Inhibition of Nogo-B reduces the progression of pancreatic cancer by regulation NF-κB/GLUT1 and SREBP1 pathways

Abstract

Pancreatic cancer (PC) is a lethal disease and associated with metabolism dysregulation. Nogo-B is related to multiple metabolic related diseases and types of cancers. However, the role of Nogo-B in PC remains unknown. In vitro, we showed that cell viability and migration was largely reduced in Nogo-B knockout or knockdown cells, while enhanced by Nogo-B overexpression. Consistently, orthotopic tumor and metastasis was reduced in global Nogo knockout mice. Furthermore, we indicated that glucose enhanced cell proliferation was associated to the elevation expression of Nogo-B and nuclear factor κB (NF-κB). While, NF-κB, glucose transporter type 1 (GLUT1) and sterol regulatory element-binding protein 1 (SREBP1) expression was reduced in Nogo-B deficiency cells. In addition, we showed that GLUT1 and SREBP1 was downstream target of NF-κB. Therefore, we demonstrated that Nogo deficiency inhibited PC progression is regulated by the NF-κB/GLUT1 and SREBP1 pathways, and suggested that Nogo-B may be a target for PC therapy.

Keywords: Cancer; Cell biology; Molecular biology.

Graphical abstract

Figure 1

Nogo-B is upregulated in PC (A) Expression of Nogo in PC and adjacent tissues was collected from TCGA and GTEx (n = 179 for PC tissues, n = 171 for adjacent tissues). (B) Association between Nogo expression and PC prognosis was analyzed by Kaplan-Meier analysis based on GEPIA data. (n = 178). (C) Expression of Nogo-B in cancer tissues and adjacent non-cancerous tissues (n = 30) were detected by IHC staining, and IHC scores were quantified by ImageJ software, scale bar: 100 μm. The data represent the mean ± SD, ∗p < 0.05, ∗∗p < 0.01.

Figure 2

Nogo-B is corrected with PC cells proliferation and migration (A–D) Pan02 and Bxpc-3 cells were transfected with Nogo-B siRNA for 24 h, then treated in serum free (SF) medium for 12 h (A), 7 days (B and C) or indicated time (D). Cell migration, proliferation was assessed using scratch assay (A), crystalline violet staining (B and C) and MTT method (D). (E–G) Nogo-B knockout cell line using CRISPR-cas9 technology was named as KO#1, KO#2. Cells were cultured in SF medium for 12 h (E and F) or 7 days (G) then used to detect cell migration or proliferation by scratch assay (E) or crystalline violet staining (G). (J–L) Knockdown/knockout or overexpressed cells were treated in SF medium for 24 h, protein expression of proliferation and apoptosis-related genes was determined by Western blot with quantitative analysis of band intensity. Scale bar: 100 μm (A, E and H), 2 mm (B and I), 1 cm (G). The data represent the mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3.

Figure 3

Knockout of Nogo-B inhibits PC proliferation and metastasis in vivo (A) Orthotopic tumors were excised 13 days after inoculation with the indicated Pan02 cells or Nogo KO Pan02 cells (left panel) and the tumors were weighted (right panel). (B) Ectopic tumors in liver were photographed (left panel) and the number of tumors were counted (right panel). (C) Orthotopic tumor sections were conducted with TUNEL staining with quantitative analysis of apoptosis cells, TUNEL positive cells were indicated by the white arrows. (D) CDH1 and CDH2 expression in orthotopic tumor sections was detected by IHC staining with quantitative analysis of MD. Scale bar: 1 cm (B), 100 μm (C), 50 μm (D). The data represent the mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 6.

Figure 4

High glucose promotes tumor growth in PC cells through Nogo-B Pan02 or Nogo-B KO Pan02 (KO#1) cells were treated with indicated concentration of glucose for 24 h. Cell proliferative and migratory abilities were determined by MTT (A), scratch assay (B) and crystalline violet staining (D). EMT, proliferation and apoptosis-related genes expression was detected by Western blot with quantitative analysis of band intensity (C and E). Scale bar: 100 μm (B), 2 mm (D). The data represent the mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3.

Figure 5

Nogo-B promotes PC progression through activation NF-κB pathway Pan02 or Nogo-B KO Pan02 (KO#1) cells were treated with indicated concentration of glucose for 24 h (A) or transfected with NF-κB overexpression plasmid or Bay for 24 h, then treated with indicated concentration of glucose for 24 h (B, D and E) or 7 days (C). Protein expression was detected by Western blot with quantitative analysis of band intensity (A, D and E), colony forming migratory abilities were assessed by scratch assay (B) and crystal violet staining (C). Scale bar: 100 μm (B), 2 mm (C). The data represent the mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3.

Figure 6

NF-κB directly regulates GLUT1 and SREBP1 (A–C) Pan02 or Nogo-B KO Pan02 (KO#1) cells were treated with indicated concentration of glucose for 24 h (A). Cells were pre-treated with Bay (B) or transfected with NF-κB overexpression plasmid (C) for 24 h, then received indicated concentration of glucose treatment for 24 h. After treatment, protein expression was detected by Western blot with quantitative analysis of band intensity. Pan02 or Nogo-B KO Pan02 (KO#1) cells were transfected with GLUT1 siRNA or SREBP1 siRNA for 24 h, then treated with indicated concentration of glucose for 24 h (D) or 7 days (E). Scale bar: 2 mm (E). The data represent the mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3.

Figure 7

NF-κB through promoter effects to GLUT1 and SREBP1 (A and B) Pan02 or Nogo-B KO Pan02 (KO#1) cells were treated with indicated concentration of glucose for 24 h. mRNA expression of GLUT1, SREBP1 was determined by qRT-PCR. (C–H) Cells were transfected with indicated plasmids for 12 h. After stabilization for 4 h, cells were treated with the indicated concentrations of glucose for 24 h. Cell lysates were collected for determination the activities of firefly and Renilla luciferases (Luc) using the Dual-Luciferase Reporter Kit. (I and J) Cells were treated with the indicated concentration of glucose for 24 h. Cellular chromatin DNA was extracted, IgG (negative control) or NF-κB antibody was used for immunoprecipitation. PCR was conducted with the corresponding primers for the NF-κB response element in GLUT1 or SREBP1 promoter. The data represent the mean ± SD, ∗p < 0.05 compared with control (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.