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

Abstract

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis-associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. 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. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. 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. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5-fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF-κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. 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.

Keywords: Atonal homolog 1; cancer stem cells; colitis-associated cancer; inflammatory bowel disease; signet ring cell.

Figure 1

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.

Figure 2

Atonal homolog 1 (Atoh1) protein stabilized by TNF-α in colon cancer acquires mucinous secretion and upregulated Lgr5 expression. (a) HD6, MUC2 and TFF3 genes were significantly upregulated in Atoh1-DLD1 cells cultured with TNF-α for 20 weeks by RT-PCR. Lgr5 gene was also upregulated in Atoh1-DLD1 cells with TNF-α. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. (b) Immunofluorescence of HD6, MUC2, TFF3 and Lgr5 showed the expression of these proteins in Atoh1-DLD1 cells with TNF-α treatment for 20 weeks. Scale bar, 20 μm.

Figure 3

Stable expression of Atonal homolog 1 (Atoh1) protein by TNF-α leads to higher malignant potential. (a) Proliferation assay revealed that Atoh1-DLD1 cells with TNF-α for 20 weeks showed slow growth. ***p < 0.001, n = 6. (b) S/G2/M phase of cells were visualized using the Fucci system. FACS analysis showed that TNF-α for 20 weeks decreased the number of Atoh1-DLD1 cells in the S/G2/M phase, whereas TNF-α increased the number of mock-DLD1 cells in the S/G2/M phase. **p < 0.01, ***p < 0.001, n = 3. (c) After 20 weeks culture with TNF-α, DLD1 cells were treated with 5-Fluorouracil (5-FU; 20 μM), oxaliplatin (L-OHP; 100 μM) or DMSO for 48 h to examine the chemoresistance. MTS assay showed that the reduction of cells in Atoh1-DLD1 cells with TNF-α by treatment with 5-FU or L-OHP was less than that of others. *p < 0.001, n = 6. (d) After treatment with 5-FU or L-OHP for 48 h, apoptosis signal was assessed by western blot. The stabilization of the Atoh1 protein by TNF-α increased the resistance to apoptosis.

Figure 4

Stable expression of Atonal homolog 1 (Atoh1) protein by TNF-α promotes cell migration. (a) Migration assay showed that the vacant area was occupied by a greater number of Atoh1-DLD1 cells cultured with TNF-α for 20 weeks than the others. Scale bar, 500 μm. (b) The ratio of the remaining vacant area is shown. The vacant area of Atoh1-DLD1 cells was smaller than that of others at 24 h after the cells were seeded. ***P < 0.001, n = 6. (c) The expression of ZEB1 in DLD1 cells was analyzed by RT-PCR. ZEB1 gene was significantly upregulated in Atoh1-DLD1 cells with TNF-α treatment for 20 weeks. **P < 0.01, ***P < 0.001, n = 3.

Figure 5

Atonal homolog 1 (Atoh1) protein was expressed in colitis-associated colorectal cancer (CAC) with NF-κB signal activation. (a) HE-staining of colonic specimen taken from patients with colorectal cancer (CRC) or CAC with ulcerative colitis (UC). Scale bar, 100 μm. Muc, mucinous adenocarcinoma; Tub, tubular adenocarcinoma. (b) Immunofluorescence staining of Atoh1 showed Atoh1 protein expression in mucinous carcinoma (MC) of CAC. Scale bar, 50 μm. Lower right frame shows a high-magnified image merged with DAPI. (c) Immunofluorescence analysis of NF-κB p65. The nuclear localization of p65 is shown in both the inflamed and cancer region of colon in patients with UC. The white arrow head indicates a cell with nuclear localization of p65. Scale bar, 50 μm. The lower right frame shows a high-magnified image merged with DAPI. (d) Immunofluorescence staining of HD6 showed the expression of HD6 only in CAC of the colon. Scale bar, 20 μm. The lower right frame shows a high-magnified image merged with DAPI.