TRIM27 promotes the Warburg effect and glioblastoma progression via inhibiting the LKB1/AMPK/mTOR axis

Abstract

Altered protein ubiquitination is associated with cancer. The novel tripartite motif (TRIM) family of E3 ubiquitin ligases have been reported to play crucial roles in the development, growth, and metastasis of various tumors. The TRIM family member TRIM27 acts as a potential promoter of tumor development in a wide range of cancers. However, little is known regarding the biological features and clinical relevance of TRIM27 in glioblastoma (GBM). Here, we report findings of elevated TRIM27 expression in GBM tissues and GBM cell lines. Further functional analysis showed that TRIM27 deletion inhibited GBM cell growth both in vitro and in vivo. Furthermore, we found that TRIM27 promoted the growth of GBM cells by enhancing the Warburg effect. Additionally, the inactivation of the LKB1/AMPK/mTOR pathway was critical for the oncogenic effects of TRIM27 in GBM. Mechanistically, TRIM27 could directly bind to LKB1 and promote the ubiquitination and degradation of LKB1, which in turn enhanced the Warburg effect and GBM progression. Collectively, these data suggest that TRIM27 contributes to GNM pathogenesis by inhibiting the LKB1/AMPK/mTOR axis and may be a promising candidate as a potential diagnostic and therapeutic marker for patients with GBM.

Keywords: AMPK; Glioblastoma; LKB1; TRIM27; Warburg effect.

Figure 1

TRIM27 is elevated in GBM tissues. (A) A heat map of differentially expressed TRIM family genes between GBM (n=169) and normal brain tissue (n=5) from the TCGA-GBM dataset. (B) Wayne plots showing eight genes that showed significantly different expression in the TCGA-GBM, GSE108474, and GSE108474 datasets. (C) TRIM27 mRNA expression in 30 GBM tissues and 6 NBT samples was determined by qRT-PCR analysis. **P<0.01. (D and E) Analysis (D) and quantification (E) of TRIM27 protein levels in GBM tissues and normal brain tissues by western blotting assay. Tubulin was used as a loading control. **P<0.01. (F) Representative immunostaining of TRIM27 protein expression in human glioma and normal brain tissue (NBT) samples (images from the Human Protein Atlas). (G and H) Representative immunostaining (G) and quantification (H) of TRIM27 in human gliomas of different grade and normal brain tissue (NBT) samples (magnification: 100 × and 200 ×). Scale bar, 50 μm. (I and J) TRIM27 protein and mRNA levels were detected in normal human astrocytes (NHAs) and glioma cell lines U251, BG7, U118MG, BG5, and LN229.

Figure 2

Effects of TRIM27 expression on GBM cell proliferation. (A) Protein levels of TRIM27 were assessed in U251 and U118MG cells transfected with shNC or shTRIM27 plasmids by western blotting assay. (B and C) The mRNA levels of TRIM27 were detected U251 and U118MG cells transfected with shNC or shTRIM27. **P<0.01. (D-G) EdU (D and E) and CCK-8 assay (F and G) showing proliferation of GBM cells following knockdown of TRIM27. Scale bar, 50 μm. *P<0.05, **P<0.01. (H-K) Representative images (H and J) and quantification (I and K) of colony formation assays using GBM cells transfected with shTRIM27 plasmids. *P<0.05, **P<0.01. (L-N) U251/shTRIM27 cells were subcutaneously injected into nude mice. Tumor volumes were measured on the indicated days. At the experimental endpoint, tumors were dissected, photographed, and weighed. Scale bar, 50 μm. n=6, *P<0.05, **P<0.01. (O) Ki67 staining using tumor tissues isolated from nude mice injected with TRIM27-silenced cells. Scale bar, 50 μm.

Figure 3

TRIM27 promotes the growth of GBM cells by enhancing glycolysis. A-D. Glucose-6-phosphate (G6P) production, glucose consumption, lactate production, and ATP levels in TRIM27-silenced GBM cells. *P<0.05. E-H. ECAR data showing the glycolytic rate and capacity in TRIM27-silenced GBM cells. Glucose (10 mM), the oxidative phosphorylation inhibitor oligomycin (1.0 μM), and the glycolytic inhibitor 2-deoxyglucose (2-DG, 50 mM) were sequentially added to each well at the indicated time points. *P<0.05. I-L. OCR results showing basal and maximum respiration in TRIM27-silenced GBM cells. Oligomycin (1.0 μM), the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP, 1.0 μM), and the mitochondrial complex I inhibitor rotenone plus the mitochondrial complex III inhibitor antimycin A (Rote/AA, 0.5 μM) were sequentially added. *P<0.05. M-P. Effects of 2-DG on the viability of TRIM27-silenced GBM cells. *P<0.05, **P<0.01.

Figure 4

TRIM27 promotes glycolysis by regulating LKB1/AMPK/mTOR pathway. (A and B) Protein levels of LKB1, total AMPK, p-AMPK, total mTOR, and p-mTOR were assessed by western blotting in TRIM27-silenced U251 cells (A) and TRIM27-overexpression U251 cells (B). (C) Western blotting showing the protein expression of TRIM27 and LKB1 in each group. (D and E) Glucose uptake, G6P production, and lactate and ATP levels were measured following knockdown of LKB1 in TRIM27-silenced U251 cells. (F and G) ECAR and OCR were measured after LKB1 knockdown in TRIM27-silenced U251 cells. (H-J) Quantification of CCK8 (H), EdU (I), and colony formation (J) assays using TRIM27-silenced U251 cells transfected with a shLKB1 construct. *P<0.05, **P<0.01. (K and L) The quantification of tumor weight (K) and tumor volume (L) in different groups. *P<0.05, **P<0.01. (M) Western blotting showing the protein expression of TRIM27 and p-mTOR in TRIM27-overexpression U251 cells treated with Met. (N and O) Glucose uptake, the production of G6P, and lactate and ATP levels were measured in TRIM27-overexpression U251 cells treated with Met. (P and Q) ECAR and OCR were measured in TRIM27-overexpression U251 cells treated with Met. (R) Quantification for CCK8 assays in TRIM27-overexpression U251 cells treated with Met. *P<0.05, **P<0.01.

Figure 5

TRIM27 interacting with LKB1 and destabilising its expression. (A and B) The interaction between TRIM27 and LKB1 was confirmed by co-IP assays in U251 and U118G cells. (C) Docking analysis for the binding of TRIM27 and LKB1. (D-G) Representative (D and F) and quantitative (E and G) results of LKB1 protein levels in TRIM27-silenced GBM cells. The cells were treated with cycloheximide (CHX, 100 mg/ml) at the indicated time points and then harvested for western blot analysis. **P<0.01. (H and I) GBM cells transfected with shTRIM27 (H) or TRIM27-overexpression plasmids (I) were treated with 10 μM MG132. The cells were then collected after 6 hours and immunoblotted with the indicated antibodies. (J and K) Lysates from U251 cells transfected with TRIM27-overexpression plasmid (J) or shTRIM27 (K) were immunoprecipitated with the anti-Ub and immunoblotted with the anti-LKB1. (L) Determination of LKB1 ubiquitination type in HEK293 cells.

Figure 6

The proposed mechanistic scheme of how TRIM27 promotes GBM progression by enhancing glycolysis via modulating the LKB1/AMPK/mTOR pathway.