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TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways

View Article: PubMed Central - PubMed

ABSTRACT

Background: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/β-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce.

Methods: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR.

Results: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1.

Conclusion: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.

Electronic supplementary material: The online version of this article (doi:10.1186/s13046-016-0426-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

TLE3 regulated p21 and p27 through the MAPK and AKT signaling pathways in CRC cells. a Western blot analyses of the expression of p-FOXO3a, total FOXO3a, phosphorylated GSK, total GSK, phosphorylated ERK, total ERK, phosphorylated AKT, total AKT, p21Cip1/WAF1 and p27Kip1 proteins in indicated human CRC cell lines. b SW620 cells with TLE3 knocking down were treated with the AKT inhibitor PF04691502 (10 μM), the ERK inhibitor GDC0994 (50 μM) or DMSO for 24 h, then harvested to examine the expression levels of the indicated proteins by western blot. c, d and e Proliferation ability of SW620 cells with TLE3 knocking down was determined by MTT assay (c), colony formation assay (d) and soft agar assay (e) after treatment with GDC0994, PF04691502 or DMSO. Error bars represent mean ± SD from 3 independent experiments. * p < 0.05
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Fig5: TLE3 regulated p21 and p27 through the MAPK and AKT signaling pathways in CRC cells. a Western blot analyses of the expression of p-FOXO3a, total FOXO3a, phosphorylated GSK, total GSK, phosphorylated ERK, total ERK, phosphorylated AKT, total AKT, p21Cip1/WAF1 and p27Kip1 proteins in indicated human CRC cell lines. b SW620 cells with TLE3 knocking down were treated with the AKT inhibitor PF04691502 (10 μM), the ERK inhibitor GDC0994 (50 μM) or DMSO for 24 h, then harvested to examine the expression levels of the indicated proteins by western blot. c, d and e Proliferation ability of SW620 cells with TLE3 knocking down was determined by MTT assay (c), colony formation assay (d) and soft agar assay (e) after treatment with GDC0994, PF04691502 or DMSO. Error bars represent mean ± SD from 3 independent experiments. * p < 0.05

Mentions: The GSEA analysis based on TCGA COAD RNA expression dataset also revealed that TLE3 level was negatively correlated with AKT activity (Additional file 4: Figure S3), indicating that TLE3 might inhibit the activation of AKT signaling pathway. Moreover, Western blot showed that the levels of phosphorylated FOXO3, GSK, ERK and AKT were decreased in SW480 and Ls174t cells with TLE3 overexpressing, whereas increased in HCT15 and SW620 cells with TLE3 knocking down in comparison with control cells (Fig. 5a). Since TLE3 significantly inhibited the G1-S phase transition as shown above, we then detected the cyclin-dependent kinases inhibitor proteins p21Cip1/WAF1 (p21) and p27Kip1 (p27) that are responsible for this transition [35, 36]. Results showed that p21 and p27 were upregulated in SW480 and Ls174t cells with TLE3 overexpressing (Fig. 5a), whereas they were down-regulated in HCT15 and SW620 cells with TLE3 knocking down (Fig. 5a). Additionally, transcriptional levels of p21 and p27 were also regulated by TLE3 (Additional file 5: Figure S4 a, b, c and d).Fig. 5


TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways
TLE3 regulated p21 and p27 through the MAPK and AKT signaling pathways in CRC cells. a Western blot analyses of the expression of p-FOXO3a, total FOXO3a, phosphorylated GSK, total GSK, phosphorylated ERK, total ERK, phosphorylated AKT, total AKT, p21Cip1/WAF1 and p27Kip1 proteins in indicated human CRC cell lines. b SW620 cells with TLE3 knocking down were treated with the AKT inhibitor PF04691502 (10 μM), the ERK inhibitor GDC0994 (50 μM) or DMSO for 24 h, then harvested to examine the expression levels of the indicated proteins by western blot. c, d and e Proliferation ability of SW620 cells with TLE3 knocking down was determined by MTT assay (c), colony formation assay (d) and soft agar assay (e) after treatment with GDC0994, PF04691502 or DMSO. Error bars represent mean ± SD from 3 independent experiments. * p < 0.05
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037636&req=5

Fig5: TLE3 regulated p21 and p27 through the MAPK and AKT signaling pathways in CRC cells. a Western blot analyses of the expression of p-FOXO3a, total FOXO3a, phosphorylated GSK, total GSK, phosphorylated ERK, total ERK, phosphorylated AKT, total AKT, p21Cip1/WAF1 and p27Kip1 proteins in indicated human CRC cell lines. b SW620 cells with TLE3 knocking down were treated with the AKT inhibitor PF04691502 (10 μM), the ERK inhibitor GDC0994 (50 μM) or DMSO for 24 h, then harvested to examine the expression levels of the indicated proteins by western blot. c, d and e Proliferation ability of SW620 cells with TLE3 knocking down was determined by MTT assay (c), colony formation assay (d) and soft agar assay (e) after treatment with GDC0994, PF04691502 or DMSO. Error bars represent mean ± SD from 3 independent experiments. * p < 0.05
Mentions: The GSEA analysis based on TCGA COAD RNA expression dataset also revealed that TLE3 level was negatively correlated with AKT activity (Additional file 4: Figure S3), indicating that TLE3 might inhibit the activation of AKT signaling pathway. Moreover, Western blot showed that the levels of phosphorylated FOXO3, GSK, ERK and AKT were decreased in SW480 and Ls174t cells with TLE3 overexpressing, whereas increased in HCT15 and SW620 cells with TLE3 knocking down in comparison with control cells (Fig. 5a). Since TLE3 significantly inhibited the G1-S phase transition as shown above, we then detected the cyclin-dependent kinases inhibitor proteins p21Cip1/WAF1 (p21) and p27Kip1 (p27) that are responsible for this transition [35, 36]. Results showed that p21 and p27 were upregulated in SW480 and Ls174t cells with TLE3 overexpressing (Fig. 5a), whereas they were down-regulated in HCT15 and SW620 cells with TLE3 knocking down (Fig. 5a). Additionally, transcriptional levels of p21 and p27 were also regulated by TLE3 (Additional file 5: Figure S4 a, b, c and d).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/&beta;-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce.

Methods: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR.

Results: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1.

Conclusion: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.

Electronic supplementary material: The online version of this article (doi:10.1186/s13046-016-0426-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus