Response to mTOR inhibition: activity of eIF4E predicts sensitivity in cell lines and acquired changes in eIF4E regulation in breast cancer.

BACKGROUND: Inhibitors of the kinase mTOR, such as rapamycin and everolimus, have been used as cancer therapeutics with limited success since some tumours are resistant. Efforts to establish predictive markers to allow selection of patients with tumours likely to respond have centred on determining phosphorylation states of mTOR or its targets 4E-BP1 and S6K in cancer cells. In an alternative approach we estimated eIF4E activity, a key effector of mTOR function, and tested the hypothesis that eIF4E activity predicts sensitivity to mTOR inhibition in cell lines and in breast tumours. RESULTS: We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition. CONCLUSIONS: Estimates of eIF4E activity predict sensitivity to mTOR inhibition in cell lines but breast tumours with high estimated eIF4E activity gain changes in eIF4E regulation in order to enhance resistance.

Expression of oestrogen receptor beta isoforms is regulated by transcriptional and post-transcriptional mechanisms.

Although ERs (oestrogen receptors) mediate breast tumour behaviour, the precise role of ERbeta remains unclear. This is mainly because analyses have been complicated by the presence in breast tissue of three ERbeta protein variants (ERbeta1, ERbeta2 and ERbeta5) that derive from differential 3' splicing. We have recently identified the first known mechanisms responsible for the differential control of isoform expression, involving regulation of translation via 5'-UTRs (untranslated regions). In the present study, we have uncovered further complexity involving the influence of multiple promoters and cross-talk between 5'- and 3'-UTRs. We demonstrate that full-length ERbeta mRNAs are transcribed from three separate promoters; two promoters are well-established within the literature, whereas the third represents a novel finding. Each promoter produces transcripts with distinct 5'-UTRs. The differential 3' splicing that produces transcripts coding for the ERbeta isoforms also defines isoform-specific 3'-UTRs. We identified exact 3'-UTR sequences for each isoform, and have shown that alternative polyadenylation sites are used in a cell-type specific manner to produce transcripts with 3'-UTRs of different lengths. Critically, we show that 5'- and 3'-UTRs combine to specify the efficiencies with which individual transcripts are translated, with 3'-UTR length having a key influence. In addition, we demonstrate how 17beta-oestradiol, a key driver of breast cancer development, affects the regulation of ERbeta expression at both transcriptional and translational levels.