Cell fate takes a slug in BRCA1-associated breast cancer

Understanding why BRCA1 mutation carriers have a predilection for developing clinically aggressive basal-like breast tumors could inform the development of targeted treatment or prevention strategies. Analysis of both mouse and human mammary epithelial cells has identified a role for BRCA1 in orchestrating differentiation. The ability to isolate discrete epithelial subpopulations from mammary tissue has recently directed attention to luminal progenitor cells - the descendants of mammary stem cells - as the likely 'cells-of-origin' in BRCA1-associated breast cancer. A new publication has confirmed the importance of aberrant luminal cells as key culprits and provided insights on how BRCA1 haploinsufficiency biases luminal cells toward a basal-like fate through aberrant expression of the transcription factor SLUG.

recog nized to have multiple other functions. In vitro cellular assays have indicated roles in regulating mammary epithelial cell proliferation and diff erentiation and in promoting luminal-to-basal lineage transdiff er entiation [1][2][3][4]. Th e ability to fractionate mammary epithelium into diff erent subtypes has enabled insights into target cells prone to tumorigenesis. Using human breast tissue, Liu et al. [5] observed that BRCA1 was required for ER-negative stem/progenitor cells to diff erentiate into mature ER-positive luminal cells. Lim et al. [6] evaluated pathologically normal primary breast tissue samples from haploinsuffi cient BRCA1 patients and identifi ed an aberrant luminal progenitor population with factor-independent growth properties. A similar observation was made in Brca1-defi cient mice [6]. Consistent with a luminal progenitor cell defect, breast tissue from BRCA1 mutation carriers generally showed an increase in this subset relative to the total epithelial population. More over, the molecular signature of luminal progenitor cells was found to be more similar to that of basal-like tumors than to that of any other tumor subtype [6]. Overall, these fi ndings indicated, but did not prove, that luminal progenitors are the 'cells-of-origin' for basallike tumors arising in BRCA1 carriers. An impor tant study by Molyneux et al. [7] conditionally deleted Brca1 in diff erent epithelial populations (hetero zygous for p53) and revealed that luminal rather than basal cells were predisposed to basal-like mammary tumors. Th ese in vivo experiments provided direct evidence that BRCA1associated breast cancers can arise from luminal ERnegative progenitors.
A recent study by Proia et al. [8] further highlighted the relevance of luminal cells in haplo insuffi cient BRCA1 human breast tissue. With an elegant in vivo assay, fresh breast epithelial cells from wild-type or BRCA1 +/mut women were simultaneously transduced with potent lentiviruses encoding mutant p53, cyclin D1, activated phosphoinositide 3-kinase (PI3K), and oncogenic K-ras and implanted into human ized mammary fat pads of nonobese diabetic/severe combined immunodefi ciency disease (NOD/SCID) mice. Whereas luminal and basallike tumors arose in mice implanted with wild-type cells, BRCA1 +/mut cells largely yielded basal-like tumors,

Abstract
Understanding why BRCA1 mutation carriers have a predilection for developing clinically aggressive basallike breast tumors could inform the development of targeted treatment or prevention strategies. Analysis of both mouse and human mammary epithelial cells has identifi ed a role for BRCA1 in orchestrating diff erentiation. The ability to isolate discrete epithelial subpopulations from mammary tissue has recently directed attention to luminal progenitor cells -the descendants of mammary stem cells -as the likely 'cells-of-origin' in BRCA1-associated breast cancer. A new publication has confi rmed the importance of aberrant luminal cells as key culprits and provided insights on how BRCA1 haploinsuffi ciency biases luminal cells toward a basal-like fate through aberrant expression of the transcription factor SLUG. indicative of a preprogrammed epithelial defect that dictates tumor phenotype. Proia et al. [8] also used epithelial subsets to demonstrate prefer en tial transformation of luminal compared to basal cells.
Gene profi ling of wild-type and BRCA1 +/mut breast epithelia generated a molecular signa ture enriched for Wnt, Notch, and melanogenesis signal ing pathways in BRCA1 +/mut tissue. Th ose fi ndings prompted an evaluation of the transcriptional repressor SLUG, which can be activated by these pathways. SLUG is a member of the SNAIL family and has been shown to have an important role in coordinating the epithelial-mesenchymal transition and pro gram ming cells toward a basal-like phenotype in breast cancer [9][10][11]. SLUG is normally expressed in the basal/stem cell-enriched population in both mice and humans [12]. Interestingly, although SLUG mRNA levels were unperturbed in BRCA1 +/mut tissue, abundant levels of SLUG protein were observed. Moreover, knockdown of BRCA1 by short interfering RNAs in breast cell lines resulted in a twofold increase in SLUG protein.
Con versely, knockdown of SLUG in breast epithelial cells biased them toward a more luminal cell fate [8]. Th us, BRCA1 appears to regulate SLUG protein stability, and this may directly infl uence the cell fate specifi cation of luminal progenitor cells. Th e precise mechanism through which BRCA1 contributes to SLUG protein stabilization remains to be elucidated. A direct interaction between SLUG with BRCA1 was not found, nor did knockdown of the BRCA1-associated RING domain-1 protein (BARD1) alter SLUG levels [8]. Th e interesting link between BRCA1 and SLUG in perturbing cell fate decisions will undoubtedly form the basis of future studies.
Despite similarities in the epithelial subsets defi ned by EpCAM and CD49f by Proia et al. [8], there are also some noteworthy diff erences. In contrast to previous authors [6,13,14], Proia et al. [8] describe two potentially distinct basal subsets: an EpCAM low popula tion ('basal/ myoepithelial') and an EpCAMpopulation ('mesen chymal' or 'basal progenitor') [15], the latter of which appears to be novel. Th is subset was found to be expanded in BRCA1 +/mut breast tissue (with no change in the luminal progenitor subset) and was attributed to diversion of luminal cells toward a basal cell fate. On the other hand, we observed a signifi cant decrease in the basal subset [6] as well as reduced numbers of mammary stem cells in mice. In addition, the mature luminal sub population contained a dramatically increased number of CK5/6expressing cells and fewer PR-positive cells, consistent with a perturbation in diff er en tiation [6]. Th ese diff ering observations may, in part, refl ect diff erent methodological approaches (magnetic beads versus fl ow cytometry for lineage depletion) and gating strategies used for cell fractionation as well as possible variation between breast samples. Of the 12 BRCA1 +/mut samples described by Proia et al. [8], at least a third of patients had prior breast cancer. Chemotherapy or endocrine therapy may modify epithelial cell composi tion. Regardless of these diff erences, both studies identify BRCA1 as a key regulator of luminal cells.
In summary, one striking consequence of BRCA1 defi ci ency in mammary epithelium appears to be perturbed cell fate specifi cation, in which luminal cells are biased toward a more basal-like phenotype [8]. Luminal progenitor cells presumably depend, in part, on BRCA1 for providing high-fi delity DNA repair, as they are highly proliferative. Altered proliferative and diff eren tiative properties, compounded by a predisposition to genomic instability, are likely to set the stage for neoplastic transformation. It seems likely that somatic gene silencing of BRCA1 through epigenetic mechanisms plays a similarly important role in sporadic basal-like breast cancer. Further elucidation of molecular pertur bations resulting from BRCA1 defi ciency will hopefully provide important clues on therapeutic targets relevant to breast cancer treatment and chemoprevention for high-risk women.