Activated K-ras and INK4a/Arf deficiency cooperate during the development of pancreatic cancer by activation of Notch and NF-κB signaling pathways

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, suggesting that novel strategies for the prevention and treatment of PDAC are urgently needed. K-ras mutations are observed in >90% of pancreatic cancer, suggesting its role in the initiation and early developmental stages of PDAC. In order to gain mechanistic insight as to the role of mutated K-ras, several mouse models have been developed by targeting a conditionally mutated K-ras(G12D) for recapitulating PDAC. A significant co-operativity has been shown in tumor development and metastasis in a compound mouse model with activated K-ras and Ink4a/Arf deficiency. However, the molecular mechanism(s) by which K-ras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated.

Methodology/principal findings: To assess the molecular mechanism(s) that are involved in the development of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency, we used multiple methods, such as Real-time RT-PCR, western blotting assay, immunohistochemistry, MTT assay, invasion, EMSA and ELISA. We found that the deletion of Ink4a/Arf in K-ras(G12D) expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found down-regulation of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice.

Conclusions/significance: Our results suggest that the activation of Notch and NF-κB together with the loss of miR-200 family is mechanistically linked with the development and progression of PDAC in the compound K-ras(G12D) and Ink4a/Arf deficient transgenic mice.

Figure 1. Notch receptors are highly expressed in KCI mice.

A, Top left panel: Tumor incidence in KCI mice (N>25). Control mice: KC and IC mice. Top right panel: Average length of tumors in KCI mice (N>20). Bottom panel: Kaplan-Meier pancreatic tumor-free survival curve for KCI mice and control animals. Control mice: combinations of KC and IC mice. B, Top panel: Microscopic examination of tumors derived from the KCI mice composed of cells which are forming ducts at places (red arrows). Focally tumor cells are in sheets. Cells are large, highly atypical, with large, pleomorphic nuclei and prominent 2–3 nucleoli (yellow arrows) per cell. Cytoplasm is eosinophilic with pale eosinophilic inclusions (green arrows) in few cells giving a rhabdoid feature to the cells. Surrounding stroma shows spindled cells with elongated nuclei (black arrows) and scattered inflammatory infiltrate comprising of neutrophils, lymphocytes and few plasma cells. Bottom panel: Ki-67 was highly expressed in tumors obtained from the KCI mice as assessed by immunohistochemistry. C, Notch signaling pathway was up-regulated at mRNA level as assessed by Real-time RT-PCR in tumors derived from the KCI mice. D, Notch pathway was highly expressed in tumors derived from the KCI mice as assessed by western blotting analysis and immunohistochemistry, respectively.

Figure 2. The expression of Notch ligands and NF-κB is upregulated in KCI mice.

A, The expression of Notch ligands and Notch downstream genes was increased at mRNA level as assessed by Real-time RT-PCR in tumors derived from the KCI mice. B, The expression of Notch ligands and Notch downstream genes was highly expressed in tumors derived from the KCI mice as assessed by western blotting analysis. C, NF-κB p65 activity was increased in tumors derived from the KCI mice by ELISA. D, Left panel, NF-κB p65 DNA-binding activity is increased in tumors derived from the KCI mice as assessed by EMSA. Right panel, phospho-p65 was highly expressed in tumors obtained from the KCI mice as assessed by immunohistochemistry.

Figure 3. The expression of Notch target genes is increased in KCI mice.

A, Western blot analysis showing the up-regulated expression of IKK, p65, and NF-κB downstream genes in tumors derived from KCI mice. B, Real-time RT-PCR showing increased expression of NF-κB downstream genes such as survivin, cyclin D1, Bcl-2, C-myc, MMP-2, and MMP-9 in the tumors derived from the KCI mice. C, The expression of miR-200 family was down-regulated in the tumors of the KCI mice as assessed by real-time RT-PCR. D, Real-time RT-PCR showing decreased expression of E-cadherin, and increased expression of vimentin, and a modest increase in the expression of ZEB1 whereas a 30-fold increased expression of ZEB2 in tumors derived from the KCI mice.

Figure 4. Inhibition of Notch pathway by Notch siRNA or GSI inhibited Rink-1 cell growth.

A, Left panel, Inhibition of Rink-1 cell growth by Notch 1–4 siRNA tested by MTT assay. The results were plotted as means ± SD of three separate experiments having six determinations per experiment for each experimental condition. Middle and Right panel: L-685,458 and DAPT were γ-secretase inhibitors (GSI), which prevent the cleavage of the Notch receptor, blocking Notch signal transduction. GSI significantly inhibited Rink-1 cell growth. Cells were seeded in 96-well plates at 5,000 cells per well and treated with GSI for 72 hours. After treatment, cell densities were determined by MTT assay. Each value represents the mean ± SD (n = 6) of three independent experiments. *P<0.05, compared to the control. B, The expression of Notch pathway was down-regulated in Rink-1 cells treated with GSI or transfected with Notch 1–4 siRNA as assessed by western blotting analysis. C, The expression of Notch target genes was down-regulated in Rink-1 cells treated with GSI or transfected with Notch-2 siRNA or Notch-4 siRNA as assessed by as assessed by real-time RT-PCR.

Figure 5. GSI induced apoptosis, inhibited migration and invasion in Rink-1 cells.

A, Top, Left panel: Cell survival of Rink-1 cells treated with GSI. Cells treated with GSI for 72 hours were evaluated by the clonogenic assay. Photomicrographic difference in colony formation in cells untreated and treated with GSI. Right panel: There was a significant reduction in the colony formation in Rink-1 cells treated with GSI compared with control cells. P values represent comparisons between cells treated with GSI and control using the paired t test. Bottom, Left panel: Characterization of apoptosis was carried out after propidium iodide (PI) and Annexin V-FITC staining with apoptosis detection kit followed by flow cytometric analysis after 48 h of GSI treatment of Rink-1 cells. The percentage of apoptotic cells increased from 10% in the control to 28–33% in GSI treated cells. Right panel: GSI induced apoptosis in Rink-1 cells. Rink-1 cells were exposed to GSI for 72 hours. Apoptosis was measured by Histone DNA ELISA. Values are reported as mean ± SD. *P<0.05, compared to the control. B, Top, Left panel, Invasion assay using GSI treated cells showing low penetration of cells through the Matrigel-coated membrane, compared with control cells. Right panel: The graphs showing the value of fluorescence from the invaded Rink-1 cells. The values indicate the comparative amount of invaded Rink-1 cells. Bottom, Wound healing assay was conducted to assess the capacity of cell migration. GSI treatment decreased the cell migration in Rink-1 cells. C, GSI inhibited the NF-κB DNA binding activity in Rink-1 cells as assessed by EMSA. D, Real-time RT-PCR and western blot analysis showed that L-685,458 inhibited the expression of Survivin, c-myc, Bcl-2, and uPA genes.

Figure 6. The miR-200b inhibited Rink-1 cell growth and Jagged-1 expression.

A, Left panel, Re-expression of miR-200b was established in Rink-1 cells by transfection with its precursor. Middle panel, Re-expression of miR-200b did not regulate the expression of Notch receptors in Rink-1 cells. Right panel, Re-expression of miR-200b regulated the expression of Jagged-1 and Jagged-2 mRNAs in Rink-1 cells. B, Left and middle panel, Re-expression of miR-200b inhibited the expression of Jagged-1 target genes at mRNA and protein levels in Rink-1 cells. Right panel, Re-expression of miR-200b inhibited Rink-1 cell growth test by MTT assay. C, Left and middle panel, Jagged-1 siRNA inhibited the expression of Jagged-1 target gene Hes-1 and Hey-1 at mRNA and protein levels in Rink-1 cells. Right panel, Jagged-1 siRNA inhibited Rink-1 cell growth test by MTT assay.

Figure 7. The schematic representation of our proposed molecular mechanism involved in the development and progression of tumors in the compound KCI transgenic mice.