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

Wang Z, Banerjee S, Ahmad A, Li Y, Azmi AS, Gunn JR, Kong D, Bao B, Ali S, Gao J, Mohammad RM, Miele L, Korc M, Sarkar FH - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Karmanos Cancer Institute, School of Medicine, Wayne State University, Detroit, Michigan, United States of America.

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.

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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.
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pone-0020537-g001: 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.

Mentions: To delineate the mechanistic role of mutated K-ras in the development and progression of PDAC, we assessed the expression of Notch pathway in the murine model. In this model, oncogenic K-ras (KrasG12D) is knocked-in into its own locus and transcriptionally silenced due to the insertion of a LoxP-Stop-LoxP element (LSL). When LSL- KrasG12D mice are bred with transgenic mice which express Cre recombinase under the control of the Pdx1 promoter, expression of Cre recombinase in pancreatic progenitor cells allows the removal of the floxed transcriptional STOP cassette, leading to the activation of the oncogenic K-ras allele. In this model, there was no tumors readily found up to 30 weeks of age in LSL- K-rasG12D; Pdx1-Cre (we called KC in this manuscript) mice, consistent with previous study [13]. We also found no evidence of PDAC in the Ink4a/Arf; Pdx1-Cre (we called IC in this manuscript) animals up to an age of 24 weeks, similar to the observation documented by other groups [13]. However, all 25 mice from LSL- K-rasG12D; Pdx1-Cre; Ink4a/Arf (we called KCI for this manuscript) group were found to have pancreatic tumors ranging in diameter from 4 to 10 mm between 45 to 80 days (Fig. 1A). The compound KCI mice with tumors became moribund (Fig. 1A, survival curve). The tumors were confirmed by histopathologic examination (Fig. 1B). The Ki-67, a known proliferation marker, was highly expressed in KCI pancreatic tumors (Fig. 1B). It has been reported that Notch signaling pathway has critical roles in the development and progression of pancreatic cancer. Therefore, we assessed the expression of Notch genes in these transgenic mice tissues. It is important to note that we focused our studies on the cleaved Notch because it is the active functional form of Notch. Therefore, Notch in our all figure legends means active cleaved Notch. We found that Notch signaling was activated in the tumors of KCI mice when compared with the pancreata of KC and IC mice, respectively (Fig. 1C, D). The expression of Notch-2 and Notch-4 was up-regulated both at the mRNA and protein levels in KCI mice. However, Notch-1 expression showed no change and Notch-3 expression was increased only at the mRNA level in the tumors of KCI mice, suggesting that roles of Notch-2, and Notch-4 could be more important in progression of pancreatic cancer. We also found that all five Notch ligands were up-regulated in the tumors derived from the KCI mice (Fig. 2A, B). To confirm these results, we evaluated the expression of Notch downstream genes such as Hes-1 and Hey-1. We found that the expression of Hes-1 and Hey-1 was increased in the tumors of KCI mice (Fig. 2A, B), which was expected based on up-regulated expression of Notch-2, Notch-4 and their ligands.


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

Wang Z, Banerjee S, Ahmad A, Li Y, Azmi AS, Gunn JR, Kong D, Bao B, Ali S, Gao J, Mohammad RM, Miele L, Korc M, Sarkar FH - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108612&req=5

pone-0020537-g001: 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.
Mentions: To delineate the mechanistic role of mutated K-ras in the development and progression of PDAC, we assessed the expression of Notch pathway in the murine model. In this model, oncogenic K-ras (KrasG12D) is knocked-in into its own locus and transcriptionally silenced due to the insertion of a LoxP-Stop-LoxP element (LSL). When LSL- KrasG12D mice are bred with transgenic mice which express Cre recombinase under the control of the Pdx1 promoter, expression of Cre recombinase in pancreatic progenitor cells allows the removal of the floxed transcriptional STOP cassette, leading to the activation of the oncogenic K-ras allele. In this model, there was no tumors readily found up to 30 weeks of age in LSL- K-rasG12D; Pdx1-Cre (we called KC in this manuscript) mice, consistent with previous study [13]. We also found no evidence of PDAC in the Ink4a/Arf; Pdx1-Cre (we called IC in this manuscript) animals up to an age of 24 weeks, similar to the observation documented by other groups [13]. However, all 25 mice from LSL- K-rasG12D; Pdx1-Cre; Ink4a/Arf (we called KCI for this manuscript) group were found to have pancreatic tumors ranging in diameter from 4 to 10 mm between 45 to 80 days (Fig. 1A). The compound KCI mice with tumors became moribund (Fig. 1A, survival curve). The tumors were confirmed by histopathologic examination (Fig. 1B). The Ki-67, a known proliferation marker, was highly expressed in KCI pancreatic tumors (Fig. 1B). It has been reported that Notch signaling pathway has critical roles in the development and progression of pancreatic cancer. Therefore, we assessed the expression of Notch genes in these transgenic mice tissues. It is important to note that we focused our studies on the cleaved Notch because it is the active functional form of Notch. Therefore, Notch in our all figure legends means active cleaved Notch. We found that Notch signaling was activated in the tumors of KCI mice when compared with the pancreata of KC and IC mice, respectively (Fig. 1C, D). The expression of Notch-2 and Notch-4 was up-regulated both at the mRNA and protein levels in KCI mice. However, Notch-1 expression showed no change and Notch-3 expression was increased only at the mRNA level in the tumors of KCI mice, suggesting that roles of Notch-2, and Notch-4 could be more important in progression of pancreatic cancer. We also found that all five Notch ligands were up-regulated in the tumors derived from the KCI mice (Fig. 2A, B). To confirm these results, we evaluated the expression of Notch downstream genes such as Hes-1 and Hey-1. We found that the expression of Hes-1 and Hey-1 was increased in the tumors of KCI mice (Fig. 2A, B), which was expected based on up-regulated expression of Notch-2, Notch-4 and their ligands.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Karmanos Cancer Institute, School of Medicine, Wayne State University, Detroit, Michigan, United States of America.

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.

Show MeSH
Related in: MedlinePlus