MT1-MMP target genes. A, clustergram of MT1-MMP and its target genes. The colored clustergram shows the expression pattern of the 11 MT1-MMP target genes and MT1-MMP itself in the 190 human tumors of the 14 most common cancer types which are included in the GCM database. Each colored square represents the normalized expression level of an individual gene in a single tumor sample using red and blue to denote high and low levels of expression, respectively. B, expression level correlation. The normalized expression level (Y axes) of MT1-MMP and the four MT1-MMP target genes (PI3Kδ, NR3C1, CK2α, and NNT) in each of 190 tumor samples (X axes) was plotted. Tumor types are shown above the plot. C, cellular mRNA levels of the identified target genes. mRNA levels were measured by qRT-PCR using the RNA samples isolated from HT, HT-MT, and HT-siRNA cells. The expression values were normalized relative to GAPDH. The levels of mRNA in HT-MT and HT-siRNA cells are shown in percentage relative to HT cells (100%).

The transactivation function of MT1-MMP. A, Western blotting of HT1080 (left) and MCF-7 cells (right) with the PI3Kδ,NR3C1, and CK2α antibodies. The incubation with the primary antibody was followed by the species-specific HRP-conjugated secondary antibody and a TMB/M substrate. Note the up-regulation of the target genes in cells expressing MT1-MMP-WT. Arrows point to the PI3Kδ,NR3C1, and CK2α bands. B, transient expression assay of the transcriptional activity of the NR3C1 promoter-luciferase chimera. HT, HT-neo, HT-MT, HT-siRNA, and HT-siRNAscr cells were each transiently transfected with both the NR3C1 promoter–firefly luciferase chimera and the pGL4.74 control vector that carried the Renilla luc gene under the control of the thymidine kinase promoter. The mean of four measurements was taken to obtain the firefly luciferase activity that was expressed as relative units defined by the ratio of firefly luciferase to Renilla luciferase activity. Where indicated, cells were coincubated for 14 h with GM6001 (50 μmol/L) before activity measurement. The activity measured in the parental HT cells was taken as 100%. Columns, mean of at least three independent transfection experiments; bars, SE. The data are statistically significant (P < 0.05). C, phosphorylated site and total protein screening. HT-MT and HT-siRNA cell lysate samples were analyzed using phosphorylated site and total kinase protein screening. A portion of the Western blot image that included pMAPK1, pMAPK3, pMAPK14, pMAP2K1/2, and GSKβ (a control). Normalized cpm of pMAPK1 (T¹⁸⁵/Y¹⁸⁷), pMAPK3 (T²⁰²/Y²⁰⁴), pMAPK14 (T¹⁸⁰/Y¹⁸²), pMAP2K1/2 (S²¹⁷/S²²¹), and GSKβ(Y²¹⁶) in HT-MT/HT-siRNA cells is 2040/3940, 620/1250, 80/230, 130/430, and 2900/2600, respectively. Normalized cpm of MAPK1, MAPK3, MAPK14, MAP2K1/2, and GSKβ total protein in HM-MT/HT-siRNA cells is 2290/960, 2190/1240, 1410/1370, 720/760, and 420/390, respectively. Normalized cpm is the trace quantity of the band normalized to a 60-s scan time. Values represent the average of the duplicate determinations performed by Kinexus.

Immunostaining of MT1-MMP, NR3C1, CK2α, and PI3Kδ in tumor xenografts. Paraffin-embedded tumor xenograft sections were stained using the antibodies to MT1-MMP, NR3C1, CK2α, and PI3Kδ. As a control, tumors were stained for α-tubulin and PKAα. FITC-labeled murine and rabbit-negative isotype control IgG were used as additional controls which were clearly negative. Nuclei were stained with DAPI. Original magnification, 400×.

Representative immunostaining of MT1-MMP, NR3C1, and PKAα in human cancer specimens arranged in TMAs. A, MT1-MMP and NR3C1 double staining. B, MT1-MMP and PKAα double staining. TMAs were stained with the antibodies to PKAα and NR3C1 (diaminobenzidine, brown) and with the antibody Ab815 to MT1-MMP (gray black) and counterstained with Nuclear red. The areas marked with squares were enlarged to facilitate the visual analysis of the colors. Bar, either 50 or 100 μm. C, analysis of the images. The selected regions (boxed) were subjected to the image analysis system using Scanscope-HT (Aperio Technology). The black and brown colors of the original image were separated using a color deconvolution algorithm (“deconvoluted and merged” panels). The separated colors are shown in the “MT1-MMP, black” and the “NR3C1, brown” panels. There is an obvious colocalization of MT1-MMP and NR3C1 in the breast carcinoma biopsies, whereas the ovarian carcinoma sample was negative in both MT1-MMP and NR3C1 markers. Original magnification, 300×.