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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.

Show MeSH

Related in: MedlinePlus

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.
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pone-0020537-g005: 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.

Mentions: To further assess the potential role of Notch pathway in pancreatic cancer, we used Rink-1 cell line which was derived from the KCI pancreatic tissues and studied the effects of inhibitors of Notch pathway. Previous studies have shown that Rink-1 cells exhibited rapid growth in vitro and formed tumors in nude mice [14]. Since Notch signaling is activated via the activity of γ-secretase, several forms of γ -secretase inhibitors including DAPT and L-685,458 have been used to inactivate Notch pathway. Therefore, we determined the cell viability of Rink-1 cells treated with GSI by the MTT assay, and the data are presented in Figure 4A. The treatment of Rink-1 cells for 72 hours with DAPT, and L-685,458 resulted in cell growth inhibition. To determine which Notch receptor could be an effective therapeutic target for pancreatic cancer, the effect of Notch 1–4 siRNA on cell growth of the pancreatic cancer cells was examined. The efficacy of GSI and Notch siRNA for knockdown of Notch protein was confirmed through western blotting. We observed that Notch protein level was barely detectable in GSI treated or Notch siRNA transfected cells (Fig. 4B). Very interestingly, only inactivation of single Notch receptor did not significantly inhibit cell growth (Fig. 4A). These results suggest that inactivation of multiple Notch receptors by GSI are good way to treat PDAC. To confirm this conclusion, we tested the expression of Notch target genes in Rink-1 cells treated with GSI or Notch siRNA. Because only Notch-2 and Notch-4 siRNA slightly inhibited cell growth, we detected the expression of Notch target genes in Rink-1 cells treated with these two siRNAs. As we expected, we found that GSI inhibited the expression of Notch target genes including Hes-1, Survivin, Bcl-2, c-myc, uPA to more degree, compared to Notch-2 siRNA or Notch-4 siRNA transfection (Fig. 4C). Therefore, we used GSI in the following experiments. Next, we tested the effects of treatment on cell viability by clonogenic assay. GSI treatment resulted in a significant inhibition of colony formation of Rink-1 cells when compared with control (Fig. 5A). Overall, the results from clonogenic assay were consistent with the MTT data, suggesting that the inactivation of Notch pathway could inhibit cell growth of Rink-1 cells.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020537-g005: 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.
Mentions: To further assess the potential role of Notch pathway in pancreatic cancer, we used Rink-1 cell line which was derived from the KCI pancreatic tissues and studied the effects of inhibitors of Notch pathway. Previous studies have shown that Rink-1 cells exhibited rapid growth in vitro and formed tumors in nude mice [14]. Since Notch signaling is activated via the activity of γ-secretase, several forms of γ -secretase inhibitors including DAPT and L-685,458 have been used to inactivate Notch pathway. Therefore, we determined the cell viability of Rink-1 cells treated with GSI by the MTT assay, and the data are presented in Figure 4A. The treatment of Rink-1 cells for 72 hours with DAPT, and L-685,458 resulted in cell growth inhibition. To determine which Notch receptor could be an effective therapeutic target for pancreatic cancer, the effect of Notch 1–4 siRNA on cell growth of the pancreatic cancer cells was examined. The efficacy of GSI and Notch siRNA for knockdown of Notch protein was confirmed through western blotting. We observed that Notch protein level was barely detectable in GSI treated or Notch siRNA transfected cells (Fig. 4B). Very interestingly, only inactivation of single Notch receptor did not significantly inhibit cell growth (Fig. 4A). These results suggest that inactivation of multiple Notch receptors by GSI are good way to treat PDAC. To confirm this conclusion, we tested the expression of Notch target genes in Rink-1 cells treated with GSI or Notch siRNA. Because only Notch-2 and Notch-4 siRNA slightly inhibited cell growth, we detected the expression of Notch target genes in Rink-1 cells treated with these two siRNAs. As we expected, we found that GSI inhibited the expression of Notch target genes including Hes-1, Survivin, Bcl-2, c-myc, uPA to more degree, compared to Notch-2 siRNA or Notch-4 siRNA transfection (Fig. 4C). Therefore, we used GSI in the following experiments. Next, we tested the effects of treatment on cell viability by clonogenic assay. GSI treatment resulted in a significant inhibition of colony formation of Rink-1 cells when compared with control (Fig. 5A). Overall, the results from clonogenic assay were consistent with the MTT data, suggesting that the inactivation of Notch pathway could inhibit cell growth of Rink-1 cells.

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