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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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Related in: MedlinePlus

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.
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pone-0020537-g006: 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.

Mentions: Recently, it has been reported that miR-200 family members target Notch pathway components, such as Jagged-1 [35], [36]. In order to examine whether miR-200 family regulate Notch pathway, we transfected miR-200b precursor into Rink-1 cells. We confirmed that the transfection of miR-200b precursor increased the relative level of miR-200b in Rink-1 cells (Fig. 6A). Over-expression of miR-200b decreased the relative mRNA levels of Jagged-1, Jagged-2 and their target genes by real time RT-PCR assay (Fig. 6A). The data from western blot analysis demonstrated that over-expression of miR-200b decreased the relative protein levels of Jagged-1 and its target gene such as Hes-1, Hey-1, and Bcl-2 (Fig. 6B). Moreover, we found that over-expression of miR-200b inhibited cell growth in Rink-1 cells (Fig. 6B). Next, we detected whether inhibition of Jagged-1 could inhibit cell growth. Jagged-1 siRNA significantly decreased the expression of Jagged-1 and its target Hes-1 and Hey-1 at mRNA and protein levels (Fig. 6C). Furthermore, we found that inhibition of Jagged-1 by Jagged-1 siRNA inhibited the Rink-1 cell growth (Fig. 6C), suggesting that Jagged-1 could be a potential target for pancreatic cancer.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108612&req=5

pone-0020537-g006: 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.
Mentions: Recently, it has been reported that miR-200 family members target Notch pathway components, such as Jagged-1 [35], [36]. In order to examine whether miR-200 family regulate Notch pathway, we transfected miR-200b precursor into Rink-1 cells. We confirmed that the transfection of miR-200b precursor increased the relative level of miR-200b in Rink-1 cells (Fig. 6A). Over-expression of miR-200b decreased the relative mRNA levels of Jagged-1, Jagged-2 and their target genes by real time RT-PCR assay (Fig. 6A). The data from western blot analysis demonstrated that over-expression of miR-200b decreased the relative protein levels of Jagged-1 and its target gene such as Hes-1, Hey-1, and Bcl-2 (Fig. 6B). Moreover, we found that over-expression of miR-200b inhibited cell growth in Rink-1 cells (Fig. 6B). Next, we detected whether inhibition of Jagged-1 could inhibit cell growth. Jagged-1 siRNA significantly decreased the expression of Jagged-1 and its target Hes-1 and Hey-1 at mRNA and protein levels (Fig. 6C). Furthermore, we found that inhibition of Jagged-1 by Jagged-1 siRNA inhibited the Rink-1 cell growth (Fig. 6C), suggesting that Jagged-1 could be a potential target for pancreatic cancer.

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