A Luminal, Steroid Receptor-Positive Breast Cancer Stem Cell that Generates Intratumoral Heterogeneity is Preserved by Progesterone and Depleted by Estradiol

Most breast cancers are luminal, depending on estrogens for growth. The remainder lack estrogen receptors, are hormone-resistant and basal-like. It is thought that breast cancers arise from self-renewing normal stem cells that have undergone malignant transformation. However currently reported cancer stem cells are hormone insensitive and generate basal, rather than luminal tumors. We now describe true luminal hormone-responsive breast cancer stem cells that express estrogen and progesterone receptors. Their self-renewal capacity is protected by progesterone. Stem cell levels are depleted by estrogens as tumors transition into differentiated and homogeneous luminal states. But, if added to estrogens, progesterone promotes heterogeneity and “rescues” the stem cells. By keeping luminal cancer stem cells “safe”, progesterone, widely used for contraception and hormone replacement, could be harmful to breast cancer survivors or women with occult disease. The cytokeratin 5 (CK5) and CD28 studies each contains 2 sample groups (CK5+, CK5–, CD28+, CD28-) assayed in triplicate, generating 12 individual samples (12 arrays). CK5+ and CK5- samples were generated from T47Dco cells maintained in 1nM β-Estradiol + 10nM Progesterone for 5 days. For CK5-staining, single cell suspensions were permeabilized and fixed with RNAlater (Ambion Inc.), and incubated with 2 µg anti-CK5 mAb (Novocastra; NCL-L-CK5) labeled with Zenon Alexa Fluor 488 (Invitrogen; Z-25002). Stained cells were centrifuged and resuspended in RNAse-free NST buffer containing DAPI and FACS sorted (Beckman Coulter XDP-100 MoFlo) based on CK5+ vs. CK5– expression. RNA was extracted using the PicoPure RNA isolation kit (Arcturus). RNA from T47Dco-derived CK5+ and CK5– cells were obtained in triplicate and expression profiled using Agilent 4x44K chips at MOgene LC (www.mogene.com). To isolate CD28+ and CD28- cell populations, T47Dco cells were treated with E+P for 5 days, stained for CD28 (Invitrogen; CD2801), FACS sorted and mRNA was extracted. CD28+ and CD28– gene profiles were generated as described above.