CREB5 mRNA is expressed higher in the GSC than in the differentiated GSC
Over the past few decades, research on the functions of CREB in the nervous system has increased.(21–25) In the central nervous system, it is believed that CREB serves as a critical regulator in several complex processes, from development to plasticity. For example, in the developing brain, CREB has been proposed as a regulator of cell survival, proliferation, and differentiation.(26–28) In contrast, in the adult brain, it is considered to play a role in learning, memory, and neuronal plasticity.(29–31) Recent research has revealed that CREB is also involved in the regulation of cancer cells' ability to transform.(32)
Previous studies have shown that when comparing glioma stem cells (GSCs) to differentiated GSCs, they have more tumorigenic potential and self-renewal ability under undifferentiated conditions.(33) Therefore, we compared the expression level of genes belonging to the CREB family by dividing them into GSC and differentiated GSC conditions using a publicly available dataset (GSE4536). Among the CREB family, CREB5 expression was the highest in undifferentiated GSCs ("NBE" conditions: serum-free DMEM/F12 media supplemented with basic FGF and EGF), and it was confirmed that the cell expressed the lowest level when differentiated ("serum" conditions: DMEM/F12 media containing 10% fetal bovine serum) (Fig. 1A, B). These findings suggest that CREB5 may also play an important role in maintaining GSCs.
CREB5 expression is correlated with poor prognosis
In this study, we investigated the clinical relevance of CREB5 in GBM patients by examining the expression patterns and survival rates of the CREB family using the Rembrandt dataset. Firstly, we compared the expression of the CREB family genes in non-tumor and GBM patients and found that several genes, including CREB5, were highly expressed in GBM (Fig. S1A). Next, we analyzed the expression of CREB family genes according to glioma grade and observed that some genes, including CREB5, were highly expressed in grade IV GBM compared to grade II or III (Fig. S1B). Finally, we evaluated the survival rates of glioma patients based on the expression levels of CREB family genes. Our investigation revealed that only high expression of CREB5 was significantly associated with decreased survival rate (Fig. S1C). The correlation between high expression of CREB5 and decreased survival rate, along with the elevated expression of CREB5 in GBM, supports the potential of CREB5 as a promising target for further investigation.
CREB5 is overexpressed in the classical subtype and highly expressed in the cellular tumor region and pseudopalisading cells around necrosis.
There are four subtypes of GBM: proneural, classical, mesenchymal, and neural. There are proneural, classical, mesenchymal, and neural types in GBM subtypes, and these have been defined through a genome-wide analysis of mRNA expression in 300 GBM patient tissues. We confirmed that CREB5 mRNA is highly expressed in the classical subtype using the Rembrandt dataset (Fig. S2A).
Using the Ivy Glioblastoma Atlas Project dataset, we confirmed where CREB5 is highly expressed in GBM tumor regions. The dataset consists of mRNA expression profiles that were gathered from various areas within tumors, including the leading edge (LE), infiltrating tumor (IT), cellular tumor (CT), perinecrotic zone (PNZ), pseudopalisading cells around necrosis (PAN), hyperplastic blood vessels in cellular tumors (HBV), and microvascular proliferation (MVP). The expression of CREB5 has been verified to be high in the cellular tumor and in the regions of pseudopalisading cells around necrosis (Fig. S2B).
CREB5 is expressed highly expressed in GSC
We conducted an experiment to confirm whether this expression pattern also occurs in the cell line we have. When comparing the expression of CREB5 mRNA in normal human astrocytes (NHA), glioma cells, and glioma stem cells, we confirmed that the expression in glioma stem cells was relatively higher (Fig. 2A). Next, we cultured glioma stem cells in fetal bovine serum for seven days to generate differentiated glioma stem cells. In differentiated glioma stem cells, the expression of stem cell markers decreased while differentiation markers increased. As expected, the expression of CREB5 decreased in differentiated glioma stem cells (Fig. 2B, C). Based on these results, we can conclude that the expression of CREB5 is significantly elevated in glioma stem cells.
Suppressing of CREB5 inhibits proliferation and self-renewal ability in GSCs
We investigated the role of CREB5 in the proliferation and self-renewal ability of glioma stem cells (GSCs). Upon silencing the expression of CREB5, we observed a decrease in proliferation in GSC11 and GSC23 (Fig. 3A, B). The Annexin V/PI assay results demonstrated that inhibition of CREB5 expression caused a shift in the distribution of GSCs towards apoptotic or necrotic regions (Fig. 3C). We also observed that a decrease in CREB5 expression led to the cessation of GSC sphere growth (Fig. 3D). Therefore, we conducted a limiting dilution assay to investigate the impact of decreased CREB5 expression on the self-renewal ability of GSCs. We confirmed a significant reduction in the self-renewal characteristics of GSCs when CREB5 expression was inhibited (Fig. 3E). These findings indicate that inhibition of CREB5 expression leads to decreased proliferation and self-renewal ability of GSCs.
The inhibition of CREB5 reduces the tumorigenic potential of GSCs in vivo
Using an in vivo orthotopic xenograft model, we evaluated the tumorigenic potential of CREB5. After reducing CREB5 expression using shCREB5 in GSC11 cells, we observed a significant decrease in tumorigenic potential compared to shNT when injected into the brains of nude mice (Fig. 4A, B). We also observed a significant increase in mouse survival time when inoculating GSCs with knockdown of CREB5 expression (Fig. 4C). These data suggest that CREB5 plays an important role in the tumorigenic potential of GSCs.
RNA-Sequencing reveals pathways and genes downregulated by shCREB5
To investigate the effects of CREB5 inhibition in glioma stem cells (GSCs) at the transcriptional level, we performed RNA-Sequencing analysis (Fig. 5A). Upon knocking down CREB5 in GSCs, we conducted a KEGG pathway analysis, which revealed a decrease in multiple cancer-associated signaling pathways (Fig. 5B).
CREB5 associates with the OLIG2 promoter
Next, we identified downstream genes that are concomitantly decreased upon CREB5 inhibition in glioma stem cells (GSCs) by representing them in a Venn diagram. The genes OLIG2, DUSP5, NFIX, ZCCHC24, VIM, and SPARCL1 were concurrently downregulated in both GSCs (Fig. 6A). OLIG2 is a gene involved in central nervous system development, and there are reports that this gene plays an important role in the maintenance of brain tumors, especially GSCs.(34–36) There is evidence that OLIG2 is highly expressed in GSCs and that its inhibition suppresses GSC proliferation.(37) Therefore, taking into account the fact that CREB5 is a transcription factor and the downregulation of OLIG2 at the transcriptional level, we investigated whether CREB5 influences the activity of the OLIG2 promoter using a luciferase reporter assay (Fig. 6B). As a result, we observed an increase in OLIG2 promoter activity upon co-transfection of the CREB5 expression vector into HEK293T cells (Fig. 6B). Furthermore, we performed a chromatin immunoprecipitation (ChIP) assay and identified multiple AP-1 sites within the OLIG2 promoter where CREB5 protein could potentially bind (Fig. 6C).(9) Additionally, we confirmed the correlation between CREB5 and OLIG2 expression in GBM by utilizing various patient datasets. We identified a positive correlation between CREB5 and OLIG2 across multiple datasets (Fig. S3). These findings confirm that OLIG2 is a direct transcriptional target of CREB5.