UBC Theses and Dissertations
The regulation of mitotic spindle orientation by BRCA1 controls the proliferation, polarization and growth arrest of human mammary epithelia Nemirovsky, Oksana
Carriers of mutations in the Breast Cancer 1 (BRCA1) gene have an increased risk to develop breast cancer, which tend to be early-onset, lack expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, and resemble basal epithelia by gene expression. The phenotypic resemblance of these tumours to normal stem/progenitor cells suggests that the loss of BRCA1 function may dysregulate stem cell maintenance and differentiation. In model organisms, a mechanism that promotes the generation of daughter cells different from a mother stem cell is the asymmetric segregation of non-genetic factors through mitotic spindle orientation. BRCA1 regulates mitotic spindle assembly through the post-transcriptional degradation of the low-penetrance breast cancer susceptibility gene product RHAMM and the abundance of RHAMM influences mitotic spindle orientation by regulating its movement along the cell cortex. This led to the hypothesis that BRCA1 is necessary for the correct orientation of the mitotic spindle in mammary epithelial cells, which controls their proliferation, polarization and growth arrest. To address this hypothesis, I studied non-malignant human mammary cell-lines and primary human progenitor cell-enriched populations. BRCA1 was silenced by shRNA introduced through lentiviral transduction. Silencing of BRCA1 in cell-lines increased both mitotic and post-mitotic abnormalities, including the loss of spindle orientation with subsequent lagging chromosomes and micronucleus formation in 2D cultures. The consequence of these defects included a significant decrease in colony-forming capacity. I then enquired whether BRCA1 is necessary for MCF10A cells to proliferate, form polarized acini, and growth arrest in 3D cultures. Control cells underwent planar division to form polarized, growth arrested acini, while BRCA1 silenced structures were larger, less polarized and more proliferative. Loss of correct spindle orientation was also observed. These results indicate that BRCA1 plays a role in maintaining the integrity of human mammary cell division. Loss of BRCA1 induces mitotic and post-mitotic consequences that impair cellular proliferative capacity and abolish ability to undergo directional division, polarization and arrest growth. These findings thus raise the possibility that breast cancer treatments aimed at counteracting the BRCA1-mediated loss of polarity may complement drugs that combat the diminished DNA repair characteristic of BRCA1-associated tumours.
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