Background:
Variations in estrogen concentration have been hypothesized to explain sex differences observed in HIV infection, clinical progression and reservoir size. Estrogen receptor alpha (ERα) is reported to directly influence HIV-latency and transcription. However, in contrast to the important role of ERα in breast cancer, the regulation of ERα in immune cells is less clear. Furthermore, in strategies to attack persistent HIV infection, the influence of latency reversal agents (LRAs) on ERα is unknown.
Methods:
To obtain a better understanding of ERα regulation in CD4+ T cells, we evaluated nuclear localization of ERα and ERα protein degradation by Fulvestrant, a selective estrogen receptor degrader (SERD), or by ARV-471, a novel, potent, first-in-human PROteolysis TArgeting Chimera (PROTAC), specific ERα-protein degrader. We next assessed the impact of different LRA classes (HDAC and BET inhibitors, PKC and STING agonist, SMAC mimetic) on ERα at both mRNA and protein levels by WB and qPCR. The breast cancer cell line T-47D was used as control and all experiments were performed in hormone-free media.
Results:
In contrast to T-47D breast cancer cells, in CD4+ T cells ERα is not primarily localized in the nucleus and β-estradiol (ER agonist) does not induce ERα nuclear translocation. Similarly, both Fulvestrant and ARV-471 do not induce degradation of ERα in CD4+ T cells. In both PBMC and CD4+ T cells, we observed a significant downregulation of ERα gene expression following 6-hour and/or 24-hour of treatment with all tested LRAs with downregulation with PMAi, PEP005 and romidepsin being the most pronounced (0.04- and 0.02-, 0.21-fold change; P < 0.01). ERα mRNA downregulation resulted in temporal decrease of ERα protein. Protein levels dropped significantly after 6-hour treatment with PMAi and after 24-hour treatment with PMAi, PEP005 and romidepsin (0.57-, 0.37-, 0.57- and 0.77-fold change; P < 0.01).
Conclusions:
Our results suggest that ERα regulation in immune cells is different than what is previously demonstrated in breast cancer. We show for the first time that in CD4+ T cells canonical nuclear translocation of ERα does not occur after exposure to estrogen and that SERD and ERα PROTAC are ineffective at ERα degradation. However, the impact of LRAs on ERα protein levels does not exclude a role of ERα in HIV latency. Collectively, these results suggest that estrogen impact on HIV transcription is unlikely due to canonical ERα nuclear mechanisms of action and merits further investigations.