The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer

Abstract

Increased expression of PRKD1 and its gene product protein kinase D1 (PKD1) are linked to oncogenic signaling in pancreatic ductal adenocarcinoma, but a direct functional relationship to oncogenic KRas has not been established so far. We here describe the PRKD1 gene promoter as a target for oncogenic KRas signaling. We demonstrate that KRas-induced activation of the canonical NF-κB pathway is one mechanism of how PRKD1 expression is increased and identify the binding sites for NF-κB in the PRKD1 promoter. Altogether, these results describe a novel mechanism governing PRKD1 gene expression in PDA and provide a functional link between oncogenic KRas, NF-κB and expression of PRKD1.

Figure 1. KRas regulates PKD1 expression by activating PRKD1 gene transcription.

(A) Sections of normal mouse pancreas, pancreata from 18 week old LSL-Kras^G12D/+;p53^R172H/+;Pdx1^cre/+(KPC) mice or pancreata from 10 week old p48^cre/+;LSL-Kras^G12D/+(KC) mice were analyzed by H&E staining and by immunohistochemistry for PKD1 expression. Shown is a characteristic area of the pancreas. (B) Whole cell lysates of indicated PDA cell lines or normal control (HPDE) were analyzed by Western blotting for expression of PKD1 (anti-PKD1). Probing lysates for β-actin (anti-β-actin) served as loading control. (C) Panc1 cells were transfected with control-shRNA or shRNA targeting expression of KRas as indicated. 48 hours after transfection cells were lysed, PKD1 was immunoprecipitated (anti-PKD1), samples separated on SDS-PAGE, and analyzed by immunoblotting for PKD1 expression (anti-PKD1). In addition, lysates were analyzed by Western blotting for efficient knockdown (anti-KRas). Probing lysates for β-actin (anti-β-actin) served as a loading control. (D,E) HPDE or BxPC3 cells were transfected with KRas^G12V or control vector as indicated. 48 hours after transfection cells were lysed, PKD1 was immunoprecipitated (anti-PKD1 antibody), samples separated on SDS-PAGE and analyzed by immunoblotting for PKD1 expression (anti-PKD1 antibody). In addition, lysates were analyzed by Western blotting for KRas knockdown (anti-KRas) and for β-actin (anti-β-actin) as a loading control. (F) Indicated cell lines were cultivated under normal growth conditions. mRNA was isolated and the expression of PKD1 and β-actin was detected by RT-PCR. (G) BxPC3 cell were transfected with KRas^G12V or control vector. 48 hours after transfection a qPCR was performed. Shown is relative fold PKD1 expression normalized to GAPDH. The asterisk indicates statistical significance. (H–J) BxPC3 (H) or HPDE (I) cells were transfected with KRas^G12V or vector control, PRKD1-luciferase reporter and renilla-luciferase reporter; Panc1 (J) cells were transfected with control-shRNA or shRNA targeting KRas (two different sequences, #1 and #2), PRKD1-luciferase reporter and renilla-luciferase reporter. 48 hours after transfection cells were lysed, and reporter gene assays performed. In addition lysates of H, I were analyzed by Western blot for KRas^G12V expression (anti-FLAG), lysates of J for knockdown of KRas (anti-KRas), as well as for β-actin (anti-β-actin) as loading controls.

Figure 2. Oncogenic Kras activates the canonical NF-κB pathway.

(A) Whole cell lysates of indicated PDA cell lines or normal control (HPDE) were analyzed by Western blotting for an activating phosphorylation of IKKα/β (anti-pS176/177-IKKα/β) or total protein (anti-IKKα/β). (B) Whole cell lysates of indicated PDA cell lines or normal control (HPDE) were analyzed by Western blotting for presence of IκBα phosphorylation (anti-pS32/36-IκBα), total IκBα, phosphorylation of p65 (anti-pS536-p65) or total p65. Probing lysates for β-actin (anti-β-actin) served as loading control. (C) HPDE cells were transfected with KRas^G12V or control vector as indicated. 24 hours after transfection lysates were analyzed by Western blotting for expression of KRas^G12V (anti-FLAG) or downregulation of IκBα (anti-IκBα), phosphorylation of p65 (anti-pS536-p65) or total p65. Probing lysates for β-actin (anti-β-actin) served as loading control. (D) HPDE cells were transfected with KRas^G12V or control vector, NF-κB-luciferase reporter and renilla-luciferase reporter, as indicated. 24 hours after transfection cells were lysed, and reporter gene assays performed. In addition lysates were analyzed by Western blot for KRas^G12V expression (anti-FLAG) as well as for β-actin (anti-β-actin). (E) Panc1 cells were transfected with control-shRNA or shRNA targeting KRas (two different sequences, #1 and #2), as indicated. 24 hours after transfection lysates were analyzed by Western blotting for expression of endogenous KRas (anti-KRas) or presence of IκBα (anti-IκBα), phosphorylation of p65 (anti-pS536-p65) or total p65. Probing lysates for β-actin (anti-β-actin) served as loading control. (F) Panc1 cells were transfected with control-shRNA or shRNA targeting KRas (two different sequences, #1 and #2), as indicated. 24 hours after transfection cells were lysed, and reporter gene assays performed. In addition lysates were analyzed by Western blot for knockdown of KRas (anti-KRas), as well as for β-actin (anti-β-actin).

Figure 3. Oncogenic Kras induces PRKD1 expression via the canonical NF-κB pathway.

(A) BxPC3 cells were transfected with vector control, KRas^G12V or p65 as indicated. 24 hours after transfection lysates were analyzed by Western blotting for expression of endogenous PKD1 (anti-PKD1). Probing lysates for KRas^G12D (anti-FLAG), p65 (anti-p65) or β-actin (anti-β-actin) served as expression or loading controls. (B) BxPC3 cells were transfected with vector control or p65 and PRKD1-luciferase and renilla-luciferase reporters. 24 hours after transfection cells were lysed, and reporter gene assays performed. In addition, probing lysates for p65 (anti-p65) or β-actin (anti-β-actin) served as expression or loading controls. (C) BxPC3 cells were co-transfected with vector control, IκBα.SD or KRas^G12V and PRKD1-luciferase and renilla-luciferase reporters. 24 hours after transfection cells were lysed, and reporter gene assays performed. Probing lysates for IκBα (anti-IκBα), KRas^G12V (anti-FLAG) or β-actin (anti-β-actin) served as expression or loading controls. (D) BxPC3 cells were co-transfected with vector control or KRas^G12V and PRKD1-luciferase and renilla-luciferase reporters and, after 5 hours, treated with BMS345541 (10 μM). 24 hours after stimulation cells were lysed, and reporter gene assays performed. Probing lysates for KRas^G12V (anti-FLAG) or β-actin (anti-β-actin) served as expression or loading controls. (E) Panc1 cells were co-transfected with PRKD1-luciferase and renilla-luciferase reporters and then treated with BMS345541 (10 μM). 24 hours after stimulation cells were lysed, and reporter gene assays performed. Probing lysates for β-actin (anti-β-actin) served as internal control. (F) Panc1 cells were transfected with control-shRNA or shRNA targeting KRas, as well as vector control or p65 and PRKD1-luciferase and renilla-luciferase reporters, as indicated. 24 hours after transfection cells were lysed, and reporter gene assays performed. Probing lysates for KRas (anti-KRas), p65 (anti-p65) or β-actin (anti-β-actin) served as expression or loading controls.

Figure 4. Mapping of the NF-κB site relevant for KRas^G12V-mediated PRKD1 promoter activation.

(A) Cells were transfected with vector control or p65 as well as full-length or indicated truncated versions of the PRKD1 promoter luciferase reporter and renilla-luciferase reporter, as indicated. 24 hours after transfection cells were lysed, and reporter gene assays performed. (B) Schematic of a NF-κB1 motif (green) in the mapped region of the PRKD1 promoter and mutational alterations performed to destruct the motif (red). (C,D) Comparison of response of wildtype and mutant PRKD1 promoter luciferase reporter to p65 or Kras^G12V. Cells were transfected with vector control or p65 (C) or vector control and Kras^G12V, as well as full-length wildtype or mutant versions of the PRKD1 promoter luciferase reporter and renilla-luciferase reporter, as indicated. 24 hours after transfection cells were lysed, and reporter gene assays performed. In addition lysates were analyzed by Western blot for expression of p65 (anti-p65) or KRas^G12V (anti-FLAG), as well as for β-actin (anti-β-actin). (E) Panc1 cells were transfected with control-shRNA or shRNA targeting expression of KRas, as indicated, for 48 hours. BxPC3 cells were transfected with vector control or KRas^G12V as indicated. Chromatin immunoprecipitation (ChIP) was performed using anti-p65 or IgG control and ChIP of p65-bound PRKD1 promoter was detected by PCR. Input controls show PCR for PRKD1 promoter and GAPDH using sheared DNA as a template. (F) Schematic of how oncogenic KRas induces the expression of PKD1 via activation of NF-κB. Red arrows take in consideration published data showing that PKD1 also can activate NF-κB downstream of mutant KRas, this indicating a potential positive feedback loop for signal amplification.

Figure 5. Correlation of p65 and PKD1 expression in human PDA.

(A) Serial sections that show a representative of 20 different patient tissue samples that were stained by H&E or IHC for expression of PKD1 or p65. Shown is a representative lesion area. The bar indicates 100 μm. (B) Shows a representative of 20 different patient tissue samples that were co-stained by IHC-IF for PKD1 (red) or phospho-S536-p65 (green). Shown is a representative lesion area. The bar indicates 100 μm. (C) Relative expression of PKD1 and pS536-p65 in n = 20 patient samples. The quantitation analysis is described in Materials & Methods.