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Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line.

Fukushima K, Tsuchiya K, Kano Y, Horita N, Hibiya S, Hayashi R, Kitagaki K, Negi M, Itoh E, Akashi T, Eishi Y, Oshima S, Nagaishi T, Okamoto R, Nakamura T, Watanabe M - Cancer Sci. (2015)

Bottom Line: We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non-mucinous form of CRC.Consequently, the treatment with TNF-α stabilized Atoh1 protein through the inactivation of GSK-3β via Akt, resulting in the mucinous form of CRC cell lines.In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

View Article: PubMed Central - PubMed

Affiliation: Departments of Gastroenterology and Hepatology, Graduate School Tokyo Medical and Dental University, Tokyo, Japan.

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Atonal homolog 1 (Atoh1) protein is stabilized in colon cancer cells by NF-κB signal activation. (a) mCherry-Atoh1 or mock DNA was transfected into DLD1 cells (Atoh1-DLD1 cells and mock-DLD1 cells). After 4 weeks in culture with inflammatory materials, the expression of mCherry-Atoh1 protein was determined by immunofluorescence using mCherry antibody and Alexa 488 antibody (green fluorescence). mCherry protein was stably expressed in the nuclei of Atoh1-DLD1 cells cultured with TNF-α, lipopolysaccharides (LPS) and flagellin. Scale bar, 50 μm. (b) Western blot analysis using Atoh1 antibody showed the expression of Atoh1 protein in Atoh1-DLD1 cells with TNF-α for 4 weeks. Endogenous Atoh1 protein was not detected in mock-DLD1 cells with TNF-α because the endogenous Atoh1 gene was not expressed in DLD1 cells. (c) Immunofluorescence staining showed nuclear localization of NF-κB p65 by treatment with TNF-α for 20 weeks. mCherry fluorescence was shown in the nuclei of only Atoh1-DLD1 cells with TNF-α, indicating the stable expression of Atoh1. Scale bar, 20 μm. (d) Western blot analysis of DLD1 cells during treatment with TNF-α for 20 weeks.
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fig01: Atonal homolog 1 (Atoh1) protein is stabilized in colon cancer cells by NF-κB signal activation. (a) mCherry-Atoh1 or mock DNA was transfected into DLD1 cells (Atoh1-DLD1 cells and mock-DLD1 cells). After 4 weeks in culture with inflammatory materials, the expression of mCherry-Atoh1 protein was determined by immunofluorescence using mCherry antibody and Alexa 488 antibody (green fluorescence). mCherry protein was stably expressed in the nuclei of Atoh1-DLD1 cells cultured with TNF-α, lipopolysaccharides (LPS) and flagellin. Scale bar, 50 μm. (b) Western blot analysis using Atoh1 antibody showed the expression of Atoh1 protein in Atoh1-DLD1 cells with TNF-α for 4 weeks. Endogenous Atoh1 protein was not detected in mock-DLD1 cells with TNF-α because the endogenous Atoh1 gene was not expressed in DLD1 cells. (c) Immunofluorescence staining showed nuclear localization of NF-κB p65 by treatment with TNF-α for 20 weeks. mCherry fluorescence was shown in the nuclei of only Atoh1-DLD1 cells with TNF-α, indicating the stable expression of Atoh1. Scale bar, 20 μm. (d) Western blot analysis of DLD1 cells during treatment with TNF-α for 20 weeks.

Mentions: To assess the stabilization of Atoh1 protein in colon cancer, we attempted to construct Atoh1 gene linked to mCherry to visualize Atoh1 protein expression. Our previous study indicated that Atoh1 protein was not detected by Atoh1 gene transfection alone in DLD1 cells derived from human sporadic CRC, in which endogenous Atoh1 gene was not expressed.13 Therefore, we tried various reagents related to inflammation to stabilize Atoh1 protein, resulting in the expression of Atoh1 protein in mCherry-Atoh1 gene-expressing DLD1 cells (Fig.1a). Among these reagents, TNF-α is one of the most important proinflammatory cytokines, with a central role in intestinal inflammation.15 Antibodies targeting TNF-α have provided the most successful approach in the clinical management of IBD.16 Therefore, we focused on the role of TNF-α in Atoh1 protein stabilization. We then confirmed the stabilization of Atoh1 protein by TNF-α (Fig.1b). To assess the effect of long-term treatment with TNF-α on cancer cells, we added TNF-α to each cell culture for 20 weeks. Fluorescence analysis showed the stable expression of Atoh1 protein in the nuclei of DLD1 cells with NF-κB signal activation by TNF-α (Fig.1c). To assess the regulation of Atoh1 protein stabilization through the phosphorylation via GSK-3β, we investigated the activation of GSK-3β by its own phosphorylation by Akt. Western blotting revealed that TNF-α treatment induced the inactivation of GSK-3β function through the phosphorylation of GSK-3β. Because Akt is well known as an upstream mediator in the signal cascade involved in GSK-3 phosphorylation,17 Akt activation was also assessed. Our results showed that the phosphorylation of Akt was induced by TNF-α. However, TNF-α did not affect any other phosphorylation enzymes in the signal cascade of GSK-3β phosphorylation (Fig.1d).


Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line.

Fukushima K, Tsuchiya K, Kano Y, Horita N, Hibiya S, Hayashi R, Kitagaki K, Negi M, Itoh E, Akashi T, Eishi Y, Oshima S, Nagaishi T, Okamoto R, Nakamura T, Watanabe M - Cancer Sci. (2015)

Atonal homolog 1 (Atoh1) protein is stabilized in colon cancer cells by NF-κB signal activation. (a) mCherry-Atoh1 or mock DNA was transfected into DLD1 cells (Atoh1-DLD1 cells and mock-DLD1 cells). After 4 weeks in culture with inflammatory materials, the expression of mCherry-Atoh1 protein was determined by immunofluorescence using mCherry antibody and Alexa 488 antibody (green fluorescence). mCherry protein was stably expressed in the nuclei of Atoh1-DLD1 cells cultured with TNF-α, lipopolysaccharides (LPS) and flagellin. Scale bar, 50 μm. (b) Western blot analysis using Atoh1 antibody showed the expression of Atoh1 protein in Atoh1-DLD1 cells with TNF-α for 4 weeks. Endogenous Atoh1 protein was not detected in mock-DLD1 cells with TNF-α because the endogenous Atoh1 gene was not expressed in DLD1 cells. (c) Immunofluorescence staining showed nuclear localization of NF-κB p65 by treatment with TNF-α for 20 weeks. mCherry fluorescence was shown in the nuclei of only Atoh1-DLD1 cells with TNF-α, indicating the stable expression of Atoh1. Scale bar, 20 μm. (d) Western blot analysis of DLD1 cells during treatment with TNF-α for 20 weeks.
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fig01: Atonal homolog 1 (Atoh1) protein is stabilized in colon cancer cells by NF-κB signal activation. (a) mCherry-Atoh1 or mock DNA was transfected into DLD1 cells (Atoh1-DLD1 cells and mock-DLD1 cells). After 4 weeks in culture with inflammatory materials, the expression of mCherry-Atoh1 protein was determined by immunofluorescence using mCherry antibody and Alexa 488 antibody (green fluorescence). mCherry protein was stably expressed in the nuclei of Atoh1-DLD1 cells cultured with TNF-α, lipopolysaccharides (LPS) and flagellin. Scale bar, 50 μm. (b) Western blot analysis using Atoh1 antibody showed the expression of Atoh1 protein in Atoh1-DLD1 cells with TNF-α for 4 weeks. Endogenous Atoh1 protein was not detected in mock-DLD1 cells with TNF-α because the endogenous Atoh1 gene was not expressed in DLD1 cells. (c) Immunofluorescence staining showed nuclear localization of NF-κB p65 by treatment with TNF-α for 20 weeks. mCherry fluorescence was shown in the nuclei of only Atoh1-DLD1 cells with TNF-α, indicating the stable expression of Atoh1. Scale bar, 20 μm. (d) Western blot analysis of DLD1 cells during treatment with TNF-α for 20 weeks.
Mentions: To assess the stabilization of Atoh1 protein in colon cancer, we attempted to construct Atoh1 gene linked to mCherry to visualize Atoh1 protein expression. Our previous study indicated that Atoh1 protein was not detected by Atoh1 gene transfection alone in DLD1 cells derived from human sporadic CRC, in which endogenous Atoh1 gene was not expressed.13 Therefore, we tried various reagents related to inflammation to stabilize Atoh1 protein, resulting in the expression of Atoh1 protein in mCherry-Atoh1 gene-expressing DLD1 cells (Fig.1a). Among these reagents, TNF-α is one of the most important proinflammatory cytokines, with a central role in intestinal inflammation.15 Antibodies targeting TNF-α have provided the most successful approach in the clinical management of IBD.16 Therefore, we focused on the role of TNF-α in Atoh1 protein stabilization. We then confirmed the stabilization of Atoh1 protein by TNF-α (Fig.1b). To assess the effect of long-term treatment with TNF-α on cancer cells, we added TNF-α to each cell culture for 20 weeks. Fluorescence analysis showed the stable expression of Atoh1 protein in the nuclei of DLD1 cells with NF-κB signal activation by TNF-α (Fig.1c). To assess the regulation of Atoh1 protein stabilization through the phosphorylation via GSK-3β, we investigated the activation of GSK-3β by its own phosphorylation by Akt. Western blotting revealed that TNF-α treatment induced the inactivation of GSK-3β function through the phosphorylation of GSK-3β. Because Akt is well known as an upstream mediator in the signal cascade involved in GSK-3 phosphorylation,17 Akt activation was also assessed. Our results showed that the phosphorylation of Akt was induced by TNF-α. However, TNF-α did not affect any other phosphorylation enzymes in the signal cascade of GSK-3β phosphorylation (Fig.1d).

Bottom Line: We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non-mucinous form of CRC.Consequently, the treatment with TNF-α stabilized Atoh1 protein through the inactivation of GSK-3β via Akt, resulting in the mucinous form of CRC cell lines.In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

View Article: PubMed Central - PubMed

Affiliation: Departments of Gastroenterology and Hepatology, Graduate School Tokyo Medical and Dental University, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus