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

Abstract

17β-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) α sub-type, which is the principal mediator of the E2 mitogenic effects. In particular, while ERα polyubiquitination controls the receptor turnover and transcriptional activity, ERα 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 ERα modulates the receptor activities, one attractive possibility is that ERα 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 ERα and their regulatory role(s) in E2-dependent cellular processes. To this purpose, initial experiments performed in vitro showed that ERα 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 ERα to non-covalently associate to Ub (i.e., L429, A430) in vitro and in cell lines. Next, we analyzed the regulatory role of the ERα-UBS in E2-dependent cellular processes. In particular, we found that even if the L429A,A430G (LAAG) mutation did not alter ERα ability to bind E2, cells expressing the mutant ERα 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 ERα-UBS mutation impairs the E2-induced activation of the PI3K/AKT as well as the E2-induced PI3K/AKT-dependent ERα Ser118 phosphorylation. The finding that this residue is not phosphorylated in LAAG ERα cells after E2 treatment further suggested an impairment of the ERα-UBS mutant ability to mediate E2 target genes expression. DNA Micro Arrays experiments definitively demonstrated that the LAAG mutant ERα 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 ERα. We found that E2 triggered CREB1 transcriptional activation through the PI3K/AKT activation and the mutations of the ERα-UBS impair this pathway. Thus, it is possible that an ERα-UBSdependent membrane complex, responsible for the E2-triggered PI3K/AKT signalling activation, controls cell proliferation through the regulation of ERα and CREB1 activation required for gene transcription. Overall, the data reported in this PhD project indicate that the ERα 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 ERα. Given the key role played by ERα in breast cancer progression, the comprehension of the regulatory role of the ERα-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.