MSX2 is an oncogenic downstream target of activated WNT signaling in ovarian endometrioid adenocarcinoma

Abstract

Ovarian endometrioid adenocarcinomas (OEAs) frequently exhibit constitutive activation of canonical WNT signaling, usually as a result of oncogenic mutations that stabilize and dysregulate the β-catenin protein. In previous work, we used microarray-based methods to compare gene expression in OEAs with and without dysregulated β-catenin as a strategy for identifying novel β-catenin/TCF target genes with important roles in ovarian cancer pathogenesis. Among the genes highlighted by the microarray studies was MSX2, which encodes a homeobox transcription factor. We found MSX2 expression was markedly increased in primary human and murine OEAs with dysregulated β-catenin compared with OEAs with intact β-catenin regulation. WNT pathway activation by WNT3a ligand or GSK3β inhibitor treatment potently induced MSX2 and ectopic expression of a dominant negative form of TCF4 inhibited MSX2 expression in ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated that β-catenin/TCF directly regulates MSX2 expression via binding to TCF binding elements in multiple regions of the MSX2 gene. Notably, ectopic MSX2 expression was found to promote neoplastic transformation of the rodent RK3E model epithelial cell line and to enhance the invasiveness of immortalized human ovarian epithelial cells in vitro and ovarian carcinoma cells in vivo. Inhibition of endogenous MSX2 expression in ovarian endometrioid cancer cells carrying a β-catenin mutation using shRNA approaches inhibited neoplastic properties of the cells in vitro and in vivo. Expression of MSX2 in selected ovarian carcinoma cells induced changes suggestive of epithelial-mesenchymal transition (EMT), but based on analysis of ovarian cell lines and primary tumor tissues, effects of MSX2 on EMT appear to be complex and context-dependent. Our findings indicate MSX2 is a direct downstream transcriptional target of β-catenin/TCF and has a key contributing role in the cancer phenotype of OEAs carrying WNT/β-catenin pathway defects.

Figure 1. MSX2 transcripts and protein are highly expressed in human and murine OEAs with dysregulated β-catenin

A) Relative expression of MSX2 in OEAs with dysregulated β-catenin (right) was compared with OEAs with intact β-catenin regulation (left), based on Affymetrix U133A microarray analysis (Probe Set1: 205555_s_at, Probe Set2: 210319_x_at); B) Representative immunohistochemical staining of MSX2 and β-catenin expression in OEAs with (OE-2T) or without (OE-20T) dysregulated β-catenin; C) Western blot analysis of MSX2 and β-actin protein expression in ovarian cancer-derived cell lines. D) Relative expression of Msx2 transcripts in murine APC−/PTEN⁻ and KRAS^mut/PTEN⁻ ovarian tumors and control ovaries based on Affymetrix Mouse430_2 microarray analysis.

Figure 2. MSX2 expression is induced by WNT signaling pathway activation and is a direct β-catenin/TCF target

A) IOSE80 (immortalized human ovarian surface epithelial) cells were treated with 50ng/ml of WNT3A or PBS for 8 h. MSX2 mRNA levels were determined by qRT-PCR and normalized to HPRT. Three independent experiments were performed (bars, mean ± SD); B) MDAH2774 cells were treated with SB216736 (GSK3β inhibitor) and harvested at the time points shown. MSX2 expression was measured by qRT-PCR and normalized to HPRT. Four independent experiments were performed (bars, mean ± SD, asterisks denote significance ranging from p =0.013 to 2.1E-05 based on Student's t test); C) MSX2 transcript and protein (inset) levels are reduced in TOV112D cells stably expressing dnTCF versus cells expressing empty vector; D) Chromatin immunoprecipitation (ChIP) assay of extracts from TOV112D cells immunoprecipitated by anti-TCF4 antibody. Inset shows schematic diagram of MSX2 locus with location of consensus TCF binding sites. Immunoprecipitated DNA was analyzed by quantitative PCR and normalized to the amount of input. Negative controls (two irrelevant [non TCF4] binding sites on the same chromosome as MSX2) and at the RPL30 locus (different chromosome) were used to demonstrate specificity.

Figure 3. MSX2 promotes neoplastic transformation of RK3E cells

A) RK3E/neo (empty vector) cells (left panel top) or RK3E cells expressing dominant negative TCF (left panel bottom) were transduced with replication defective retroviruses containing vector alone (pPGS), MSX2, or β-cat/S33Y. Two to three weeks after infection with the indicated retroviruses, plates were fixed, stained, and photographed. Quantified data shown (right panel) are from three independent experiments (bars, mean ± SD); B) RK3E cells with vector alone (pPGS), two clones of RK3E cells expressing MSX2 (MSX2-C1 and MSX2-C2) derived from a focus formation assay, and RK3E cells stably over-expressing β-cat/S33Y were assessed for their ability to grow in soft agar. After 4 weeks, dishes were stained with methylene blue and colonies were photographed (upper panel) and counted (bottom left panel) using the Image J program (http://rsb.info.nib.gov/ij/). Data shown represent the mean number of colonies from two independent experiments, each in triplicate; bars, mean ± SD. Western blot analysis of RK3E cells after transduction with MSX2, activated β-catenin (S33Y) or empty vector (bottom right panel). β-actin expression was evaluated as a loading control.

Figure 4. shRNA-mediated knockdown of endogenous MSX2 inhibits cell proliferation and tumor growth

A) Western blot analysis of lysates from TOV112D and TOV21G cells after stable transduction with virus expressing two different shRNAs targeting MSX2 and a non-silencing control shRNA (shc). MSX2 protein levels were quantified with the Image J program and normalized to β-actin. The ratios were normalized to control shRNA. B) Polyclonal TOV112D and TOV21G cells stably expressing either of two MSX2 shRNAs or control shRNA were plated at 1×10⁴/cm² in 6-well plates and grown in complete medium. Cell numbers were determined by counting with a hemocytometer on the indicated days. Three independent experiments were performed, each in triplicate (data shown represent mean ± SEM; C) TOV112D cells stably expressing either of two MSX2 shRNAs or control shRNA were grown in soft agar for 4 weeks. Number of colonies was counted by Image J after staining with methylene blue (left panel). Data shown (right panel) represent mean number of colonies from two independent experiments, each in triplicate; bars, mean ± SD. D) Xenograft tumor growth in nude mice after s.c injection of TOV112D cells expressing MSX2 shRNAs or control shRNA. Three or four mice per group were injected on both flanks. Data shown represent mean ± SEM.

Figure 5. MSX2 stimulates invasion of IOSE 80 cells and is associated with EMT-like changes

A) Representative photographs of control (neo) and MSX2 expressing IOSE80 cells that invaded through Matrigel in transwell invasion assays (upper panel). The number of cells penetrating the membrane was counted in five fields from two independent experiments each in triplicate (lower panel). Columns and error bars represent mean ± SD (p=.001 based on Mann-Whitney U-test). B) Representative photomicrographs of control (neo) and MSX2 expressing PEO4 ovarian carcinoma cells on the chick CAM are shown. CAM tissues were evaluated by light microscopy (H&E staining, left panels), or fluorescence microscopy (center and right panels: immunostaining for chick laminin, red; DAPI, blue; and GFP, green). Dashed lines in center panels indicate the CAM surface and dashed rectangles indicate portion shown at higher magnification in right panels. C) Phase-contrast photographs of cultured IOSE, PEO4, and OVCA433 cells showing fibroblast-like morphological change in cells expressing MSX2 compared to control (neo). D) Western blot analysis of N-cadherin, E-cadherin, Vimentin, MSX2 and β-actin (loading control) in PEO4, IOSE80 and OVCA433 cells expressing vector (neo) or MSX2.