Summary Gene expression signatures relating mammary stem cell populations to breast cancers have focused on adult tissue. Here, we identify, isolate and characterize the fetal mammary stem cell (fMaSC) state since the invasive and proliferative processes of mammogenesis resemble phases of cancer progression. fMaSC frequency peaks late in embryogenesis, enabling more extensive stem cell purification than achieved with adult tissue. fMaSCs are self-renewing, multipotent, and co-express multiple mammary lineage markers. Gene expression, transplantation, and in vitro analyses reveal putative autocrine and paracrine regulatory mechanisms including ErbB and FGF signaling pathways impinging on fMaSC growth. Expression profiles from fMaSCs and associated stroma exhibit significant similarities to basal-like and Her2+ intrinsic breast cancer subtypes. Our results reveal significant links between development and cancer and provide resources to identify new candidates for diagnosis, prognosis and therapy.

SummaryLittle is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers.

Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.

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Equilibrium and molecular structures, relative energies of conformers of gaseous cysteine (Cys, C, Cys-SH) and gaseous cysteine sulfenic acid (Cys-SOH), and the mechanisms of the reaction of Cys-SOH with 3-hydroxy-5,5-dimethyl-2-cyclohexen-1-one, the enol tautomer of 5,5-dimethyl-1,3-cyclohexadione (dimedone), have been studied using BD(T), CCSD(T), and QCISD(T) with the cc-pVTZ basis set and using MP2 and the density functionals B3LYP, B3PW91, PBE1PBE, PBEh1PBE, M062X, CAM-B3LYP, and WB97XD with the 6-311+G(d,p) basis set. The structures of the six lowest energy conformers of gaseous Cys-SOH are compared with the six lowest energy conformers of gaseous cysteine (Cys-SH). The relative stability of the six lowest energy conformers of Cys-SH and Cys-SOH are influenced by the interplay among many factors including dispersive effects, electronic effects, electrostatic interactions, hydrogen bonds, inductive effects, and noncovalent interactions. The mechanism of the addition of the lowest energy conformer of cysteine sulfenic acid (Cys-SOH) to dimedone may proceed through a six-membered ring transition state structure and through cyclic hydrogen-bonded transition state structures with one water molecule (8-membered ring), with two water molecules (10-membered ring), and with three water molecules (12-membered ring). Inclusion of one and two water molecules in the transition state structures lowers the activation barrier, whereas inclusion of a third water molecule raises the activation barrier.