and growth are hallmark characteristics of human gliomas. Many genetic changes lead to uncontrolled growth through dysregulation of proteins directly involved in cell cycle progression and cell apoptosis [
30]. GO and GSEA analysis indicated that TAGLN2 might indeed promote growth through functions in cell cycle progression and cell survival. In vitro and in vivo experiments supported this analysis.
TAGLN2 knockdown in glioma cells induced cell cycle arrest at G0-G1 and cell apoptosis, and reduced growth in orthotopic xenografts. The fact that knockdown of
TAGLN2 leads to reduced cell proliferation in vitro and in vivo renders the gene/pathway as a potential molecular target for therapy. Moreover, to uncover the potential molecular mechanisms by which TAGLN2 promote glioma development, we detect the expression change of FoxM1, an oncogenic transcriptional factor that regulates some key mediators of cell cycle progression, including CDK2, cyclin B1, cyclin D1, p21 and p27 [
20]. Meanwhile, recent studies have revealed that the oncogenic potential of FoxM1 is determined by its capacity to transactivate target genes that are implicated in different phases of cancer development [
31], including cancer survival, invasiveness, EMT process, and angiogenesis. In addition, a variety of oncogenic genes including Survivin, c-Myc, β-catenin, and Snail were identified to be regulated by FoxM1. In the present study,
TAGLN2 knockdown led to significantly reduced levels of FoxM1, as well as downstream oncogenic factors including CDK2, cyclin B1, and cyclin D1, c-Myc and Survivin. Meanwhile, tumor suppressor p21 and p27 were induced after
TAGLN2 depletion. These data suggest that TAGLN2 activates FoxM1 signaling during gliomagenesis. However, further investigation is necessary to elucidate the mechanism of TAGLN2 regulation of FoxM1 axis in glioma.
Consistent with our current study, TAGLN2 has been reported to be up-regulated and possess proto-oncogenic functions in a variety of cancers. For example, high expression of TAGLN2 in tumor-derived lung cancer endothelial cells was associated with clinical stage, tumor size, and tumor development in lung cancer tissues [
10]. In addition, TAGLN2 overexpression has been reported to be correlated to lymph node metastasis and histological neural invasion of bladder [
32], colorectal [
8], esophageal [
33], and gastric [
34] cancer. Similarly, the elevated expression of TAGLN2 was observed in uterine cervical squamous cell carcinoma (SCC), while suppression of TAGLN2 in human uterine SCC cells significantly inhibited tumor growth and invasion [
35]. However, in gynecological malignancies, decreased expression of TAGLN2 was found in metastatic cells in comparison to primary tumors [
36]. Meanwhile, TAGLN2 was suggested to be negatively correlated with breast cancer metastasis, and metastatic breast cancer cell line exhibited down-regulation of TAGLN2 protein [
37]. These contradictory results reveal a complex role of TAGLN2 in tumorigenesis, urging further investigation.
Based on its potentially crucial role in promoting glioma development, we were also interested in identifying upstream regulators of TAGLN2 expression. It is well known that TGFβ is a multifunctional cytokine that modulates biological processes, such as cell stemness, angiogenesis, cell growth, and immune function [
38,
39]. In addition, studies have shown that TGFβ2 overexpression promotes motility, invasion and EMT in cells from some cancer types [
40], thus indicating a role for the TGFβ2/Smad signaling pathway in tumorigenesis. In our study, we observed a strong correlation between
TGFβ2 and
TAGLN2 mRNA levels in cases from TCGA and CGGA databases, and in cell culture, TGFβ2 induced TAGLN2 protein in U87MG and U251 cells. The TGFβ type I receptor specific inhibitor SB431542 prevented TAGLN2 induction, and therefore further supported a role for TGFβ2/Smad signaling in the regulation of TAGLN2 in glioma. However, the precise molecular mechanisms of the cross-talk between TAGLN2 and TGFβ2 signaling in gliomas require further investigation.