Abstract A242: R-spondin 2 drives Wnt signaling and tumor formation in breast and liver cancer.

Background: R-spondins are secreted agonists of Wnt signaling that function in development and promote tissue stem cell proliferation. Rspo2 was identified as a candidate oncogene in intestinal tumors via a Sleeping Beauty transposon insertional mutagenesis screen in mice (1). Further, oncogenic activation of RSPO2 and RSPO3 mediated by recurrent genomic rearrangements has been identified in human colorectal cancer (2). The purpose of the current study was to determine if R-spondins function as oncogenes in other cancer types characterized by active Wnt signaling. Methods: R-spondin mRNA expression was determined in primary human breast tumors and adjacent normal tissue by quantitative RT-PCR. Affymetrix microarrays were used to assay gene expression and classify by molecular subtype human hepatocellular carcinomas compared to pre-malignant lesions and normal livers. RSPO2 was depleted or overexpressed in breast cell lines by stable transduction with anti-RSPO2 shRNA or RSPO2 cDNA. Resulting changes in gene expression and proliferation were measured by qRT-PCR and MTS assay respectively. RSPO2 was somatically overexpressed in murine liver by hydrodynamic injection and Fah selection in an Fah-null model. Subsequent gene expression studies were conducted by qRT-PCR and immunohistochemistry. Results: RSPO2 was highly expressed in 12% of primary human breast tumors compared to adjacent normal tissue. Similarly, RSPO2 expression was elevated in the subset of primary human liver cancers with activated Wnt/beta-catenin signaling. In human breast cancer cells with elevated RSPO2 expression, RSPO2 knockdown decreased Wnt signaling and proliferation, while RSPO2 overexpression in a non-tumorigenic breast epithelial cell line potentiated Wnt signaling. Overexpression of RSPO2 in the mouse liver increased Wnt signaling and promoted an enlarged liver phenotype and tumor formation. Conclusions: These data strongly suggest that RSPO2 is a driver of human breast and liver cancer. Future work will further characterize signaling pathways implicated in RSPO-driven phenotypes, and develop targeted therapy to inhibit RSPO signaling. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A242. Citation Format: Caitlin B. Conboy, Bobbi R. Tschida, Hsiangyu Hu, Michael B. Burns, Nuri A. Temiz, Timothy Kuka, Vincent W. Keng, Sara Toffanin, Reuben S. Harris, Josep Llovet, Timothy K. Starr, David A. Largaespada. R-spondin 2 drives Wnt signaling and tumor formation in breast and liver cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A242.