Fig 1: Schematic model of the netrin-1-induced growth inhibition of PDACNetrin-1 is expressed in the exocrine acini of normal pancreatic tissue, and this expression is impaired in early-stage PDAC. Netrin-1 inhibits PDAC tumorigenesis by interacting with the UNC5b receptor and stimulating FAK phosphorylation. Activated FAK induces the production of nitric oxide (NO) and promotes the expression, 3-nitrotyrosine modification and dephosphorylation of the protein phosphatase PP2A, leading to significantly enhanced PP2A activity. PP2A suppresses MEK/ERK/c-Jun signaling and down-regulates integrin β4 expression, facilitating the in vivo growth arrest of PDAC cells. Interestingly, in this study, that netrin-1 also mildly induced c-Raf phosphorylation, which could promote MEK/ERK signaling. Together, our data suggest that netrin-1 protects against PDAC and, at the same time, imply that netrin-1 intricately regulates PDAC progression in different cells and/or distinct stages of PDAC tumorigenesis.
Fig 2: Netrin-1 expression is decreased in early-stage PDAC samples(A) Netrin-1 immunohistochemical staining (ab122903, Abcam) in normal pancreatic tissue and stage I–IV pancreatic ductal adenocarcinoma (PDAC). Three representative graphs of each stage are shown (200×). The dashed circles show representative acini that are positive for netrin-1 staining in the normal pancreatic tissue. It is difficult to observe the acinar cells and netrin-1 staining in the stage I and II PDAC samples. The ductal expression of netrin-1 becomes evident in stage III and IV PDACs. (B) Statistical analyses of the netrin-1 expression level in the normal pancreatic tissues (N = 10) and the total PDAC tumors (N = 61) on the immunostained tissue array (**P < 0.01). The line refers to the group median. (C) Stage-specific analysis of netrin-1 expression in the PDAC samples (N = 30 for stage I, N = 24 for stage II, and N = 7 for stages III/IV) compared with that in the normal pancreas tissues (N = 10) on the immunostained tissue array (*P < 0.05, **P < 0.01). The line refers to the group median.
Fig 3: Netrin-1 inhibits PDAC growth by decreasing integrin β4 expression(A–B) Real-time RT-PCR (A) and western blotting (B) analyses of integrin β4 expression in control (ctrl) and netrin-1-over-expressing MiaPaCa II cells (ntn1+). GAPDH was used as the internal control for both analyses. (C–D) Real-time RT-PCR (C) and western blotting (D) analyses of integrin β4 expression in MiaPaCa II cells treated with the indicated concentrations of netrin-1 for 48 hr. GAPDH was used as the internal control for both analyses. (E) Western blotting analysis of integrin β4 expression in control GFP RNAi (RiGFP) and ITGB4 RNAi (RiITGB4) MiaPaCa II cells. GAPDH was used as the internal control. (F) Representative xenograft tumors formed by GFP RNAi (asterisk) and ITGB4 RNAi (arrowhead) MiaPaCa II cells in SCID-beige mice (bar, 1 cm). (G) Statistics for the weights of the xenograft tumors formed by the control RiGFP (N = 7) and RiITGB4 (N = 7) MiaPaCa II cells (***P < 0.001) in SCID-beige mice. (H) Growth curves of the RiGFP and RiITGB4 MiaPaCa II xenograft tumors in Balb/c NU/NU mice. The tumor volumes were measured every other day and calculated using the formula v = 0.5 ab2 (a: long diameter; b: short diameter); the total 28-day result is shown (error bars represent the standard deviation of the measurements, *P < 0.05, **P < 0.01).
Fig 4: Netrin-1 down-regulates integrin β4 expression through the UNC5b receptor and the activation of FAK(A) Real-time PCR analysis for the expression of netrin-1 receptors in MiaPaCa II cells. GAPDH was used as an internal control. (B–C) The anti-UNC5b antibody selectively blocks the integrin β4-suppressing effect of netrin-1. The receptors on the MiaPaCa II cells were blocked by their respective antibodies before the cells were treated with the indicated concentrations netrin-1; integrin β4 expression in the cells was then detected by real-time RT-PCR (***P < 0.001, **P < 0.01) (B) and western blotting (C). GAPDH was used as an internal control. (D) Netrin-1 interacts with UNC5b but not integrin β4 in the myc-netrin-1-over-expressing MiaPaCa II cells. An anti-myc antibody to pull down the myc-tagged netrin protein in the netrin-1-over-expressing MiaPaCa II cells, followed by immunoblotting analysis of the integrin β4 (itgb4), UNC5b and netrin-1 (ntn1) levels in the precipitate. (E) Western blotting analysis of UNC5b expression in the UNC5B RNAi (RiUNC5B) and control GFP RNAi (RiGFP) MiaPaCa II cells. (F) Netrin-1 suppresses integrin β4 expression in the control GFP RNAi cells but not the UNC5B RNAi cells. Unc5b-knockdown and control MiaPaCa II cells were treated with recombinant netrin-1, and the integrin β4 expression was detected before and after netrin-1 treatment by western blotting. (G) Western blotting analysis of the FAK and phospho-FAK levels in the netrin-1-over-expressing MiaPaCa II cells, and the MiaPaCa II cells treated with the indicated concentrations of recombinant netrin-1 for 2 hr. The vector-transfected and the untreated MiaPaCa II cells were used as the respective negative controls, and GAPDH was used as an internal control. (H) UNC5b RNAi abrogates netrin-1-induced FAK phosphorylation. The FAK and phospho-FAK levels in the control and UNC5b RNAi MiaPaCa II cells were detected before and after netrin-1 stimulation by western blotting. (I) FAK inhibition abrogates the netrin-1-induced down-regulation of integrin β4. Western blot analysis of the phospho-FAK and integrin β4 levels in MiaPaCa II cells treated with the indicated concentrations of recombinant netrin-1 in the presence or absence of FAK inhibitor 14. (J) FAK inhibition abrogates the netrin-1-induced growth arrest of MiaPaCa II cells in Matrigel, as analyzed by the 3D growth of the control and netrin-1-over-expressing MiaPaCa II cells in Matrigel in the presence of 20 nM FAK inhibitor 14. Scale bars, 50 μm.
Fig 5: Netrin-1 induces nitric oxide (NO)-stimulated PP2A activation and suppresses the MAPK pathway to down-regulate integrin β4 expression(A) The western blotting analysis of the MAPK pathway (C-RAF/MEK/ERK) and PI3K-AKT pathway in the netrin-1-treated MiaPaCa II cells shows that MEK/ERK signals in the MAPK pathway were reduced and the PI3K-AKT pathway was unchanged. (B) Western blotting assay showing reduced MEK/ERK signals and decreased integrin β4 expression in the netrin-1-over-expressing MiaPaCa II cells. (C) A ChIP analysis (upper panel) and western blotting assay (below panel) of the phosphorylated c-Jun (pc-Jun) levels in the netrin-1-over-expressing MiaPaCa II cells showed reduced pc-Jun levels and decreased pc-Jun recruitment to the integrin β4 gene promoter upon netrin-1 over-expression. (*P < 0.05). (D) RT-PCR and western blotting assay showing increased PP2A expression and decreased phosphorylation and enhanced 3-nitrotyrosine modification of PP2A in the netrin-1-over-expressing MiaPaCa II cells. (*P < 0.05). (E) A western blotting assay of the netrin-1-over-expressing MiaPaCa II cells treated with the PP2A inhibitor okadaic acid showed that the repression of MEK/ERK signaling and integrin β4 expression depends on PP2A activation. (F) NO production was increased in the netrin-1-over-expressing and recombinant netrin-1-treated MiaPaCa II cells. FAK inhibitor PF-562271 blocks the netrin-1-induced NO production. (*P < 0.05). (G) Western blotting assay of the netrin-1-over-expressing MiaPaCa II cells treated with the NO scavenger PITO. The NO scavenger abolished the netrin-1-induced changes in PP2A expression and phosphorylation, the MEK/ERK signals, c-Jun phosphorylation and integrin β4 expression. GAPDH was used as an internal control for all western blotting assays.
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