Abstract P6-04-10: Components of cap-independent translation as novel targets in breast cancer

Abstract

Abstract Background: The majority of cellular mRNAs are translated in a cap-dependent manner, involving ribosome assembly at 5’ 7-methylguanylate cap structures followed by scanning and initiation at a favorable AUG codon. Alternatively, mRNA structural elements termed internal ribosome entry sites (IRESs) facilitate translation in a cap-independent manner, and have been identified in mRNAs encoding many canonical cellular oncogenes, including c-MYC, c-SRC, XIAP, Aurora-A kinase, and IGF1R. IRES trans-activating factors (ITAFs) are accessory proteins that augment or inhibit translation mediated by specific IRES sequences, via binding to IRES structures and/ or other translational machinery. In addition, conversion of ribosomal RNA (rRNA) uracil bases to pseudouracil enhances IRES-mediated translation. Methods: Analysis of publically available microarray datasets of breast cancer and normal breast tissue clinical specimens revealed that expression of several ITAFs is upregulated at the mRNA level in malignant tissues compared to normal controls. mRNAs encoding the ITAFs polypyrimidine tract binding protein 1 (PTBP1), splicing factor proline/ glutamine rich (SFPQ), poly-RC binding protein 2 (PCBP2), and interleukin enhancer binding factor 2 (ILF2, also NF45) were significantly upregulated (p < 0.05), as well as mRNA encoding the dyskeratosis congenita 1 (DKC1) rRNA-modifying enzyme. Independent analysis of an 855 human breast tumor dataset revealed that high mRNA expression of DKC1 and ILF2 are enriched in basal-like breast tumors compared to other intrinsic subtypes (t-test, p<0.01). Kaplan-Meier analyses revealed that high expression of DKC1 or ILF2 mRNAs were both significantly associated with decreased relapse-free survival duration (p = 0.0007 and p = 0.002, respectively). To investigate the biological impact of ITAF and DKC1 silencing, PTBP1, PCBP2, SFPQ, and DKC1 knockdown was accomplished using pGIPZ and pTRIPZ lentiviral shRNA vectors. Silencing of these genes and concomitant reduction in protein levels (measured by western blot) led to significant proliferation impairment in MDA-MB-231, MDA-MB-436, and MCF7 breast cancer cells in culture, using Alamar Blue assay. In addition, 4T1 mouse mammary carcinoma cells express DKC1, PTBP1 and SFPQ, and may serve as a useful model in immunocompetent mice. Conclusions and future directions: Future experiments will include investigation of protein-level expression in archival human specimens using immunohistochemistry, as well as investigation of the effects of ITAF/ DKC1 silencing in human xenografts using NOD/ SCID mice. Based on their increased expression in malignant breast tissues and activity in translation of oncoproteins, cellular ITAF genes and DKC1 gene may represent novel therapeutic targets for intervention. In particular, the increased expression of DKC1 enzyme and ILF2 in basal-like breast cancers implies a possible therapeutic opportunity in triple-negative breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-10.