mRNA translation is induced by the IGF-I downstream mediator AIB1 in breast cancer cells

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 389-405-Signaling Originating from Membrane Receptors
Basic/Translational
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-391
Aleksandra M Ochnik*, Mark S Peterson, Svetlana V Avdulov and Douglas Yee
Masonic Cancer Center, University of Minnesota, Minneapolis, MN
Abstract:

The insulin-like growth factor (IGF-I) tyrosine kinase signalling axis activates multiple downstream effectors of the AKT- (PI3K) and Ras-pathways (mTOR, S6K1) to control mRNA translation (eIF4E-BP1) in breast cancer [1]. AIB1, a member of the nuclear p160 steroid receptor co-activator family is a downstream regulator of these pathways [2]. A “switch” mechanism to bypass cap-dependent translation (CDT) to promote cap-independent translation (CIT) occurs in aggressive disease [3]. Thus, we hypothesize that IGF signalling acts via AIB1 to regulate mRNA translation to promote breast tumor growth and metastasis. Estrogen receptor (ER) positive (MCF7L) and triple negative breast cancer (TNBC; ER, progesterone receptor, human epidermal growth factor receptor 2, negative, MDA-MB-231 (231), MDA-MB-435 (435) and LCC6 (a metastatic sub-line of the 435 cells) wild-type, stably expressing AIB1 and scrambled control short hairpin RNA (shAIB1 and shCON) cells were created. A bicistronic reporter expression construct (pCDNA3-rLuc-IRES-fLuc) that measures CDT and CIT was transfected into the cells to measure mRNA translation in addition with either empty vector (EV) or AIB1 expression plasmids. Post-transfection, cells were treated for 24h in serum free media plus and minus IGF-I (10nM) and/or kinase inhibitors: rapamycin (Rap; 10nM/mTOR); H89-dihydrochloride (H89; 10μM/s6K1); UO126 (UO; 10μM/MEK) and LY294002 (LY; 10nM/PI3K). Polyribosomal RNA was stratified using sucrose gradients and fractionated and qRT-PCR was performed. Target protein phosphorylation was measured by immunoblot. IGF-I increased CDT and CIT compared to vehicle control in EV and AIB1 transfected MCF7L cells (p<0.01). This effect was partially seen in 435 cells and only in AIB1 transfected 231 cells (p<0.05). mRNA translation increased in AIB1 compared to EV transfected cells and was reduced in shAIB1 vs. shCON 231 and LCC6 cells (p<0.01). IGF-I-induced CDT decreased in AIB1 transfected cells by the inhibitor co-treatments except in 231 cells where only LY was inhibitory (p<0.05). Similarly inhibitors decreased IGF-I-induced CIT in MCF7L cells yet only H89, LY and UO in 435 cells and UO in 231 cells (p<0.05). Reduced IGF-I-induced target protein phosphorylation by the inhibitor co-treatments was observed. Ribosome-bound AIB1, 4E-BP1 and E2F1 (a transcription factor) mRNA reduced (4.2, 2.1 and 1.9; fold change respectively) and total-RNA of BYSL (2-fold, a ribosome biogenesis activator) in 24h IGF-I treated shAIB1 vs. shCON LCC6 cells. These effects were matched with increased EIF4E and hypophosphorylated eIF4E-BP1 and reduced S6K1 protein. Thus, AIB1 enhanced IGF-I-induced translation and this mostly occurred via the AKT-pathway for CDT and Ras-pathway for CIT. Combinatorial therapies to target downstream mediators of IGF-signalling may be viable future therapies in AIB1-dependent luminal and TNBC.

1. Sachdev, D. and D. Yee. Mol Cancer Ther, 2007. 6(1): p. 1-12 2. Lahusen, T., et al., Breast Cancer Res Treat, 2009. 116(2): 225-37 3. Braunstein, S., et al., Mol Cell, 2007. 28(3): 501-12

Nothing to Disclose: AMO, MSP, SVA, DY

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Susan G. Komen for the Cure Susan G. Komen for the Cure