The E3 ubiquitin ligase NEDD4 is translationally upregulated and facilitates pancreatic cancer

Abstract

Aim: To determine the regulation and function of the neural precursor cell expressed developmentally down regulated protein 4 (NEDD4) in PDAC and to determine its dependency on phosphatase and tensin homolog (PTEN) and PI3K/AKT signaling.

Methods: We investigated the expression of NEDD4 and the tumor suppressor PTEN in normal immortalized human pancreatic duct epithelial cell line and pancreatic adenocarcinoma (PDAC) cell lines. We further evaluated whether RNAi-mediated depletion of NEDD4 can attenuate PDAC cell proliferation and migration. We subsequently determined the crosstalk between NEDD4 expression and the PTEN/PI3K/AKT signaling pathway. Finally, we determined the mechanism behind differential NEDD4 protein expression in pancreatic cancer.

Results: The expression of NEDD4 was heterogeneous in PDAC cells, but was significantly higher compared to normal pancreatic ductal epithelial cells. Analogically, PTEN was decreased in the PDAC cells. A combination of MTT assay, wound healing migration assay, and transwell invasion assays confirmed that depletion of NEDD4 decreased the proliferation and migration ability of PDAC cells. Western blot and immunofluorescence results revealed that NEDD4 could affect PTEN/PI3K/AKT signaling pathway in PDAC cells. Polysomal profiling revealed that higher NEDD4 protein expression in PDAC cells was due to undefined mechanism involving translational activation.

Conclusions: Our results reveal a novel mechanism of upregulation of NEDD4 expression in PDAC. Our findings indicate that NEDD4 potentially plays a critical role in activating the PI3K/AKT signaling pathway by negatively regulating PTEN levels in PDAC cells, which promotes pancreatic cancer cell proliferation and metastasis. Therefore, NEDD4 may be a potential therapeutic target in PDAC.

Keywords: NEDD4; PDAC; PI3K/AKT; PTEN; pancreatic ductal adenocarcinoma.

Figure 1. NEDD4 is heterogeneously expressed in PDAC cells and cross-talk with the PTEN/PI3K/AKT signaling pathway

A. The expression of NEDD4 and PTEN in indicated PDAC cell lines, and the normal immortalized human pancreatic duct epithelial cell line, HPDE6c7. The numbers below the NEDD4 and PTEN blots show the relative quantification as obtained by the Image J-mediated densitometry analyses of the blots shown on the left, normalized to GAPDH expression. B. Western blot analysis demonstrating the effect of siRNA-related depletion of NEDD4 on PI3K/Akt activation, PTEN expression, and epithelial (E-cadherin) and mesenchymal cell marker (vimentin) expression.

Figure 2. NEDD4 may affect PTEN/PI3K/AKT signaling pathways

NEDD4, PTEN, p-AKT, E-cadherin and vimentin were detected by immunofluorescence. Depletion of NEDD4 negatively affected PTEN expression. A decreased phosphorylation level of AKT associated with acquisition of E-cadherin and loss of vimentin (200x) was also observed.

Figure 3. Depletion of NEDD4 inhibited proliferation, migration and invasion in PDAC cells

A. Cell proliferation was analyzed by MTT. Indicated cells were pretreated with NEDD4 siRNA and proliferation was compared at 24, 48, and 72 hours. Depletion led to a significant inhibition of cell proliferation. B, C. Wound-healing and transwell invasion assays, respectively, revealed inhibitory effect on cell migration and invasion ability, similarly to proliferation inhibition experiment. *P<0.05.

Figure 4

A. Quantitative real-time PCR (qRT-PCR)-based determination of change in steady-state NEDD4 mRNA using total mRNA obtained from indicated cells. All panels are representative of a minimum of three experimental replicates. B. Inhibition of proteasome-mediated degradation results did not increase NEDD4 protein levels in the HPDE6c7 cells. Cells were incubated with MG-132 for 8 hours and immunoblotted for the indicated proteins. GAPDH served as a loading control.

Figure 5

A, B. Representative polysome trace obtained by fractionation of HPDE6c7 (A) and PANC-1 (B) cells. C. QRT-PCR-based determination of change in polyribosomal bound NEDD4 mRNA using non-polyribosomal and polyribosomal enriched mRNA obtained from the indicated cell lines. The data has been plotted relative to relative NEDD4 mRNA in the HPDE6c7 cells. All panels are representative of a minimum of three experimental replicates. *P<0.05 compared to HPDE6c7 cells.