ROR1 activates CREB to regulate tumor-cell growth.

(A) Relative expression of selected genes by MDA-MB-231 cells transduced with either Ct-shRNA (white bars) or ROR1-shRNA (black bars), as assessed via quantitative RT-PCR. The numbers on the y-axis represent fold difference in specific gene expression relative to GAPDH. Error bars indicate S.E.M. (n = 3 experiments). _* indicates P<0.05, _** indicates P<0.01 and _*** indicates P<0.001 (Student's t test). (B) Immunoblot analyses for proteins listed on the right margin, using lysates from MDA-MB-231 control cells versus cells silenced for ROR1. (C) Immunoblot analysis of CREB phosphorylation at ser-133 (p-CREB), total CREB (t-CREB), ROR1, or β-Actin for MDA-MB-231 cells with or without ROR1 silencing, as indicated on the top of each panel. The phospho-specific antibody recognizes phospho-serine-133 CREB. (D) Tumors extracted from mice engrafted with MDA-MB-231 control cells or ROR1-shRNA-transduced cells were examined for p-CREB by immunohistochemistry staining (n = 3) or Bcl-2, Cyclin D1 and β-Actin protein expression by immunoblot analysis. The scale bar in the left panels represents 35 µm. The antibody staining is in red and the nuclear counterstain is in blue. The intensity of p-CREB was quantified and data were represented as mean intensity ± S.E.M from 3 tumor tissues per group. (E) MDA-MB-231 cells infected with Ad-GFP or Ad-ACREB were examined for expression of proteins by immunoblot analyses. (F) MDA-MB-231tumor cells with or without ROR1 expression were transfected with Ad-GFP or Ad-ACREB and then monitored for cell growth. The numbers of viable cells (after 72 hours culture) are represented by the height of each bar in the graph. The error bars provide the S.E.M. of triplicate samples. P indicates the statistical significance as assessed by Student's t test. (G) Representative images of breast cancer tissues stained for phospho-CREB (p-CREB) (top panels) or ROR1 (bottom panels). Tissue-bound antibody is shown in red and the nuclear counterstain with hematoxylin is in blue. The scale bar in the top left picture represents 35 µM. Tissues were scored as 0 (none of the cells within the sample bound to the mAb); 1 (low-level binding of the mAb to the tumor cells or low-to-moderate-level binding of the mAb on less than 50% of tumor cells); 2 (moderate-level staining on more than 50% of tumor cells or high-level staining of the tumor cells). Red arrows point to the tumor cells, blue arrow points to stromal cells and green arrow points to lymphocytes.