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Regulatory mechanisms governing mammary epithelial and progenitor cell growth

University of British Columbia

The processes involved in mammary gland development are intimately linked with those that drive breast oncogenesis. Regulation of growth, tissue polarity and genome stability are a few of the factors the maintain homeostasis in breast epithelium and prevent malignant progression. In this work, a series of clonal 184-hTERT cell lines were generated that modeled the in vitro growth characteristics of bi-potent mammary progenitor cells; they were dependent upon fibroblasts for low-density growth and formed dual-lineage acini in 3D culture. These lines were subsequently used in a genome-wide siRNA screen to identify the factors that regulate fibroblast-driven epithelial cell growth. Fibroblasts constitute the majority of cells within stroma, which plays a major role in supporting mammary progenitor cell growth. From this screen, 49 surface and secreted factors were identified that putatively transduce the signals emanating from the fibroblasts that are required for epithelial cell growth. These factors were more potent than any of the previously described growth factor receptors. When assessed in primary tissue, Gpr39, Scarb2, Ntn1, Efna4, Nptx1, and Ctnna1 were found to have the greatest effect on overall progenitor cell growth, while SerpinH1 differentially suppressed luminal progenitor cells, and Nkain4 and Kcnj5 differentially suppressed bi-potent progenitor cells. Further profiling of these lines identified the planar cell polarity protein Celsr1 as differentially regulated under fibroblast-dependent conditions. Silencing of Celsr1 increased the number of bi-potent progenitor cells detected in the colonyforming assay. Furthermore, it induced branching morphogenesis within normally spherical acini and disrupted the apical polarity of these structures in 3D culture. Within this system, Celsr1 is suspected of signaling through Shisa4. This is the first description of a noncanonical Celsr1 interactor. Finally, a curious variant line was identified amongst the collection of 184-hTERT cells generated for this work. This line harbours mitotic spindle and cell cycle checkpoint defects, and rapidly gains chromosome 20 during passaging. Through an elimination process, de novo promoter hypomethylation and subsequent overexpression of CENPI was identified as likely being responsible for this phenotype. This is the first description of CENPI deregulation and one of a few descriptions of gene promoter hypomethylation resulting in genome instability.

Text

2013-06-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/35593

Pathology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/