Back to Journals » OncoTargets and Therapy » Volume 13

Coronin 3 Promotes the Development of Oncogenic Properties in Glioma Through the Wnt/β-Catenin Signaling Pathway

Authors Wang M, Li Q, Yu S, Zhang Z, Qiu P, Zhang Y, Yang W, Xu G, Xu T

Received 16 April 2020

Accepted for publication 25 June 2020

Published 7 July 2020 Volume 2020:13 Pages 6661—6673


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Yong Teng

Min Wang,1 Qi Li,1 Shengyuan Yu,1 Zexiang Zhang,2 Peng Qiu,1 Yubao Zhang,1 Wei Yang,1 Guangming Xu,1 Tongjiang Xu1

1Department of Neurosurgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250014, People’s Republic of China; 2Department of Gastrointestinal Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250014, People’s Republic of China

Correspondence: Tongjiang Xu
Department of Neurosurgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250014, People’s Republic of China
Tel +86-531-87938911

Purpose: Evidence indicates that the actin-binding protein Coronin 3, which is aberrantly expressed in various cancers, is associated with cancer development and progression. However, little is known about the role of Coronin 3 in glioma tumorigenesis. Here, we aimed to explore the biological function and regulatory mechanism of Coronin 3 in glioblastoma (GBM).
Materials and Methods: Coronin 3 level in human GBM clinical samples and cell lines was investigated. The shRNA knockdown strategy was used to assess the tumor characteristics of GBM cell lines. The role of β-catenin in Coronin 3-mediated oncogenic phenotypes was evaluated.
Results: Coronin 3 was found to be highly upregulated in glioma cell lines. Furthermore, knockdown of Coronin 3 significantly inhibited the growth of glioma cells both in vivo and in vitro and suppressed the expression of Wnt/β-catenin pathway genes, including β-catenin, Cyclin D1, and c-Myc. Moreover, we demonstrated that Coronin 3 regulates the expression of β-catenin in glioma. Our results revealed that Coronin 3-stimulated tumor growth was β-catenin-dependent.
Conclusion: Our study reveals a new molecular mechanism of Coronin 3 in promoting glioma growth and development through regulating the Wnt/β-catenin signaling pathway.

Keywords: glioblastoma, Coronin 3, proliferation, oncogenic property, Wnt/β-catenin


Glioblastoma (GBM), the most aggressive form of malignant glioma, shows poor prognosis and a median survival time of fewer than 15 months.1 In contrast to other solid tumors, GBM is characterized by diffuse infiltrative growth around the normal brain parenchyma.2 The biological behavior of gliomas is determined by the rate of GBM cell proliferation, invasion, and tumor migration.3 However, the mechanisms involved in these processes remain elusive. In an effort to develop potential effective targeted therapies, recent studies have focused on understanding the molecular pathogenesis underlying glioma formation and progression.

Coronin is an actin-binding protein, and the proteins belonging to the coronin family differ both in terms of structure and function.4 Functional studies have shown that it can regulate multiple cellular responses.5 Coronin 3 (Coronin 1C, CRN2) is a member of the coronin protein family that is expressed ubiquitously—localized to lamellipodia—which reportedly interacts with the actin-related protein 2/3 complex.6,7 In recent years, aberrant expression of Coronin 3 has been identified to be responsible for the metastatic behavior of many malignancies, such as head and neck squamous cell carcinoma,8 breast cancer,9 and hepatocellular carcinoma.10 Breast cancer patients and cell lines exhibited increased Coronin 3 expression, which was associated with cancer cell invasion and increased metastatic risk.11 Upregulation of Coronin-1C enhanced the migration of human HCC cells, whereas suppressed cell migration and proliferation were observed in Coronin-1C-knockdown cells.12 Although only a few studies on Coronin 3 alteration in gliomas have been reported, Coronin 3 overexpression detected in glioma samples correlated with the degree of malignancy.13 However, the oncogenic properties of Coronin 3 and its molecular mechanisms in glioma cells have not been fully understood.

Wnt/β-catenin signaling pathway has been reported to regulate many aspects of cell behavior, such as cell-fate decision, cell migration, and cell differentiation.14,15 Upon stabilization, active β-catenin accumulates in the cytosol and is translocated to the nucleus, where it forms a transcription complex with T cell factor (TCF)/lymphoid enhancer factor (LEF) to activate the transcription of downstream target genes.16 Wnt/β-catenin has been reported to promote GBM cell migration and metastasis,17 yet the detailed mechanism of how Coronin 3 activates the oncogenic signaling pathway remains unknown. Therefore, we aimed to ascertain Coronin-3 expression in gliomas and to provide substantial evidence for the role of Coronin-3 in the growth and invasion of glioma cells and the underlying molecular mechanisms. The results of this study are expected to contribute to the understanding of the functions of Coronin-3 in GBM tumorigenesis.

Materials and Methods

Human Tissue Specimens and Cell Lines

The GBM tissues and specimens used in this study were obtained by surgical resection from 23 patients at the Neurosurgery Department of Shandong Provincial Hospital from 2017 to 2019, and the study was approved by the Shandong Provincial Hospital Ethics Committee. Histopathological diagnoses and grading of the glioma samples were independently verified by two neuropathologists, based on WHO classification of tumors (2007). None of the patients had received chemotherapy, immunotherapy, or radiotherapy prior to specimen collection. There were five cases of Grade I, seven cases of Grade II, six cases of Grade III, and five cases of Grade IV glioma specimens. Five samples of non-tumor brain tissue were removed during intracranial decompression surgery. The samples were immediately frozen and stored at –80 °C until analysis. The normal human astrocyte (NHA) cell and human glioblastoma cell lines (LN-18, A-172, U87, SNB19, LN-229, and U251MG) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, USA) and 100 U/mL penicillin (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37 °C in an atmosphere containing 5% CO2.

Immunohistochemistry (IHC)

IHC was performed on polyformalin-fixed and paraffin-embedded glioma tissues and healthy brain tissues. Tissue sections were incubated with primary antibodies against Coronin 3 (ab1571; 1:200; Abcam), β-catenin (9562S; 1:300; CST), and Ki67 (ab15580; 1:200; Abcam) at 4 °C overnight, followed by incubation with an HRP-conjugated secondary antibody. Diaminobenzidine was used for color reactions and images were acquired using a bright field microscope (Leica, Germany).

Western Blotting

Total protein from tissues and cells was extracted using RIPA buffer containing 1% phenylmethylsulphonyl fluoride. Protein concentration was determined before loading using a BCA protein assay kit (Beyotime Bio Corp, China). Equal amounts of protein (20 μg) were loaded onto a 12% SDS polyacrylamide gel. The membrane was blocked using 5% bovine serum albumin (BSA) and probed with primary antibodies against Coronin 3 (ab1571; 1:200; Abcam, MA, USA) overnight at 4 °C and then incubated with an HRP-conjugated secondary antibody at 25 °C for 1 h. Finally, protein bands were visualized using the ECL detecting system (Applygen, Beijing). The integrated density values were normalized against those of GAPDH.

Plasmid Construction and Transfection

Scrambled negative control (SC) or Coronin 3 shRNA1/2 were purchased from Addgene (Beijing, China) and cloned into the pLKO.1 vector. The plasmid construct encoding human Coronin 3, was prepared by PCR amplification and subsequently subcloned into the pCDH-CMV-MCS-EF1-copGFP construct designed by Addgene (Beijing, China). 293FT cells were transfected with shRNA plasmids or overexpression plasmids. Lipofectamine 2000 (Life Technologies, USA) was used for all transfections according to the manufacturer’s instructions. Forty-eight hours after transfection, the culture supernatant was collected and used to infect GBM cells. Transfections were confirmed by Western blotting before various experiments.

Cell Growth Curve Analysis

Cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, 2 × 106 cells were seeded in each well of a 96-well plate and were cultured overnight. The CCK-8 reagent was added at 24, 48, 72, 96, or 120 h and incubated at 37 °C for 1 h. Each assay was independently repeated three times in triplicate.


The cells were fixed with 4% paraformaldehyde at 37 °C for 1 h, permeabilized with 0.5% Triton X-100 for 10 min, and blocked in 5% BSA for 1 h at 25 °C. Then, the cells were probed with 5% BSA containing primary antibody anti-β-catenin (MAB8814, CST) overnight at 4 °C, followed by incubation with 488-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA, USA) for 1 h at room temperature in the dark. After washing with 0.1M PBS for three times, the cells were stained with DAPI (H-1200,Vector laboratories). Then, cells were mounted on glass slides, and the fluorescence was visualized by laser scanning confocal microscopy (Olympus, Tokyo, Japan).

EdU Staining

Cells were seeded in 96-well plate and incubated with 50 μM of 5-ethynyl-2′-deoxyuridine (EdU, RiboBio; R11053), according to the manufacturer’s instructions. Subsequently, the DNA content was stained with DAPI and visualized under a laser scanning confocal microscope (Olympus, Tokyo, Japan).

Colony Formation Assay

Cells were seeded in 6-well plates (1 × 103 cells/well) and cultured in DMEM with 10% FBS. After 6 days, the cells were washed with phosphate-buffered saline, fixed with methanol, stained with 1% crystal violet, and observed under a microscopy (Olympus, Tokyo, Japan).

Wound Scratch Assay

Cells were seeded in a 6-well plate, and after reaching 90% confluence, the cell monolayer was scraped with a pipette tip, and re-incubated in with serum-free medium. Images were acquired at 0 h and 24 h after wounding using an inverted microscopy (Olympus, Tokyo, Japan) to assess wound closure.

Cell Migration and Invasion Assay

Cell migration and invasion were detected using the Transwell assay (Beyotime, Shanghai, China). Cells were seeded on uncoated (for migration assays) or Matrigel-coated (for invasion assays) upper chambers (BD Bioscience, New Jersey, USA). For the migration assay, the lower chamber was filled with a medium containing 10% FBS. For the invasion assay, cells were seeded in a serum-free medium in the upper chamber, which was pre-coated with Matrigel. Next, the lower chamber was filled with the complete medium. After 24 h of incubation at 37 °C, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Eight random visual fields were counted per chamber using an inverted microscope (Olympus, Tokyo, Japan).

T-Cell Factor (TCF)-Responsive Luciferase Reporter Assay

The effect of Coronin 3 on Wnt/β-catenin signaling was detected using a pair of luciferase reporter constructs, as previously described.18 Briefly, TOPflash and FOPflash (Millipore) were used; TOPflash contains 3 TCF/LEF sites—upstream of a thymidine kinase promoter—and a firefly luciferase gene, while the FOPflash construct contains 3 mutated TCF/LEF sites, and is used as a control for measuring nonspecific reporter activation (Promega, Madison, WI). Luciferase activity was measured using a dual-luciferase assay system kit (Promega), according to the manufacturer’s protocol, with the Renilla reniformis (sea pansy) luciferase activity as an internal control. Relative luciferase activity was expressed as normalized-fold change to controls.

Glioma Xenograft Mouse Models

Approximately 5 × 105 active U87 cells infected with lentiviruses containing shRNA or empty vector were resuspended in 200 μL PBS and subcutaneously injected into groups of seven nude mice. After 35 days of injection, the mice were euthanized, and xenografts were excised, weighed, and photographed.

Hematoxylin and Eosin Staining

The xenografts were cut in small pieces to ensure thorough fixation. Next, the tumors were fixed in 4% paraformaldehyde for 24 h, stored in 70% ethanol overnight, and embedded in paraffin. Next, 5-μm-thick sections were prepared with a microtome (Leica, Germany) and deparaffinized with 100% xylene, and rehydrated using descending ethanol series. Finally, the sections were stained with hematoxylin and eosin (Sangon Biotech).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism version 7.0 for Windows (San Diego, CA, USA). The data are presented as the mean ± SD or mean ± SEM. The Student’s t-test was used to compare the test and control groups, and comparisons between the two groups were determined by the least significant difference using the one-way analysis of variance method. P < 0.05 indicated a significant difference.


Coronin 3 is Highly Expressed in GBM and Human GBM Cell Lines

Human GBM clinical samples—including glioma tissues (Grade I–IV) and non-tumor healthy brain tissues—were collected to examine Coronin 3 expression by IHC. The results confirmed that Coronin 3 expression was higher in the GBM tissues than in healthy tissues, and that its expression increased with the GBM histological grade (Figure 1A). Concordantly, Western blotting revealed that the expression of Coronin 3 was higher in human GBM tissues (n=4) than that in the paired adjacent non-tumor tissues (Figure 1B). We further investigated the level of Coronin 3 in different subtypes of GBM cell lines. Western blotting revealed that Coronin 3 expression was markedly upregulated in all the tested GBM cell lines compared with that in primary NHA (Figure 1C). Collectively, these results indicate that Coronin 3 is upregulated in GBM.

Figure 1 Coronin 3 is overexpressed in glioblastoma (GBM) cells. (A) Immunohistochemistry for Coronin 3 in GBM (WHO Grade I–IV) and non-tumor (normal) tissues. Scale bar represents 50 μm. (B) Western blotting for Coronin 3 in four matched GBM tissues (T) and adjacent normal brain tissues (N) (n = 8). Coronin 3 expression is normalized to that of GAPDH. ***P < 0.001. (C) Western blotting for Coronin 3 in GBM cell lines compared with that in normal human astrocytes (NHA) (n = 6). Coronin 3 expression is normalized to that of GAPDH. ***P < 0.001 vs NHA. (D) Western blotting for evaluating the efficiency of Coronin 3 knockdown in U87 and LN-229 cells (n = 6). Coronin 3 expression is normalized to that of GAPDH. ***P < 0.001 vs SC-shRNA. One-way ANOVA was used to analyze the data, which are shown as the mean ± SEM.

Knockdown of Coronin 3 in GBM Cells Results in Decreased Cell Proliferation

To further assess the role of Coronin 3 in GBM cells, U87 and LN-229 cell lines with stable shRNA knockdown of Coronin 3 were generated. The transfection efficacy of Coronin 3-shRNA constructs was verified by Western blotting (Figure 1D). CCK-8 assay was used to examine the effect of Coronin 3 knockdown on the proliferative ability of GBM cells. The results revealed that the growth rate of U87 and LN-229 cells transfected with Coronin 3-shRNA was significantly decreased compared with that of the scrambled control (Figure 2A). The EdU incorporation assay was performed to examine the effect of Coronin 3 knockdown on DNA replication, as a more specific evaluation of proliferation. U87 and LN-229 cells transfected with Coronin 3-shRNA revealed a significantly reduced number of EdU-positive cells compared with that in cells transfected with the scrambled control (Figure 2B and C). Concordantly, colony formation assay showed that knockdown of Coronin 3 could suppress the proliferation of U87 and LN-229 cells (Figure 2D).

Figure 2 Knockdown of Coronin 3 inhibits the proliferation of glioblastoma (GBM) cells in vitro. (A) Effect of Coronin 3 knockdown on cell proliferation at different time points in U87 and LN-229 cells (n = 7). ***P < 0.001 vs SC-shRNA. (B) Cell proliferation analyzed by EdU incorporation in U87 and LN-229 cells transfected with SC-shRNA and Coronin 3-shRNA. Scale bar represents 50 μm. (C) Percentage of EdU-positive cells (n = 16 vision fields). **P < 0.01, ***P < 0.001 vs SC-shRNA. (D) Effect of Coronin 3 knockdown on colony formation by cells (n = 18 vision fields). ***P < 0.001 vs SC-shRNA. One-way ANOVA was used to analyze the data, which are shown as the mean ± SD.

Knockdown of Coronin 3 Inhibits GBM Cell Invasion and Migration in vitro

To further investigate the effect of Coronin 3 on GBM cell invasion and migration, we employed a wound healing assay and a Transwell invasion assay. The wound area was much smaller in SC-shRNA-transfected U87 and LN-229 cells compared with that in Coronin 3-shRNA-transfected cells 24 h after wounding, indicating that Coronin 3 promoted GBM cell migration (Figure 3A). Moreover, the downregulation of Coronin 3 inhibited the migration and invasion of U87 and LN-229 cells, as determined by the Transwell assay (Figure 3BD). These results suggest that the downregulation of Coronin 3 inhibits the invasion and migration of GBM cells.

Figure 3 Coronin 3 knockdown suppresses glioblastoma (GBM) cell migration and invasion in vitro. (A) Wound healing assay was used to evaluate the motility of GBM cell lines. The representative images of migration were acquired 24 h after wounding. Scale bar represents 100 μm (n = 15 vision fields). (B) Transwell cell migration and invasion assays were used to investigate the effect of Coronin 3 knockdown on GBM cell migration and invasion. Scale bar represents 50 μm. (C and D) Quantification of U87 and LN-229 cells showing migration and invasion (n = 12 vision fields). ***P < 0.001 vs SC-shRNA. One-way ANOVA was used to analyze the data, which shown as the mean ± SD.

The Signaling Basis for Coronin 3 Function in Tumorigenesis

Next, we investigated the molecular mechanism underlying the tumor-promotive role of Coronin 3. Upon downregulation of Coronin 3, the protein levels of MMP-9, integrin β1, cyclin D1, c-Myc, and β-catenin, which are the key signaling intermediaries of the canonical Wnt pathway, were significantly decreased (Figure 4AF). These changes were corroborated by a marked decrease in the immunofluorescence of β-catenin in Coronin 3-shRNA-transfected U87 and LN-229 cells compared to that in SC-shRNA-transfected cells (Figure 4G).

Figure 4 Wnt/β-catenin pathway was downregulated in Coronin 3-knocked down glioblastoma (GBM) cells. (A) Representative Western blots of MMP-9, integrin β1, cyclin D1, c-Myc, and β–catenin. (BF) Quantification of MMP-9, integrin β1, cyclin D1, c-Myc, and β-catenin expression, where expression is normalized to that of GAPDH (n = 6). ***P < 0.001 vs SC-shRNA. (G) Immunofluorescence and fluorescent signal intensity analysis for β-catenin in U87 and LN-229 cells. Nuclei were labeled by DAPI. ***P < 0.001 vs SC-shRNA. Scale bar represents 100 μm. One-way ANOVA was used to analyze the data, which are shown as the mean ± SD.

Coronin 3 Regulates the Expression of β-Catenin

To thoroughly test the relationship between Coronin 3 and β-catenin, we obtained U87 and LN-229 cells stably expressing pcDNA-Coronin 3. We observed that the overexpression of Coronin 3 remarkably increased the expression of β-catenin protein, compared with that in the control cells (Figure 5A and B). Furthermore, the effect of Coronin 3 on Wnt/β-catenin signaling was detected using a pair of luciferase reporters containing TCF sites. Dual-luciferase reporter assay also revealed that Coronin 3 overexpression significantly promoted the activity of firefly luciferase that carried wild-type but not mutant TCF binding sites in U87 as well as LN-229 cells (Figure 5C and D). These findings indicated that Coronin 3 drives the expression of β-catenin.

Figure 5 Coronin 3 interacts directly with β-catenin. (A and B) Coronin 3 overexpression markedly enhanced the and protein level of β-catenin in U87 and LN-229 cells. GAPDH served as an internal control (n = 7). (C and D) U87, and LN-229 cells were transfected with firefly luciferase reporter plasmids containing either wild-type or mutant TCF binding sites. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity (n = 8). **P < 0.01 vs pc-DNA-Con, ***P < 0.001 vs pc-DNA-Con. The t-test was used to analyze the data, which are shown as the mean ± SD.

Coronin 3 Exerts Its Oncogenic Effect by Promoting the Wnt/β-Catenin Signaling Pathway

The role of β-catenin in the development of Coronin 3-induced oncogenic phenotypes was evaluated. First, rescue experiments with an activator of the Wnt/β-catenin pathway—lithium chloride (LiCl)19—were performed to verify the involvement of the Wnt/β-catenin signal in Coronin 3-induced oncogenic phenotypes. The results showed that the reduced growth rate of Coronin 3-shRNA1-transfected U87 cells was reversed after LiCl treatment (Figure 6A). Besides, the metastatic ability of U87 cells was enhanced in response to LiCl treatment, as evidenced by the results of the Transwell assay (Figure 6B). Next, we examined whether blocking the activity of β-catenin with the β-catenin inhibitor IWP-2,20 could result in the attenuation of the effect of Coronin 3 upregulation. As expected, Coronin 3 overexpression promoted the proliferation, migration, and invasion of U87 cells. However, treatment with the β-catenin inhibitor IWP-2 significantly attenuated the promotive effect of Coronin 3 overexpression on the proliferation, migration, and invasion of U87 cells (Figure 6C and D). Taken together, our findings suggest that Coronin 3 promotes the development of oncogenic phenotypes in GBM cells, partly via promoting Wnt/β-catenin signaling.

Figure 6 Wnt/β-catenin signaling is involved in mediating Coronin 3-induced promotion of oncogenic properties. (A) Treatment with LiCl (30 mM) reversed the anti-proliferation effect of Coronin 3-shRNA. ***P < 0.001 vs SC-shRNA. ##P < 0.01 vs Coronin 3-shRNA (n = 8). (B) Treatment with LiCl reversed the anti-migration and anti-invasion effect of Coronin 3-shRNA. ***P < 0.001 vs SC-shRNA. ###P < 0.001 vs Coronin 3-shRNA (n = 16 vision fields). (C) Treatment with β-catenin inhibitor IWP-2 (1 μM) attenuated the promotive effect of pcDNA-Coronin 3 on proliferation. ***P < 0.001 vs pcDNA-Con. ##P < 0.01 vs pcDNA-Coronin 3 (n = 8). (D) Treatment with β-catenin inhibitor IWP-2 attenuated the pro-migration and pro-invasion effect of pcDNA-Coronin 3. ***P < 0.001 vs pcDNA-Con. ###P < 0.001 vs pcDNA-Coronin 3 (n = 16 vision fields). One-way ANOVA was used to analyze the data, which are shown as the mean ± SD.

Knockdown of Coronin 3 Inhibits GBM Growth in vivo

To explore the effects of Coronin 3 on tumor growth in vivo, U87 cells stably transfected with Coronin 3-shRNA or SC-shRNA were subcutaneously injected into nude mice to establish xenograft models. The tumors formed in mice transfected with Coronin 3-shRNA grew significantly slower than those in SC-shRNA-transfected mice, indicating that Coronin 3 knockdown inhibited tumor growth (Figure 7A and B). We then performed HE and Ki67 staining on tumors from each group of mice with xenograft tumors. H&E staining revealed decreased mitosis in tumors from mice injected with cells stably expressing Coronin 3-shRNA (Figure 7C). Ki67 expression was significantly weaker in tumors derived from mice injected with cells stably expressing Coronin 3-shRNA (Figure 7C). These results are consistent with those in vitro, which firmly validated the oncogenic role of Coronin 3 in the tumorigenesis of GBM. To further investigate whether the Wnt/β-catenin pathway plays a vital role in the Coronin 3-induced tumor growth in vivo, we performed IHC, and found that the downregulation of Coronin 3 reduced the expression of β-catenin (Figure 7C and D). A summary figure illustrates the key findings of the study (Figure 7E).

Figure 7 Knockdown of Coronin 3 suppressed glioma growth in vivo. U87 cells transfected with Coronin 3-shRNA or SC-shRNA were injected into nude mice (n = 8 per group). (A) Tumors from the xenograft were assessed for volume after 35 days of injection. (B) Images of the dissected tumors. (C) Representative images of H&E staining and immunohistochemistry of Ki-67 and β-catenin are shown. Scale bar represents 100 μm. (D) Semi-quantitative analysis of β-catenin positive rate (n = 12 sections from 3 mice). (E) A summary figure to illustrate key findings of the study. ***P < 0.001 vs SC-shRNA. One-way ANOVA was used to analyze the data, which are shown as the mean ± SD.


Coronin 3 displays highly co-operative binding to actin filaments; it is a transcriptionally dynamic gene whose expression is known to be aberrantly upregulated in multiple types of clinically aggressive cancer.21 Its expression was reportedly increased in diffuse hepatocellular carcinoma and was known to be correlated with the degree of tumor malignancy.12,22 Coronin 3 is also known to be highly expressed in gastric cancer tissue and to be positively correlated with tumor metastasis.23 Concordant with these reports, Coronin 3 knockdown is known to result in significantly attenuated migration of HeLa and HEK 293 cells.24 However, there are very few studies on the deregulation of Coronin 3 in gliomas, and its functional mechanism in GBM is elusive. Thal et al first reported that the expression of Coronin 3 in glioma tissues was significantly higher than that in healthy brain tissues, and that it was related to malignancy.13 Ziemann et al further proved that Coronin 3 promotes GBM progression via modulation of the actin cytoskeleton.25 However, there are other plausible mechanisms to explain the GBM growth observed in this study, and these should therefore be targeted in the future to develop novel therapeutic approaches.

In our study, we focused on the regulatory mechanisms of Coronin 3 in GBM. Our results showed that Coronin 3 was markedly overexpressed in malignant gliomas, and that its expression level were positively correlated to tumor grade, which confirmed that Coronin 3 might be associated with the development and progression of gliomas; this suggests that Coronin 3 functions as an oncogene. Western blotting demonstrated that the expression of Coronin 3—in the tumor tissue as well as GBM cell lines—was increased, which indicates that the expression of Coronin 3 is correlated with the growth and invasiveness of tumor cells. Therefore, we hypothesized that the downregulation of Coronin 3 might serve as a potential strategy for glioma treatment.

Reorganization of the actin cytoskeleton is the primary mechanism underlying the motility of cells and is essential for most types of cell migration.26 Coronin 3 has been reported to be actively involved in mediating cell migration and progression.27 In the present study, Coronin 3 knockdown using shRNA transfection resulted in significantly reduced Coronin 3 expression in glioma cells, as indicated by Western blotting. In vitro, Coronin 3 downregulation resulted in significantly inhibited proliferation, migration, and invasion, which suggested that Coronin 3 expression was associated with the metastatic potential of glioma. Next, we demonstrated that the knockdown of Coronin 3 resulted in the downregulation of the Wnt/β-catenin signaling pathway by reducing the expression of the pathway intermediaries at the protein level, suggesting that Wnt/β-catenin pathway is regulated by Coronin 3 in gliomas. Concordantly, the dual-luciferase reporter assay revealed β-catenin to be a direct target of Coronin 3, which indicated that Coronin 3 knockdown inhibits the Wnt/β-catenin signal pathway via β-catenin.

Wnt/β-catenin signaling pathway is a major pathway that is activated during the carcinogenesis of glioma.28 This pathway contributes to GBM pathology at multiple levels, including tumor initiation, maintenance of stem-cell status, invasion, and angiogenesis.29,30 Although GBMs do not harbor genetic alterations in the components of the Wnt/β-catenin pathway, aberrant activation of Wnt signaling appears to be achieved mainly via epigenetic silencing of the negative regulators of the Wnt/β-catenin pathway and overexpression of the positive regulators.31 Coronin 3 has been shown to promote the development of neuroblastomas, but the mechanism in gliomas has not been elucidated fully. Therefore, we speculated that Coronin 3 played a role in the development of glioma by regulating the Wnt/β-catenin signaling pathway. This study identified Coronin 3 as a positive regulator of the Wnt/β‐catenin signaling pathway in GBM. We confirmed our hypothesis by observing the tumor phenotypes in response to LiCl-induced activation of the Wnt signaling pathway in Coronin 3-shRNA-transfected GBM cells, and IWP-2-induced inhibition of the Wnt signaling in pc-DNA-Coronin 3-transfected GBM cells. Several actin-binding proteins have been reported to regulate the actin dynamics around the E-cadherin–β-catenin complex.32,33 As a conserved actin-binding protein that governs the cellular actin dynamics, Coronin 3 may also be modulated by β-catenin to regulate actin filament assembly and disassembly. At the cell-cell contacts, β-catenin also links the cytoplasmic domain of cadherin-type adhesion receptors to Coronin 3,34 which allows the cells to interact via robust intercellular adhesion junctions. These potential interaction mechanisms between Coronin 3 and β‐catenin should be further investigated.


In summary, our study suggests that Coronin 3 expression might serve as a significant independent prognostic factor of high-grade glioma. Further, Coronin 3 promotes glioma cell proliferation, invasion and migration, thereby leading to the activation of the Wnt/β-catenin signal pathway and promoting the development of glioma phenotypes in vitro and in vivo. Our results suggest that Coronin 3 plays an important role in gliomagenesis and may serve as a potential target for glioma therapy.


GBM, glioblastoma; TCF, T cell factor; LEF¸ lymphoid enhancer factor.

Data Sharing Statement

We declare that materials described in the manuscript, including all relevant raw data, will be freely available upon reasonable request.


The authors declare that they have no competing interests.


1. Ferguson SD, Lariviere MJ, Mansour N, Lesniak MS. Malignant glioma: chemovirotherapy. Tumors Cent Nerv Syst. 2011;1:357–364.

2. D’Alessandro G, Catalano M, Sciaccaluga M, Chece G, Limatola C. KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo. Cell Death Dis. 2013;4:e773. doi:10.1038/cddis.2013.279

3. Cheng YC, Tsai WC, Sung YC, Chang HH, Chen Y. Interference with PSMB4 expression exerts an anti-tumor effect by decreasing the invasion and proliferation of human glioblastoma cells. Cell Physiol Biochem. 2018;45:819–831. doi:10.1159/000487174

4. Roadcap DW, Clemen CS, Bear JE. The role of mammalian coronins in development and disease. Subcell Biochem. 2008;48:124–135.

5. Clemen CS, Rybakin V, Eichinger L. The coronin family of proteins. Sub Cell Biochem. 2008;48:1–5.

6. Rosentreter A, Hofmann A, Xavier CP, Stumpf M, Noegel AA, Clemen CS. Coronin 3 involvement in F-actin-dependent processes at the cell cortex. Exp Cell Res. 2007;313:878–895. doi:10.1016/j.yexcr.2006.12.015

7. Lizaka M, Han H-J, Akashi H, et al. Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins. Cytogen Cell Gene. 2000;88:221–224. doi:10.1159/000015555

8. Walk EL, Hong W, Garcia MV, Bear JE, Weed SA. Abstract 4752: cortactin and coronin 1B cooperate to promote tumor cell invasion in head and neck squamous cell carcinoma. Cancer Res. 2011;71:4752–4762.

9. Wang J, Tsouko E, Jonsson P, et al. Abstract P4-07-12: miR-206 inhibits cell migration through direct targeting of the actin-binding protein coronin 1C in triple-negative breast cancer. Cancer Res. 2013;73:1690–1702.

10. Wu L, Peng CW, Hou JX, et al. Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation. J Exp Clin Cancer Res. 2010;29:17. doi:10.1186/1756-9966-29-17

11. Castagnino A, Castro-Castro A, Marie Irondelle M, et al. Coronin 1C promotes triple-negative breast cancer invasiveness through regulation of MT1-MMP traffic and invadopodia function. Oncogene. 2018;37:6425–6441. doi:10.1038/s41388-018-0422-x

12. Wang ZG, Jia MK, Cao H, Bian P, Fang XD. Knockdown of Coronin-1C disrupts Rac1 activation and impairs tumorigenic potential in hepatocellular carcinoma cells. Oncol Rep. 2012;29:1066‒1072.

13. Thal D, Xavier CP, Rosentreter A, et al. Expression of coronin-3 (coronin-1C) in diffuse gliomas is related to malignancy. J Pathol. 2008;214:415–424. doi:10.1002/path.2308

14. Luu H, Zhang R, Haydon R, et al. Wnt/β-catenin signaling pathway as novel cancer drug targets. Curr Cancer Drug Targets. 2004;4:653–671. doi:10.2174/1568009043332709

15. Wang X, Meng X, Sun X, et al. Wnt/β-catenin signaling pathway may regulate cell cycle and expression of cyclin A and cyclin E protein in hepatocellular carcinoma cells. Cell Cycle. 2009;8:1567–1570. doi:10.4161/cc.8.10.8489

16. Leung JY, Kolligs FT, Wu R, Zhai Y, Fearon ER. Activation of AXIN2 expression by beta-catenin-T cell factor. A feedback repressor pathway regulating Wnt signaling. J Biol Chem. 2002;277:21657–21665. doi:10.1074/jbc.M200139200

17. Zhang L, Liu H, Mu X, Cui J, Peng Z. Dysregulation of Fra1 expression by Wnt/β-catenin signaling promotes glioma aggressiveness through epithelial-mesenchymal transition. Biosci Rep. 2017;37:BSR20160643. doi:10.1042/BSR20160643

18. Chen L, Li M, Li Q, Wang CJ, Xie SQ. DKK1 promotes hepatocellular carcinoma cell migration and invasion through β-catenin/MMP7 signaling pathway. Mol Cancer. 2013;12:157. doi:10.1186/1476-4598-12-157

19. Hao HP, Wen LB, Li JR, et al. LiCl inhibits PRRSV infection by enhancing Wnt/β-catenin pathway and suppressing inflammatory responses. Antivir Res. 2015;117:99–109. doi:10.1016/j.antiviral.2015.02.010

20. Guo YZ, Xie XL, Fu J, Xing GL. SOX9 regulated proliferation and apoptosis of human lung carcinoma cells by the Wnt/β-catenin signaling pathway. Eur Rev Med Pharmacol Sci. 2018;22(15):4898–4907. doi:10.26355/eurrev_201808_15626

21. Chan KT, Roadcap DW, Holoweckyj N, Bear JE. Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin. Biochem J. 2012;444:89–96. doi:10.1042/BJ20120209

22. Wu L, Hou JX, Peng CW, et al. Increased coronin-1C expression is related to hepatocellular carcinoma invasion and metastasis. Chin J Hepatol. 2010;18:516.

23. Gui R, Qifei T, Yanxin A, et al. Coronin 3 promotes gastric cancer metastasis via the up-regulation of MMP-9 and cathepsin K. Mol Cancer. 2012;11:67. doi:10.1186/1476-4598-11-67

24. Samarin SN, Koch S, Ivanov AI, Parkos CA, Nusrat A. Coronin 1C negatively regulates cell–matrix adhesion and motility of intestinal epithelial cells. BBRC. 2010;391:0–400.

25. Ziemann A, Simon H, Ridhirama B, et al. CRN2 enhances the invasiveness of glioblastoma cells. Neuro Oncol. 2013;5:548–561.

26. Yamazaki D, Kurisu S, Takenawa T. Regulation of cancer cell motility through actin reorganization. Cancer Sci. 2005;96:379–386. doi:10.1111/j.1349-7006.2005.00062.x

27. Sun Y, Shang Y, Ren G, et al. Coronin3 regulates gastric cancer invasion and metastasis by interacting with Arp2. Cancer Biol Ther. 2014;15:1163–1173. doi:10.4161/cbt.29501

28. Liu C, Tu Y, Sun X, et al. Wnt/beta-Catenin pathway in human glioma: expression pattern and clinical/prognostic correlations. Clin Exp Med. 2011;11:105–112. doi:10.1007/s10238-010-0110-9

29. Tao Q, Wu C, Xu R, et al. Diallyl trisulfide inhibits proliferation, invasion and angiogenesis of glioma cells by inactivating Wnt/β-catenin signaling. Cell Tissue Res. 2017;370(3):379‒390. doi:10.1007/s00441-017-2678-9

30. Wang G, Shen J, Sun J, et al. Cyclophilin A maintains glioma-initiating cell stemness by regulating Wnt/β-catenin signaling. Clin Cancer Res. 2017;23(21):6640‒6649. doi:10.1158/1078-0432.CCR-17-0774

31. Arnés M, Casas Tintó S. Aberrant Wnt signaling: a special focus in CNS diseases. J Neurogenet. 2017;31:1–7. doi:10.1080/01677063.2017.1338696

32. Bahloul A, Simmler MC, Michel V, Leibovici M, Petit C. Vezatin, an integral membrane protein of adherens junctions, is required for the sound resilience of cochlear hair cells. EMBO Mol Med. 2009;1:125–138. doi:10.1002/emmm.200900015

33. Lilien J, Balsamo J. The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of β-catenin. Curr Opin Cell Biol. 2005;17:459–465. doi:10.1016/

34. Gottardi CJ. Adhesion signaling via catenins. Bone. 2010;47:S18. doi:10.1016/j.bone.2010.04.013

Creative Commons License This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

Download Article [PDF]