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Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in Apc(Min/+) mice.

Brudvik KW, Paulsen JE, Aandahl EM, Roald B, Taskén K - Mol. Cancer (2011)

Bottom Line: Treatment of Apc(Min/+) mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas.Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE(2)-induced β-catenin phosphorylation and c-Myc upregulation.Based on our findings we suggest that PGE(2) act through PKA to promote β-catenin nuclear translocation and tumor development in Apc(Min/+) mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership and Biotechnology Centre, University of Oslo, Oslo, Norway.

ABSTRACT

Background: The adenomatous polyposis coli (APC) protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E(2) (PGE(2)) - PI-3 kinase pathways. Recent reports show that PGE(2)-induced phosphorylation of β-catenin by protein kinase A (PKA) increases nuclear translocation indicating two mechanisms of action of PGE(2) on β-catenin homeostasis.

Findings: Treatment of Apc(Min/+) mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE(2)-induced β-catenin phosphorylation and c-Myc upregulation.

Conclusion: Based on our findings we suggest that PGE(2) act through PKA to promote β-catenin nuclear translocation and tumor development in Apc(Min/+) mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

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PKA antagonist reduces tumor load in ApcMin/+ mice. A. Images of developing adenomas representative of each treatment acquired by transillumination microscopy are shown. White bars = 500 μm. B. Number (B; left panel) and size (B; mid panel) of spontaneously occurring tumors and distribution of tumor size in the treatment groups (B; right panel), tumor numbers/size class). Box plots with median (horizontal line); 25-75% (box) and 2.5-97.5% (bar) percentiles are shown for each treatment group (n = 7-10 as indicated). All tumors from the same animals were pooled when comparing tumor size distribution between the groups (n = 41-585) as indicated. Mann-Whitney Rank Sum test was used to compare the groups (SigmaPlot 11.0 (CA, USA). C. Number (C; left panel) and size (right panel) of spontaneously occurring tumors in the small intestine relative to the distance from the ventricle.
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Figure 1: PKA antagonist reduces tumor load in ApcMin/+ mice. A. Images of developing adenomas representative of each treatment acquired by transillumination microscopy are shown. White bars = 500 μm. B. Number (B; left panel) and size (B; mid panel) of spontaneously occurring tumors and distribution of tumor size in the treatment groups (B; right panel), tumor numbers/size class). Box plots with median (horizontal line); 25-75% (box) and 2.5-97.5% (bar) percentiles are shown for each treatment group (n = 7-10 as indicated). All tumors from the same animals were pooled when comparing tumor size distribution between the groups (n = 41-585) as indicated. Mann-Whitney Rank Sum test was used to compare the groups (SigmaPlot 11.0 (CA, USA). C. Number (C; left panel) and size (right panel) of spontaneously occurring tumors in the small intestine relative to the distance from the ventricle.

Mentions: To more closely delineate the effect of PKA in the COX-2 - PGE2 pathway active in colorectal cancer, we treated ApcMin/+ mice with the PKA antagonist Rp-8-Br-cAMPS for 6 weeks using earlier established doses (see Additional file 1, Supplementary information) and compared the result with that of treatment with the COX inhibitor indomethacin, previously shown to inhibit tumor development in the ApcMin/+model [20]. Phosphate buffered saline (PBS) was used as vehicle control for the Rp-8-Br-cAMPS. Examination revealed that indomethacin reduced the number and area of tumors in the small intestine of the ApcMin/+ mice compared to PBS (from 47 to 3 tumors per mouse and from 0.44 mm2 to 0.10 mm2 tumor area; P < 0.001; Figure 1A, B). The PKA antagonist Rp-8-Br-cAMPS did not significantly reduce the number of adenomas (47 versus 43 tumors; P = 0.368, Figure 1A, B), but reduced the tumor area by 36% (from 0.44 mm2 to 0.28 mm2; P < 0.001; Figure 1). Specifically, tumor load was reduced in the distal part of the small intestine (Figure 1C). The differential effect of COX inhibitor and PKA antagonist on tumor numbers and tumor size indicated to us that the mechanisms of action could be distinct and were examined in more detail in the following.


Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in Apc(Min/+) mice.

Brudvik KW, Paulsen JE, Aandahl EM, Roald B, Taskén K - Mol. Cancer (2011)

PKA antagonist reduces tumor load in ApcMin/+ mice. A. Images of developing adenomas representative of each treatment acquired by transillumination microscopy are shown. White bars = 500 μm. B. Number (B; left panel) and size (B; mid panel) of spontaneously occurring tumors and distribution of tumor size in the treatment groups (B; right panel), tumor numbers/size class). Box plots with median (horizontal line); 25-75% (box) and 2.5-97.5% (bar) percentiles are shown for each treatment group (n = 7-10 as indicated). All tumors from the same animals were pooled when comparing tumor size distribution between the groups (n = 41-585) as indicated. Mann-Whitney Rank Sum test was used to compare the groups (SigmaPlot 11.0 (CA, USA). C. Number (C; left panel) and size (right panel) of spontaneously occurring tumors in the small intestine relative to the distance from the ventricle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3278393&req=5

Figure 1: PKA antagonist reduces tumor load in ApcMin/+ mice. A. Images of developing adenomas representative of each treatment acquired by transillumination microscopy are shown. White bars = 500 μm. B. Number (B; left panel) and size (B; mid panel) of spontaneously occurring tumors and distribution of tumor size in the treatment groups (B; right panel), tumor numbers/size class). Box plots with median (horizontal line); 25-75% (box) and 2.5-97.5% (bar) percentiles are shown for each treatment group (n = 7-10 as indicated). All tumors from the same animals were pooled when comparing tumor size distribution between the groups (n = 41-585) as indicated. Mann-Whitney Rank Sum test was used to compare the groups (SigmaPlot 11.0 (CA, USA). C. Number (C; left panel) and size (right panel) of spontaneously occurring tumors in the small intestine relative to the distance from the ventricle.
Mentions: To more closely delineate the effect of PKA in the COX-2 - PGE2 pathway active in colorectal cancer, we treated ApcMin/+ mice with the PKA antagonist Rp-8-Br-cAMPS for 6 weeks using earlier established doses (see Additional file 1, Supplementary information) and compared the result with that of treatment with the COX inhibitor indomethacin, previously shown to inhibit tumor development in the ApcMin/+model [20]. Phosphate buffered saline (PBS) was used as vehicle control for the Rp-8-Br-cAMPS. Examination revealed that indomethacin reduced the number and area of tumors in the small intestine of the ApcMin/+ mice compared to PBS (from 47 to 3 tumors per mouse and from 0.44 mm2 to 0.10 mm2 tumor area; P < 0.001; Figure 1A, B). The PKA antagonist Rp-8-Br-cAMPS did not significantly reduce the number of adenomas (47 versus 43 tumors; P = 0.368, Figure 1A, B), but reduced the tumor area by 36% (from 0.44 mm2 to 0.28 mm2; P < 0.001; Figure 1). Specifically, tumor load was reduced in the distal part of the small intestine (Figure 1C). The differential effect of COX inhibitor and PKA antagonist on tumor numbers and tumor size indicated to us that the mechanisms of action could be distinct and were examined in more detail in the following.

Bottom Line: Treatment of Apc(Min/+) mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas.Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE(2)-induced β-catenin phosphorylation and c-Myc upregulation.Based on our findings we suggest that PGE(2) act through PKA to promote β-catenin nuclear translocation and tumor development in Apc(Min/+) mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership and Biotechnology Centre, University of Oslo, Oslo, Norway.

ABSTRACT

Background: The adenomatous polyposis coli (APC) protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E(2) (PGE(2)) - PI-3 kinase pathways. Recent reports show that PGE(2)-induced phosphorylation of β-catenin by protein kinase A (PKA) increases nuclear translocation indicating two mechanisms of action of PGE(2) on β-catenin homeostasis.

Findings: Treatment of Apc(Min/+) mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE(2)-induced β-catenin phosphorylation and c-Myc upregulation.

Conclusion: Based on our findings we suggest that PGE(2) act through PKA to promote β-catenin nuclear translocation and tumor development in Apc(Min/+) mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

Show MeSH
Related in: MedlinePlus