
==== Front
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Neoplasia
Neoplasia (New York, N.Y.)
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S1476-5586(24)00092-7
10.1016/j.neo.2024.101050
101050
Original Research
FABP4 facilitates epithelial-mesenchymal transition via elevating CD36 expression in glioma cells
You Zhongsheng ab1
Hu Zihao f1
Hou Chongxian ab1
Ma Chengcheng abcdef
Xu Xiangdong ab
Zheng Yaofeng ab
Sun Xinlin ab
Ke Yiquan ab
Liang Jianli ab
Xie Zijing ab
Shu Lingling shull@sysucc.org.cn
cde⁎
Liu Yang lynanchang@hotmail.com
ab⁎⁎
a Key Laboratory of Neurosurgery in Guangdong Province, Southern Medical University, Guangzhou 510060, PR China
b Department of Neuro-oncological Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510060, PR China
c State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, PR China
d Department of Hematological Oncology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China
e State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, PR China
f School of Medicine, Nankai University, Tianjin, PR China
⁎ Corresponding author at: State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, PR China. shull@sysucc.org.cn
⁎⁎ Corresponding author at: Key Laboratory of Neurosurgery in Guangdong Province, Southern Medical University, Guangzhou 510060, PR China. lynanchang@hotmail.com
1 These authors contributed equally to the work.

06 9 2024
11 2024
06 9 2024
57 10105026 5 2024
17 8 2024
3 9 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• We determine the elevated FABP4 expression in glioblastoma with tissue samples and public databases including TCGA and CGGA.

• Our results revealed the critical role of FABP4 in EMT process.

• We delineated the FABP4-CD36 signaling pathway in glioma EMT.

• FABP4-induced CD36 expression primarily activated non-canonical TGFβ signaling.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis. A better understanding of mechanisms concerned in glioma invasion might be critical for treatment optimization. Given that epithelial-mesenchymal transition in tumor cells is closely associated with glioma progression and recurrence, identifying pivotal mediators in GBM EMT process is urgently needed. As a member of Fatty acid binding protein (FABP) family, FABP4 serves as chaperones for free fatty acids and participates in cellular process including fatty acid uptake, transport, and metabolism. In this study, our data revealed that FABP4 expression was elevated in human GBM samples and correlated with a mesenchymal glioma subtype. Gain of function and loss of function experiments indicated that FABP4 potently rendered glioma cells increased filopodia formation and cell invasiveness. Differential expression genes analysis and GSEA in TCGA dataset revealed an EMT-related molecular signature in FABP4-mediated signaling pathways. Cell interaction analysis suggested CD36 as a potential target regulated by FABP4. Furthermore, in vitro mechanistic experiments demonstrated that FABP4-induced CD36 expression promoted EMT via non-canonical TGFβ pathways. An intracranial glioma model was constructed to assess the effect of FABP4 on tumor progression in vivo. Together, our findings demonstrated a critical role for FABP4 in the regulation invasion and EMT in GBM, and suggest that pharmacological inhibition of FABP4 may represent a promising therapeutic strategy for treatment of GBM.

Keywords

FABP4
Glioblastoma
Epithelial-mesenchymal transition
Tumor invasion
CD36
Abbreviations

FABP4 fatty acid binding protein 4

GBM glioblastoma

EMT epithelial-mesenchymal transition

TCGA The Cancer Genome Atlas

CGGA Chinese Glioma Genome Atlas

CCK-8 Cell counting Kit-8

TGFβ transforming growth factor
==== Body
pmcIntroduction

Gliomas are cancerous brain tumors that originate from glia cells. Glioblastoma multiforme (GBM) is the most progressive type with adverse prognosis, despite multimodal treatment [1]. GBMs were diagnosed previously according to histologic findings involving microvascular proliferation and necrosis [2]. The up-to-date classification of tumors of central nervous system defines GBM primarily based on molecular features [3]. The highly invasive ability of GBM cells was regarded as a vital factor associated with treatment resistance and recurrence. Therefore, further investigations on molecular research deciphering GBM invasiveness are warranted.

Epithelial-mesenchymal transition (EMT) describes a biological process that epithelial cells acquire mesenchymal characteristics and has been well-validated to be closely associated with tumor invasion and metastasis [4]. Moreover, EMT has also been reported as a key regulator of invasive state in glioblastoma [5]. Previous studies have defined several key molecules including TGFβ and Wnt as key inducers in EMT [6]. During EMT process, epithelial molecules such as E-cadherin and cytokeratins were downregulated while mesenchymal markers, such as N-cadherin and vimentin were upregulated in tumor cells.

The adipokine adipocyte fatty acid-binding protein (A-FABP or FABP4), primarily expressed in adipocytes, is implicated in the regulation of lipid metabolism [7]. In fact, FABP4 has been reported to be highly expressed and involved in cancer progression in various types of tumors, including prostate cancer [8], hepatocellular carcinoma [9] and ovarian cancer [10]. FABP4 was reported to be a target of VEGF and capable to regulate endothelial cell proliferation [11]. Downregulation FABP4 in vivo would exert antiangiogenic and antitumor effects [12]. Moreover, Huang et.al revealed that FABP4 enhanced prostate cancer progression and invasion by inducing matrix metalloproteinases (MMPs) and cytokine production [8]. Gharpure et.al demonstrated that FABP4 mediated metastasis-related signaling pathways and contributed to poor outcome in ovarian cancer [10]. A study from Miao and colleagues indicated that FABP4 deactivates NF-κB-IL1α pathway and promoted migration phenotype in neuroblastoma cells [13]. Furthermore, Li and colleagues previously revealed a pro-growth role of FABP4 via mediating Wnt10b expression in glioblastoma cells [14].

In the present study, we hypothesized that FABP4 promotes EMT process in GBM and that this EMT induction is mediated by upregulation of CD36. We tested the physiologic relevance of FABP4-incuced EMT in a series of in vitro experiments as well as clinical specimens.

Materials and methods

Glioma sample and cell culture

Glioma samples were obtained from consenting patients diagnosed as glioma (WHO I-IV). 54 paraffin-embedded glioma samples, of which 2, 19, 18 and 15 samples were respectively classified as WHO grade I, II, III and IV, as well as corresponding clinicopathological information were obtained from patients with surgical operation in the department of neuro-oncological surgery at Zhujiang Hospital from 2020 to 2023.

For the establishment of primary glioma cells, glioblastoma tissues were enzymatically digested using Accutase (Sigma) for 30 min at 37 ℃, and primary glioma cells were cultured in DMEM (Gibco, USA) medium supplemented with 10 % fetal bovine serum (Gibco) and antibiotics.

shRNA and lentivirus

shRNAs for FABP4 were designed and provided by Sangon Biological Engineering Technology and Service Co., Ltd. and Sigma-Aldrich. FABP4 expression was silenced by transducing glioma cells with lentivirus expressing shRNAs, and was overexpressed with lentivirus expressing full length of FABP4 ORF. ShRNA as well as PCR sequences for FABP4, CD36 and GAPDH were provided in Supplementary Table 1. For the construction of FABP4-expressing lentiviral vector, the coding sequence of FABP4 was amplified by RT-PCR and the PCR product was cloned into the GV341 vector plasmid. Primer sequence was listed in Supplementary Table 1.

Cell proliferation and invasion assay

Cell proliferation was measured via using CCK-8 and Edu assay as previously described [15]. Invasion assay was performed by using cell culture insert with 8-um pores in 24-well plates (Costar, USA). Briefly, Matrigel-coated inserts with DMEM medium were added with 1×105 glioma cells and placed in a 24-well plate for 24 h. Invaded cells on the bottom side were fixed and stained with 0.05 % Crystal Violet. Cell counting was performed that arbitrary fields from each of the triplicate assays were measured under phase-contrast microscope at 100x magnification (Nikon Instrument Inc.) and then quantified by ImageJ software.

Immunological analysis

Human FABP4 ELISA kit (R&D Systems) was used to determine the protein concentration of FABP4 from culture media according to manufacturer's instruction. supernatants of medium from glioma cells cultured for 24 h were collected and analyzed by Elisa kit.

Immunoblot Analysis assays

The immunoblot assay was performed as described previously [16]. Lysates from glioma cells was processed with immunoblot analysis using antibodies which is listed in Supplementary Table 2. Adherent glioma cells were harvested in lysis buffer containing protease inhibitors (Roche, Germany) under ice. Prepared cell lysis solution with sample buffer were separated by 8–12 % SDS-PAGE and transferred to PVDF membrane (Sigma) in Tris-Glycine transfer buffer at constant current. Following transfer, the membranes were washed and incubated with specific primary antibodies at 4 ℃ overnight, followed by secondary antibodies incubation. Beta-actin protein is used as a blot loading control in the present study. In some cases, target blots correspond to the different membranes due to the similar molecular weight proteins. And original immunoblotting images were provided in the supplementary data titled "Uncropped blots".

Tissue immunohistochemical and immunofluorescence staining

Tissue IHC and IF staining were performed as previously reported [17,18]. Briefly, specimens of surgical GBM tissues and xenograft samples were fixed, embedded and sectioned followed by immuno-staining. sections were evaluated using a fluorescence microscope (Nikon), followed by analyzing with NIS-Element Viewer software. Antibodies used for targeted proteins were listed in Supplementary Table 2.

In silico analysis

Expression profiles of mRNAs associated with glioma and corresponding clinical data were retrieved from glioma datasets including TCGA (https://portal.gdc.cancer.gov/), CGGA (http://www.cgga.org.cn/) and Ivy GAP (https://glioblastoma.alleninstitute.org/) database. Gene expression analysis, survival analysis, correlation analysis and gene set enrichment analysis (GSEA) were conducted via R studio, and Gliovis [19]. In detail R packages including DESeq2 and ClusterProfiler are used in the bio-informatics analysis. For survival analysis in TCGA and CGGA datasets based on FABP4 expression level, glioma patients with complete overall survival information, mRNA sequencing data and WHO Grade classification data were included in cohorts. A total of 667 glioma patients were included in TCGA cohorts and 633 patients in CGGA cohorts. P value smaller than 0.05 was considered statistically significant.

In vivo xenograft assay

Five to eight-week-old Balb/c male mice were purchased from the Central Animal Facility of Southern Medical University. Glioma cells (1 × 105 cells in 0.1 ml PBS) stably transfected with mCherry-LUC vector were orthotopically injected into the brain of Balb/c nude mice according to Ozawa's instruction [20]. Each group included 8 mice.

Statistical analysis

All statistical analyses in this study were performed using Prism 8.0 (GraphPad Software Inc., USA) and R software. Data were expressed as Mean ± SD. Sample size for each study was determined based on literature documentation of similar well-characterized experiments. Statistical significance was assessed by Student's t test or one-way ANOVA with Bonferroni correction for multiple comparisons. P value smaller than 0.05 was considered statistically significant. Statistical outlier analysis was calculated using the GraphPad Outlier calculator.

Results

FABP4 is highly expressed in GBM tissue

FABP4 expression was examined in human glioma tissues. Immunostaining suggested that FABP4 was upregulated in human GBM samples, comparing with lower grade gliomas (Fig. 1a). Increased FABP4 expression in GBM tissues was further validated in human protein atlas (HPA) database (Fig. 1b). We collected 54 glioma samples from Zhujiang hospital and found that FABP4 was mostly expressed in high grade glioma specimens, especially glioblastoma (Fig. 1c). Moreover, when clinicopathological data including age, gender, tumor size and glioma grade were analyzed, we interestingly found that FABP4 expression solely correlated with tumor grade (Fig. 1d). FABP4 expression was measured in GBM cell lines and primary cells. FABP4 expression levels varied in different glioma cells (Fig. 1e and f). And secreted FABP4 production from culture media was also detected (Fig. 1g). Together, our findings indicated that FABP4 is upregulated in GBM, compared with lower glioma grade.Fig. 1 FABP4 is abundantly expressed in GBM samples. (a) Representative MRI, H&E and FABP4 IHC staining images of specimens from patients with GBM and lower grade glioma. Fluorescence staining for FABP4 of GBM and lower grade glioma patients. (b) IHC images of high and low grade glioma from Human Protein Atlas datasets. Scale bar= 50/20 μm. (c) IHC score of FABP4 in human glioma specimens of different grades. (d) Correlation of FABP4 expression with gender, age, tumor size and tumor grade. (e) Protein level of FABP4 in primary glioma cells and cell lines. (f) Fluorescence staining of FABP4 in glioma cells. (g) Secretion level of FABP4 in glioma cells. Data are expressed as Mean ± SD.**p < 0.01, ***p < 0.001. H&E, hematoxylin and eosin. IHC, Immunohistochemistry.

Fig 1

FABP4 is associated with mesenchymal phenotype in glioma tissue

When FABP4 and mesenchymal markers were examined in human GBM samples, immunostaining assays indicated that FABP4 were closely correlated with vimentin and α-SMA expression (Fig. 2a and b). Role of FABP4 in GBM subtypes was further evaluated in GBM datasets. TCGA, CGGA and Ivy_GAP GBM datasets revealed a similar conclusion that FABP4 was dramatically upregulated in mesenchymal subtype, rather than classical and proneural subtypes (Fig. 2c). Moreover, gene expression of mesenchymal markers ACTA2 and S100A4 were positively associated with FABP4 (Fig. 2d). These results revealed a close association between FABP4 and mesenchymal phenotype in glioma.Fig. 2 FABP4 is closely associated with mesenchymal subtype in GBM. (a) Fluorescence staining of FABP4 and vimentin in GBM samples. (b) Fluorescence staining of FABP4 and α-SMA in GBM samples. (c) FABP4 mRNA expression in different molecular subtypes of GBM samples from TCGA, CGGA and Ivy database. (d) Correlation between the expression of FABP4 and EMT-related markers (ACTA2 and S100A4) in TCGA GBM dataset was evaluated. Results are represented as Mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 2

FABP4 enhances the invasive potential of glioma cells

To explore the effect of FABP4 in glioma cell invasion, we artificially upregulated or downregulated FABP4 in primary glioma cells and examined in vitro biological characteristic. Since actin cytoskeleton remodeling is required for cell invasion and EMT process [21], actin staining by phalloidin was used to evaluate cell invasiveness in the present study. Upregulated FABP4 expression significantly increased filopodia formation in glioma cells (Fig. 3a). Loss of function assay indicated that FABP4 downregulation dramatically impaired the filopodia formation (Fig. 3b). Similarly, number of filopodia growed when treated with recombinant human FABP4 protein (Fig. 3c). Next, the role of FABP4 in glioma cell invasion was analyzed using a Transwell system. Elevated FABP4 expression contributed significantly to enhanced cell invasiveness (Fig. 3d). Gene set enrichment analysis (GSEA) based on TCGA GBM datasets revealed that cancer cell invasiveness signature was significantly correlated to FABP4 expression (Fig. 3e).Fig. 3 FABP4 confers increased invasiveness in glioma cells. (a) primary glioma cells were transfected with empty vector or FABP4-expressing lentiviral vector, fluorescent staining of FABP4 and Phalloidin were assessed. White arrowheads indicate filopodia on glioma cells. (b) Glioma cells were treated with PBS or rhFABP4, FABP4 and Phalloidin expressions were evaluated. (c) primary glioma cells with scrambled shRNA or shFABP4 were stained with FABP4 and Phalloidin. (d) Invasion abilities of glioma cells with FABP4 expression were assessed. (e) Gene set enrichment analysis (GSEA) was performed according to FABP4 expression in TCGA GBM dataset showed enriched pathways associated with cell invasiveness. Results are represented as Mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3

FABP4 promotes EMT process in glioma cells

To further explore FABP4-mediated biological function, GBM patients from TCGA database were clustered into two groups based on FABP4 expression and differential gene expression was performed (Fig 4a). EMT-related genes such as FN1, ACTA2 were significantly upregulated in high-FABP4 group (Fig 4a). Gene set enrichment analysis (GSEA) between high and low FABP4 expression groups was conducted. EMT-associated pathway was significantly enriched in high-FABP4 group (Fig. 4b and Supplementary Fig. 1). Immunoblot and immunofluorescence assay revealed that FABP4 upregulation in primary glioma cells led to increased expression of N-Cadherin, vimentin, α-SMA (Fig. 4c and d). Conversely, FABP4 downregulation resulted in decreased expression of N-Cadherin and vimentin (Fig. 4e). And immunofluorescence staining in glioma cells revealed a similar result (Fig. 4f). These data suggested that FABP4 contributed to EMT process in glioma cells.Fig. 4 FABP4 induces EMT process in GBM. (a) differential gene expression analysis between high and low FABP4 groups from TCGA GBM dataset. Several key molecules related to EMT are indicated. (b) GSEA analysis reveals a significant correlation between FABP4 expression and EMT process in TCGA GBM dataset. (c) Expression of EMT-related molecules in glioma cells that transfected with FABP4-lentiviral vector or treated with recombinant human FABP4 protein. (d) Immunofluorescence analysis of N-Cadherin and Vimentin in glioma cells with FABP4 over-expression. (e) Immunoblot analysis of N-Cadherin and Vimentin in glioma cells when FABP4 is downregulated. (f) Immunofluorescence analysis of N-Cadherin in glioma cells treated with shRNA targeting FABP4. Scale bar=20 μm. Results are represented as Mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Fig 4

FABP4 accelerates EMT process via mediating CD36 expression

To reveal the FABP4-mediated molecules, we carried out protein interaction analysis via STRING and identified ten molecules (Fig. 5a). Among these potential targets, CD36 and PPARG mRNA expression were significantly upregulated in high-FABP4 group in TCGA GBM dataset (Fig. 5b and Supplementary Fig. 2). Interestingly, our results indicated that manipulated upregulation or downregulation of FABP4 influenced CD36 expression in both transcriptional and protein levels, while had no significant impact on PPARG (Fig. 5c and d). In CGGA and TCGA glioma datasets, CD36 expression was strongly associated with tumor grade (Fig. 5e and Supplementary Fig. 3a), and was elevated in mesenchymal subtypes (Fig. 5f and Supplementary Fig. 3b). Additionally, CD36 could serve as an independent survival factor predicting poor prognosis in glioma patients (Fig. 5g and Supplementary Fig. 3c). A significantly positive correlation between FABP4 and CD36 gene expression was also found in glioma datasets (Fig. 5h). FABP4-induced CD36 expression was further strengthened by immunofluorescence method (Fig. 5i). Moreover, when CD36 was knocked down in glioma cells, it impaired FABP4-induced expression EMT-related proteins (Fig. 5j), which was also revealed in immunostaining method (Fig. 5k). Furthermore, CD36 knockdown also diminished FABP4-induced filopodia formation in glioma cells (Fig. 5l).Fig. 5 FABP4-induced EMT is mediated in CD36-dependent manner. (a) Protein-protein interaction analysis of FABP4 via using STRING database. (b) CD36 mRNA expression in high or low FABP4 group from TCGA GBM dataset. (c) Immunoblot analysis of CD36 and PPARG in glioma cells with FABP4 upregulation or downregulation. (d) Transcriptional levels of CD36 and PPARG in glioma cells upon FABP4 overexpression. (e) CD36 mRNA expression in glioma samples with different grades. (f) CD36 mRNA expression in three GBM subtypes. (g) Kaplan-Meier survival analysis for CD36 in patients with glioma from TCGA dataset. (h) Correlation between FABP4 and CD36 in TCGA glioma dataset. (i) Immunofluorescence staining of CD36 in glioma cells with FABP4 upregulation. (j) Indicated proteins in glioma cells treated with exogenous FABP4 followed by CD36 knockdown. (k) Immunofluorescence staining of N-Cadherin and Vimentin in FABP4-expressed glioma cells treated with CD36 downregulation. (l) Glioma cells with stable FABP4 expression are treated with CD36 knockdown, F-actin staining is detected with phalloidin. White arrowheads indicate filopodia on glioma cells. Scale bar=50 μm. Results are represented as Mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Fig 5

CD36 promotes EMT process via activating non-canonical TGFβ pathways

Based on The Cancer Genome Atlas (TCGA) project, The Cancer Proteome Atlas (TCPA) provides protein expression data and analysis covering major signaling pathways using reverse-phase protein arrays (RPPAs). In silico analysis of GBM TCPA data revealed that SERPINE1 (PAI.1) and Fibronectin were highly expressed in samples with high FABP4 expression (Fig. 6a). TGFβ1 has been well recognize as a classic and strong promoter of EMT [22]. And binding of TGFβ1 with receptors initiates canonical and (or) non-canonical signaling pathways [23]. While TGFβ treatment caused increased p-Smad3 expression, those with stable CD36 expression did not affect p-Smad3 expression (a critical indicator of the canonical TGFβ-smad2/3 signaling), however, led to activation of RhoA/ROCK and ERK pathways (non-canonical TGFβ signaling) (Fig. 6b and Supplementary Fig. 4). Upregulated CD36 expression in glioma cells resulted in higher fluorescence intensity of ROCK1 that revealed by ICC (Fig. 6c). Furthermore, immunoblotting assays demonstrated that FABP4 treatment effectively increased CD36 expression and activation of RhoA/ROCK and JNK signaling (Fig. 6d and e), while had no impact on p-Smad3 expression (Fig. 6e). Moreover, as the fact that FABP4 protein stimulated glioma cell invasion, CD36 knockdown would impair pro-invasive capacity of FABP4 (Fig. 6f).Fig. 6 CD36 enhances EMT process by activating non-canonical TGFβ pathways. (a) Association between CD36 mRNA level and EMT proteins (PAI.1, fibronectin) in TCPA. (b) Primary glioma cells with empty vector or CD36 transfection were treated with 100 pM TGFβ1 for 6 h, indicated proteins of canonical and non-canonical TGFβ pathways were examined. (c) Immunofluorescence staining of ROCK1 and CD36 in glioma cells with EV or CD36 transfection. (d) PGCs with (without) CD36 knockdown were treated with recombinant human FABP4 proteins at different time point, expressions of indicated proteins were analyzed by immunoblotting. (e) PGCs with CD36 downregulation were stimulated with rhFABP4, immunofluorescence stainings of ROCK1 and p-Smad3 were evaluated. (f) PGC-shScr or PGC-shCD36 was added with rhFABP4, cell invasiveness and filopodia formation were measured. Results are represented as Mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 6

FABP4 mediates EMT process in intracranial xenografts

To demonstrate in vivo effect of FABP4, intracranial xenograft model was established using shFABP4 glioma cells or shScr glioma cells as control group. Bioluminescent imaging revealed that mice with shFABP4-tumor cells exhibited a comparatively reduced size of tumor mass in brain (Fig. 7a and b). More importantly, FABP4 downregulation led to decreased expression of N-Cadherin and vimentin (Fig. 7c). While intracranial xenograft with shScr glioma cells showed an irregular tumor edge, shFABP4-exnograft exhibited a smooth tumor edge, indicating that targeting FABP4 effectively impaired tumor invasiveness (Fig. 7d).Fig. 7 Targeting FABP4 effectively reduces tumor progression and EMT in a xenograft model. (a) Luminescent imaging of representative nude mice xenografts from mCherry-LUC-labeled shScr (n = 5) or shFABP4 PGC#2 (n = 5) at day 3 and 25. (b) Luminescent signal intensity of GBM-bearing mice in two groups were evaluated. (c) Representative immunofluorescence images of FABP4, N-Cadherin, Vimentin and α-SMA. Scale bar=20 μm. (d) Sample sections from control (upper) and shFABP4 tumors (bottom) were immunofluorescently labeled with mCherry. (e) Schematic diagram illustrating the FABP4-CD36 signaling pathway in EMT regulation. Results are represented as Mean ± SD of biologically triplicate assays. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 7

Discussion

In the present study, we provided evidences demonstrating that FABP4 exhibits a pivotal role in EMT and cell invasiveness of GBM cells. Mechanistic investigation indicated that FABP4 promotes EMT process primarily via elevating CD36 expression, which subsequently augmented non-canonical TGFβ signaling pathways (Fig. 7e).

Our results revealed that FABP4 was abundantly expressed in GBM, when compared to lower grade glioma. FABP4 has recently been reported to be upregulated in multiple kinds of tumors and closely associated with progression and adverse prognosis [9,10,[24], [25], [26]]. In this research, FABP4 expression in collected glioma samples was determined by IHC staining. IHC results suggested that FABP4 staining intensity was significantly associated with tumor grade. The Cancer Genome Atlas network firstly classified GBM into four molecular subtypes including proneural, neural, mesenchymal and classical [27]. Molecular classification was simplified to proneural, mesenchymal and classical subtypes in further researches. Among these, the mesenchymal subtype correlated with more aggressive, angiogenic, inflammatory features, and thus a worse prognosis [28]. Proneural GBM subtype is linked to less proliferative and related to a long survival time. The classic subtype is characterized by a high rate of EGFR amplification, while lacked IDH1 abnormality, which is frequently observed in proneural and mesenchymal subtypes. Our data showed that FABP4 was closely related with mesenchymal phenotype, indicating an association between FABP4 and aggressive feature. These evidences together revealed FABP4 as an unfavorable role in GBM progression and prognosis.

In vitro experiment results showed that FABP4 expression enhanced GBM cell invasive ability, while has no significant effect on cell proliferation. GSEA analysis based on TCGA database also confirmed the pro-invasion feature of FABP4. FABP4-induced cell invasiveness was recently reported in colon cancer, via increasing free fatty acids transportation [29]. Our data showed that FABP4 was primarily concerned with GBM cell invasion, rather than cell proliferation. However, FABP4 was reported to regulate endothelial cell proliferation and angiogenic capacity, indicating that FABP4 exerted different biological effects among different cell types.

According to previous studies, FABP4 expression could regulate tumor cell proliferation, invasion and metastasis through multiple signaling pathways. For instance, FABP4 in prostate cancer promoted AKT and ERK signaling activation, which resulted in tumor cell invasiveness [8]. Interestingly, reports from Li et.al indicated that FABP4 accelerates glioma cell proliferation in a Wnt10-dependent manner [14], suggesting a critical role of Wnt/β-catenin signaling in FABP4-induced molecular pathways, as Wnt10 have been regarded as a canonical β-catenin-dependent Wnt ligand [30]. Yet the report from Li et.al did not provide sufficient evidences that FABP4 triggers canonical Wnt/β-catenin signaling in glioma cells. The downstream pathways of FABP4-Wnt10b thus need to be further explored. Combined with these findings, robust FABP4 expression in glioblastoma cells could lead to upregulated Wnt10b and CD36 gene expression. Further investigation would be focused on the interactions of theses FABP4-induced molecules. In fact, CD36 is an adipocyte progenitor marker, most likely through facilitating lipid uptake [31]. FABP4-Wnt10b In a research on determining the impact of Wnt10b on the function of brown adipose tissue, authors established Wnt10b+/− mice and obtained primary brown adipocyte [32]. Their data showed significant decrease of CD36 gene expression in brown adipocyte with Wnt10b knockdown [32], indicating a interaction between Wnt10 and CD36. Based on these findings, we would speculate that FABP4 might activate Wnt10-CD36 signaling to modulate glioma cell invasion. Protein interaction analysis using STRING database was conducted to determine FABP4-regulated molecules. FABP1, PPARG, SCARB2, GOT2, LIPE, ADIPOQ, CD36, LPL, PLIN1 and CEBPA were listed as potential candidates for FABP4-mediated targets. Among these, CD36 and PPARG was the sole one that influenced by FABP4 and elevated in mesenchymal GBM subtype. Previous studies have defined CD36 as a promoter in cancer EMT process. CD36 in cervical cancer synergized with TGFβ to promoted EMT [33]. And CD36 was significantly associate with EMT in hepatocellular carcinoma [34]. Moreover, CD36 was reported to be a potential marker to identify glioblastoma stem cell population. Multi-dimensional analysis suggested that CD36 was consistently expressed in glioma-spheres [35]. CD36 in endothelial cell is a receptor for thrombospondin-1 and evolved in angiogenesis regulation [36]. Both CD36 and FABP4 are required for fatty acid transportation across the vessel wall and are responsive to endothelial Notch signaling [37]. And CD36 in endothelial cells is critical for FA uptake [38]. Although a study from Berger and colleagues suggested that CD36 reduction by FABP4 regulated fat mass homeostasis [39]. Interaction between CD36 and FABP4 in angiogenesis or fat acid metabolism were rarely reported. Interestingly, a recent report revealed that fatty acid metabolism in breast cancer is mediated by CD36 through direct interaction with FABP4 [40]. And we provided evidences in the present study that FABP4-CD36 axis in glioma cells promotes tumor invasion. While mechanism regarding interaction between FABP4 and CD36 requires further investigation.

In summary, our study depicted a pro-invasive role of FABP4 in glioblastoma cells. We provided evidence that FABP4 expression was associated with EMT process and thus contributed to tumor progression. Moreover, our data demonstrated that FABP4-mediated EMT was primarily regulated via upregulating CD36 expression and subsequently activated non-canonical TGFβ signaling pathways. Therefore, FABP4 is a potential therapeutic target in glioma and deserves further investigation.

Data availability

All data generated during this study are included either in the main article or in the supplementary information files.

Disclosure

None of the authors of this manuscript is a current Editor or Editorial Board Member of Cancer Science.

Funding information

This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China (No. 82002631 ), President Foundation of ZhuJiang Hospital, 10.13039/501100010096 Southern Medical University (No. yzjj2022ms07 ) and Guangzhou Basic and Applied Basic Research Scheme (No. 2024A04J4879).

Ethics statement

Animal study was performed with the permission of the Animal Care and Use Committee of Southern Medical University. Research protocol and informed consent were approved by the Ethical Committee of Zhujiang Hospital. Registry and the Registration No of the study/trial: N/A.

CRediT authorship contribution statement

Zhongsheng You: Software, Project administration, Methodology, Investigation, Data curation, Conceptualization. Zihao Hu: Visualization, Validation, Project administration, Methodology. Chongxian Hou: Methodology, Investigation, Formal analysis, Data curation. Chengcheng Ma: Software. Xiangdong Xu: Resources, Project administration. Yaofeng Zheng: Software. Xinlin Sun: Resources. Yiquan Ke: Visualization, Validation, Supervision. Jianli Liang: Formal analysis. Zijing Xie: Project administration. Lingling Shu: Visualization, Validation, Supervision, Funding acquisition, Conceptualization. Yang Liu: Writing – review & editing, Writing – original draft, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101050.
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