Abstract 5495: Identification of chemosensitivity nodes and small molecule inhibitors for breast cancer stem cell proliferation and survival
Authors: Zhang Fang , Rothermund Kristi , Prochownik Edward V. , Lazo John S. 
Published: February 2011

Cancer stem cells (CSCs) are believed to be important in breast cancer initiation and propagation. Thus, drugs targeting genes that are functionally significant for breast CSC proliferation and survival might offer promise for more effective breast cancer therapy. Therefore, we developed a CSC-like cell line, MCF7-Oct3/4, by incorporating a segment of Oct3/4 promoter to MCF7 cells. These cells shared many common properties of breast CSC but remained in an undifferentiated state for an extended period of time and could be expanded indefinitely, all attributes that are important for high-throughput screening. It is our hypothesis that MCF7-Oct3/4 cells express genes, which, when inhibited preferentially inhibit the growth and/or survival of these cells versus their non-CSC counterparts. Thus, inhibition of these genes will lead to selective depletion of the CSC population.

Using MCF7-Oct3/4 cells, we screened a human druggable genome siRNA library containing 16,560 siRNAs targeting 5,520 genes in a one-gene, one-well format (3 siRNAs against one gene pooled in one well). Cells were transfected with library siRNAs at 30 nM using a wet-reverse transfection method and incubated for 72 hours before cell viability was accessed by CellTiter Blue Cell Viability Assay. The percent (%) inhibition of each siRNA pool was calculated using in-plate positive and negative controls and ranked from high to low. The top 450 siRNA pools (% inhibition &gt; 20%) were selected for secondary screening against both MCF7-Oct3/4 and isogenic MCF7 cells. The % inhibition of these pooled siRNAs for the two cell lines were then compared to identify siRNAs that preferentially inhibit MCF7-Oct3/4 cells. We have identified 63 siRNAs with &gt; 3 fold greater % inhibition for MCF7-Oct3/4 than for MCF7 cells. Among these hits, 21 siRNAs had &gt; 50% inhibition for MCF7-Oct3/4 cells and had &gt; 5 fold greater % inhibition for MCF7-Oct3/4 than for MCF7 cells. These 21 hit siRNAs were further validated by transfecting the two cell lines with the 3 individual siRNAs. Fourteen siRNAs had at least 2 siRNAs that selectively inhibited MCF7-Oct3/4 cells. We also screened 1,280 pharmaceutically active compounds against both MCF7-Oct3/4 and MCF7 cells and compared the % inhibition of each compound for the two cell lines. We have identified and validated 4 compounds that had significantly greater inhibition against MCF7-Oct3/4 cells compared with MCF7 cells. These hit compounds were also tested on CSC-like cells derived from three other breast cancer cell lines. All 4 compounds exhibited selective inhibition for CSC-like cells derived from at least two other cell lines.

In conclusion, the MCF7-Oct3/4 cell model is a valuable and robust tool for using high-throughput screening to identify genes that appeared to be important for breast CSC proliferation and survival as well as small molecule compounds that selectively inhibit breast CSCs.

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 5495.