Common breast cancers and rarer breast cancers such as metaplastic carcinomas appear to originate from two different cell types, scientists claim. Studies in a human in mouse (HIM) model suggest that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer including estrogen receptor-positive and estrogen receptor-negative tumors that have, respectively, luminal and basal-like characteristics. In contrast, transformation of CD10+ cells results in the development of rare metaplastic tumors.
The Tufts University School of Medicine-led team say they have in addition found CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Charlotte Kuperwasser, M.D., and colleagues at Tufts, Unviersity of North Carolina, and Harvard Medical School, report their findings in PNAS in a paper titled “Defining the cellular precursors to human breast cancer.”
Molecular classification of tumors has shown that ER+ and ER- tumors generally retain expression of markers of the two major differentiation states of normal human breast tissue: luminal and basal/myoepithelial (ME), the researchers explain. ER+ tumors express hormone receptors and genes characteristic of luminal epithelial cells including EpCAM and Mucin 1 (Muc1). ER- tumors lack estrogen-responsive genes and express makers characteristic of basal/ME cells such as CD49f and the EGF receptor.
The notion exists, therefore, that ER+ luminal-type breast cancers may be derived from luminal progenitor cells, whereas ER− basal-like breast cancers may be derived from basal/ME progenitor cells. The situation is complicated because ER− tumors also encompass a number of rare cancers. In these cases the tumor cells not only lack ER-responsive and luminal genes but also exhibit features of alternate cell types not found in normal breast epithelium.
Moreover, there have been conflicting reports regarding the phenotypes of stem/progenitor cells and their relationship to differentiated cell progeny. Initial reports suggested that EpCAM+MUC1− or EpCAM+CD49f+ cells contained populations with bipotent potential. Other studies, however, indicated that stem/progenitor cells are found within the EpCAM−CD49f+ population.
Moreover, the team adds, “because EpCAM and CD49f are also used to identify populations of differentiated luminal and basal/ME cells, no unique set of cell surface markers can currently discriminate progenitor cells from more mature cells.”
To try and gain more insight into the nature of progenitor cells responsible for different types of breast cancer, the team therefore used an HIM model to evaluate mammary epithelial cells enriched within the two major breast epithelial lineages, to see if they do give rise to distinct tumor subtypes.
Flow cytometry analysis of reduction mammoplasty tissues for EpCAM and CD49f expression indicated that four populations of epithelial cells could be identified: EpCAMhi CD49f−, EpCAMhiCD49f+, EpCAMloCD49f+, and EpCAM−CD49f+. About 74.4% of CD10+ cells (CD10 is a well-established marker of basal/ME cells) were found in the EpCAMloCD49f+ population.
The team then used an immunomagnetic approach to to enrich for basal/ME-lineage cells (CD10+) and luminal-lineage cells that were EpCAM+CD10−, which they referred to as simply EpCAM+. They subsequently confirmed that bead sorting for CD10 efficiently enriched for basal/ME EpCAMloCD49f+ cells, while bead sorting for EpCAM efficiently recovered the EpCAMhi luminal populations containing both CD49 + and CD49f−cells.
Unsorted primary human mammary epithelial cells (HMECs) plated in serum-free mammary epithelial growth medium formed colonies that grew in suspension as well as distinct luminal, ME, and mixed adherent colonies that could be distinguished morphologically and by expression of CK14 and CK8/18. Growth of cell colonies in vitro under adherent conditions confirmed that luminal EpCAM+ cells preferentially grew as suspension colonies, while basal/ME lineage CD10+ cells preferentially grew as adherent colonies.
The team then modified the HIM model to create human breast cancer tissues in vivo by introducing oncogenes into freshly dissociated epithelial cells derived from reduction mammoplasty tissues before injection into humanized mammary fat pads. Breast tumors derived from unsorted breast epithelial cells, exhibited a mixed phenotype that contained both luminal and basal features, which “suggested the possibility that these tumors were derived from a mixture of transformed basal/ME- and luminal-lineage epithelial cells,” the authors note.
Transformation of luminal EpCAM+ (including both CD49f+ or CD49f-) cells led primarily to the formation of ductal carcinomas with redominant luminal features. Interestingly, transformation of the CD49f− subset of luminal cells resulted in the development of tumors with significantly higher expression of ERα and reduced expression of basal CK14, when compared with CD49f+ tumors.
In contrast with EpCAM+ tumors, however, those derived from CD10+ cells exhibited pronounced squamous, metaplastic, and giant cell differentiation, which was associated with a notable lack of ERα expression, a significant decrease in luminal CK expression, and marked expression of the basal marker CK14.
The researchers carried out global gene expression analyses on tumor RNA from unsorted, EpCAM+, or CD10+ cells and from tumors derived from EpCAM+/CD49f+ and EpCAM+/CD49f− cells. This showed that tumors arising from EpCAM+ or CD10+ cells could be segregated from one another, and that tumors derived from unsorted cells clustered more closely with tumors arising from CD10+ cells than with those derived from EpCAM+ cells. In addition, while tumors derived from EpCAM+ /CD49f+ and EpCAM+/CD49f− cells could be distinguished from unsorted or CD10+sorted cells, they couldn’t be distinguished from tumors derived from bulk EpCAM+ cells.
The team then derived a CD10 signature based on the genes that were differentially expressed between tumors derived from CD10+ sorted cells, compared with tumors derived from all EpCAM+ cells (including EpCAM+/CD49f+ and EpCAM+/CD49f− cells). “Interestingly, the CD10 signature was most enriched in claudin-low tumors, which are associated with metaplastic mesenchymal and mammary stem cell like characteristics,” they write.
“Altogether, these results suggest that EpCAM+ epithelial cells serve as precursors for differentiated ER+ and ER− ductal carcinomas, whereas CD10+ cells serve as precursors for rare and undifferentiated metaplastic/claudin-low carcinomas.”
The findings hint that breast epithelial cells within the CD10+ population might contain cells with metaplastic potential, before neoplastic transformation. Cultured CD10+ cells, but not EpCAM+ cells, resulted in the emergence of a subset of variant HMEC (vHMEC) cells that lacked expression of many genes associated with mammary differentiation. This subset of cells was also able to form skin-like tissues in 3-D human skin equivalent (HSE) cultures that also displayed stratified layers and expression of skin markers.
To determine whether these metaplastic breast epithelial cells were indeed precursors to metaplastic breast cancer, immortalized vHMECs were transformed with oncogenes and resulting tumors examined. The transformed vHMECs did indeed generate tumors that exhibited mixed epidermal and metaplastic features including squamous, spindle-cell, medullary, and giant cell differentiation.
“Our results strongly imply that the great majority of human breast cancers are likely derived from EpCAM+ luminal epithelial cells because EpCAM+ cells were able to give rise to both ER+ and ER− tumors, indicating that basal-like tumors need not originate from basal/ME progenitor cells,” the researchers conclude. Moreover, given the histological and molecular similarities between tumors derived from CD10+ cells and vHMECs, “these results strongly support the notion that the cellular precursors to rare metaplastic breast carcinomas may reside within the CD10+ cell population.”