Abstract 4213: Lentiviral shRNA screen testing a gene signature of breast cancer stem cells using high throughput mammosphere assays

Abstract

Abstract One in 8 women in the US suffer from breast cancer and current treatments have reduced the rate of cancer dependent deaths, the major problem facing this field is the high likelihood of relapse within 5-10 years of initial therapy. Relapse might be caused by the presence of cancer stem cells in the tumor which are not inhibited by traditional therapies and thus may require therapies that target this subpopulation of cells. We have recently published a gene signature which is derived from an overlap between CD44+/CD24-/low vs. all other cell subpopulations, and cancer-derived Mammosphere (MS) vs. bulk tumor which we reasoned will best represent the tumor-initiating or “cancer stem cells. We found that 477 genes were differentially expressed in the combination group of which 185 of these were highly expressed in CD44+/CD24-/low cells and in MS, a highly significant overlap (p<1.0E-9, one-sided Fisher's exact). The rest of the 292 genes demonstrated a reduction in expression in the CD44+/CD24-/low cells and cancer-derived MS vs. all other cells and bulk tumors, respectively (p<5.0E-5, one-sided Fisher's exact). We then proceed to test our gene signature by screening this entire list of genes for its role in MS. We created a library comprises 14 plates containing approximately 1193unique shRNA constructs, with 3-4 shRNA constructs per gene, thereby targeting 493 unique genes. The library is designed and constructed on a “one gene, one well” structure, such that each sequence validated lentiviral shRNA construct targeting an individual gene is located in a unique well of a 96-well plate. Using SUM159 cells that were transduced with individual lentiviruses representing our previously identified genes (efficiency ∼80%), along with a positive control lentivirus targeting Bmi1, a polycomb group gene known to be required for self-renewal of many stem/progenitor cell types, a non-specific negative control lentivirus targeting firefly luciferase, as well as untreated cells. In each of the transduced population cells will be plated in a 96 well dish at a density of 2,000 cells/well in serum-free medium supplemented with B27 nutrients, bFGF, EGF. MSs were allowed to form for 4 days, and then counted using Gelcount (Oxford Optornix). The MSs were trypsinized and replated in the same plate to form secondary mammospheres and primary and secondary mammosphere formation efficiency was calculated. The primary & secondary MSFE was determined for all the shRNAs and we identified 151 shRNAs while the secondary MS formation identified 63 shRNA's of which 13 shRNAs were common that demonstrated significant change (p<0.05) in MS formation with respect to control. This gives us a set of targets that need to be validated by testing them in another cell line, which in turn will provide pharmaceutical targets to eliminate cancer stem cells. Top 5 targets will be tested in tumor xenografts to test clinical relevance of these therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4213.