TRACKING MAMMARY EPITHELIAL CELL LINEAGE AND CELL DIVISIONS IN THE NORMAL MAMMARY GLAND AND MAMMARY NEOPLASIA USING TRANSGENIC MICE
Open Access
- Author:
- Mathers, Jessica
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 21, 2011
- Committee Members:
- Edward Joseph Gunther, Dissertation Advisor/Co-Advisor
Edward Joseph Gunther, Committee Chair/Co-Chair
Sarah Bronson, Committee Member
Gary Alan Clawson, Committee Member
Jiyue Zhu, Committee Member - Keywords:
- mammary stem cell
breast cancer
mammary gland - Abstract:
- With as many as 1 in 8 women diagnosed with breast cancer in their lifetime, breast cancer is the most commonly diagnosed cancer in women in the Western Hemisphere and the second most common cause of cancer-related death in females. Decades of study have uncovered causative exposures and mutations that transform normal mammary epithelial cells (MECs) into breast cancer cells. Nonetheless, the cellular mechanisms that define the clinical behavior of breast cancers remain incompletely defined. Lineage commitment pathways yield diverse MEC cell types within the breast, and recent findings suggest these MEC lineage hierarchies may persist within breast cancers, perhaps helping to explain the cellular heterogeneity seen in tumors. To extend these studies, models are needed that permit cell fate tracking in the discrete MEC compartments of both normal and malignant mammary tissue. In this work, we describe novel transgenic mouse models that permit temporally-regulated, MEC compartment-restricted expression of a histone-fused eGFP (H2B-eGFP) reporter in both normal and malignant mammary epithelium. Transactivator transgenes expressed in either the luminal or basal layer of mammary ducts drove widespread H2B-eGFP labeling of luminal or basal MECs, respectively. We tested whether the H2B-eGFP reporter could be used to track cell divisions within labeled MEC compartments. Indeed, when H2B-eGFP transgene expression was switched off in pulse-chase experiments, washout of label depended on partitioning of labeled histones between daughter cells during MEC proliferation. Moreover, the H2B-eGFP nuclear label was readily detectable in live MECs, enabling live cell imaging of MECs propagated in culture. H2B-eGFP labeling was used for short-term lineage tracing of MECs during lobuloalveolar development. Hormones of pregnancy trigger the development of bi-layered alveolar outgrowths that are believed to arise from luminal progenitor cells. Contrasting with this model, we found that labeled cells from both the luminal and basal MEC compartments contribute to alveolar outgrowths. Furthermore, both luminal and basal MECs proliferated while contributing to alveologenesis, and did not merely migrate into alveoli or become incorporated as “bystanders”. These findings clarify a lineage commitment pathway operative during a key, hormonally-driven stage of mammary gland development. In separate studies, we labeled either basal or luminal MECs residing in the secretory epithelium of lactating mice to examine whether a subset of these MECs persist throughout the mammary gland remodeling program triggered by weaning. Remarkably, substantial numbers of both luminal and basal MECs survived mammary gland involution and contributed to remodeled ducts. These findings have implications for understanding how a lactation-involution cycle protects against breast cancer in rodents and humans. In other studies, the H2B-eGFP labeling strategy was applied in the context of Wnt1-driven transgenic mouse models of breast cancer. Here, we sought to use pulse-chase H2B-eGFP labeling of tumor cells to identify a relatively slow-cycling sub-population of tumor cells, as relatively quiescent tumor cells have been proposed to be treatment-refractory and enriched in tumor-propagating potential. Each transactivator drove reproducible, compartment-restricted H2B-eGFP labeling of a large fraction of MECs within Wnt1-initiated mammary hyperplasia, as expected. In contrast, the fraction of tumor cells labeled in Wnt1-initiated mammary cancers showed marked tumor-to-tumor variability, suggesting that the genetic and epigenetic events that cooperate in tumorigenesis sometimes interfere with transgene-mediated labeling. Notably, both the luminal- and basal-MEC-directed transactivators were capable of driving H2B-eGFP labeling of a subset of tumor cells from MMTV-Wnt1 transgenic mice. This finding supports the contention that Wnt1 initiates “mixed-lineage” mammary tumors comprised of both luminal and basal tumor cells, suggesting that Wnt1 transforms a bi-potent MEC progenitor. Tumors that labeled efficiently were studied further by quantifying H2B-eGFP washout on a per-cell basis in pulse-chase experiments. Mammary cancers typically were comprised of tumor cell populations that were heterogeneous with respect to rates of proliferation. Together, this work sets the stage for prospective studies that will compare the biological potential of luminal- versus basal-type tumor cells (i.e., those labeled using the contrasting compartment-restricted transactivators) and rapid- versus slow-cycling tumor cells (i.e., those that have depleted versus retained H2B-eGFP label). Breast cancers can recur many years after eradication of all clinically-detectable disease, but the cellular mechanisms that maintain disease dormancy remain undefined. H2B-eGFP labeling was applied in the context of reversible Wnt1-driven mammary tumors to investigate the cellular mechanisms that maintain tumor dormancy in a mouse breast cancer model. Here, H2B-eGFP and Wnt1 were co-expressed such that switching off both transgenes simultaneously initiated washout of incorporated H2B-eGFP label and regression of Wnt1-dependent mammary cancers. Subclinical lesions comprised of dormant mammary cancer persisted long after tumor regression, and these lesions frequently harbored tumor cells that retained bright H2B-eGFP label, indicating they had ceased proliferating. These label-retaining cells represent a candidate quiescent tumor cell population that may serve as a critical link between primary mammary cancer and subsequent disease relapse.