WNT/β-catenin mediates radiation resistance of mouse mammary progenitor cells

Abstract

It has been speculated that stem cells may represent the cellular origins of cancer because they exist quiescently for long periods of time and could accumulate multiple mutations over the lifespan of an organism, ultimately giving rise to tumors when stimulated to proliferate (1). Recently, it was reported that highly tumorigenic cells with properties consistent with those of stem/progenitor cells can be isolated from human breast cancers (2). These data suggest that cancer stem cells may exist in human breast cancer and that they may have different biologic features than other, more differentiated cells that constitute the majority of the cells in human breast cancers. Conceivably, cancer stem cells may be more resistant to conventional breast cancer therapies, which may ultimately result in recurrence or metastasis even when remarkable initial responses are observed clinically (3).

Although the elucidation of sensitive stem and progenitor cell markers in the mammary gland and in breast tumors has been relatively recent, the normal tissue response of stem and progenitor cells to radiation, an integral component of multidisciplinary breast cancer therapy, has been the subject of several decades of radiobiological data. The development of a simple, reproducible in vivo clonogenic assay in the jejunum, where the effects of radiation on stem cells can be easily measured, led to the conclusions that, in the small intestine, the cells at position 4–5 in the crypt now identified as the stem cells are exquisitely sensitive to radiation (4). However, a second population of potential stem cells, elsewhere called transiently amplifying cells or progenitors, exists that can be called into action in the event of lethal damage to the stem cells. At low doses of radiation the crypt of the small intestine contains four to five clonogenic regenerating cells, but at higher doses of radiation up to 30–40 potential clonogenic regenerators can be called into play (4). The elucidation of stem/progenitor cell markers in the mammary gland now allow this work to be tested at the cellular level in the mammary gland. We hypothesize that mammary gland progenitors may be resistant to radiation and that this resistance is mediated by the β-catenin stem cell survival signaling pathway.

β-Catenin is an essential component of both intercellular junctions and the canonical Wnt signaling pathway, which has been implicated in stem cell survival (5). In recent studies, activation of β-catenin in granulocyte–macrophage progenitors in chronic myelogenous leukemia appeared to enhance their self-renewal activity and leukemic potential (3). In addition, studies in the intestine and mammary gland have linked β-catenin signaling to stem cell survival and tumorigenesis (6–8).

Several methods are currently being used to isolate and study mammary stem/progenitor cells, including long-term bromodeoxyuridine labeling to identify label-retaining cells, Hoechst dye efflux to identify side population (SP) properties, and the potential stem/progenitor cell–cell surface markers, such as stem cell antigen 1 (Sca1) and α6- and β1-integrins (9–11). In the hematopoietic system, cells that efflux Hoechst 33342 dye have been shown to comprise a small fraction of bone marrow, which are capable of recapitulating the bone marrow in irradiated mice, establishing their functional capacity as hematopoietic stem cells (12). These cells are represented as a SP on flow cytometry analysis present in both mouse and human mammary glands (13–16). In hematopoietic stem cells, the efflux of Hoechst dye is due to the presence of a family of drug-effluxing protein pumps, including the breast cancer resistance protein-1 (BCRP1)/ABCG₂ transporter, that may be responsible for drug resistance in many types of cancer (17). In the mouse mammary gland, the SP phenotype depends on several members of the ABC transporter family (18). Outgrowth experiments using SP are confounded by the toxicity of the Hoechst dye (14); however, the SP fraction in mouse mammary gland is enriched for long-term bromodeoxyuridine label-retaining cells, as well as Sca1-positive cells, which have the capacity to generate functional mammary outgrowths in transplantation experiments (13). Shackleton et al. (10) recently confirmed outgrowth potential of a Sca1 ^{population but demonstrated that the majority of outgrowth potential of the Sca1 population in fact lies in the small, Sca1-positive cells. Taken together, these data support the SP and Sca1 phenotypes as useful surrogates for stem-like/progenitor cells. A thoughtful review of the mammary SP phenotype was published recently (}19), and review of the literature regarding both SP and Sca1 suggests that each of these represents markers useful for isolating potential downstream progenitors (9) rather than the more primitive stem cells isolated as described by Shackleton et al. (10) as lin^CD24CD29.

Although it has been speculated that stem or progenitor cells in the mammary gland are more resistant to conventional cancer therapies, this relationship has not been explicitly demonstrated. Here we demonstrate that progenitors in murine mammary epithelial cell (MEC) culture are enriched by clinically relevant doses of ionizing radiation and that this enrichment is enhanced by β-catenin stabilization. In human breast cancer MCF-7 cells, radiation induced enrichment of both SP progenitors and a lin^{CD24CD29 subpopulation. These findings from normal and genetically manipulated mouse mammary glands may have important implications for the evaluation of new and current cancer therapies and the development of future new anti-stem-cell-targeted therapies.}

Acknowledgments

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