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

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.

Figure 1

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

Figure 2

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

Figure 3

Tumorigenicity assay in nude mice. (a) SW620-ctrl and SW620-rP1 cells were injected subcutaneously into nude mice (n=4). Photographs of the tumors and mice were taken at the end of the study. (b) IGFBP-rP1 overexpression showed a lower growth rate (Mann–Whitney U-test). Points and bars represent the average±S.D. (c) Weights of xenograft tumors with and without IGFBP-rP1 overexpression. (d) Photographs of tumors and nude mice after SW480-sh2 and SW480-shNC cells were injected subcutaneously (n=4). (e) IGFBP-rP1 knockdown increased the tumor growth rate in nude mice (Mann–Whitney U-test). Points and bars represent the average±S.D. (f) The average tumor was heavier in the SW480-sh2 group than the SW480-shNC group. (g and h) EMT markers examined by western blot in harvested mouse tumor samples (GAPDH as a loading control)

Figure 4

IGFBP-rP1 inhibited tumor metastasis in NOD/SCID mice. (a) Representative gross images and hematoxylin and eosin-stained lung sections from animals injected intravenously with control or IGFBP-rP1 overexpressing SW620 cells. Arrows, curve, and triangle: metastatic nodes in the lung. Scale bars, 9 mm (right top) and 8 mm (right bottom). (b) Box plot showing the number of lung metastatic nodules. P, by Mann–Whitney U-test. (c) Representative images of pleural metastasis of animals injected intravenously with control SW620 cells. (d) Quantification of the pleural nodules in each experimental group. P, by Mann–Whitney U-test. (e) Representative H&E images of pleural invasion and costal cartilage invasion tissue were shown, respectively. Scale bars, 6 mm (right) and 800 μm (left)

Figure 5

IGFBP-rP1 inhibits TGF-β receptor expression and its downstream signaling in SW620 and SW480 cells. (a) SW620 cell lysates from blk, vector, and IGFBP-rP1 cells were analyzed the indicated proteins by western blot. (b) SW480 cell lysates from scramble shRNA and IGFBP-rP1 shRNA cells were analyzed the indicated proteins by western blot. (c) Western blot of the indicated antibodies in two SW480-IGFBP-rP1 knockdown cell clones and controls untreated or treated with 10 ng/ml SB431542 for 48 h

Figure 6

IGFBP-rP1 blocks TGF-β1-induced EMT in HT29 cells. (a) HT29 cells were incubated with TGF-β1 (10 ng/ml) for the indicated times. The levels of P-smad2/3 were assessed by western blot. (b) Expression of EMT markers assessed by western blot. (c) TGF-β1 promoted cell migration determined by transwell migration assay. The results were shown as mean+S.D. from triplicate experiments. (d, e) Western blot of the indicated antibodies and transwell migration assay in HT29 cells and controls untreated or treated with 1 μg/ml rhIGFBP-rP1 and/or 10 ng/ml TGF-β1. The results were shown as mean+S.D. from triplicate experiments