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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.

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Related in: MedlinePlus

IGFBP-rP1 overexpression blocks EMT in CRC. (a and b) Well-established EMT markers were analyzed by western blot after IGFBP-rP1 transfection in SW620 and CW2 cells. E-cadherin expression was markedly induced and the mesenchymal markers N-cadherin, vimentin, and MMP9 were decreased in IGFBP-rP1-transfected cells (-rP1) compared with control vector-transfected (-ctrl) and parental cells (-blk). Equal loading was confirmed by GAPDH. (c) SW620 cells were treated with rhIGFBP-rP1 protein (4 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. (d) Immunofluorescence images of SW620 cells stained for E-cadherin, β-catenin, and F-actin. The images were taken at × 630 (for E-cadherin and β-catenin ) and × 1000 (for F-actin). Arrow: Lamellipodia and microspike formation. DAPI and β-catenin staining was showed seperately and then the merged images were showed. (e and f) Wound-healing and transwell motility assays for SW620-ctrl and SW620-rP1 cells. The motility was drastically decreased in IGFBP-rP1 transfected SW620 cells ( × 100). Cell motility detemined by wound-healing assay was quantified as an inverse ratio of gap distance (GD) at 48 h relative to that at 0 h. *P< 0.05. The data were expressed as mean+S.D. of three independent experiments
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fig1: IGFBP-rP1 overexpression blocks EMT in CRC. (a and b) Well-established EMT markers were analyzed by western blot after IGFBP-rP1 transfection in SW620 and CW2 cells. E-cadherin expression was markedly induced and the mesenchymal markers N-cadherin, vimentin, and MMP9 were decreased in IGFBP-rP1-transfected cells (-rP1) compared with control vector-transfected (-ctrl) and parental cells (-blk). Equal loading was confirmed by GAPDH. (c) SW620 cells were treated with rhIGFBP-rP1 protein (4 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. (d) Immunofluorescence images of SW620 cells stained for E-cadherin, β-catenin, and F-actin. The images were taken at × 630 (for E-cadherin and β-catenin ) and × 1000 (for F-actin). Arrow: Lamellipodia and microspike formation. DAPI and β-catenin staining was showed seperately and then the merged images were showed. (e and f) Wound-healing and transwell motility assays for SW620-ctrl and SW620-rP1 cells. The motility was drastically decreased in IGFBP-rP1 transfected SW620 cells ( × 100). Cell motility detemined by wound-healing assay was quantified as an inverse ratio of gap distance (GD) at 48 h relative to that at 0 h. *P< 0.05. The data were expressed as mean+S.D. of three independent experiments

Mentions: To directly investigate the potential role of IGFBP-rP1, we stably overexpressed it in CRC cell lines and evaluated its effect on EMT. We found that IGFBP-rP1 overexpression increased the expression levels of the epithelial cell marker E-cadherin and blocked that of the mesenchymal markers N-cadherin, vimentin, and MMP9 in SW620 and CW2 cells (Figures 1a and b). As IGFBP-rP1 is a secreted protein, we sought to determine whether exogenous IGFBP-rP1 similarly changed EMT marker protein levels. In agreement with the changes in IGFBP-rP1-overexpressing cells, addition of recombinant human IGFBP-rP1 protein (rhIGFBP-rP1) resulted in high E-cadherin and low mesenchymal markers levels in SW620 cells (Figure 1c). Meanwhile, upregulated E-cadherin accumulated in the cell-to-cell junctions, and nuclear β-catenin re-localized to the membrane and cytoplasm of SW620 cells, as expected (Figure 1d). Furthermore, SW620-IGFBP-rP1 cells exhibited organized F-actin fibers as well as reduced stress fibers and lamellipodia at the periphery compared with the control cells and the parental cells (Figure 1d). Functionally, overexpression of IGFBP-rP1 decreased the motility ability (Figures 1e and f) of SW620 cells. Collectively, these data indicate that IGFBP-rP1 is a negative regulation of EMT in CRC.


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 overexpression blocks EMT in CRC. (a and b) Well-established EMT markers were analyzed by western blot after IGFBP-rP1 transfection in SW620 and CW2 cells. E-cadherin expression was markedly induced and the mesenchymal markers N-cadherin, vimentin, and MMP9 were decreased in IGFBP-rP1-transfected cells (-rP1) compared with control vector-transfected (-ctrl) and parental cells (-blk). Equal loading was confirmed by GAPDH. (c) SW620 cells were treated with rhIGFBP-rP1 protein (4 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. (d) Immunofluorescence images of SW620 cells stained for E-cadherin, β-catenin, and F-actin. The images were taken at × 630 (for E-cadherin and β-catenin ) and × 1000 (for F-actin). Arrow: Lamellipodia and microspike formation. DAPI and β-catenin staining was showed seperately and then the merged images were showed. (e and f) Wound-healing and transwell motility assays for SW620-ctrl and SW620-rP1 cells. The motility was drastically decreased in IGFBP-rP1 transfected SW620 cells ( × 100). Cell motility detemined by wound-healing assay was quantified as an inverse ratio of gap distance (GD) at 48 h relative to that at 0 h. *P< 0.05. The data were expressed 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

fig1: IGFBP-rP1 overexpression blocks EMT in CRC. (a and b) Well-established EMT markers were analyzed by western blot after IGFBP-rP1 transfection in SW620 and CW2 cells. E-cadherin expression was markedly induced and the mesenchymal markers N-cadherin, vimentin, and MMP9 were decreased in IGFBP-rP1-transfected cells (-rP1) compared with control vector-transfected (-ctrl) and parental cells (-blk). Equal loading was confirmed by GAPDH. (c) SW620 cells were treated with rhIGFBP-rP1 protein (4 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. (d) Immunofluorescence images of SW620 cells stained for E-cadherin, β-catenin, and F-actin. The images were taken at × 630 (for E-cadherin and β-catenin ) and × 1000 (for F-actin). Arrow: Lamellipodia and microspike formation. DAPI and β-catenin staining was showed seperately and then the merged images were showed. (e and f) Wound-healing and transwell motility assays for SW620-ctrl and SW620-rP1 cells. The motility was drastically decreased in IGFBP-rP1 transfected SW620 cells ( × 100). Cell motility detemined by wound-healing assay was quantified as an inverse ratio of gap distance (GD) at 48 h relative to that at 0 h. *P< 0.05. The data were expressed as mean+S.D. of three independent experiments
Mentions: To directly investigate the potential role of IGFBP-rP1, we stably overexpressed it in CRC cell lines and evaluated its effect on EMT. We found that IGFBP-rP1 overexpression increased the expression levels of the epithelial cell marker E-cadherin and blocked that of the mesenchymal markers N-cadherin, vimentin, and MMP9 in SW620 and CW2 cells (Figures 1a and b). As IGFBP-rP1 is a secreted protein, we sought to determine whether exogenous IGFBP-rP1 similarly changed EMT marker protein levels. In agreement with the changes in IGFBP-rP1-overexpressing cells, addition of recombinant human IGFBP-rP1 protein (rhIGFBP-rP1) resulted in high E-cadherin and low mesenchymal markers levels in SW620 cells (Figure 1c). Meanwhile, upregulated E-cadherin accumulated in the cell-to-cell junctions, and nuclear β-catenin re-localized to the membrane and cytoplasm of SW620 cells, as expected (Figure 1d). Furthermore, SW620-IGFBP-rP1 cells exhibited organized F-actin fibers as well as reduced stress fibers and lamellipodia at the periphery compared with the control cells and the parental cells (Figure 1d). Functionally, overexpression of IGFBP-rP1 decreased the motility ability (Figures 1e and f) of SW620 cells. Collectively, these data indicate that IGFBP-rP1 is a negative regulation of EMT in CRC.

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