Deubiquitinase USP33 promotes the glycolysis and growth of osteosarcoma by modifying PFKFB3 ubiquitination and degradation

Abstract

Osteosarcoma (OS) is the most common malignant tumor of the bone tissue with the lowest survival rate among all pediatric cancers. OS cells grow vigorously under malnutrition; however, the mechanism by which they adapt to metabolic stress via metabolic reprogramming remains undefined. Here, we demonstrated that USP33, a member of the DUBs family, was significantly upregulated in the tissues of patients with OS compared to normal tissues. Moreover, high USP33 expression was significantly associated with poor survival. Functional assays suggested that USP33 promoted OS cell growth through the induction of aerobic glycolysis. Additionally, we confirmed that 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) was critical for USP33-induced proliferation and aerobic glycolysis in OS cells, and the protein expression levels of PFKFB3 and USP33 were positively correlated in the OS tissues. Mechanistically, USP33 stabilized the expression of PFKFB3 by suppressing the ubiquitin mediated PFKFB3 degradation. Collectively, these findings reveal a mechanism by which OS cells survive in a dystrophic tumor microenvironment, with the USP33-PFKFB3 axis as a critical driver of aerobic glycolysis and OS proliferation. Furthermore, these findings reveal novel insights into the adaptation of cancer cells to metabolic stress in OS.

Keywords: Osteosarcoma; PFKFB3; USP33; deubiquitinase; ubiquitination.

Figure 1

Overexpression of USP33 in osteosarcoma (OS) is strongly associated with the poor prognosis of patients. A, B. The USP33 mRNA levels in OS tissues and the adjacent normal tissues was analyzed by qRT-PCR. ***P < 0.001. C. The USP33 protein levels in OS tissues and adjacent normal tissues were determined by western blot. **P < 0.01. D. Representative images of IHC and H&E staining of USP33 in OS tissues and adjacent normal tissues. Scale bar, 20 μm. E, F. According to the expression level of USP33, Kaplan-Meier plots showed the probability of progression-free and overall survival of patients with OS.

Figure 2

USP33 promotes osteosarcoma (OS) cell proliferation. A, B. The USP33 expression levels in normal hfoBI-19 osteoblasts and four OS cell lines determined by qRT-PCR and western blot. C. The protein levels of USP33 in USP33 knockdown Saos-2 and U2OS stable cells determined by western blot. D, E. The mRNA levels of USP33 in USP33 knockdown Saos-2 and U2OS stable cells determined by qRT-PCR. F, G. The viability of USP33 knockdown Saos-2 and U2OS stable cells determined by CCK-8 assay. *P < 0.05, **P < 0.01. H. The protein levels of USP33 in p-USP33-overexpressing 143B and MG-63 stable cells determined by western blot. I, J. The viability of p-USP33-overexpressing 143B and MG-63 stable cells determined by CCK-8 assay. *P < 0.05, **P < 0.01. K. Colony formation analysis of USP33 knockdown U2OS stable cells. **P < 0.01. L. Colony formation analysis of USP33 knockdown MG-63 stable cells. **P < 0.01.

Figure 3

USP33 promotes osteosarcoma (OS) proliferation by enhancing the Warburg effect. A. Cellular glucose consumption, G6P levels, ATP levels and lactate generation in USP33 knockdown U2OS cells. *P < 0.05. B. Cellular glucose consumption, G6P levels, ATP levels and lactate generation in p-USP33-overexpressing MG-63 stable cells. *P < 0.05. C, D. ECAR data suggested the capacity and glycolytic rate in USP33 knockdown U2O cellss. *P < 0.05. E, F. OCR outcomes reveal maximum respiration and basal respiration in USP33 knockdown U2O cells. G, H. ECAR data exhibiting the capacity and glycolytic rate in p-USP33-expressing MG-63 cells. *P < 0.05. I, J. OCR outcomes reveal maximum respiration and basal respiration in p-USP33-expressing MG-63 cells.

Figure 4

USP33 positively modulates the protein levels of PFKFB3 in osteosarcoma (OS) cells. A. Western blot analysis of PFKFB3 and USP33 expression in USP33 knockdown U2OS cells. B. Western blot analysis of PFKFB3 and USP33 expression in p-USP33-expressing MG-63 cells. C. qRT-PCR analysis for USP33 and PFKFB3 mRNA expression in USP33 knockdown U2OS cells. D. qRT-PCR analysis for the mRNA expression of PFKFB3 and USP33 in p-USP33-expressing MG-63 cells. E, F. PFKFB3 protein expression in OS tissues and adjacent normal tissues determined by western blotting. G. Scatter plots exhibited a positive association between PFKFB3 and USP33 protein level in OS tissues. *P < 0.05.

Figure 5

USP33 interacts with PFKFB3 and stabilises the expression of PFKFB2. A, B. Co-IP analyse indicated the interaction between PFKFB3 and USP33 in MG-63 and U2OS cells. C, D. 3D structure of PFKFB3 and USP33. USP33 and PFKFB3 were reflected in red and blue, respectively. E, F. The plasmid p-USP33 was transfected into MG-63 cells. Subsequently, MG-63 cells were exposed to CHX at a specific time, and the degradation of PFKFB3 was detected using Western blot analysis. *P < 0.05. G. MG132 was added to MG-63 cells, and the expression of USP33 was altered. The protein expression level of PFKFB3 was determined using Western blot. H. p-USP33 plasmid was transfected into MG-63 cells in the presence of MG132. Western blot analysis was performed to examine PFKFB3 ubiquitination. I. The shUSP33 plasmid was transfected into U2OS cells in the presence of MG132. Western blot analysis was performed to examine PFKFB3 ubiquitination.

Figure 6

PFKFB3 is critical for the USP33-induced increase of aerobic glycolysis and proliferation in osteosarcoma (OS) cells. A. The expression of PFKFB3 and USP331 in various groups was determined by western blot. B. CCK-8 analysis of U2OS cells transfected by a specific plasmid. *P < 0.05. C. Colony formation analysis of U2OS cells that were transfected by a specific plasmid. *P < 0.05. D. G6P levels, cellular glucose consumption, lactate formation and ATP levels in the indicated group. *P < 0.05. E. ECAR showed the capacity and glycolytic rate in the indicated group. *P < 0.05. F. OCR displayed the maximum respiration and the basal respiration in the indicated group. G, H. Tumor volume quantification or tumor weight determination was performed in the different groups. *P < 0.05. I. Diagram of the regulatory mechanism of USP33 in promoting the glycolysis and the growth of OS by modifying PFKFB3 ubiquitination and degradation.