Abstract 4188: Conditional epithelial cell-specific knockout of CCN6/Wisp3 disrupts normal development of the virgin murine mammary gland
Authors: Martin Emily E. , Huang Wei , Guan Jun-Lin , Kleer Celina G. 
Published: August 2015

Abstract
Background: Breast cancer remains one of the top causes of morbidity and mortality among cancer patients in the U.S. Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. Our laboratory has shown that CCN6/Wisp3, a secreted extracellular matrix-associated protein which belongs to the CCN family, is lost in 80% of IBC compared to stage-matched non-IBC tumors. We have shown previously that CCN6 knockdown in benign human breast cells leads to an epithelial-to-mesenchymal transition and increased resistance to anoikis. Further, CCN6 overexpression in breast cancer cells reduces invasion and growth in vivo and in vitro. However, the consequences of CCN6 knockout in the mammary gland have never been explored. We hypothesized that the conditional deletion of CCN6 in mammary epithelial cells in mice may lead to defects in mammary gland development and may induce mammary tumors.
Methods: We created a novel transgenic mouse model in which CCN6 is conditionally knocked out in mammary epithelial cells using the Cre-Lox recombination system. We collected groups of CCN6 knockout mice and littermate controls at 8 weeks, 4 months, and 12 months of age to observe the effect of CCN6 knockout on the development of the virgin mammary gland and on de novo tumor formation in aged mice. We also collected groups of CCN6 knockout and control mice at early pregnancy, late pregnancy, lactation and involution timepoints. Mammary whole mounts were prepared in order to characterize the phenotype, and portions of the inguinal gland were collected for immunohistochemical, protein and RNA analysis.
Results: Conditional CCN6 knockout in the murine mammary gland results in defective mammary gland development in pubertal (8-week-old) and post-pubertal (4-month-old) virgin female mice compared to controls. At 8 weeks of age, virgin CCN6 knockout mice exhibit significantly fewer terminal end buds and fewer bifurcated terminal end buds. At 4 months of age, virgin CCN6 knockout mice have fewer lobuloalveolar units and a hypobranching phenotype compared to controls. We observed no phenotypic differences in the pregnancy and lactation timepoints between CCN6 knockout mice and controls, and no differences in pup weight or average litter size, suggesting that CCN6 is not critical for pregnancy and lactation. CCN6 knockout did not lead to spontaneous tumorigenesis in aged mice.
Conclusion: We provide new evidence that CCN6 is an important signaling molecule in the mammary gland in vivo, as CCN6 is necessary for normal mammary development in virginal animals. We are currently analyzing the effects of CCN6 knockout in mammary gland involution after lactation, and the signaling pathways affected by CCN6 knockout in vivo. Further studies are needed to investigate effect of CCN6 knockout in breast cancer in vivo; to this end, we are currently crossing our CCN6 knockout line with the oncogenic MMTV-Pymt transgenic line.
Citation Format: Emily E. Martin, Wei Huang, Jun-Lin Guan, Celina G. Kleer. Conditional epithelial cell-specific knockout of CCN6/Wisp3 disrupts normal development of the virgin murine mammary gland. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4188. doi:10.1158/1538-7445.AM2015-4188