Zero to one:normal derived human ER+ cells in culture-proliferating

Cell culture technology is used to model structural and functional properties of human organs under normal and pathological conditions “in a dish”. The most obvious reason to culture human breast-derived cells is our fundamental desire to understand and ultimately treat breast cancer. Highly reproducible serum-free formulations for long-term propagation of normal human breast epithelial cells have existed for more than three decades and have served to complement the insight gained from a vast number of established breast cancer cell lines. The unspoken dichotomy in the experimental approach, however, has lied in the puzzling fact that normal-derived cells show a more myoepithelial expression profile, while breast cancer cells show more of a luminal profile making these difficult to compare experimentally. Moreover, normal estrogen receptor positive (ER+) luminal cells, thought to be equivalents to the most frequent form of human breast cancer, the ER+ subtype, completely fail to grow under standard culture conditions. One might choose to ignore this fact since breast homeostasis relies on a stem cell hierarchy and stem cells reside in the myoepithelial compartment which, if given the right conditions, can differentiate into ER+ luminal cells. The problem with this is that myoepithelial cells in culture, for unknown reasons, fail to behave like myoepithelial cells in vivo. This review summarizes some of the progress that has been made in the field with regard to the ER+ luminal breast epithelial lineage, especially within a human context, and its relevance to human breast cancer

Zero to one: normal derived human ER+ cells in culture-proliferating

Cell culture technology is used to model structural and functional properties of human organs under normal and pathological conditions “in a dish”. The most obvious reason to culture human breast-derived cells is our fundamental desire to understand and ultimately treat breast cancer. Highly reproducible serum-free formulations for long-term propagation of normal human breast epithelial cells have existed for more than three decades and have served to complement the insight gained from a vast number of established breast cancer cell lines. The unspoken dichotomy in the experimental approach, however, has lied in the puzzling fact that normal-derived cells show a more myoepithelial expression profile, while breast cancer cells show more of a luminal profile making these difficult to compare experimentally. Moreover, normal estrogen receptor positive (ER+) luminal cells, thought to be equivalents to the most frequent form of human breast cancer, the ER+ subtype, completely fail to grow under standard culture conditions. One might choose to ignore this fact since breast homeostasis relies on a stem cell hierarchy and stem cells reside in the myoepithelial compartment which, if given the right conditions, can differentiate into ER+ luminal cells. The problem with this is that myoepithelial cells in culture, for unknown reasons, fail to behave like myoepithelial cells in vivo. This review summarizes some of the progress that has been made in the field with regard to the ER+ luminal breast epithelial lineage, especially within a human context, and its relevance to human breast cancer

Establishment of a normal-derived estrogen receptor-positive cell line comparable to the prevailing human breast cancer subtype

Understanding human cancer increasingly relies on insight gained from subtype specific comparisons between malignant and non-malignant cells. The most frequent subtype in breast cancer is the luminal. By far the most frequently used model for luminal breast cancer is the iconic estrogen receptor-positive (ER(pos)) MCF7 cell line. However, luminal specific comparisons have suffered from the lack of a relevant non-malignant counterpart. Our previous work has shown that transforming growth factor-β receptor (TGFβR) inhibition suffices to propagate prospectively isolated ER(pos) human breast luminal cells from reduction mammoplasties (HBEC). Here we demonstrate that transduction of these cells with hTERT/shp16 renders them immortal while remaining true to the luminal lineage including expression of functional ER (iHBEC(ERpos)). Under identical culture conditions a major difference between MCF7 and normal-derived cells is the dependence of the latter on TGFβR inhibition for ER expression. In a breast fibroblast co-culture model we further show that whereas MCF7 proliferate concurrently with ER expression, iHBEC(ERpos) form correctly polarized acini, and segregate into proliferating and ER expressing cells. We propose that iHBEC(ERpos) may serve to shed light on hitherto unappreciated differences in ER regulation and function between normal breast and breast cancer

Propagation of oestrogen receptor-positive and oestrogen-responsive normal human breast cells in culture

Investigating the susceptibility of oestrogen receptor-positive (ER(pos)) normal human breast epithelial cells (HBECs) for clinical purposes or basic research awaits a proficient cell-based assay. Here we set out to identify markers for isolating ER(pos) cells and to expand what appear to be post-mitotic primary cells into exponentially growing cultures. We report a robust technique for isolating ER(pos) HBECs from reduction mammoplasties by FACS using two cell surface markers, CD166 and CD117, and an intracellular cytokeratin marker, Ks20.8, for further tracking single cells in culture. We show that ER(pos) HBECs are released from growth restraint by small molecule inhibitors of TGFβ signalling, and that growth is augmented further in response to oestrogen. Importantly, ER signalling is functionally active in ER(pos) cells in extended culture. These findings open a new avenue of experimentation with normal ER(pos) HBECs and provide a basis for understanding the evolution of human breast cancer

Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity

Abstract Tumorigenesis is increasingly considered to rely on subclones of cells poised to undergo an epithelial to mesenchymal transition (EMT) program. We and others have provided evidence, however, that the tumorigenesis of human breast cancer is not always restricted to typical EMT cells but is also somewhat paradoxically conveyed by subclones of apparently differentiated, non-EMT cells. Here we characterize such non-EMT-like and EMT-like subclones. Through a loss-of-function screen we found that a member of the E3 ubiquitin ligase complexes, FBXO11, specifically fuels tumor formation of a non-EMT-like clone by restraining the p53/p21 pathway. Interestingly, in the related EMT-like clone, FBXO11 operates through the BCL2 pathway with little or no impact on tumorigenesis. These data command caution in attempts to assess tumorigenesis prospectively based on EMT profiling, and they emphasize the importance of next generation subtyping of tumors, that is at the level of clonal composition