Biology of Mammary Gland Development and Tumorigenesis

Division Of Basic Sciences - Nci

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Abstract

Long-label-retaining mammary epithelial cells (LREC) were labeled with 5-ethynl-2-deoxyuridine (EdU) in two ways. First, EdU was given upon two consecutive days per week during weeks 4 through 10 and analyzed for label retention at 13 weeks of age. Alternatively, EdU was given for 14 consecutive days beginning at 28 days of age and ending at 46 days of age. Analyses were conducted at greater than 91 days of age (13 weeks). Many more LREC were detected following the second labeling method and their distribution among the subsequently developed ducts indicated that these cells were distributed into portions of the gland that developed subsequent to the ending of EdU treatment. These observations may have important meaning with respect to the previously demonstrated retention of regenerative capacity throughout the mouse mammary gland despite age or reproductive history. These results suggest LREC may represent long-lived progenitor cells that are responsible for mammary gland homeostasis and act as multipotent stem cells capable of mammary gland regeneration upon fragment transplantation into epithelium denuded mammary fat pads. It is widely accepted that mitochondria arose from an endosymbiotic relationship between an Archaebacterium that endocytosed an alpha-Proteobacterium approximately 2 billion years ago. Through this primordial phenomenon a eukaryotic precursor, exhibiting aerobic and anaerobic respiration characteristics, became the antecedent for all eukaryotic organisms. Mitochondria, through evolutionary progression, have retained their ability to produce ATP for multicellular organisms but exist in different forms (derived from fission and fusion). During this transformation, they have gained a myriad of additional functions. We hypothesize that the sequence of mitochondrial DNA (mtDNA) is conserved throughout clonal expansion in tissues and tumors. Mouse mammary tumor virus (MMTV)-DNA insertion in somatic DNA is random. DNA insertion into somatic genomes can be used to determine clonality of normal mammary outgrowths during serial passages in CzechII mice. Mouse mtDNA was extracted, using a novel approach, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments, which were serially transplanted in epithelium-free, surgically-cleared mammary fat pads. Next generation deep sequencing was used to determine if mtDNA sequence was conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA sequences are maintained during serial passage of clonal, phenotypically heterogeneous normal and neoplastic cellular populations and are present consistently in both metastatic clones and in individual tumor transplants grown from the original metastatic tumor. During metastasis and transplantation one of the two mutations in the mitochondria DNA became more prevalent indicating that the mutations were present within different mitochondrial genomes. We conclude that mtDNA somatic mutations are conserved during clonal expansion and thus mtDNA could be a stable biomarker for solid tumors. The mechanism by which this is achieved appears to be through mtDNA replication and heteroplasmic shifting leading to homoplasmy reached in a specific tumor. This conclusion is based upon the observation of mtDNA sequence variation or lack thereof in a phenotypically heterogenous cellular population comprised of the progeny of a single cellular antecedent. Further studies such as direct replacement of mitochondria carrying a marked genome in a clonogenic cell and examination of the mtDNA from the subsequent clonal population is required for final proof. Nonetheless our sequence evidence is compelling and has possible translational applications. Mammary progenitor cells have been postulated to have a role in breast cancer is located in the breast of females and It distinguishes mammals from other animals by secretion of milk or lactation which provides nourishment for their young. Mammary gland architecture is comprised of cell types which has been determined to stem from a hierarchal process. In 1996, Smith and colleagues demonstrated, through limited dilution transplantation studies, that the hierarchal process begins with mammary stem cells, which gives rise to the two major cell types in the mammary gland: ductal and alveolar progenitor epithelial cells. The ductal progenitor cell gives rise to the ductal luminal cells, which mitotically divide without hormone stimulation until puberty. After puberty, hormones prompt expansion of the ductal epithelial branching that spans the mammary fat pad. The ductal progenitor epithelial cells also give rise to specialized cap cells, found only at the apex of the terminal end bud which then gives rise to myoepithelial cells. The alveolar progenitor cells, unlike the ductal progenitor cells, have been shown to give rise to secretory alveolar cells and myoepithelial cells, which are necessary for lactation and milk secretion in the mouse. Elucidation of the underlying mechanisms responsible for the differentiation and stemness of mouse mammary progenitor cells have been of broad interest and paramount to understanding the heterogeneity of mammary development and tumorigenesis. Consequently, studies have isolated and characterized mouse mammary progenitor cell lines such as: COMMA-1D, NSP2 and SP3, but have not parsed the various transcriptomes that may play a role in progenitor differentiation and mammary gland development. Our study reports that there are not only significant differences (3 Fold; P.05), in genetic expression between normal mouse mammary control cell lines, In Vitro: Balb/C and Comma-1D but also in the NSP2 (mouse mammary ductal limited, alveolar-incompetent) and the SP3 (mouse mammary duct and alveolar-competent) progenitor cell lines. Our laboratory previously presented evidence that ductal and alveolar development was the province of two distinct mammary progenitors. Our current evidence supports these earlier observations and extends these to molecular and biological pathways involved in the development of normal mammary gland and the genes necessary for ductal versus alveolar differentiation in mammary epithelium.