Abstract 1816: Blocking synthesis of oncogenic proteins in breast tumor cells through pharmacologic inhibition of the IRES
Authors: Christos Vaklavas, Zheng Meng, E. Lucile White, Lynn Rasmussen, Joseph A. Maddry, Miranda Nebane-Akah, William E. Grizzle, Kurt R. Zinn, Andra R. Frost, Cecil R. Stockard, Sharon L. Samuel, Maaike Everts, Richard J. Whitley, Scott W. Blume
Journal: Cancer Research (2012)

Abstract Many genes involved in the control of cell proliferation and survival, i.e. those most important to cancer biology, are now known to be regulated specifically at the translational (RNA to protein) level. An intriguing translation-regulatory element is the internal ribosomal entry site (IRES), which provides a mechanism by which oncogene-associated mRNAs can be translated independently of the global controls on protein synthesis. Tumor cells may rely on IRES-mediated translation of key oncogenic proteins to promote their own survival and proliferation under adverse conditions or exposure to cytotoxic chemotherapy. We have previously shown that the mRNA for type 1 insulin-like growth factor receptor (IGF1R) contains an IRES immediately upstream of the initiation codon. We have characterized the RNA-binding proteins that regulate the IRES, and elucidated the mechanism by which the IRES recruits the 40S ribosome. We have accumulated evidence that IGF1R IRES activity may be aberrantly upregulated in human breast cancer cells and tissues. Based on these observations, we hypothesized that IRES-mediated translation of key oncogenic proteins could be a rational target in cancer therapeutics with pharmacologic potential. Using T47D breast cancer cells genetically engineered to produce firefly luciferace under control of the IGF1R IRES, and a similar construct from which the IRES has been deleted as a control, a high throughput screen of 135000 compounds was performed. Four promising lead compounds with distinct chemical scaffolds have been identified and further validated in dose response titrations. Their specificity in blocking IRES-mediated translation was shown in reticulocyte lysates using a bicistronic construct containing the IGF1R IRES. The compounds inhibited de novo synthesis of IGF1R in T47D and SUM159 breast cancer cells with an IC50 on Western blots between &amp;lt;2 and 10 µg/mL. Consistent with the importance of IGF1R in tumor cell survival, the IRES inhibitors induced massive apoptosis in the same cell lines with a toxic concentration-90 in the range of &amp;lt;1 - 8 µg/mL. In contrast, normal human mammary epithelial cells tolerated prolonged exposure to IGF1R IRES inhibitors. A high degree of synergism (3+ to 4+) was observed when the IRES inhibitor was combined with a cytotoxic chemotherapeutic agent (doxorubicin), with combination indices of 0.153 - 0.334 and dose reduction indices for doxorubicin of 5.0-14.9. Furthermore, these compounds were shown to completely block the clonogenic survival and the formation of mammospheres in both ER positive and negative breast cancer cell lines, suggesting that cancer stem cells are sensitive to IRES inhibition. The pharmacologic inhibition of IRES-mediated translation is a novel strategy to modulate oncogene expression and could represent a new paradigm in cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1816. doi:1538-7445.AM2012-1816