Cell Cycle-Related FAM64A Could be Activated by TGF-β Signaling to Promote Glioma Progression

Abstract

Gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. FAM64A, a cell cycle-related gene, has been found to promote cell proliferation in various tumors, including gliomas. However, the regulatory mechanism and clinical significance of FAM64A in gliomas remain unclear. In this study, we investigated FAM64A expression in gliomas with different grades and constructed FAM64A silenced cell lines to study its functions. Our results demonstrated that FAM64A was highly expressed in glioblastoma (P < 0.001) and associated with a poor prognosis (P < 0.001). Expression profiles at the single-cell resolution indicated FAM64A could play a role in a cell-cycle-dependent way to promote glioma cell proliferation. We further observed that FAM64A silencing in glioma cells resulted in disrupted proliferation and migration ability, and increased cell accumulation in the G2/M phase (P = 0.034). Additionally, TGF-β signaling upregulates FAM64A expression, and SMAD4 and FAM64A co-localize in high-grade glioma tissues. We found FAM64A knockdown inhibited TGF-β-induced epithelial-mesenchymal transition in glioma. Our findings suggest that FAM64A could serve as a diagnostic and therapeutic target in gliomas.

Keywords: Cell cycle; EMT; FAM64A; Glioma; Single-cell RNA-seq; TGF-β.

Fig. 1

FAM64A expression positively correlates with tumor grade. A FAM64A mRNA expression of gliomas in different WHO grades. B FAM64A expression in glioblastoma is higher than in other histological types (astrocytes, oligodendrocytes, oligoastrocytoma). C Kaplan–Meier survival curves of TCGA-GBMLGG cohorts of FAM64A in high expression and low expression, with 95% confidence bands. D FAM64A was detected by immunoblotting in normal brain tissues and glioma samples. E FAM64A protein expression in glioma samples with different grades was assessed by quantitative analysis. F Nomogram plot to predict patients’ prognosis based on the FAM64A expression, Grade, age, and IDH status. For example, a patient who was diagnosed with gliomas at the age of 50 years (50 points), with IDH mutation (0 points), grade 2 (0 points), and the normalized FAM64A expression of 4 (20 points), has a total of 70 points. The cumulative points correspond to a survival probability of 87% and 77% at 3 and 5 years respectively. G The sensitivity of temozolomide is inversely correlated with FAM64A expression. Data represents the mean ± SD of triplicate samples, *P < .05, **P < .01, ***P < .001, t test was used to compare two individual groups

Fig. 2

FAM64A plays a role in the proliferation of glioma cells. A Umap visualization of GSE131928 scRNA dataset. Colors are coded for the clustering output of annotated cell groups (Monocytes/Macrophage/Microglia cells, Glia/Glioma cells, and T cells) and the FAM64A expression. B shFAM64A U87 cells have lower viability after 48 h and 72 h compared with control groups. C Immunofluorescent quantitation of the percentage of Ki67-positive cells in FAM64A silencing U87 cells and control groups. D KEGG enrichment analysis of TCGA GBM cohort with different FAM64A expressions

Fig. 3

FAM64A knockdown leads to S phase arrest. A Umap visualization of neoplasm cells, colors are coded for the different cell cycles and FAM64A expression. B RNA velocity analysis overlaid on the umap plot of neoplasm cells. The blue rectangle represents the initiation stage and the arrows represent predicted developmental directions. C The cell cycle populations were measured by flow cytometry. D Cell cycle-related proteins in U87 scramble controls and FAM64A silencing groups (shFAM64A#1 and shFAM64A#2). Control groups exhibited higher E2F1, cyclin D1, GADD45, and Phospho-Rb expression compared with FAM64A silencing groups. The protein level of E2F1, cyclin D1, cyclin B1, GADD45, CDK2, Phospho-Rb, and Rb in cells was determined by western blotting, normalized by β-TUBULIN

Fig. 4

FAM64A could be induced by TGF-β signaling. A Heatmap showing the TGF-β signaling pathway network among cells (T cells, TAMs, FAM64A highly expressed neoplasm cells, FAM64A lowly expressed neoplasm cells). TAMs are both the strongest TGF-β signaling senders and receptors. B Transcription factor prediction for FAM64A. TGF-β associated transcription factors have a high regulatory potential score. C Enhanced TGF-β signaling promoted proliferation of U87 cells and induced FAM64A expression. D The concentration of TGF-β and expression of FAM64A, SMAD3, and SMAD4 in glioma samples. E Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope. High-grade glioma has a higher expression of FAM64A and SMAD4. F FAM64A co-localizes with SMAD4, a transcription factor of the TGF-β signaling pathway

Fig. 5

TGF-β induced EMT is dependent on FAM64A. A TGF-β signaling upregulated SMAD3 and SMAD4 in U87 scramble controls and FAM64A silencing groups (shFAM64A#1 and shFAM64A#2). TGF-β of low concentration (10 ng/mL) could promote N-Cadherin expression. N-Cadherin expression decresased with the elevated TGF-β concentrations (50 ng/mL and 200 ng/mL). Control groups exhibited higher N-cadherin expression compared with FAM64A silencing groups (Paired t-test, P = 0.0090, 0.0037, respectively). The protein level of N-Cadherin, SMAD3, and SMAD4 in cells was determined by western blotting, normalized by β-TUBULIN. B TGF-β signaling upregulated SMAD3 and SMAD4 in U251 scramble controls and FAM64A silencing groups (shFAM64A#1 and shFAM64A#2). TGF-β of low concentration (10 ng/mL) could promote N-Cadherin expression. However, N-Cadherin expression decresased with the elevated TGF-β concentrations (50 ng/mL and 100 ng/mL). Control groups exhibited higher N-cadherin expression compared with FAM64A silencing groups (Paired t-test, P = 0.0234, 0.0417, respectively). The protein level of N-Cadherin, SMAD3, and SMAD4 in cells was determined by western blotting, normalized by β-TUBULIN. C Cell migration ability was determined by cell scratch assay. FAM64A silencing U87 cells have a higher migration ability compared with scramble control groups

Fig. 6

Schematic diagram displaying TGF-β plays an important role in the tumor EMT process. The TGF-β signaling activates downstream transcription factors (SMAD2, SMAD3, and SMAD4) and upregulated the expression of FAM64A. FAM64A could promote the EMT process in glioma