Abstract LB-143: DNp63 governs metastatic outgrowth of breast cancer stem cells

Abstract

Abstract Background: In the human mammary gland p63 isoforms control mammary epithelial cells fate and DNp63, which lacks the amino-terminal trans-activation domain, counterbalances the full-length isoform TAp63 to allow maturation of epithelia and the maintenance of the myoepithelial/basal cells features. Mammary gland tissue's adult stem cells retain self-renewal and multi-lineage differentiation ability and, by acquiring a malignant behavior, are in charge of tumor seeding. A correlation between Cancer Stem Cells (CSCs) in primary lesions and increased metastatic dissemination has been reported, however, still little is known about the unique profile of distant metastasis’ cell origin. Herein, we show for the first time that breast CSCs (BCSCs) expressing DNp63 are capable to generate metastasis in a preclinical model, while TAp63 can be solely tumorigenic and unable to serial xenografting. We anticipate novel farsighted implications for a prognostic role of p63 and for its targeting to prevent tumor growth and metastatic disease. Methods: BCSCs were freshly isolated from the histological uninvolved resection of breast cancer tissues. Obtained cells were plated in ultra-low flask in serum-free media in presence of bFGF and EGF. Cells were treated with 1mmol/l doxorubicin hydrochloride. Synthetic genes encoding DNp63 and TAp63 were inserted into the p-TWEEN EGFP lentiviral expression vector. Stable p63 knockdown was produced by lentiviral transduction of the pLentilox 3.7 vector carrying shp63 and scramble sequences. For the tumorigenic and metastagenic assay BCSCs (3×105) were suspended in matrigel and injected orthotopically or in the sub-renal capsule of NOD/SCID mice, respectively. Tumors were measured with a caliper each week and volume was calculated by the formula: π/6 x larger diameter x (smaller diameter)2. Metastasis formation was followed over time and visualized by in vivo imaging. Summary: Herein, we demonstrate that luminal and basal BCSCs differ in the content of p63 isoforms, DNp63expression was higher in the stem-like compartment of basal BCSCs, compared to TAp63 mainly expressed in bulk primary cells. DNp63 increased the G0-G1 phase in both luminal and basal BCSCs, enhanced doxorubicin resistance and induced a mesenchymal switch. Despite both TAp63 and DNp63 overexpressing BCSCs formed subcutaneous xenografts in NOD/SCID mice, DNp63 cells exclusively retain capabilities of serial xenografting. Thus, suggesting that exogenous expression of TAp63 could promote transition from stem cells to progenitor cells. In a metastatic preclinical model TAp63 overexpression hampered metastatic potential of BCSCs, while DNp63 overexpressing cells gained capabilities to colonize distant organs such as lung and kidney. Conclusions: These findings state DNp63 as a major regulator of stemness and invasiveness in malignant breast tissues and offer new insights on p63 role as a prognostic biomarker and therapeutic target. Citation Format: Alice Turdo, Simone Di Franco, Antonina Benfante, Maria Luisa Colorito, Marco Bonanno, Miriam Gaggianesi, Daniela Barcaroli, Francesco Dieli, Jan Paul Medema, Vincenzo De Laurenzi, Giorgio Stassi, Matilde Todaro. DNp63 governs metastatic outgrowth of breast cancer stem cells. [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 LB-143. doi:10.1158/1538-7445.AM2015-LB-143