The immunoregulatory protein B7-H3 promotes aerobic glycolysis in oral squamous carcinoma via PI3K/Akt/mTOR pathway

Abstract

OSCC (oral squamous carcinoma) is one of most common malignant cancer. Although previous studies have found abnormal expression of B7-H3 in human OSCC, the exact role and molecular mechanism of B7-H3 in OSCC remain unknown. In this study, we investigated the role of B7-H3 in glucose metabolic reprogramming of OSCC cells in vitro and in vivo. We first detected the expression of B7-H3 in OSCC samples. Next, siRNAs and overexpression short-hairpin RNA of B7-H3 were transfected into SCC25 and Cal27 cells, and cell proliferation, migration and invasion were analyzed via CCK8, colony formation and transwell assays. Then glycolysis flux was determined through measuring glucose uptake and lactate production, and mRNA and protein expression levels were determined by real-time quantitative PCR and western blot respectively. The results presented here showed B7-H3 was upregulated in OSCC samples compared with normal tissues, and the expression level was associated with tumor size and nodal metastasis. B7-H3 affects OSCC cell proliferation, migration and invasion. We also found that B7-H3 promoted the Warburg effect, evidenced by increase glucose uptake and lactate production. We further demonstrated that B7-H3 enhanced OSCC glycolysis through the upregulation of HIF-1α and its downstream targets, Glut1 and PFKFB3, which are key factors in glycolysis. Mechanically, we demonstrated that B7-H3 regulates HIF-1α expression through PI3K/Akt/mTOR pathway. Metabolic imaging of human OSCC cancer xenograft in mice confirmed that B7-H3 enhanced tumor glucose uptake, glycolysis promoted genes expression and tumor growth. Taken together, our results have unveiled a mechanism that B7-H3 drives OSCC progression through enhancing of glycolytic metabolic program in OSCC.

Keywords: B7-H3; OSCC; PI3K/Akt/mTOR pathway; glycolysis; immunoregulatory protein.

Figure 1

Upregulation of B7-H3 in OSCC specimens. A B7-H3 protein expression in human OSCC and adjacent healthy tissues was assessed by IHC (weakly expressed in normal tissues, moderately expressed in OSCC1, intensely expressed in OSCC2). B B7-H3 mRNA expression in human OSCC and adjacent healthy tissues was assessed by qRT-PCR. B7-H3 mRNA expression was upregulated in human OSCC than adjacent healthy tissue. C and D The association between B7-H3 mRNA expression and the clinicopathological characteristics in human OSCC was analyzed. High expression of B7-H3 mRNA in human OSCC was associated with advanced T stage and lymph node metastasis. E B7-H3 mRNA expression in OSCC cell lines was upregulated than normal epithelial cell lines. F B7-H3 protein expression in OSCC cell lines was upregulated than normal epithelial cell lines.

Figure 2

B7-H3 silencing suppressed OSCC cell proliferation, migration and invasion. A B7-H3 mRNA was evaluated by qRT-PCR 48 h after siRNA transfection. siB7-H3 1 transfection significantly decreased B7-H3 expression at the mRNA level in SCC25 and Cal27 cells. B B7-H3 protein was evaluated by western blot 72 h after siRNA treatment. SiB7-H3 1 transfection significantly decreased B7-H3 expression at the protein level in SCC25 and Cal27 cells. C The proliferation rate was observed by CCK8 assays at 2 days and 5 days after B7-H3 silencing. B7-H3 silencing remarkably slowed the proliferation rate. D Colony formation assays showed that B7-H3 silencing inhibited the colony formation of SCC25 and Cal27 cells. E The migratory ability was evaluated by transwell assays. Silencing B7-H3 reduced the migration of SCC25 and Cal27 cells. F Invasion was evaluated with Matrigel-coated transwell assays. Silencing B7-H3 reduced the invasive ability of SCC25 and Cal27 cells.

Figure 3

B7-H3 overexpressing promoted OSCC cell proliferation, migration and invasion. A OSCC cell lines transfected with shRNA and selected with puromycin were observed under a fluorescence microscope: the transfection rates in both cell lines were more than 90%. B, C Overexpression of B7-H3 by shRNA in SCC25 and Cal27cells was validated by qRT-PCR and western blot. D Proliferation was determined by CCK8 assays at 2 days and 5 days after seeding. B7-H3 overexpression remarkably increased the proliferation rate. E Colony formation assays showed that B7-H3 overexpression promoted colony formation in OSCC. F Migration was evaluated by transwell assays. Overexpression of B7-H3 increased the migratory ability of SCC25 and Cal27 cells. G Invasion was evaluated with Matrigel-coated transwells. Overexpression of B7-H3 increased the invasive ability of SCC25 and Cal27 cells.

Figure 4

B7-H3 regulated aerobic glycolysis in OSCC cells. Glucose uptake and intracellular lactate production were detected in B7-H3-silenced cells and B7-H3-overexpressing cells. A, B B7-H3 silencing suppressed glucose uptake of and intracellular lactate production in SCC25 and Cal27 cells. C, D Overexpression of B7-H3 promotes glucose uptake and intracellular lactate production in SCC25 and Cal27.

Figure 5

B7-H3 regulates glycolysis through HIF-1α and its downstream targets. QRT-PCR and western blot were used to determine the mRNA and protein expression of HIF-1α and key factors regulated by B7-H3 silencing and overexpression. A Silencing B7-H3 downregulated the mRNA level of PFKFB3 and Glut1 without affecting HIF-1α mRNA level. B Overexpression of B7-H3 upregulated the mRNA of PFKFB3 and Glut1 without affecting HIF-1α mRNA level. C Silencing B7-H3 downregulated the protein level of HIF-1α and its downstream targets PFKFB3 and Glut1. D Overexpression of B7-H3 upregulated the protein level of HIF-1α and its downstream targets PFKFB3 and Glut1. E Western blot analysis of HIF-1α, PFKFB3 and Glut1 expression in B7-H3-overexpressing cells when HIF-1α was silenced by siRNA. Silencing HIF-1α downregulated the expression of PFKFB3 and Glut1 in B7-H3-overexpressing cells. F, G Glucose uptake and intracellular lactate production were detected in B7-H3-overexpressing cells when HIF-1α was silenced. Silencing HIF-1α decreased glucose uptake and lactate production in B7-H3-overexpressing cells.

Figure 6

B7-H3 regulates aerobic glycolysis of OSCC through PI3K/Akt/mTOR signaling. A The protein expression of PI3K, Akt, and mTOR 72 h after siRNA treatment using siB7-H3 1 or siNC was determined via western blot. Silencing B7-H3 downregulated the expression of p-PI3K, p-Akt and p-mTOR. B The protein expression levels of PI3K, Akt, and mTOR in B7-H3-overexpressing and normal control cells were determined via western blot. Overexpression of B7-H3 upregulated the expression levels of p-PI3K, p-Akt and p-mTOR. C Protein expression of Akt, mTOR, HIF-1α and downstream glycolysis key factors in B7-H3-overexpressing cells was determined via western blot 3 h after blockade of the PI3K/Akt signaling pathway using LY294002, API-2 and rapamycin.

Figure 7

B7-H3 promotes glucose uptake and tumor growth in OSCC tumor xenografts. Cal27 B7-H3-overexpressing OSCC cells were subcutaneously implanted in athymic nude mice (n=4/group). A Images of the tumor specimens. B Tumor volume was monitored by caliper measurements for 6 weeks after injection. C Average tumor weight measured at 6 weeks after injection. Mice in the B7-H3-overexpressing group had a larger tumor weight. D Glucose uptake was measured in vivo at 6 weeks after tumor cell injection using the fluorescent probe 2-DG-750, as described in Materials and Methods (n=3/group; two of the mice died before fluorescent imaging). IVIS images of 2-DG-750 uptake in each individual mouse. Average of total flux (photons/second) indicates the intensity of 2-DG-750 uptake at the primary site of cell injection. Higher 2-DG-750 uptake in the B7-H3-overexpressing group was detected. E The expression levels of B7-H3, HIF-1α and PFKFB3 in implanted tumors were detected by IHC. Intense B7-H3 staining in the B7-H3-overexpressing group and weak B7-H3 staining in the control group were observed at the end point. Similarly, the expression levels of HIF-1α and PFKFB3 in the B7-H3-overexpressing group were higher than those in the control group.