Eukaryotic translation initiation factor 4E (eIF4E) selectively promotes translation of mRNAs with atypically long and structured 5′-UTRs and has been implicated in drug resistance. Through genome-wide transcriptome and translatome analysis we revealed eIF4E overexpression could promote cellular activities mediated by ERα and FOXM1 signalling pathways. Whilst eIF4E overexpression could enhance the translation of both ERα and FOXM1, it also led to enhanced transcription of FOXM1. Polysome fractionation experiments confirmed eIF4E could modulate the translation of ERα and FOXM1 mRNA. The enhancement of FOXM1 transcription was contingent upon the presence of ERα, and it was the high levels of FOXM1 that conferred Tamoxifen resistance. Furthermore, tamoxifen resistance was conferred by phosphorylation independent eIF4E overexpression. Immunohistochemistry on 134 estrogen receptor (ER + ) primary breast cancer samples confirmed that high eIF4E expression was significantly associated with increased ERα and FOXM1, and significantly associated with tamoxifen resistance. Our study uncovers a novel mechanism whereby phosphorylation independent eIF4E translational reprogramming in governing the protein synthesis of ERα and FOXM1 contributes to anti-estrogen insensitivity in ER + breast cancer. In eIF4E overexpressing breast cancer, the increased ERα protein expression in turn enhances FOXM1 transcription, which together with its increased translation regulated by eIF4E, contributes to tamoxifen resistance. Coupled with eIF4E translational regulation, our study highlights an important mechanism conferring tamoxifen resistance via both ERα dependent and independent pathways.

Breast cancer is one of the prevalent causes of cancer in women. Two thirds of breast cancer patients are ER-positive and can be benefited from tamoxifen treatment. However, 50% of the patients will eventually develop the resistance. Identifying molecular targets associated with tamoxifen resistance would help in designing better therapeutic strategies. Previous study from our group identify a novel factor BQ323636.1 (BQ), a spliced variant of NCOR2, could confer tamoxifen resistance. From our in vivo study, nuclear enrichment of BQ in primary breast tumor was found to be associated with tamoxifen resistance. However, the molecular mechanism governed the nuclear import of BQ and was not clear. From bioinformatics prediction, we identified a functional nuclear localization signal (NLS) on BQ. We therefore performed a protein screening to search for the import receptor which could interact with BQ. KPNA1 was found to interact with BQ. To further validate such an interaction, we fused the NLS to GPF reporter protein (GFP-NLS) and performed a co-immunoprecipitation. The result showed that the GFP-NLS was able to interact with KPNA1. Knockdown of KPNA1 by RNAi could prevent the nuclear import of BQ. This confirmed the functional role of KPNA1 in the nuclear import of BQ. In addition, knockdown of KPNA1 in BQ overexpressing breast cancer cells could reverse the tamoxifen resistance. The results highlighted the functional significance of KPNA1 in the tamoxifen resistance. Subsequently, we investigated the functional role of BQ in the nucleus. We found BQ could deplete the gene repressive function of NCOR2. We found that NCOR2 could interact with HSF4 and should repress the expression of HIF1α. In the presence of BQ, the repressive function of NCOR2 on HIF1α was compromised, leading to up-regulation of HIF1α. To further validate the effect of HIF1α in the tamoxifen resistance, HIF1α inhibitor was employed and found that the treatment of the inhibitor could reduce the resistance in BQ overexpressing breast cancer cells. Therefore, our study uncovered the role of KPNA1 in the development of tamoxifen resistance through which KPNA1 could promote the nuclear import of BQ. Subsequently, BQ could lead to the up-regulation of HIF1α which also play significant role in the development of tamoxifen resistance. Citation Format: Ka Chun Mok, Ho Tsoi, Ui-Soon Khoo, Ellen Man. The molecular mechanism for producing BQ323636.1 in Tamoxifen resistance breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5903.

Breast cancer is one of the prevalent causes of cancer in women and its occurrence has been rising in the last few decades. Two thirds of breast cancer patients are ER-positive and can receive tamoxifen (TAM) treatment. Unfortunately, many of the patients eventually develop resistance to tamoxifen. Identifying a molecular marker associated with tamoxifen resistance would help in designing better therapeutic strategies to overcome this. In eukaryotes, translation initiation is the most regulated step. Abnormal translation machinery underlies a variety of human diseases including cancers. Eukaryotic translation initiation factor 4E (eIF4E) is the least abundant initiation factor and is believed to be the rate limiting step for translation initiation. Overexpression of eIF4E in cells selectively promotes translation of mRNAs involved in cell proliferation, apoptosis and tumor progression. Recently, eIF4E overexpression has been implicated in drug resistance in melanoma1. We hypothesized that eIF4E may also be involved in tamoxifen resistance. The aim of this study was to characterize the role of eIF4E in tamoxifen resistance and identify novel molecular targets that may be translationally regulated. Immunohistochemistry (IHC) staining of eIF4E showed up-regulation in human breast cancer samples compared with non-tumor counterparts. In silico analysis predicted that ERá and FOXM1 have a long and highly structured 5’-UTR, which are sensitive to the expression level of eIF4E. Polysomal fractionation confirmed that ERá and FOXM1 mRNA were more actively translated in MCF7 than in MCF10A. The correlation between eIF4E and ERá/FOXM1 were further confirmed in tissue microarray (TMA). The role of eIF4E in conferring TAM resistance was studied by MTT assays. Overexpression of eIF4E induced TAM resistance while knockdown eIF4E sensitized cells to TAM. Additionally, overexpression of eIF4E upregulated ERá/FOXM1 expression at protein level while knockdown eIF4E downregulated their expression at protein level. Our results show that eIF4E induced TAM resistance possibly through translational regulation of ERá/FOXM1. eIF4E is a promising biomarker for anti-estrogen drug responsiveness and a future therapeutic target for treating drug resistant breast cancer patients. Reference: 1. Zhan Y, et al. The role of eIF4E in response and acquired resistance to vemurafenib in melanoma J Invest Dermatol. 2015 May;135(5):1368-76. (2178 characters without spaces) Citation Format: Chun Gong, Ka Chun Mok, Yuen-Nei CHEUNG, Pui-Sum Man, EWF Lam, Ui-Soon Khoo. Translational regulation by eIF4E and its contribution to tamoxifen resistance in 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 4761.