Breast Disease

Profoundly different patterns of potency and division are exhibited by mammalian embryonic and adult stem cells. Additional confusion surrounds stem-cell surrogates, cache and reserve cells having some characteristics of stem cells and not others. Mystification may have been introduced historically with the concepts of determinate and regulative development, but, hopefully, the muddle can be resolved by tracing the evolution of stem cells in Metazoa.

Blastomeres in marine sponges, cnidarians, lophotrochozoans, small ecdysozoans (e.g., Caenorhabditis elegans), and some deuterostomes (e.g., echinoderms and ascidians) exhibit determinative development. Their larval and adult cells have narrow potencies, sometimes coupled to virtually unlimited proliferation, and function in the growth, maintenance and regulation of body size.

The embryos of larger arthropods and deuterostomes with well-provisioned eggs or viviparity, on the other hand, exhibit regulative development, while their larval “set-aside” or adult stem cells function in the growth, maintenance, and regulation of organ size coupled to constrained proliferation and cell turnover.

Mammalian embryonic stem cells would seem adapted to rapid proliferation, functioning in part to enclose yolk or to acquire access to maternal resources. The cellular products of embryonic stem cells routinely come under global influences and give rise to the cells of germ layers and organ rudiments. Mammalian adult stem cells resemble the blastomeres of planktonic and benthic organisms with small eggs and may have evolved in mature organisms as an adaptation to the growth and maintenance of tissues via proliferation and the regulation of organ size via cell loss (e.g., terminal differentiation). Cancer stem cells, instrumental in metastasis, would seem to ignore mechanisms normally functioning in the removal of excess cells.

Strategies for regenerative therapies in adult mammals, therefore, might be based on stimulating growth of adult stem cells or their surrogates in specific tissues rather than on introducing embryonic stem cells into adults. Likewise, strategies for the containment of cancer might be based on promoting normal pathways of cell loss, the basal mode for handling excess cells.

Cancer stem cells either could be rare or common in tumors, constituting the major distinction between the two fundamentally opposed theoretical models of tumor progression: A newer and restrictive stem cell propagation model, in which the stem cells are a small and special minority of the tumor cells, and a standard older model, an unrestricted cell proliferation theory, in which many or most tumor cells are capable of indefinite generations of cell division.

Stem cells of tumors are difficult to quantitate using functional assays, and the validity of the most common assays is seriously questioned. Nonetheless, stem cells are an essential component of any tumorigenesis model. Alternative approaches to studying tumor stem cells should be explored.

Cell populations can be conceived of as having a genealogy, a relationship of cells to their ancestral lineage, from the zygote to the adult cells or neoplasms. Models using ancestral trees thus offer an anatomic and genetic means to “observe” stem cells independent of artificial conditions.

Ancestral trees broaden our attention backward along a lineage, to the zygote stage, and thereby add insight into how the mutations of tumors accumulate. It is possible that a large fraction of mutations in a tumor originate from normal, endogenous, replication errors (nearly all being passenger mutations) occurring prior to the emergence of the first transformed cell.

Trees can be constructed from experimental measurements – molecular clocks – of real human tissues and tumors. Detailed analysis of single-cell methylation patterns, heritable yet slightly plastic, now can provide this information in the necessary depth.

Trees based on observations of molecular clocks may help us to distinguish between competing theories regarding the proliferative properties among cells of actual human tumors, to observe subtle and difficult phenomena such as the extinction of stem lineages, and to address the origins and rates of mutations in various normal, hormone-stimulated, aging, or neoplastic tissues.

The simple concept that cancers arise from the transformation of a normal stem cell, the stem cell origination theory, is sometimes superficially and confusingly referred to as “the stem cell theory”. This concept is compatible with but not a requisite assumption for both of the major competing theories of tumor progression, and plays essentially no role in clarifying the nature of tumor progression.

The existence of immortal DNA strands (IDSs) in distributed stem cells (DSCs) of adult human tissues was first inferred by Cairns. Cairns deduced the existence of IDSs by connecting two seemingly disparate observations – one his own and the other belonging to Lark. Cairns noted a mathematical discrepancy between predicted human tissue cell mutation rates and human cancer incidence. He integrated this insight with Lark's earlier discovery of non-random mitotic chromosome segregation in both plant root tip cells and mouse fetal fibroblast cultures to predict the existence of IDSs as the essential elements of a mutation-defense mechanism in DSCs. Since Cairns' seminal prediction, several laboratories have identified IDSs in diverse mammalian cells with DSC properties. Past studies focused on the potential roles of IDSs as originally envisioned in DSC genetic fidelity or in the maintenance of the DSC phenotype. Another possible consequence of IDSs, aging, has received little attention. Herein, the potential for cumulative chemical modifications and decompositions (i.e., “age spots”) of IDSs in DSCs to act as a major determinant of human aging is considered. If accrued chemical alterations of IDSs prove to be essential determinants of aging, then a means to restore IDSs may yield new strategies for tissue rejuvenation.

The somatic DNA strand-specific imprinting to effect gene regulation and selective chromatid segregation model was previously proposed to produce developmentally nonequivalent sister cells in mitosis. Such a mechanism might explain generation of stem-cell pattern of cell division in eukaryotes. The developmentally controlled process involves a pair of homologous chromosomes at a specific cell division to establish embryonic left-right body axis asymmetry. As a result, visceral organs in the two sides of vertebrate's body develop asymmetrically. The model was specifically proposed to explain the well-known axis randomization phenotype of the left-right dynein mutant mice where one-half of animals develop with standard visceral organ's positioning and the balance develops with the inverted arrangement. The model postulated that the specific dynein, a microtubule-based molecular motor protein, promotes the selective chromatid segregation process in mitosis. Thus, random segregation involving sister chromatids of a pair of specific chromosomes leads to axis randomization of the mutant. Moreover, the model uniquely predicts that 50 percent mutant embryos should produce symmetrical cell divisions because of random segregation; consequently, their either visceral side would develop as mirror image of the other side resulting in embryonic lethality. In view of this prediction, validity of prominent body axis-determination models is scrutinized here. Results supporting the cell-type regulated chromosome 6 and chromosome 7 selective chromatids segregation phenomenon existing in mouse cells are reviewed. Published results with the mutant mice are consistent with the chromosome segregation model for axis determination.

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent. This conclusion is based upon the maintenance of retroviral insertion sites within the somatic DNA of successive transplant generations derived from a single mammary fragment. In addition, dissociation of these clonal dominant glands and implantation of dispersed cells at limiting dilution demonstrated that both duct-limited and lobule-limited outgrowths were developed as well as complete, fully differentiated glands. Thus, transplantation has revealed three distinct mammary epithelial progenitors in the mouse. Recently, using cre-lox conditional activation of reporter genes, the lobule-limited progenitor was lineally marked by lacZ expression. In situ, these cells were shown to regenerate secretory lobules upon successive pregnancies. In transplant studies, they demonstrated the capacity for self-renewal and contributed to the new generation of all of the epithelial cell types among mammary secretory lobules. Using this conditional activation model, cells isolated from other tissues of the WAP-Cre/Rosa26/lacZReporter mice, co-mingled with normal wild type mammary epithelial cells and transplanted into epithelium-divested mammary fat pads, were shown to be amenable to redirection of their cell fate by interaction with the mammary microenvironment in vivo. This suggests the ascendancy of the microenvironment over the intrinsic nature of somatic stem cells.

Adult stem cells reside in a specialized microenvironment, the niche, which controls their behavior. As mammary stem cells, and consequently their niches, are still poorly defined, we look at better-characterized adult mammalian stem cell niches in the hematopoietic system and the skin. We attempt to define the mammary stem cell niche functionally, based on the widely used mammary fat pad reconstitution assay.

We note that the concept of the niche needs to be extended from the specialized microenvironment described in the hematopoietic system, to a model that takes into account the macroenviroment, as recently shown in the skin, and systemic clues as we will illustrate for the mammary gland where the reproductive hormones are major determinants of stem cell activation. In fact, in the mammary gland a special type of stem cells is determined only during pregnancy.

Reproductive hormones act on hormone receptor positive cells, sensor cells, in the mammary epithelium to induce paracrine signaling that leads to activation of stem cells. Some of the downstream mediators are in common with other niches such as Wnt and possibly Notch signaling. Other signals are specific to the mammary gland such as amphiregulin and RANKL.

Breast carcinoma cells and embryonic progenitors similarly implement stem cell-associated signaling pathways to sustain continued growth and plasticity. Indeed, recent studies have implicated signaling pathways, including those associated with the Notch, and Transforming Growth Factor-Beta (TGF-β) superfamilies, as instrumental to both embryological development and breast cancer progression. In particular, Nodal, an embryonic morphogen belonging to the TGF-β superfamily, and its co-receptor, Cripto-1, are requisite to both embryogenesis and mammary gland maturation. Moreover, these developmental proteins have been shown to promote breast cancer progression. Here, we review the role of Nodal and its co-receptor Cripto-1 during development and we describe how this signaling pathway may be involved in breast cancer tumorigenesis. Moreover, we emphasize the potential utility of this signaling pathway as a novel target for the treatment and diagnosis of breast cancer.