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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

Overexpression of TLE3 repressed the proliferation and tumorigenesis of human CRC cells. a Overexpression of TLE3 in SW480 and Ls174t cells. α-Tubulin was used as a loading control. b Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by MTT assays. c Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by colony formation assays. d Overexpression of TLE3 repressed anchorage-independent growth ability of SW480 and Ls174t cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW480/vector or SW480/TLE3 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. p < 0.05. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05
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Fig2: Overexpression of TLE3 repressed the proliferation and tumorigenesis of human CRC cells. a Overexpression of TLE3 in SW480 and Ls174t cells. α-Tubulin was used as a loading control. b Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by MTT assays. c Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by colony formation assays. d Overexpression of TLE3 repressed anchorage-independent growth ability of SW480 and Ls174t cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW480/vector or SW480/TLE3 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. p < 0.05. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05

Mentions: To investigate the potential role of TLE3 in the progression of human CRC, stable CRC cell lines SW480 and Ls174t of TLE3 overexpression were established (Fig. 2a). In comparison with control cells, the proliferation of SW480 and Ls174t cells was inhibited by TLE3 overexpression as determined by MTT and colony formation assays (Fig. 2b and c). Anchorage-independent growth activity was examined using soft agar formation assays, whose results showed that TLE3 overexpression also repressed the proliferation of SW480 and Ls174t cells in soft agar, as both the number and size of colonies were decreased in comparison with control cells (Fig. 2d). To confirm this effect in vivo, tumorigenesis assays by subcutaneous injection were performed in nude mice. The TLE3 overexpression group exhibited remarkably slower tumor growth and smaller tumor volume in comparison with the control group (Fig. 2e, n = 6). In addition to the difference of tumor volume, much lower Ki-67 index was found in tumors formed by TLE3 overexpression group than that in the control group, as detected by IHC analysis of Ki-67 (Fig. 2f).Fig. 2


TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways
Overexpression of TLE3 repressed the proliferation and tumorigenesis of human CRC cells. a Overexpression of TLE3 in SW480 and Ls174t cells. α-Tubulin was used as a loading control. b Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by MTT assays. c Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by colony formation assays. d Overexpression of TLE3 repressed anchorage-independent growth ability of SW480 and Ls174t cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW480/vector or SW480/TLE3 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. p < 0.05. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037636&req=5

Fig2: Overexpression of TLE3 repressed the proliferation and tumorigenesis of human CRC cells. a Overexpression of TLE3 in SW480 and Ls174t cells. α-Tubulin was used as a loading control. b Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by MTT assays. c Overexpression of TLE3 repressed cell proliferation of SW480 and Ls174t cells by colony formation assays. d Overexpression of TLE3 repressed anchorage-independent growth ability of SW480 and Ls174t cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW480/vector or SW480/TLE3 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. p < 0.05. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05
Mentions: To investigate the potential role of TLE3 in the progression of human CRC, stable CRC cell lines SW480 and Ls174t of TLE3 overexpression were established (Fig. 2a). In comparison with control cells, the proliferation of SW480 and Ls174t cells was inhibited by TLE3 overexpression as determined by MTT and colony formation assays (Fig. 2b and c). Anchorage-independent growth activity was examined using soft agar formation assays, whose results showed that TLE3 overexpression also repressed the proliferation of SW480 and Ls174t cells in soft agar, as both the number and size of colonies were decreased in comparison with control cells (Fig. 2d). To confirm this effect in vivo, tumorigenesis assays by subcutaneous injection were performed in nude mice. The TLE3 overexpression group exhibited remarkably slower tumor growth and smaller tumor volume in comparison with the control group (Fig. 2e, n = 6). In addition to the difference of tumor volume, much lower Ki-67 index was found in tumors formed by TLE3 overexpression group than that in the control group, as detected by IHC analysis of Ki-67 (Fig. 2f).Fig. 2

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