Iinteractions between estrogen receptor alpha activities and the ubiquitin-based signalling network

17?-estradiol (E2) controls a plethora of physiological processes but plays also a critical role in breast cancer progression, thus the fine comprehension of the mechanisms that control E2-induced cell proliferation would help to identify new putative druggable targets for the treatment of breast cancer. The ubiquitin (Ub)- system is gaining much attention for cancer therapies because it allows to build a complex interactions network that is critical for signal transduction to many cellular processes. The Ub-based network depends on non-covalent binding between ubiquitinated proteins and proteins that possess an ubiquitin binding domain (UBD). Interestingly, recent papers reported that the Ub-system deeply impacts the E2 signalling by modulating the estrogen receptor (ER) ? sub-type, which is the principal mediator of the E2 mitogenic effects. In particular, while ER? polyubiquitination controls the receptor turnover and transcriptional activity, ER? monoubiquitination is required for the E2-dependent activation of rapid signalling to cell proliferation. Even if it has not been clarified how the Ub modification on ER? modulates the receptor activities, one attractive possibility is that ER? could recognize and transduce the Ub modification on itself or on interacting proteins through an UBD. Thus, the main goal of the present PhD project was to understand the noncovalent Ub-binding abilities of ER? and their regulatory role(s) in E2-dependent cellular processes. To this purpose, initial experiments performed in vitro showed that ER? has two different Ub-binding surfaces (UBSs): in A/B and E domains. By focusing on the E domain Ub-binding ability we identified the structural determinants required for ER? to non-covalently associate to Ub (i.e., L429, A430) in vitro and in cell lines. Next, we analyzed the regulatory role of the ER?-UBS in E2-dependent cellular processes. In particular, we found that even if the L429A,A430G (LAAG) mutation did not alter ER? ability to bind E2, cells expressing the mutant ER? did not proliferate after E2 treatment. Because the E2-dependent cell proliferation depends on the activation of extra-nuclear signalling kinases, we evaluated the activating phosphorylation of some signalling pathways activated by E2. Our results indicate that the ER?-UBS mutation impairs the E2-induced activation of the PI3K/AKT as well as the E2-induced PI3K/AKT-dependent ER? Ser118 phosphorylation. The finding that this residue is not phosphorylated in LAAG ER? cells after E2 treatment further suggested an impairment of the ER?-UBS mutant ability to mediate E2 target genes expression. DNA Micro Arrays experiments definitively demonstrated that the LAAG mutant ER? was less transcriptionally active than the wt receptor. Interestingly, the Ingenuity Pathway Analysis helped us identifying CREB1 as another transcription factor activated by E2 through wt but not throught LAAG ER?. We found that E2 triggered CREB1 transcriptional activation through the PI3K/AKT activation and the mutations of the ER?-UBS impair this pathway. Thus, it is possible that an ER?-UBSdependent membrane complex, responsible for the E2-triggered PI3K/AKT signalling activation, controls cell proliferation through the regulation of ER? and CREB1 activation required for gene transcription. Overall, the data reported in this PhD project indicate that the ER? possesses an UBS on its E domain that plays a critical role for E2-induced nuclear and extranuclear signalling to cell proliferation. In conclusion, our findings open new avenues in the field of E2-activated molecular mechanisms to physiological effects that now have to include also the noncovalent Ub-binding abilities of ER?. Given the key role played by ER? in breast cancer progression, the comprehension of the regulatory role of the ER?-UBS on E2 mitogenic effects reveals new putative druggable target. In this respect, the interference of the UBD:Ub interaction by using specific small molecules has been already proposed as a future pharmacological target against cancer.