Slit-2 induces a tumor-suppressive effect by regulating beta-catenin in breast cancer cells

Abstract

SLIT-2 is considered as a candidate tumor suppressor gene, because it is frequently inactivated in various cancers due to hypermethylation of its promoter region and allelic loss. However, the exact mechanism of its tumor-suppressive effect has not been elucidated. Here, we observed that Slit-2-overexpressing breast cancer cells exhibited decreased proliferation and migration capabilities compared with control cells under in vitro conditions. These results were confirmed in vivo in mouse model systems. Mice injected with MCF-7/Slit-2 cells showed a 60-70% reduction in tumor size compared with mice injected with MCF-7/VC cells both in the absence and presence of estrogen. Upon further elucidation, we observed that Slit-2 mediates the tumor-suppressive effect via a coordinated regulation of the beta-catenin and PI3K signaling pathways and by enhancing beta-catenin/E-cadherin-mediated cell-cell adhesion. Our study for the first time reveals that Slit-2-overexpressing breast cancer cells exhibit tumor suppressor capabilities through the novel mechanism of beta-catenin modulation.

FIGURE 1.

Slit-2-overexpressing MCF-7 cells show decreased proliferation and chemotactic capability. Slit-2 expression (A, upper panel) and Robo-1 expression (A, second panel) in MCF-7/VC and MCF-7/Slit-2 clones were analyzed by Western blotting using anti-c-myc and anti-Robo-1 antibodies. Equal protein was confirmed in each sample by stripping and re-probing the blot with anti-β-Actin antibody (A, third panel). Slit-2-myc expression also analyzed in cell culture supernatant concentrates. Cell culture supernatants were concentrated by using Amicon Ultra-100K filters (A, lower panel). B, cells were subjected to a proliferation assay after seeding in 96-well plates in DMEM supplemented with 0.2% fetal bovine serum and 50 ng/ml EGF. Then the number of viable cells was quantified at various time points by using the CellTiter 96®Aqueous kit (Promega), as per the manufacturer's instructions. C, cells were subjected to chemotaxis assay toward CXCL12 (50 ng/ml) by using chemotaxis chambers as described under “Experimental Procedures.” D and E, for the soft agar colony formation assay, cells were mixed in medium containing 0.4% Ultra pure agarose (Invitrogen) and layered on top of 1 ml of medium containing 0.8% agarose in 6-well culture plates. Then cells were incubated with 1 ml of DMEM supplemented with 10% fetal bovine serum. After 2 weeks of incubation, the colonies were stained with 0.005% Crystal Violet for 1 h and counted by using a Nikon Diaphot 300 inverted microscope. All of the above experiments were repeated three times, and a representative one is shown. LPF = low power field. ^*, p < 0.05 for all experiments. VC, Vector Control-MCF-7; Slit-2 (c1), Slit-2-overexpressing MCF-7 clone 1; Slit-2 (c2), Slit-2-overexpressing MCF-7 clone 2; SN, cell supernatant concentrate.

FIGURE 2.

Slit-2 may induce its function through an autocrine manner. A, control siRNA (solid line) and Robo-1 siRNA-transfected (dotted line) MCF-7/Slit-2 (c2) cells were stained using anti-Robo-1 antibody and analyzed by flow cytometry. Cells stained with control IgG (filled area) represent the antibody control. E and F, control siRNA and Robo-1 siRNA-transfected MCF-7/Slit-2 (c2) cells were subjected to a proliferation assay as described under “Experimental Procedures.” The experiments were done in triplicate and are presented as the mean ± S.E. The data are representative of three different experiments. ^*, p < 0.05 for all experiments.

FIGURE 3.

Slit-2 overexpression suppresses the tumorigenic capability of MCF-7 breast cancer cells in in vivo models. MCF-7/VC and MCF-7/Slit-2 (c2) cells were injected (3 × 10⁶ cells) into the flanks of female SCID mice (n = 5). One group of mice was injected subcutaneously with 2.5 μg of β-estradiol 17-valerate in 50 μl of Sesame oil twice a week. Tumor size was measured by using digital calipers once a week, and volume was determined according to the formula (W2 × L)/2. A, the graph represents the tumor volume in mice injected with MCF-7/VC or MCF-7/Slit-2 (c2) cells with or without estrogen treatment. Tumor volume was assessed up until 5 weeks after injection. B, the experiments in panel A were repeated in nude mice with or without estrogen treatment. Tumor size was measured by using digital calipers once a week for 6 weeks, and volume was determined as indicated above. At the end of the fifth week in the case of SCID mice (C) and at the end of the sixth week in the case of nude mice (D), the tumor volume was analyzed and calculated by using the Micro CT scan imaging system. ^*, p < 0.05 for all experiments. E, photographs of the tumors derived from SCID mice 5 weeks after the injection of MCF-7/VC or MCF-7/Slit-2 9 (c2) cells in the presence or absence of estrogen. F, photographs of representative nude mice 6 weeks after the injection of MCF-7/VC or MCF-7/Slit-2 (c2) cells in the presence or absence of estrogen. G, Micro CT-scanned photographs of SCID mice 5 weeks after the injection of MCF-7/VC or MCF-7/Slit-2 (c2) cells in the presence or absence of estrogen. UN = untreated.

FIGURE 4.

Increased β-catenin degradation was observed in MCF-7/Slit-2 cells compared with vector control (VC) cells. Both MCF-7/Slit-2 and MCF-7/VC cells were lysed and Western blotted with β-catenin (A, upper panel) or phospho-β-catenin (p-β-catenin) (serine 45) (B, upper panel) antibodies. Equal protein was confirmed in each sample by stripping and re-probing the blots with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or anti-β-actin antibodies (A and B, lower panels). The cell lysates were immunoprecipitated with anti-β-catenin antibody and Western blotted with anti-GSK-3β antibody (C, upper panel) or anti-ubiquitin (Ub) antibody (D, upper panel). Equal protein was confirmed in each sample by Western blotting with anti-β-catenin antibody (C and D, lower panels). AbC, antibody control; VC, vector control; TCL, total cell lysates. E, un-transfected (UN), control siRNA-transfected (NT), and β-catenin-siRNA-transfected MCF-7 cells lysed, and lysates were analyzed for β-catenin expression by Western blotting by using anti-β-catenin antibody. F, soft agar colony formation assay was preformed with control siRNA-transfected (NT) and β-catenin-siRNA transfected MCF-7 cells as mentioned above. G, tumors derived from MCF-7/VC and MCF-7/Slit-2 cells were excised, minced, and homogenized in modified radioimmune precipitation assay buffer. The lysates were analyzed for β-catenin expression by Western blotting by using anti-β-catenin antibody. All of the above experiments were repeated three times, and a representative one is shown.

FIGURE 5.

Slit-2-overexpressing cells show decreased nuclear translocation of β-catenin as well as increased expression of E-cadherin, enhanced intercellular adhesions, and association between β-catenin and E-cadherin. Nuclear extracts (NE) and cytoplasmic extracts (CE) were collected from both MCF-7/VC and MCF-7/Slit-2 cells by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology) as per the manufacturer's protocol. The extracts were subjected to Western blotting using anti-β-catenin antibody (A, upper panel). The purity of fractionation and equal loading of protein in each lane were determined with Oct-1 antibody (A, lower panel). B, MCF-7/VC and MCF-7/Slit-2 cells were cultured in chamber slides. Cells were fixed and treated with rabbit anti-β-catenin antibody. After washing, cells were probed with Alexa 568-tagged anti-rabbit IgG antibody, and the slides were mounted by using Prolong Gold Antifade with DAPI (Invitrogen), and then examined under a Zeiss confocal microscope. The pictures were acquired by using LSM 510 software. C, nuclear fluorescent staining was quantitated by using Volocity software at the Beth Israel Deaconess Medical Center Microscopy Core Facility. The percentage of nuclear fluorescent staining was calculated by considering the fluorescence of the MCF-7/VC cells as 100%. Both MCF-7/Slit-2 and MCF-7/VC cells were lysed, and the cell lysates were immunoprecipitated with anti-β-catenin antibody and Western blotted with anti-E-cadherin antibody (D, upper panel). Equal protein was confirmed in each sample by stripping and re-probing the blot with anti-β-catenin antibody (D, lower panel). All of the above experiments were repeated three times, and a representative one is shown. ^*, p < 0.05 for all experiments. E and F, MCF-7/VC and MCF-7/Slit-2 cells were cultured in chamber slides. For panel E, cells were fixed and treated with mouse monoclonal anti-E-cadherin antibody. After washing, cells were probed with Alexa 568-tagged anti-mouse IgG secondary antibody. For panel F, cells were treated with mouse monoclonal anti-E-cadherin antibody and rabbit anti-β-catenin antibody, then cells were washed and probed with Alexa 568-tagged anti-mouse IgG and fluorescein isothiocyanate-labeled anti-rabbit IgG secondary antibodies. For both panels E and F, the slides were mounted by using Prolong Gold Antifade with DAPI (Invitrogen) and examined under a Zeiss confocal microscope. The pictures were acquired by using LSM510 software. All of the above experiments were repeated three times, and a representative one is shown.

FIGURE 6.

Slit-2-overexpressing cells exhibit decreased β-catenin/TCF transcriptional activity. A, TCF luciferase constructs (0.5 μg), containing the wild-type (pTOPFLASH) or mutant (pFOPFLASH, Upstate, Charlottesville, VA) TCF binding sites, were transfected into MCF-7/VC and MCF-7/Slit-2 cells (5 × 10⁵ per well). Transfection experiments were carried out in triplicate using Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. The cells cotransfected with 0.1 μg of pRL-TK Renilla luciferase vector (Promega) were taken as an internal control. The cells were incubated for 48 h following the transfection and then untreated (UN) or treated with EGF (100 ng/ml) for 24 h. Cells were lysed and the activities of firefly and Renilla luciferase were assayed and measured in a Wallac Victor² luminometer (PerkinElmer Life Sciences) using reagents from the Dual Luciferase kit (Promega), as mentioned under “Experimental Procedures.” The firefly (TOPFLASH or FOPFLASH) luciferase activity was corrected for Renilla luciferase activity (pRL-TK) to control for transfection efficiency. TOPFLASH activity was also normalized to the FOPFLASH activity. Data are expressed as the mean of triplicate values of the normalized TOPFLASH activity. Control, untreated value is taken as 100%, and other sample values were compared accordingly. All of the above experiments were repeated three times, and a representative one is shown. B, nuclear extracts (NE) and cytoplasmic extracts (CE) were collected from both MCF-7/VC and MCF-7/Slit-2 cells by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology) as per the manufacturer's protocol. The extracts were subjected to Western blotting using anti-TCF-4 antibody (B, upper panel). The purity of fractionation and equal loading of protein in each lane was determined with Oct-1 antibody (B, lower panel). Both MCF-7/Slit-2 and MCF-7/VC cells were lysed, and the cell lysates were Western blotted with anti-MMP-2 (C, upper panel), anti-MMP-9 (C, second panel), and anti-cyclin D1 (C, third panel) antibodies. Equal protein was confirmed in each sample by stripping and re-probing the blot with anti-β-actin antibody (C, lower panel).

FIGURE 7.

Slit-2 transiently transfected MDA-MB-231 cells show decreased proliferation, β-catenin, and cyclin D1 expression and increased β-catenin/E-cadherin association. pcDNA 3.1/V5-His-Slit-2 plasmid and vector control plasmids were transiently transfected to MDA-MB-231 cells as mentioned under “Experimental Procedures.” Cells were lysed and analyzed for Slit-2-V5 expression by Western blotting using anti-V5 antibody (A) or subjected to proliferation assay by using the CellTiter 96®Aqueous kit (Promega), as per the manufacturer's instructions (B). C, cells were lysed, and the cell lysates were Western blotted with anti-β-catenin antibody or anti-cyclin D1 antibody or (D) lysates were immunoprecipitated with anti-β-catenin antibody and Western blotted with anti-E-cadherin antibody (D, upper panel). Equal protein was confirmed in each sample by stripping and re-probing the blot with anti-β-catenin antibody or anti-β-actin antibody (C and D, lower panels). All of the above experiments were repeated three times, and a representative one is shown. ^*, p < 0.05 for all experiments.

FIGURE 8.

Slit-2-overexpressing cells show decreased phosphorylation of Akt and GSK-3β. MCF-7/Slit-2 and MCF-7/VC cells were lysed, and the cell lysates were Western blotted with anti-phospho-Akt (p-Akt) (A, upper panel) or anti-phospho-GSK-3β (p-GSK-3β) (B, upper panel) antibodies. In panel A, the MCF-7/Slit-2 (+) and MCF-7/VC (-) cells were untreated or treated with EGF (100 ng/ml) for various time points, as indicated. Equal protein was confirmed in each sample by stripping and re-probing the blots with anti-Akt antibody or anti-GSK-3β antibody (A and B, lower panels).