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Mammary Stem Cell Project

Breast Cancer Remains a Serious Health Risk

The American Cancer Society estimates that more than 180,000 Americans (~270 people every day) will be diagnosed with breast cancer this year. The term breast cancer describes a diverse array of malignancies that affect mammary tissue, each with a different outcome and prognosis. Although recent advances in treatment and diagnosis have resulted in declines in overall breast cancer death rates, some forms of breast cancer still carry a high mortality rate. Forty thousand Americans are expected to succumb to the disease this year. Moreover, survivors of breast and other cancers may live with constant fears of relapse from occult cancer cells capable of re-growing tumors. Clearly, the molecular characterization of the cells which initiate and perpetuate cancer would allow earlier and more sensitive detection of disease, more accurate prognoses, more effective treatment and the development of truly curative therapies.

Tumors May Be Propagated By Cancer Stem Cells, But Their Relationship With Normal Stem Cells is Unknown

Recent studies in human hematopoietic, neuronal, and mammary malignancies have indicated that a limited subset of cells within a tumor are capable of clonogenic re-growth of the malignancy using xenografts into immune-compromised mice [1-4]. These cells fall under the operational designation of "cancer stem cells" as these cells can regenerate the phenotypic heterogeneity of the original tumor as a xenograft and has the ability to continue to regrow a tumor upon serial transplantation (Clarke et al., Cancer Research 2006). Although normal tissue stem cells share these characteristics, the relationship between these purported "cancer stem cells" and the stem cells that generate the corresponding healthy tissue remains to be determined. For instance, it is possible that the cancer stem cell originated from a proliferative progenitor that mutationally acquired the regenerative attributes of a normal tissue stem cell. Alternatively, it may be that the normal tissue stem cell was itself a target cell for tumor initiation. Direct experiments to distinguish these possibilities have yet to be performed, in part because definitive and functionally relevant markers for stem cells in most organs have not been rigorously established.

Elegant studies in several adult tissues have confirmed the presence of normal, organ-specific stem cells that contain the capacity to proliferate and differentiate into all the lineages of a given tissue. Although they are non-cancerous, these adult tissue stem cells may remain quiescent for long periods, and in some instances, have been shown to encode transport proteins that can export a variety of drugs [5-14]. If "cancer stem cells" share these properties, they may be difficult to detect, long-lived, and intrinsically resistant to conventional therapies that rely on cell proliferation for their cytotoxic effects.

To treat cancers more effectively and ultimately cure the disease, it is imperative to develop a more complete understanding of organ-specific, tissue stem cells and determine whether such cells contribute directly to cancer or share important functional similarities with the cells that perpetuate cancer in its primary, dormant or metastatic forms.

Mammary transplantation of donor material

The Mammary Gland Contains Tissue Stem Cells But Definative Molecular Markers Are Lacking

Evidence for the existence of mammary stem cells has been steadily mounting [11, 15-18]. Although there are currently no highly specific or functionally relevant molecular markers of these cells, classic transplant assays demonstrated that rare cells distributed throughout the adult mammary gland have the capacity to regenerate an entire functional gland when transplanted into the cleared fat pad of a recipient mouse (Figure 1)[16]. Moreover, serial transplantations of gland fragments generated functional clonal outgrowths up to 10 generations, suggesting that individual cells exist in mice that are capable of self-renewal, and of generating all the lineages of the mature mammary gland [15, 18]. These are the two hallmarks of a mammary stem cell.

Based on dilution experiments, it was estimated that approximately 1/2500 epithelial cells derived from a pubertal mouse are stem cells [18]. In order to facilitate the further characterization of putative mammary stem cells, attempts have been made to obtain enriched populations. Fractionation of cells from adult mouse mammary glands based on particular cell surface markers enabled significant enrichment for cells capable of regenerating a functional mammary gland after transplantation into de-epithelialized mammary fat pads [11, 17]. However, comparison of the existing reports reveals no consensus concerning the molecular signatures that define mammary stem cells, and it is also unclear how closely they correspond to the presumptive cancer stem cells described above.

The Source of Mammary Stem Cells

Classic studies show that cells from mouse mammary rudiments can generate functional glands when transplanted into the cleared fat pads of adult recipient mice [16]. Thus an intriguing possibility is that the cells which originally establish the gland share identity with the adult tissue stem cell. Were such embryonic mammary stem cells extant in adults, they could contribute to either the cyclical development of the normal adult gland, or be diverted to neoplasia by cumulative mutagenic events. These ideas echo those of Cohnheim who observed that the "tumour is …a fault or irregularity of the embryonic rudiment," a theme reiterated by Pierce et al., in 1978 who suggested, "Cancer is a problem of developmental biology." And, it is this close parallel between the mechanics of normal tissue development and neoplastic transformation, which have led us to our current approach in seeking out, and characterizing the stem cells which may form the basis of both processes.

H2BGFP is incorporated into the   chromatin of living cells

Specific Aims

The Specific Aims of this project are to identify and characterize the stem cells that generate the mouse mammary gland, and to determine the signal transduction pathways that contribute to normal mammary development. We will also investigate whether these stem cells or proliferative progenitors contribute to the initiation and perpetuation of breast cancer, and whether specific signal transduction pathways employed during embryonic development are reactivated during cancer progression.

Strategy

Development and validation of H2BGFP

We are developing new strategies based on molecular genetic tools that we originally devised to label chromatin in living cells (Figure 2) [19, 20]. We are extending this approach to generate state of the art genetic strategies to label cells and track cell lineages in living mice (e.g. Figure 3). By utilizing this labeling technique, we will be able to identify if quiescence is a property of mammary stem cells. The labeled cells will be analyzed using recently developed micro-scale cell sorting together with high-throughput molecular profiling to identify genes with functional relevance for stem cell maintenance, proliferation or differentiation. The validation of the role of candidate genes in stem cell biology will be tested using in situ molecular profiling and classical mammary repopulation assays.

Attempts to isolate mammary stem cells have typically utilized the complex cellular environment of the adult mouse mammary gland. The concentration of cells able to regenerate entire functioning mammary glands has been estimated to be approximately 1/2500, but current efforts to isolate these stem cells have resulted in low levels of purity that render characterization highly problematic [11, 17, 18]. To overcome this issue, our genetic system labels cells using functional properties of both the mammary gland itself as well as a putative property of stem cells (described above). In addition, the application of these molecular tools and reagents derived from these studies to mouse models of human breast cancer will provide a powerful approach to the study of cancer pathology and the role of stem cells and their proliferative descendants in the physiology of mammary tumorigenesis in mice and in humans.

References:

  1. Al-Hajj, M., et al., Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A, 2003. 100(7): p. 3983-8.
  2. Miyamoto, T., I.L. Weissman, and K. Akashi, AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci U S A, 2000. 97(13): p. 7521-6.
  3. Singh, S.K., et al., Cancer stem cells in nervous system tumors. Oncogene, 2004. 23(43): p. 7267-73.
  4. Singh, S.K., et al., Identification of a cancer stem cell in human brain tumors. Cancer Res, 2003. 63(18): p. 5821-8.
  5. Clarke, M.F., et al., Cancer Stem Cells--Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Res, 2006. 66(19): p. 9339-44.
  6. Alvi, A.J., et al., Functional and molecular characterisation of mammary side population cells. Breast Cancer Res, 2003. 5(1): p. R1-8.
  7. Bhattacharya, S., et al., Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay. Invest Ophthalmol Vis Sci, 2003. 44(6): p. 2764-73.
  8. Goodell, M.A., et al., Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med, 1996. 183(4): p. 1797-806.
  9. Goodell, M.A., et al., Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med, 1997. 3(12): p. 1337-45.
  10. Hierlihy, A.M., et al., The post-natal heart contains a myocardial stem cell population. FEBS Lett, 2002. 530(1-3): p. 239-43.
  11. Spangrude, G.J. and G.R. Johnson, Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci U S A, 1990. 87(19): p. 7433-7.
  12. Stingl, J., et al., Purification and unique properties of mammary epithelial stem cells. Nature, 2006. 439(7079): p. 993-7.
  13. Uchida, N., et al., ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood, 2004. 103(12): p. 4487-95.
  14. Welm, B.E., et al., Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol, 2002. 245(1): p. 42-56.
  15. Zhou, S., et al., The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med, 2001. 7(9): p. 1028-34.
  16. Daniel, C.W., et al., The in vivo life span of normal and preneoplastic mouse mammary glands: a serial transplantation study. Proc Natl Acad Sci U S A, 1968. 61(1): p. 53-60.
  17. Deome, K.B., et al., Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res, 1959. 19(5): p. 515-20.
  18. Shackleton, M., et al., Generation of a functional mammary gland from a single stem cell. Nature, 2006. 439(7072): p. 84-8.
  19. Smith, G.H., Experimental mammary epithelial morphogenesis in an in vivo model: evidence for distinct cellular progenitors of the ductal and lobular phenotype. Breast Cancer Res Treat, 1996. 39(1): p. 21-31.
  20. Kanda, T., K.F. Sullivan, and G.M. Wahl, Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol, 1998. 8(7): p. 377-85.
  21. Wong, E.T., et al., Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange. Nucleic Acids Res, 2005. 33(17): p. e147.

Personnel

Jennifer C. Lin Jennifer C. Lin, Ph.D. obtained a B.S. in 1997 in Biotechnology from Rutgers University, New Jersey. In 2003, She received a Ph.D. in Molecular and Developmental Biology from Stony Brook University, New York. She was a postdoctoral fellow at The Scripps Research Institute from 2003-2006. Jen joined the Wahl Lab in 2007.

Jennifer C. Lin CV
Benjamin T. Spike Benjamin T. Spike, Ph.D. carried out his undergraduate studies in Southern California and in Goettingen, Germany, receiving his Bachelor’s degrees in European History and Molecular Biology from the University of California, San Diego in 1997. He went on to receive his M.S. in Biology under the mentorship of Dr. Wahl in 1998. And after earning a Ph.D. in Cancer Biology from the University of Chicago in the summer of 2007, Ben returned to the Wahl Lab.

Benjamin T. Spike CV
Dannielle Engle Dannielle Engle graduated from Northwestern University in 2005 with a BA in Biological Sciences. After starting her Ph.D. program at UCSD, she joined the Wahl Lab in the spring of 2006.

Dannielle Engle CV