Adrenal tumors provide insight into the role of cortisol in NK cell activity.

Elevated glucocorticoid (GC) activity may limit tumor immune response and immune checkpoint inhibitor (ICI) efficacy. Adrenocortical carcinoma (ACC) provides a unique test case to assess correlates of GC activity, as approximately half of ACC patients exhibit excess GC production (GC+). ACC multi-omics were analyzed to identify molecular consequences of GC+ and assess the rationale for combining the glucocorticoid receptor (GR) antagonist relacorilant with an ICI. GC status, mRNA expression, and DNA mutation and methylation data from 71 adrenal tumors were accessed via The Cancer Genome Atlas. Expression of 858 genes differed significantly between GC- and GC+ ACC cases. KEGG pathway analysis showed higher gene expression of three pathways involved in steroid synthesis and secretion in GC+ cases. Fifteen pathways, most related to NK cells and other immune activity, showed lower expression. Hypomethylation was primarily observed in the steroid synthesis pathways. Tumor-infiltrating CD4+ memory (P = 0.003), CD8+ memory (P < 0.001), and NKT-cells (P = 0.014) were depleted in GC+ cases; tumor-associated neutrophils were enriched (P < 0.001). Given the pronounced differences between GC+ and GC- ACC, the effects of cortisol on NK cells were assessed in vitro (NK cells from human PBMCs stimulated with IL-2 or IL-12/15). Cortisol suppressed, and relacorilant restored, NK cell activation, proliferation, and direct tumor cell killing. Thus, GR antagonism may increase the abundance and function of NK and other immune cells in the tumor microenvironment, promoting immune response in GC+ ACC and other malignancies with GC+. This hypothesis will be tested in a phase 1 trial of relacorilant + ICI.

RGD-dependent binding of TP508 to integrin alphavbeta3 mediates cell adhesion and induction of nitric oxide.

TP508, a 23-amino acid RGD-containing synthetic peptide representing residues 508 to 530 of human prothrombin, mitigates the effects of endothelial dysfunction in ischaemic reperfusion injury. The objective of this study was to investigate whether TP508 binds to members of the integrin family of transmembrane receptors leading to nitric oxide synthesis. Immobilised TP508 supported adhesion of endothelial cells and alphavbeta3-expressing human embryonic kidney cells in a dose- and RGD-dependent manner. Soluble TP508 also inhibited cell adhesion to immobilised fibrinogen. The involvement of alphavbeta3 was verified with function-blocking antibodies and surface plasmon resonance studies. Adhesion of the cells to immobilised TP508 resulted in an induction of phosphorylated FAK and ERK1/2. In endothelial cells, TP508 treatment resulted in an induction of nitric oxide that could be inhibited by LM609, an alphavbeta3-specific, function-blocking monoclonal antibody. Finally, TP508 treatment of isolated rat aorta segments enhanced carbachol-induced vasorelaxation. These results suggest that TP508 elicits a potentially therapeutic effect through an RGD-dependent interaction with integrin alphavbeta3.

The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASxalpha strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.

Agonist-activated glucocorticoid receptor inhibits binding of heat shock factor 1 to the heat shock protein 70 promoter in vivo.

We have previously shown that activation of glucocorticoid receptor (GR) signaling in stressed cells will cause inhibition of the heat shock response as mediated by heat shock transcription factor 1 (HSF1). In that work, a full-length human heat shock protein 70 (Hsp70) promoter was used to measure HSF1 transactivity, and the data suggested inhibition of HSF1 through the transactivation or transrepressive properties of GR. Here, we show that the inhibitory effect of glucocorticoid agonist (dexamethasone) upon Hsp70 promoter activity is rapid, occurring within 1 h of hormone addition. Moreover, addition of hormone during the first hour of recovery from stress was sufficient to inhibit HSF1. Thus, dexamethasone is able to rapidly reverse HSF1 transactivity, suggesting a transrepressive mode of action for GR. Yet, GR transrepression of HSF1 by analysis of putative negative glucocorticoid response elements in the Hsp70 promoter was not found. To further investigate the in vivo nature of this fast-acting mechanism, we used the chromatin immunoprecipitation assay with primers specific to the human Hsp70 promoter. Dexamethasone inhibited HSF1 binding at the Hsp70 promoter in response to heat or chemical shock (sodium arsenite). Moreover, dexamethasone also blocked promoter binding by a constitutively active mutant of HSF1 (hHSF1-E189) expressed under nonstress conditions. In all cases, inhibition of HSF1 recruitment to the promoter by dexamethasone was blocked by the GR antagonist RU486, a result that was consistent with promoter activity based on chloramphenicol acetyl transferase gene expression. The ability of dexamethasone to prevent HSF1 recruitment to the promoter was fast acting (occurring in as little as 15 min), and the hormone also caused release of HSF1 already bound to the promoter. Although these results suggest GR can effectively prevent HSF1 binding to Hsp promoters, fractionation and Western blot experiments showed that stress-activated HSF1 was not released from the nucleus in response to hormone. Thus, this effect of dexamethasone is either specific to the Hsp70 promoter or causes shunting of HSF1 to other high-affinity nuclear sites. These observations provide evidence of a novel mechanism for attenuation of the heat shock response by glucocorticoids: prevention or reversal of HSF1 recruitment to Hsp promoters through the rapid actions of GR.

Enhancement of glucocorticoid receptor-mediated gene expression by constitutively active heat shock factor 1.

To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stress-activated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE(2)E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE(2)E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the existence of HSF1-regulated genes that serve to elevate the response of steroid receptors under stress conditions.