Abstract 2018: Investigation of ITAF's role in the pathophysiology of breast cancer

Abstract

Abstract A subset of cellular mRNAs can initiate cap-independent translation via internal ribosome entry sites (IRES) structures, thus maintaining translation when cap-dependent translation is compromised. Many of these IRES harboring mRNAs encode canonical oncoproteins such as c-MYC, VEGF and EGFR. Cells use IRES-mediated translation under conditions that are common to the tumor microenvironment, including nutrient deprivation, hypoxia, inflammation, and DNA damage, leading to the hypothesis that cancer cells use cap-independent translation to survive pro-apoptotic pressure. To investigate this possibility, we profiled IRES trans-activating factors (ITAFs), which are accessory proteins that regulate IRES activity in the cells. ITAFs are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that participate in diverse nuclear functions, including RNA splicing, DNA repair, and transcription. During cap-independent translation, ITAFs egress into the endoplasmic reticulum (ER) to form a complex with the 40S ribosome and other accessory proteins to regulate translation of client IRES harboring mRNAs. We hypothesize that ITAFs could influence the pathophysiology of breast cancer by controlling the translation of onco-and antiapoptotic proteins. In this study, we profiled the expression of two ITAFs, interleukin enhancer binding protein 2 (ILF2) and splicing factor proline/glutamine-rich (SFPQ) in normal and breast cancer specimens, and investigated the biological effects of these ITAFs in both cancer cell lines and preclinical in vivo models using knockout studies. Additionally, we investigated the impact of their subcellular localization in cancer cells and the translation of their client mRNAs under ER stress using immunoblots as well as polysome and immunofluorescence analysis. Finally, we investigated the impact of inhibiting nuclear pore translocation on the function of ITAFs in breast cancer cells, using a selective inhibitor of nuclear export (SINE) compound. Analysis of microarray datasets from cancer and normal tissue specimens (The Cancer Genome Atlas, METABRIC, and University of North Carolina datasets) revealed that expression of several ITAFs is upregulated in malignant tissues compared to normal controls (p<0.05) and associated with survival outcome (p<0.03). Silencing of ITAFs led to significant proliferation impairment in breast cancer cells in 2-D culture (p<0.001), soft agar assays (p< 0.01) and pre-clinical in vivo models (p<0.05). In cancer cell lines, treatment with an ER stressor led ITAFs to egress to the ER, with increased translation of client IRES-harboring mRNAs despite impaired global cap-dependent translation. Finally, the SINE compound prevented ITAFs from translocating to the ER and decreased translation of client mRNAs. These data suggest that ITAFs impact the pathophysiology of breast cancer, and may represent novel therapeutic or diagnostic targets. Citation Format: Jessica Bockhorn, Toby M. Ward, J. Chuck Harrell, Xiaofei Liu, Gaby Fuchs, Mark D. Pegram. Investigation of ITAF's role in the pathophysiology of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2018.