Increased Radiation Resistance of Mouse Mammary Side Population Stem/Progenitor Cells: Role of Wnt/ ß -Catenin Signaling Wendy A. Woodward MD-PhD, Mercy S. Chen BS, Thomas A. Buchholz MD, and Jeffrey Rosen PhD U.T. M.D. Anderson Cancer Center and Baylor College of Medicine INTRODUCTION Mammary Stem Cells: Long replicative potential Capacity to self-renew and proliferate Attractive candidate for cell origin of cancer Required in adult mammary gland –Fulfill demands of pregnancy- dependent epithelial turnover (tissue renewal) –Respond to damage (tissue repair) Hypothesis: Breast cancers arise from cancer stem cells, which may be resistant to conventional therapy, and are therefore a critical determinant of recurrence Rationale: Understanding the pathways involved in stem cell survival may identify new targets for molecular therapeutics METHODS Primary mouse mammary epithelial cells (MECs) and immortalized Comma D (CD) mammary epithelial cells were irradiated in 60 mm culture dishes using a 137 cesium cell irradiator. MECs were isolated from 6-8 week old wild type Balb/c mice, from mice containing a floxed exon III ß- catenin allele, which generates stabilized ß- catenin upon excision in culture with an adenovirus-driven Cre recombinase, from transgenic Wnt-1mice with mammary hyperplasias, from wild type (WT) MECs in mice of the same background as Wnt-1 transgenic mice. MECs were maintained in stem cell promoting media after plating for 2 days and irradiated on day 4. On day 5 cells were trypsinized and stained for 60 min with Hoechst dye prior to analysis using flow cytometry. CD cells were transduced with control ß-galactosidase, amino- terminal, stabilized ß-catenin, or dominant- negative ß-engrailed retroviruses. CD cells were transplanted into the cleared fat pads of Balb/c mice 48h after transduction, and outgrowths were biopsied after 8 weeks. RESULTS: Radiation increases the proportion of stem-like progenitor cells in a mouse mammary cell lines, CommaD cells (CD), and in primary mammary epithelial cells(MECs) Radiation leads to a greater increase in stem-like progenitor cells in CD cells and MECs expressing stabilized ß-catenin Outgrowths from transplanted CD cells expressing stabilized ß-catenin fill more of the fat pad compared to outgrowths from CD cells expressing ß-galactosidase, but form disorganized and hyperplastic appearing outgrowths. Outgrowths from CD cells expressing ß-engrailed appear to lack myoepithelial cells Fig. 2 Floxed exon III ß-catenin allele generates stabilized ß-catenin upon excision in culture with an adenovirus-driven Cre recombinase (A). Clinically relevant doses of radiation lead to a greater increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cells expressing stabilized ß- catenin than in control cells (B). Fig. 3 Clinically relevant doses of radiation lead to a greater increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cells from transgenic Wnt-1 mice with mammary hyperplasias. Fig. 1 Clinically relevant doses of radiation increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cell culture. Magnitude of increase varies by mouse strain. CONCLUSIONS: Irradiation of immortalized cells and primary mammary epithelial cells increases the percentage of stem- like cells, likely due to stem cell radioresistance relative to the differentiated cells Expression of ß -Catenin, a stem cell survival factor, further increases the percentage of radiation-induced stem-like cells and promotes mammary outgrowth from immortalized cells A. B. A. B. 5’ LTR  +  -catenin mut IRES-GFP 3’ LTR Amp pMSCV-  -catenin mut - IRES - GFP pMSCV- IRES - GFP 5’ LTR  + GFP IRES-GFP 3’ LTR Amp C. Fig 4. CD cells were transduced with MSCV vector containing IRES-GFP or a stabilized ß -catenin and IRES-GFP(A). Cells were sorted by flow cytometry and positive cells were cultured to enrich for GFP+ cells (B). Clinically relevant doses of radiation lead to a greater increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cells expressing stabilized ß-catenin than in control cells (C). Figure 5. 10,000 CD cells transduced with stabilized ß-galactoidase ß -catenin or ß -engrailed were transplanted into cleared mammary fat pads. ß -catenin outgrowths were larger and filled more of the fat pad at 8 weeks. ß -Galactosidase outgrowths were roughly normal appearing on H&E sections. ß -Catenin outgrowths contain normal branching and ductal elements but were highly disorganized and contain abnormal areas of undifferentiated cells (inset). ß -engrailed outgrowths were small and characterized by marked dilation of of the ducts with irregular or absent myoepithelial cells(inset: immunohistochemistry of same section with smooth muscle actin antibody). ß-Galactosidase ß -Catenin ß -Engrailed (Dominant negative) Harada et al., EMBO J. 18:5931, 1999 Baylor cow parade M. D. Anderson Cancer Center