Abstract #3069: Progestin-dependent conversion of ER+PR+ CK5- luminal to ER\#8722;PR\#8722;CK5+ basal-like breast cancer stem cells

Abstract

Addition of progestins (P) to estrogens (E) for hormone replacement therapy (HRT) appears to increase the risk of developing breast cancer. The mechanisms remain unexplained. We recently demonstrated in solid tumor models of luminal cytokeratin 18 (CK18+)-positive, estrogen (ER+) and progesterone (PR+) receptor-positive breast cancer, that P expands a rare subpopulation of cells that are ER\#8722;PR\#8722; and express the basal, and stem cell marker, CK5. The ER-PR-CK5+ cells are CD44+, contain the multidrug resistance protein BCRP1, and exhibit tumorigenic properties of cancer stem cells (Horwitz et al. 2008 PNAS 105:5774). We speculated that P in HRT expands breast cancer stem cells in women with occult microdisease. Objective: To define mechanisms by which P expands ER\#8722;PR\#8722;CK5+ tumor-initiating, basal-like cells in ER+PR+CK5- luminal breast cancer. Results: ER+PR+ (T47D, MCF7, BT474), and ER+PR- (T47D-Y) luminal breast cancer cells are all CK5\#8722; in vitro. P treatment for 24h induces CK5 in 5-15% of the PR+ cells but not in the PR- cells. Antiprogestins block the P-dependent increase in CK5. Estrogens, androgens, or glucocorticoids have no effect on CK5. Thus in luminal ER+PR+ cells, P acting through PRs are absolutely required for CK5 induction. However, the newly activated CK5+ cell subpopulation is PR-. To explain this, PR+ and PR\#8722; cells with different fluorescent tags were treated with P in cocultures. CK5 was induced only in the PR+ cells. This indicates that CK5 is activated in the same cells that contain PR; there is no paracrine signal. PRs must be subsequently extinguished in these cells. Cycloheximide blocks CK5 induction by P indicating an indirect PR transcriptional effect. To assess the mechanism, the human CK5 promoter was linked to luciferase. In luminal PR+ breast cancer cells, P increases luciferase activity 20-fold. However ERK and Akt kinase pathway inhibitors suppress P-dependent activation of the CK5 promoter, suggesting that cell surface signaling mediates the P effects. To track living CK5+ cells, the CK5 promoter was linked to ZsGreen fluor, cloned into a lentivirus vector, and used to infect PR+ breast cancer cells. P for 24h induces ZsGreen fluorescence in a subpopulation of cells. Once induced, CK5 expression is irreversible; cells remain green fluorescent despite P withdrawal. Conclusions: Progestins induce a subset of ER+PR+CK5- luminal cells to acquire basal ER\#8722;PR\#8722;CK5+ stem cell-like properties. This phenotypic switch is permanent. We have proposed that in women with occult, undiagnosed breast cancer, P in HRT expands a cancer stem cell subpopulation. This deleterious effect of P might be prevented by kinase inhibitors.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3069.