Abstract
BRCA1 deficiency may perturb the differentiation hierarchy present in the normal mammary gland and is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome. Oriented cell division is a mechanism known to regulate cell fates and to restrict tumor formation. We now show that the cell division axis is altered following shRNA-mediated BRCA1 depletion in immortalized but non-tumorigenic, or freshly isolated normal human mammary cells with graded consequences in progeny cells that include aneuploidy, perturbation of cell polarity in spheroid cultures, and a selective loss of cells with luminal features. BRCA1 depletion stabilizes HMMR abundance and disrupts cortical asymmetry of NUMA-dynein complexes in dividing cells such that polarity cues provided by cell-matrix adhesions were not able to orient division. We also show that immortalized mammary cells carrying a mutant BRCA1 allele (BRCA1 185delAG/+) reproduce many of these effects but in this model, oriented divisions were maintained through cues provided by CDH1+ cell-cell junctions. These findings reveal a previously unknown effect of BRCA1 suppression on mechanisms that regulate the cell division axis in proliferating, non-transformed human mammary epithelial cells and consequent downstream effects on the mitotic integrity and phenotype control of their progeny.
Highlights
BRCA1 deficiency is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome [1], resembling the cells that constitute the outer “basal” layer of the normal adult human mammary gland
In a first set of experiments, we examined the effect of lenti-short hairpin RNAs (shRNA)-mediated BRCA1 depletion in a subline of the non-tumorigenic, but immortalized, MCF-10A human mammary epithelial cell line that stably expresses a TUBA1B-RFP fusion protein from the endogenous TUBA1B locus (Figure 1A)
We have discovered a new and critical role of BRCA1 in the preservation of the cell division axis for proliferative, primary human mammary progenitor cells and immortalized, non-tumorigenic mammary cells
Summary
BRCA1 deficiency is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome [1], resembling the cells that constitute the outer “basal” layer of the normal adult human mammary gland. This compartment of the normal gland contains primitive cells able to regenerate normal-appearing, bilayered mammary structures in vivo and colonies in vitro that contain cells with variable numbers of luminal as well as basal features [2, 3]. The location, content, and activity of these dynein complexes are established by biochemical gradients of Ran-GTP at chromosomes and polo-like kinase 1 (PLK1) at spindle poles [10] or kinetochores [12], as well as a spindle pole located complex of hyaluronan-mediated motility receptor (HMMR) and dynein light chain 1 [13]
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