BCL9 promotes tumor progression by conferring enhanced proliferative, metastatic, and angiogenic properties to cancer cells

Abstract

Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances beta-catenin-mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.

Figure 1

BCL9 is overexpressed in multiple myeloma and colon carcinoma. A, high BCL9 mRNA expression in multiple myeloma primary tumors (MMPT) as well as multiple myeloma cell lines (MMCL) compared with normal plasma cells (NPC; left) and in colon carcinoma primary tumors (CCPT) as well as colon carcinoma cell lines (CCCL) compared with normal colonic mucosa (NCM) and normal crypt cells (Ncr; right). Immunoblot showing increased BCL9 protein expression in multiple myeloma (left) and colon carcinoma (right) cell lines (B) and primary tumors (C). Note that BCL9 protein expression in normal plasma cells and normal colonic mucosa is negligible. Igλ loading control is shown for normal plasma cells and multiple myeloma cells. Normal plasma cells secrete high levels of immunoglobulin, whereas multiple myeloma cells secrete varying immunoglobulin levels. MM1S (a multiple myeloma cell line) and Colo320 (a colon carcinoma cell line) were used as BCL9-expressing positive controls in the primary tumor Western blots.

Figure 2

Gain-of-function and loss-of-function validation of BCL9 in tumor cell lines. A, BCL9 overexpression (left) with lentiviral BCL9 GFP and knockdown (right) with lentiviral BCL9 shRNA in multiple myeloma cell line MM1S and colon carcinoma cell line Colo320 as documented by immunoblot analysis. B, confocal immunofluorescence confirming knockdown of BCL9 expression in MM1S and Colo320 cells. Control shRNA and BCL9 shRNA expressing stable cells were sorted, fixed, and stained with indicated antibodies.

Figure 3

BCL9 enhances tumor cell proliferation, migration, and invasion. Effect of BCL9 knockdown on tumor cell proliferation (A), anchorage-independent soft-agar colony formation (B), cell migration (C), and cell invasion (D) in MM1S and Colo320 cells. MM1S cells stably overexpressing BCL9 GFP shows change in colony morphology and increase in cell migration compared with control GFP cells. Bars, SE.

Figure 4

BCL9 knockdown increases survival and decreases tumor metastasis in xenograft mouse models of cancer. A, Kaplan-Meier survival curve of mice engrafted with MM1S (multiple myeloma cells) as well as Colo320 (colon carcinoma cells) control shRNA and BCL9 shRNA cells. Whole-body imaging and histologic analysis of engrafted mice with Colo320 (B) and MM1S (C) cells. Note decreased tumor burden in the liver and kidneys of mice with Colo320 BCL9 shRNA knockdown cells (white arrows) and peridural space (PS) of mice engrafted with MM1S BCL9 shRNA cells (black arrows). SC, spinal cord; BM, bone marrow.

Figure 5

BCL9 is involved in tumor metastasis and angiogenesis. A, heat map of expression profiles of MM1S and Colo320 stably expressing control shRNA or BCL9 shRNA. Genes altered on knockdown of BCL9 are indicated. Down-regulation of CD44 and VEGF, known Wnt target genes, are indicated in red and by arrows. B, BCL9, c-Myc, cyclin D1, CD44, and VEGF expression in MM1S and Colo320 cells following BCL9 shRNA knockdown as measured by quantitative reverse transcription-PCR. *, P < 0.01; **, P < 0.0001. Nontargeting control shRNA was used as control. C, down-regulation of BCL9 by lentiviral BCL9 shRNA decreases c-Myc and cyclin D1 but not β-catenin protein expression in MM1S and Colo320 cells. D, decreased metastasis, tumor nodules (white arrows), and CD44 (black arrows) expression in mice engrafted with Colo320 BCL9 knockdown cells compared with control. E, BCL9 knockdown decreases VEGF secretion in MM1S and Colo320 cells as measured by ELISA. F, down-regulation of BCL9 decreases host angiogenesis in mice injected with MM1S BCL9 shRNA cells as measured by decreased mouse CD34 staining (black arrows). Mouse anti-CD34 stains the endothelial blood vessel cells. The number of blood vessels formed in mice injected with MM1S BCL9 shRNA is significantly decreased.

Figure 6

Immunohistochemical analysis of BCL9 expression in multiple myeloma and colon carcinoma primary tumors. A, increased nuclear localization of BCL9 in multiple myeloma (MM) compared with normal bone marrow (NBM) plasma cells. Normal bone marrow cells are costained with anti-CD138 (red) and anti-BCL9 (brown) or anti-β-catenin (brown). Arrows, cells in the insets. B, nuclear BCL9 expression in colon carcinoma. The dashed black line demarcates normal colon (black arrows) and tumor (red arrows). Note that normal colon crypt cells are negative for BCL9 (top inset, black arrow), whereas some stromal cells are positive (blue arrow). Nuclear β-catenin expression in adjacent tissue sections is also shown (bottom). Areas in the black boxes are enlarged (right, bottom inset). C, up-regulation of nuclear BCL9 (red arrows) and β-catenin (blue arrows) in the invasive regions of colon carcinoma. D, increased nuclear BCL9 (brown staining and red arrows) and β-catenin staining (brown staining and black arrows) in colonic adenoma polyps. Areas in the black and yellow boxes are enlarged below. Note the absence of BCL9 expression and conspicuous cytoplasmic and membrane β-catenin expression in the stalk (bottom inset). The blue staining seen in the stalk and some stromal cells (yellow arrows) corresponds to counterstaining with hematoxylin.