Abstract 2458: Genetically induced cancer stem cells (CSCs) in mouse mammary gland from Ink4a/Arf-knockout mammary cells transduced with H-rasG12V
Authors: Kai Kazuharu , Saya Hideyuki , Hortobagyi Gabriel N. , Ueno Naoto T. 
Published: February 2011

The Ink4a/Arf locus has crucial roles in senescence, one of the cellular programs that prevent tumorigenesis. However, how this locus relates to cellular plasticity, stemness and differentiation, is not fully understood. Furthermore, how knockout (KO) of Ink4a/Arf affects tumorigenesis, specifically the acquiring of CSC traits, also has not been elucidated. The purpose of this study was to establish an induced-CSC model that can be used to analyze the molecular mechanism of CSCs evolving in mouse mammary cells (MMCs). First, to investigate influences of Ink4a/Arf-KO in mouse mammary stem/luminal progenitor cells, especially in regard to cellular plasticity in vivo, CD29highCD24+ mammary stem and CD29middleCD24+ luminal progenitor cells from wild-type or Ink4a/Arf-KO MMCs were sorted and inoculated into cleared fat pads. Compared with repopulated mammary glands from wild-type CD29highCD24+ cells, those from Ink4a/Arf-KO CD29highCD24+ cells were significantly less branching and had smaller terminal end buds. In fat pads inoculated with Ink4a/Arf-KO CD29middleCD24+ cells, no repopulation was observed, whereas repopulation with wild-type CD29middleCD24+ cells was observed in 2 of 5 inoculations. These results indicate that Ink4a/Arf-KO disturbs the plasticity of mammary stem/progenitor cells. Next, to investigate what kind of genetic events stimulate Ink4a/Arf-KO cells into transformation, we transduced two well-known oncogenic drivers, c-myc and H-rasG12V, into Ink4a/Arf-KO MMCs. In this experiment, we cultured Ink4a/Arf-KO cells in floating culture supplemented with fibroblast growth factor/epidermal growth factor and without serum. Then, these mammosphere cells transduced with oncogenes were inoculated into the fat pads of syngenic mice. In the case of mock- and c-myc-transduced cells, no tumors were observed. Meanwhile, tumors were observed in 6 of 8 mice with H-rasG12V-transduced cells. These tumors were positive for estrogen receptor/CK18 and negative for CK5/CK14; therefore, they seemed to derive from luminal progenitor cells. Indeed, they had broad expression of CD61, a marker of such cells. We could see the disseminated tumor cells in distant organs, lung, spleen, liver, and lymph node. These tumors, in contrast to mammospheres from Ink4a/Arf-KO cells, had lost expression of E-cadherin, suggesting that activating H-ras transforms Ink4a/Arf-KO cells from epithelial to mesenchymal. Furthermore, individual transformed cells were highly tumorigenic; in 3 of 8 mice, injection of only 20 cells into cleared fat pads initiated tumors. Almost all these cells had a CD44+CD24− expression profile. In summary, we successfully induced CSCs though two sequential cellular steps, disturbance of cellular plasticity and epithelial-to-mesenchymal transition. This proof-of-concept on how to induce CSCs can shed light for further CSC research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2458.