Differential Gene Expression Profiles between N-Terminal Domain and Ligand-Binding Domain Inhibitors of Androgen Receptor Reveal Ralaniten Induction of Metallothionein by a Mechanism Dependent on MTF1

Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein–protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy.

Restrained expression of canine glucocorticoid receptor splice variants α and P prognosticates fatal disease outcome in SIRS

Glucocorticoids play a central role in the inflammatory response and alleviate the symptoms in critically ill patients. The glucocorticoid action relies on the glucocorticoid receptor (GR) which translocates into the nucleus upon ligand-binding and regulates transcription of a battery of genes. Although the GR is encoded by a single gene, dozens of its splice variants have been described in diverse species. The GRα isoform encodes the full, functionally active protein that is composed of a transactivation, a DNA-binding, and a C-terminal ligand-binding domain. The second most highly expressed receptor variant, the GR-P, is formed by an intron retention that introduces an early stop codon and results in a probably dysfunctional protein with truncated ligand-binding domain. We described the canine ortholog of GR-P and showed that this splice variant is highly abundant in the peripheral blood of dogs. The level of cGRα and cGR-P transcripts are elevated in patients of SIRS and the survival rate is increased with elevated cGRα and cGR-P expression. The ratio of cGRα and cGR-P mRNA did not differ between the survivor and non-survivor patients; thus, the total GR expression is more pertinent than the relative expression of GR isoforms in assessment of the disease outcome.

Recurrent activating mutations in AHR act as drivers of urinary tract cancer

Bladder cancer is a common cancer with a high recurrence rate and with limited progress in treatment and survival of patients with advanced stages of the disease. The tumorigenesis of bladder cancer is still poorly understood, but identification of genetic contributors to its development may provide leads for targeted therapeutic approaches. By an in-depth analysis of whole-genome sequencing data of metastasized urinary tract cancer samples, we identified a novel recurrent in-frame deletion of exons 8 and 9 in the aryl hydrocarbon receptor (AHR) gene, encoding a ligand-activated transcription factor with context-specific physiological functions. The deletion (AHRΔe8-9) is highly specific to urinary tract cancer and occurs in 10.7% of metastatic lesions. Additionally, a recurrent AHR hotspot point mutation and AHR gene amplifications were identified indicating that alterations in the AHR gene are a key cancer driver event for urinary tract cancer adding up to a prevalence of ~19% (40 out of 206). The hotspot point mutation results in an amino acid change (AHRQ383H) in the ligand-binding domain of the protein and causes altered ligand affinities such as AHRQ383H activation upon incubation with the AhR antagonist CH-2233191. The in-frame deletion of exons 8 and 9 disrupt the ligand-binding domain and result in a constitutive nuclear localization of the protein and ligand-independent transcriptional activation. The constitutive activation of the AhR pathway by the hAHRΔe8-9 mutant in mouse bladder organoids induces dedifferentiation and enables anchorage independent growth. In conclusion, our results indicate that AhR is a key proto-oncogene and cancer driver gene in urinary tract cancer and emphasize the potential of the AhR pathway as a target for therapeutic interventions.

The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution and quantitative fluorescence microscopy in living cells. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work unveils likely pathophysiologically relevant quaternary assemblies of the nuclear receptor with important implications for glucocorticoid action and drug design.