Abstract P2-04-05: Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones

All Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+PR+ ones. We have identified two such Luminal-derived ER–PR– cells: The first, we call "Luminobasal" (LB), are ER–PR– and cytokeratin 5 (CK5)-positive. The second, we call "Double Negative" (DN), are ER–PR– and CK5–. Currently, neither is targeted for treatment. Luminobasal cells: To address the relationships between true Luminal ER+PR+CK5– and Luminobasal ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from solid tumor xenografts starting from the same parental T47Dco Luminal human breast cancer cell line. Cells were gene profiled and unique immunohistochemical (IHC) biomarkers for each were confirmed. Interaction dynamics studies show that in mixed-cell 3D colonies, pLUM or Luminal MCF-7 cells suppress growth of pLB cells. Similarly, in mixed-cell solid tumor xenografts, pLUM cells suppress pLB proliferation. Alarmingly, in mixed-cell models, monotherapy of a pLUM or MCF-7 subpopulation with the antiestrogen Fulvestrant inadvertently expands the number of pLB cells. This suggested that pLB cells also need to be treated. An 89 drug FDA-approved oncology library and high throughput screening methods were therefore used to show that pLB cells are specifically targeted by the EGFR inhibitors Gefitinib and Erlotinib. We then showed, in both mixed-cell 3D colonies and mixed-cell solid mouse tumors, that combination therapy using Fulvestrant plus Gefitinib constitutes a robust treatment strategy that targets both cell populations simultaneously. Double Negative cells: DN cells are EGFR– and therefore unaffected by EGFRi. However they express the Luminal marker Claudin-3 (CLD3). Among other conditions, DN cells arise as a discrete CLD3+ subpopulation within CLD3– pLB xenografts. This allowed us to use CLD3 for FACS isolation and purification of DN cells from xenografts. Purified cells were gene expression profiled, which showed that DN cells have a unique genomic signature that distinguishes them from LUM and LB cells. They express diagnostic markers suitable for IHC and FACS-sorting, some of which also present potential therapeutic targets. We propose that heterogeneous Luminal breast cancers may be best treated with combination therapies that include endocrine, EGFRi and/or DN inhibitors. Importantly, optimizing combination regimens that eradicate or suppress not only ER+PR+ cells but also diverse ER–PR– cells in Luminal disease requires that primary tumors be pre-screened for appropriate biomarkers, including but not limited to ER, PR, CK5 and EGFR. Citation Format: Allison L Scaling, Aaron J Knox, Maurcio P Pinto, Brian S Bliesner, James M Haughian, Hany A Abdel-Hafiz, Kathryn B Horwitz. Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-05.