Limits...
IGFBP-rP1 suppresses epithelial-mesenchymal transition and metastasis in colorectal cancer.

Zhu S, Zhang J, Xu F, Xu E, Ruan W, Ma Y, Huang Q, Lai M - Cell Death Dis (2015)

Bottom Line: Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined.Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression.Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China [2] Key Laboratory of Disease Proteomics of Zhejiang Province, Hangzhou, Zhejiang, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

Show MeSH

Related in: MedlinePlus

IGFBP-rP1 knockdown promotes EMT in SW480 cells. (a) The effectiveness of shRNA interference was confirmed by real-time RT-PCR and western blot analyses. The data were expressed as mean+S.D. of three independent experiments. (b) The expression of EMT markers was detected by western blot in scrambled control cells (shNC) and two stable IGFBP-rP1 knockdown cell clones (sh2 and sh3). (c) Nuclear expression of β-catenin detected by western blot in two SW480/shIGFBP-rP1 cell lines. Relocalization of β-catenin from adherens junctions of the membrane to cytoplasm and nucleus detected by immunofluorescence. (d) Wound-healing and transwell motility assays for SW480 cells expressing shRNA directed against IGFBP-rP1 or scrambled control shRNA ( × 100). *P<0.05. The data were expressed as mean+S.D. of three independent experiments. (e and f) The addition of rhIGFBP-rP1 protein to stable IGFBP-rP1-knockdown cells. SW480-sh2 cells were treated with rhIGFBP-rP1 protein (1 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. Transwell motility assay was performed in SW480-sh2 cells treated with rhIGFBP-rP1 ( × 100) and value was shown as mean+S.D. of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4385937&req=5

fig2: IGFBP-rP1 knockdown promotes EMT in SW480 cells. (a) The effectiveness of shRNA interference was confirmed by real-time RT-PCR and western blot analyses. The data were expressed as mean+S.D. of three independent experiments. (b) The expression of EMT markers was detected by western blot in scrambled control cells (shNC) and two stable IGFBP-rP1 knockdown cell clones (sh2 and sh3). (c) Nuclear expression of β-catenin detected by western blot in two SW480/shIGFBP-rP1 cell lines. Relocalization of β-catenin from adherens junctions of the membrane to cytoplasm and nucleus detected by immunofluorescence. (d) Wound-healing and transwell motility assays for SW480 cells expressing shRNA directed against IGFBP-rP1 or scrambled control shRNA ( × 100). *P<0.05. The data were expressed as mean+S.D. of three independent experiments. (e and f) The addition of rhIGFBP-rP1 protein to stable IGFBP-rP1-knockdown cells. SW480-sh2 cells were treated with rhIGFBP-rP1 protein (1 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. Transwell motility assay was performed in SW480-sh2 cells treated with rhIGFBP-rP1 ( × 100) and value was shown as mean+S.D. of three independent experiments

Mentions: We next determined whether knockdown of IGFBP-rP1 reversed its inhibitory role in EMT. We used short-hairpin RNA (shRNA) to stably knock down IGFBP-rP1 in SW480 cells, which have detectable endogenous IGFBP-rP1 expression (Figure 2a). To rule out clone-to-clone variations, two clones named SW480-sh2 and SW480-sh3 were selected. The knockdown efficiency of IGFBP-rP1 was further confirmed at the mRNA and protein levels (Figure 2a). IGFBP-rP1 downregulation induced EMT, leading to augmented N-cadherin, vimentin, and MMP9 protein levels as well as a significant decrease in ZO-1 expression (Figure 2b). Nuclear expression of β-catenin detected by western blot exhibited increased nuclear β-catenin levels in two SW480/shIGFBP-rP1 cell lines. Similarly, immunofluorescence showed that knockdown of IGFBP-rP1 results in relocalization of β-catenin from adherens junctions of the membrane to cytoplasm and nucleus (Figure 2c). Functionally, knockdown of IGFBP-rP1 dramatically increased the migration ability of SW480 cells (Figure 2d). In a complementary experiment, we determined whether the re-expression of IGFBP-rP1 rescued the EMT process induced by silencing it, and found that addition of rhIGFBP-rP1 protein restored ZO-1 expression as well as rescued the increased Vimentin, N-cadherin, and MMP9 expression triggered by IGFBP-rP1 knockdown in SW480 cells (Figure 2e). Consistently, the elevated transwell migratory cells were also reduced due to the restoration of IGFBP-rP1 (Figure 2f). Taken together, the functional role of IGFBP-rP1 in inhibiting EMT in CRC is further strengthened.


IGFBP-rP1 suppresses epithelial-mesenchymal transition and metastasis in colorectal cancer.

Zhu S, Zhang J, Xu F, Xu E, Ruan W, Ma Y, Huang Q, Lai M - Cell Death Dis (2015)

IGFBP-rP1 knockdown promotes EMT in SW480 cells. (a) The effectiveness of shRNA interference was confirmed by real-time RT-PCR and western blot analyses. The data were expressed as mean+S.D. of three independent experiments. (b) The expression of EMT markers was detected by western blot in scrambled control cells (shNC) and two stable IGFBP-rP1 knockdown cell clones (sh2 and sh3). (c) Nuclear expression of β-catenin detected by western blot in two SW480/shIGFBP-rP1 cell lines. Relocalization of β-catenin from adherens junctions of the membrane to cytoplasm and nucleus detected by immunofluorescence. (d) Wound-healing and transwell motility assays for SW480 cells expressing shRNA directed against IGFBP-rP1 or scrambled control shRNA ( × 100). *P<0.05. The data were expressed as mean+S.D. of three independent experiments. (e and f) The addition of rhIGFBP-rP1 protein to stable IGFBP-rP1-knockdown cells. SW480-sh2 cells were treated with rhIGFBP-rP1 protein (1 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. Transwell motility assay was performed in SW480-sh2 cells treated with rhIGFBP-rP1 ( × 100) and value was shown as mean+S.D. of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385937&req=5

fig2: IGFBP-rP1 knockdown promotes EMT in SW480 cells. (a) The effectiveness of shRNA interference was confirmed by real-time RT-PCR and western blot analyses. The data were expressed as mean+S.D. of three independent experiments. (b) The expression of EMT markers was detected by western blot in scrambled control cells (shNC) and two stable IGFBP-rP1 knockdown cell clones (sh2 and sh3). (c) Nuclear expression of β-catenin detected by western blot in two SW480/shIGFBP-rP1 cell lines. Relocalization of β-catenin from adherens junctions of the membrane to cytoplasm and nucleus detected by immunofluorescence. (d) Wound-healing and transwell motility assays for SW480 cells expressing shRNA directed against IGFBP-rP1 or scrambled control shRNA ( × 100). *P<0.05. The data were expressed as mean+S.D. of three independent experiments. (e and f) The addition of rhIGFBP-rP1 protein to stable IGFBP-rP1-knockdown cells. SW480-sh2 cells were treated with rhIGFBP-rP1 protein (1 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. Transwell motility assay was performed in SW480-sh2 cells treated with rhIGFBP-rP1 ( × 100) and value was shown as mean+S.D. of three independent experiments
Mentions: We next determined whether knockdown of IGFBP-rP1 reversed its inhibitory role in EMT. We used short-hairpin RNA (shRNA) to stably knock down IGFBP-rP1 in SW480 cells, which have detectable endogenous IGFBP-rP1 expression (Figure 2a). To rule out clone-to-clone variations, two clones named SW480-sh2 and SW480-sh3 were selected. The knockdown efficiency of IGFBP-rP1 was further confirmed at the mRNA and protein levels (Figure 2a). IGFBP-rP1 downregulation induced EMT, leading to augmented N-cadherin, vimentin, and MMP9 protein levels as well as a significant decrease in ZO-1 expression (Figure 2b). Nuclear expression of β-catenin detected by western blot exhibited increased nuclear β-catenin levels in two SW480/shIGFBP-rP1 cell lines. Similarly, immunofluorescence showed that knockdown of IGFBP-rP1 results in relocalization of β-catenin from adherens junctions of the membrane to cytoplasm and nucleus (Figure 2c). Functionally, knockdown of IGFBP-rP1 dramatically increased the migration ability of SW480 cells (Figure 2d). In a complementary experiment, we determined whether the re-expression of IGFBP-rP1 rescued the EMT process induced by silencing it, and found that addition of rhIGFBP-rP1 protein restored ZO-1 expression as well as rescued the increased Vimentin, N-cadherin, and MMP9 expression triggered by IGFBP-rP1 knockdown in SW480 cells (Figure 2e). Consistently, the elevated transwell migratory cells were also reduced due to the restoration of IGFBP-rP1 (Figure 2f). Taken together, the functional role of IGFBP-rP1 in inhibiting EMT in CRC is further strengthened.

Bottom Line: Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined.Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression.Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pathology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China [2] Key Laboratory of Disease Proteomics of Zhejiang Province, Hangzhou, Zhejiang, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

Show MeSH
Related in: MedlinePlus