Targeting COL5A1 enhances anoikis thus attenuating malignancy of glioblastoma via inhibiting the Wnt/β-catenin signaling pathway

Abstract

Purpose: As one of the most prevalent primary brain tumors, glioblastoma (GBM) is characterized by its severe malignancy and extremely poor prognosis. Recent studies have demonstrated that targeting anoikis and malignancy showed impressed efficiency for treatment in a wide range of solid tumors, however, relevant research on GBM still remains unclarified.

Methods: In this study, genes related with malignancy and anoikis of GBM were identified by utilizing the Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA) and the Molecular Signatures Database (MSigDB). Subsequently, the role of the key gene was validated via proliferation, invasion and migration experiments both in conditions with and without attachment. Moreover, RNA sequencing analysis was employed to reveal further mechanisms.

Results: Here, Type V collagen alpha 1 (COL5A1) was identified as a critical gene associated with anoikis and poor outcomes. Additionally, COL5A1 knockdown induced significant reduction in malignancy of GBM both in vitro and in vivo. Moreover, cell anoikis was remarkable enhanced by reduced expression of COL5A1 after low-attachment cell culture. Mechanically, RNA sequencing analysis revealed that the activity of the Wnt/β-catenin signaling pathway was diminished following COL5A1 knockdown, which indicated that COL5A1 reduced anoikis via regulating Wnt/β-catenin signaling pathway thus promoted malignancies of GBM cells.

Conclusion: These findings demonstrated the novel evidence that COL5A1 serves as an essential regulatory factor influencing both anoikis and malignancy of GBM cells by regulating Wnt/β-catenin signaling pathway, indicating that COL5A1 could be a novel prognosis-related biomarker and potential therapeutic target for GBM.

Keywords: COL5A1; Anoikis; Glioblastoma; Malignancy; Wnt/β-catenin signaling pathway.

Fig. 1

COL5A1 is a critical gene associated with malignancy and anoikis in GBM. (A) Identification of genes associated with GBM prognosis utilizing the TCGA and CGGA databases. (B) Common genes exhibiting HR < 1 and HR > 1 in both TCGA and CGGA databases. (C-D) WGCNA of common prognostic genes and anoikis scores based on the TCGA database. (E-F) WGCNA of common prognostic genes and anoikis scores based on the CGGA database. (G) Genes within the most significantly correlated module, demonstrating an anoikis score of 0.52, as identified using the TCGA database. (H) Genes within the most significantly correlated module, demonstrating an anoikis score of 0.81, as identified using the CGGA database. (I) Common genes associated with malignancy and anoikis across both databases

Fig. 2

COL5A1 plays a crucial role in the malignancy of GBM both in vivo and in vitro. (A) The mRNA and protein expression levels of COL5A1 in paired GBM and adjacent tissues. (B) The mRNA and protein expression levels of COL5A1 in NHA and various GBM cell lines. (C) Quantitative PCR results for control (shNT) and knockdown (shCOL5A1) groups in U373 and U87 cells. (D) Western blot results for control (shNT) and knockdown (shCOL5A1) groups in U373 and U87 cells. (E) Proliferation capabilities were evaluated over a 96-hour period for NT and KD groups. (F) Colony formation assays comparing NT and KD groups in both U373 and U87 cell lines. (G) Invasion abilities among different cell groups were assessed. (H-I) Wound healing assays presented the migratory capacities of NT and KD cells. (J) Kaplan-Meier survival curves illustrating poor outcomes of NT groups. H&E staining images depicting brain tissue samples from mice with GBM following intracranial transplantation are provided

Fig. 3

The reduction in GBM aggressiveness resulting from the absence of COL5A1 was enhanced under low-attachment conditions. (A) The expression of COL5A1 was found to be elevated in low-attachment conditions in both U373 and U87 cell lines. (B-C) CCK8 assays were conducted to assess the proliferation capacity of cells cultured under normal and low-attachment conditions over a period of 96 h. (D-E) The migration capabilities of cells cultured in both normal and low-attachment environments were evaluated. (F-G) Matrigel invasion assays demonstrated the effects on invasion ability across different attachment conditions

Fig. 4

COL5A1 knockdown enhances anoikis in GBM cells. (A) NT and KD GBM cells were cultured in low-attachment 96-well plates for a duration of 2 days. Calcein AM/PI staining revealed an increase in anoikis within the COL5A1 knockdown groups. Calcein AM stained viable cells green, while PI staining dead cells red. (B) The counts of stable cell spheres with diameters exceeding 50 μm were recorded. (C, E) Apoptotic cells were quantified using flow cytometry analysis. Compared to attachment conditions, general apoptosis was found to be elevated under low-attachment conditions. Furthermore, anoikis was significantly enhanced in the KD groups. Q1 (upper-left, 7-ADD⁺/PE⁻): Cellular debris or mechanically damaged cells; Q2 (upper-right, 7-ADD⁺/PE⁺): Late apoptotic cells; Q3 (lower-left, 7-ADD ⁻/PE⁻): Viable/normal cells; Q4 (lower-right, 7-ADD⁻/PE⁺): Early apoptotic cells. (D, F) Apoptosis markers including cleaved-caspase3, Bcl-2, and Bax in GBM cells exhibited results consistent with those obtained from flow cytometry analysis

Fig. 5

COL5A1 regulated anoikis via the Wnt/β-catenin signaling pathway. (A) A total of 286 significantly differentially expressed genes (log₂|FC| > 2, adjusted P < 0.05) were identified using the limma package in R programming. (B) KEGG analysis indicated the relevant pathways associated with COL5A1. (C) GESA demonstrated that the Wnt/β-catenin signaling pathway was significantly suppressed following COL5A1 knockdown. (D) The suppression of this signaling pathway was validated through western blot analysis. (E) The anoikis ratio decreased upon activation of the Wnt/β-catenin signaling pathway, as evidenced by Calcein AM/PI staining. Under low-attachment conditions, cells treated with LiCl (5 mM) formed more stable spheres with diameters exceeding 50 μm. (F-I) Flow cytometry analysis utilizing 7-ADD and PI demonstrated the impact of LiCl (5 mM) on apoptosis in U373 and U87 cell lines. Q1 (upper-left, 7-ADD⁺/PE⁻): Cellular debris or mechanically damaged cells; Q2 (upper-right, 7-ADD⁺/PE⁺): Late apoptotic cells; Q3 (lower-left, 7-ADD ⁻/PE⁻): Viable/normal cells; Q4 (lower-right, 7-ADD⁻/PE⁺): Early apoptotic cells