Intracellular Retention of Estradiol is Augmented by GRAM Domain Containing Protein ASTER-B in Breast Cancer Cells.

Estrogens are naturally occurring steroid hormones that also act as the primary mitogens for estrogen receptor-positive (ER+) breast cancers. While elevated blood levels of estrogens have been associated with poor prognosis, the relationship between circulating hormone levels in the blood are related to intracellular hormone concentrations. Here, we observed that MCF-7 cells acutely treated with 17ß-estradiol (E2) retain a substantial amount of the hormone even upon removal of the hormone from the culture medium. Moreover, global patterns of E2-dependent gene expression are sustained for hours after acute E2 treatment and hormone removal. While circulating E2 is sequestered by sex hormone binding globulin (SHBG), the mechanisms of intracellular E2 retention are poorly understood. We found that a mislocalized GRAM-domain containing protein ASTER-B in the nucleus, which is observed in a subset of patients, is associated with higher cellular E2 retention. Accumulation and retention of hormone are related to the steroidal properties of E2. Finally, we observed that nuclear ASTER-B-mediated E2 retention is required for sustained hormone-induced ERα chromatin occupancy at enhancers and gene expression, as well as subsequent cell growth responses. Our results add intracellular hormone retention as a mechanism controlling E2-dependent gene expression and downstream biological outcomes. S ignificance: This study advances our understanding of how estradiol can be accumulated and retained intracellularly to drive a pro-proliferative gene expression program in ER+ breast cancer cells. Mechanistically, intracellular E2 retention is mediated in part by mislocalized, nuclear ASTER-B, which is aberrantly localized to the nuclei of cancer cells in some breast cancer patients.

A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells.

Mono(ADP-ribosyl)ation (MARylation), a post-translational modification (PTM) of proteins, is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and an integral component of the ribosome, is MARylated by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. We mapped and confirmed the sites of MARylation, which occur on three acidic residues within blades 4 and 5 of ß-propeller domain of RACK1, a chaperone that shuttles and anchors proteins where needed. Site-specific MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 to stress granules with key components, such as G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1 to dissociate the stress granules and return RACK1 and the 40S ribosomal subunit to the cytoplasm, allowing for a restoration of translation. Collectively, our results highlight the discovery of a PARP14/TARG1-regulated RACK1 MARylation cycle that controls stress granule assembly and disassembly in ovarian cancer cells.