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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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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.
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pone-0020537-g003: 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.

Mentions: It has been reported that Notch pathway stimulates NF-κB activity in cervical cancer cells by associating with the IKK signalosome through IKKα [29]. Previous study has shown that Notch pathway regulates the IKKα expression in pancreatic cancer [30]. Thus, we investigated the expression of IKK protein in the tumors of KCI mice. We found that all IKK family members such as IKKα, IKKβ and IKKγ were activated in the tumors of KCI mice (Fig. 3A). To further explore the effects of NF-κB activation, we examined the expression levels of certain NF-κB target genes including COX-2, cyclin D1, MMP-9, MMP-2, Bcl-2, c-myc, and survivin by real-time RT-PCR and western blotting, respectively, using the tumor tissues obtained from the compound KCI transgenic animals. Real-time RT-PCR and western blot analysis showed that the expression of these genes was activated in the tumors of KCI mice (Fig. 3A, B). We also found that the expression of Stat3 was activated in the tumors form KCI mice (data not shown). It is well known that these genes play critical roles in cell growth, invasion and metastasis. Therefore, these results further support the role of NF-κB in tumor growth and progression in the compound KCI mice.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020537-g003: 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.
Mentions: It has been reported that Notch pathway stimulates NF-κB activity in cervical cancer cells by associating with the IKK signalosome through IKKα [29]. Previous study has shown that Notch pathway regulates the IKKα expression in pancreatic cancer [30]. Thus, we investigated the expression of IKK protein in the tumors of KCI mice. We found that all IKK family members such as IKKα, IKKβ and IKKγ were activated in the tumors of KCI mice (Fig. 3A). To further explore the effects of NF-κB activation, we examined the expression levels of certain NF-κB target genes including COX-2, cyclin D1, MMP-9, MMP-2, Bcl-2, c-myc, and survivin by real-time RT-PCR and western blotting, respectively, using the tumor tissues obtained from the compound KCI transgenic animals. Real-time RT-PCR and western blot analysis showed that the expression of these genes was activated in the tumors of KCI mice (Fig. 3A, B). We also found that the expression of Stat3 was activated in the tumors form KCI mice (data not shown). It is well known that these genes play critical roles in cell growth, invasion and metastasis. Therefore, these results further support the role of NF-κB in tumor growth and progression in the compound KCI mice.

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