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.
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Data Availability
The datasets used and analyzed during the current study are available from Gliovis (http://gliovis.bioinfo.cnio.es/) and GEO database (https://www.ncbi.nlm.nih.gov/geo/). Analysis scripts are available from the corresponding author on reasonable request.
Abbreviations
- TGF-β :
-
Transforming growth factor b
- GBM:
-
Glioblastoma
- LGG:
-
Lower grade glioma
- TCGA:
-
The cancer genome atlas
- scRNA-seq:
-
Single cell RNA-seq
- IDH:
-
Isocitrate dehydrogenase
- PLK:
-
Polo-like kinase
- TME:
-
Tumor microenvironment
- EMT:
-
Epithelial-mesenchymal transition
- TMZ:
-
Temozolomide
- MGMT:
-
O6-methylguanine-DNA methyltransferase
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Acknowledgements
The bioinformatic analysis was supported by the Medical Research Data Center of Fudan University.
Funding
This work was supported by the National Natural Science Foundation of China (82072784, 8210113482, 82103690), National Key R&D Program of China (2022YFC3401600), and CAMS Innovation Fund for Medical Sciences (2022-I2M-C&T-B-112). The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.
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MF, JZ and LZ have contributed equally to this study. WH, WW, and YM conceived the general framework of this study and revised the manuscript. JZ, MF and LZ performed the cell experiments. MF, YF, QW, XF, LZ, and JZ analyzed data. MF and LZ interpreted the results. FF collected clinical samples. MF, LZ, and JZ wrote the manuscript. All authors have read and approved the final manuscript.
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The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Huashan Hospital (Approval number: KY2015-256).
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Glioma tissues used in the current study were resected in 2020 from patients in the Department of Neurosurgery, Huashan Hospital of Fudan University. Normal brain tissues were gathered from traumatic brain injury patients. Written informed consent were obtained from all patients for use of tissue sample.
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10571_2023_1348_MOESM1_ESM.tif
Supplementary file1 (TIF 33731 KB)— Suppl. Fig. 1 (A) Pan-cancer analysis of FAM64A. Most TCGA tumors had a higher accumulation of FAM64A than the adjacent non-tumor tissues. (B) IDH wildtype gliomas expressed higher FAM64A compared with IDH mutant gliomas in TCGA-GBMLGG cohort. (C) FAM64A in mesenchymal subtype is higher than neural and proneural subtype. (D) FAM64A expression in negative correlation with the sensitivity of BI-2536, a PLK inhibitor. Data represents the mean ± SD of triplicate samples, *P < .05, **P < .01, ***P < .001, t test was used to compare two individual groups
10571_2023_1348_MOESM2_ESM.tif
Supplementary file2 (TIF 26981 KB)— Suppl. Fig. 2 (A) Umaps of GSE131928 dataset before and after removing batch effect using harmony algorithm. And the batch effect was very apparent. (B) Cell groups (Monocytes/Macrophage/Microglia cells, Glia/Glioma cells, and T cells) were verified by classic gene markers (T cells: CD3D, CD3E, CD4; Glia/Glioma cells: OLIG2, FA2H, CNP, GFAP; Monocytes/Macrophages/Microglias: CD68, CD163, CD14, FPR1). (C) FAM64A knock-down efficiency of stable cell lines was validated by immunoblotting. ShFAM64A represented the FAM64A silencing U87 cells and shScramble is the control group. (D) shFAM64A U251 cells have lower viability after 48h and 72h compared with control groups
10571_2023_1348_MOESM3_ESM.tif
Supplementary file3 (TIF 30471 KB)— Suppl. Fig. 3 (A) Heatmap for output of copy number variation of glia/glioma cells via inferCNV algorithm. The T cell and TAMs are set as references. (B) Copy number variation (CNV) visualization of GSE131928 scRNA dataset. Color is coded for different clustering of inferCNV algorithm. CNV score for Group 18 is low. Therefore, other groups were included in the following analysis
10571_2023_1348_MOESM4_ESM.tif
Supplementary file4 (TIF 26265 KB)— Suppl. Fig. 4 (A) Network plot of TGF-β signaling crosstalk. TAMs have a very strong interaction with FAM64A highly expressed neoplasm cells via the TGF-β signaling pathway (the directional red and green bands mean the crosstalk between TAMs and FAM64A highly expressed neoplasm cells). (B) Ligand-receptor plot of TGF-β signaling pathway. TGFB1-(TGFBR1+TGFBR2) contribute most to the signaling flow between TAMs and neoplasm cells. (C) Integrated genome browser visualization of tag density profiles for ChIP-Seq Smad2, ChIP-Seq Smad3, and ChIP-Seq Smad4. (D) FAM64A mRNA expression in control group and Smad3 knock out groups in GSE125116. (E) TGF-β concentration in WHO IV gliomas is higher than normal brain tissues, WHO II and III gliomas significantly. (F) Representative images of IHC staining for FAM64A in different pathological grades of gliomas. (G) TGF-β signaling upregulated SMAD3 and SMAD4 in HA-MG 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 decreased with the elevated TGF-β concentrations (20 ng/mL and 40 ng/mL). Control groups exhibited higher N-cadherin expression compared with FAM64A silencing groups (Paired t-test, P= 0.0171, 0.0963, respectively). The protein level of N-Cadherin, SMAD3, and SMAD4 in cells was determined by western blotting, normalized by β-TUBULIN. Data represents the mean ± SD of triplicate samples, *P < .05, **P < .01, ***P < .001, t test was used to compare two individual groups
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Fu, M., Zhang, J., Zhang, L. et al. Cell Cycle-Related FAM64A Could be Activated by TGF-β Signaling to Promote Glioma Progression. Cell Mol Neurobiol 43, 2975–2987 (2023). https://doi.org/10.1007/s10571-023-01348-2
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DOI: https://doi.org/10.1007/s10571-023-01348-2