Factors influencing ER subtype-mediated cell proliferation and apoptosis

&nbsp; The aim of the current thesis is to elucidate the role of estrogen receptor (ER)&alpha;and ER&beta;in cell proliferation and apoptosis induced by estrogenic compounds. Special attention is paid to the importance of the receptor preference of the estrogenic compounds, the cellular ER&alpha;/ER&beta;ratio, the role of coregulators, and ER-mediated induction of protein expression. In <strong>chapter 1 </strong>estrogenic compounds and their interaction with estrogen receptors are described and the two different estrogen receptors, ER&alpha;and ER&beta;, are introduced. It is described how estrogenic compounds eventually exert biological effects through coregulator recruitment upon ER binding, transcription initiation, and protein expression. <strong>Chapter 2 </strong>describes under which conditions T47D-ER&beta;breast cancer cells with tetracycline-dependent ER&beta;expression and constant ER&alpha;expression best mimic ER&alpha;/ER&beta;ratios in breast and other estrogen-sensitive tissues in vivo in rat as well as in human. At protein and mRNA level, ER&alpha;and ER&beta;levels and ratios are determined in T47D-ER&beta;cells exposed to a range of tetracycline concentrations and in rat and human breast, prostate, and uterus or endometrium. The ER&alpha;/ER&beta;ratio in rat mammary gland and in human breast tissue can be mimicked by exposing the T47D-ER&beta;cells to &gt;150 ng/ml tetracycline, but the ER&alpha;/ER&beta;ratio of other estrogen-sensitive rat and human tissues can also be mimicked. The ER&alpha;/ER&beta;ratios in MCF-7 and native T47D cells are high due to a lack of ER&beta;expression and therefore do not reflect ratios in rat and human tissues. It is demonstrated how these different tissues might vary in their proliferative response towards 17&beta;-estradiol (E2) by exposing T47D-ER&beta;cells to E2 under defined tetracycline concentrations. In <strong>chapter 3 </strong>the modulation of the interaction of ER&alpha;and ER&beta;with coregulators in the ligand-dependent responses induced by estrogenic compounds is investigated. To this end, selective ER&alpha;and ER&beta;agonists are characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ER&alpha;and ER&beta;in ER-selective reporter gene assays, and subsequently tested for stimulation of cell proliferation in T47D-ER&beta;cells with variable ER&alpha;/ER&beta;ratio and for ligand-dependent modulation of the interaction of ER&alpha;and ER&beta;with coregulators using the Microarray Assay for Real-time Coregulator &ndash; Nuclear receptor Interaction (MARCoNI) with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ER&alpha;/ER&beta;ratio and receptor selectivity of the compounds on stimulation of cell proliferation. ER&alpha;agonists activate cell proliferation whereas ER&beta;seems to suppress ER&alpha;-mediated cell proliferation. The responses in the MARCoNI assay reveal that the modulation of the interaction of ER&alpha;or ER&beta;with coregulators by a specific agonist are very similar indicating only a limited number of differences upon ER&alpha;or ER&beta;activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists are more substantial and can be used to classify the different agonists by hierarchical clustering. The results obtained corroborate that the ultimate effect of the model compounds on proliferation of estrogen-responsive cells depends on the intrinsic relative potency of the agonist towards ER&alpha;and ER&beta;and the cellular ER&alpha;/ER&beta;ratio whereas differences in the modulation of the interaction of the different ERs with coregulators for a given ligand might also contribute to the compound-specific pharmacology. Based on ligand-dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay is able to classify the different ER&alpha;and ER&beta;agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER-selective reporter gene or proliferation assays. It is concluded that differences in the modulation of the interaction of ER&alpha;and ER&beta;with coregulators contribute to the ligand-dependent responses but do not fully explain the differences in pharmacology between ER-mediated responses by the different estrogenic compounds. To investigate if this is also the case for ER antagonists, <strong>chapter 4 </strong>handles the modulation of the interaction of ER&alpha;and ER&beta;with coregulators in the ligand-dependent responses induced by the ER antagonistic compounds 4-hydroxytamoxifen (4OHT) and fulvestrant. Comparison of these results to ligand-dependent interaction of ER&alpha;and ER&beta;with coregulators expressed in modulation index (MI) profiles for the ER agonist E2 elucidates whether differences in the (ant)agonist-dependent interaction of ER&alpha;and ER&beta;with coregulators contribute to the differences in (ant)agonist responses. To this end, the selected ER antagonistic compounds are first characterized for intrinsic relative potency reflected by IC50 and maximal efficacy towards ER&alpha;and ER&beta;using ER-selective U2OS reporter gene assays, and subsequently tested for ligand-dependent modulation of the interaction of ER&alpha;and ER&beta;with coregulators using the MARCoNI assay. Results obtained with the U2OS reporter gene assays indicate a preference of 4OHT to bind ER&beta;and find fulvestrant to be less ER-specific. The responses in the MARCoNI assay reveal that ER&alpha;- and ER&beta;-mediated interaction with coregulators expressed in MI profiles are similar for 4OHT and fulvestrant and generally opposite to the MI profile of the ER agonist E2. Hierarchical clustering with Euclidian distance as the cluster distance metric, based on the MI profiles, is able to clearly discriminate the two compounds with ER antagonistic properties from the ER agonist E2. Taken together the data reveal that modulation of the interaction of ERs with coregulators discriminates ER agonists from antagonists but does not discriminate between the preferential ER&beta;antagonistic compound 4OHT and the less specific ER antagonist fulvestrant. It is concluded that differences in modulation of the interaction of ER&alpha;and ER&beta;with coregulators contribute to the differences in ligand-dependent responses induced by ER agonists and ER antagonists, but the importance of the subtle differences in modulation of the interaction of ERs with coregulators between the ER antagonistic compounds 4OHT and fulvestrant for the ultimate biological effect remains to be established. To further investigate whether 4OHT, the active metabolite of the breast cancer drug tamoxifen, exerts ER&alpha;/ER&beta;ratio-dependent effects on cell proliferation and apoptosis, in <strong>chapter 5 </strong>the results of a quantitative proteomics study are described. This is of importance given that the ER&alpha;/ER&beta;ratio usually increases in tumorous tissue compared to normal tissue due to decreased ER&beta;expression. ER&alpha;/ER&beta;ratio-dependent effects of 4OHT on cell proliferation and apoptosis of the T47D-ER&beta;human breast cancer cell line with tetracycline-dependent ER&beta;expression are detected. In the cells expressing only ER&alpha;decreased cell proliferation and increased apoptosis is induced by 4OHT, which is opposite to the effects detected in cells expressing ER&alpha;and ER&beta;, where increased cell proliferation and decreased apoptosis upon 4OHT exposure is found. Post-translational modifications like acetylation, methylation, and phosphorylation of several ribosomal and mitochondrial protein groups are induced by 4OHT, mostly in T47D-ER&beta;cells with both ER&alpha;and ER&beta;expressed. Altogether the results suggest that effects of 4OHT on major biological functions like cell proliferation and apoptosis in the T47D-ER&beta;cells are affected by the ER&alpha;/ER&beta;ratio. 4OHT may have differential cellular effects, being more effective in reducing cell proliferation and increasing apoptosis if ER&alpha;dominates and ER&beta;expression levels are low since 4OHT then antagonizes ER&alpha;. <strong>Chapter 6 </strong>presents a discussion on the implications of the mechanisms of action of several estrogenic compounds discussed in this thesis. Altogether the results of the present thesis have elucidated the action of different estrogenic compounds, their interaction with the two ER subtypes, and the subsequent recruitment or rejection of coregulators, as well as the resulting effects on cell proliferation and apoptosis, and these results emphasize the importance of the ER&alpha;/ER&beta;ratio for the ultimate effects of estrogenic compounds on cell proliferation and apoptosis