Glucocorticoid Receptor Regulates and Interacts with LEDGF/p75 to Promote Docetaxel Resistance in Prostate Cancer Cells.

Patients with advanced prostate cancer (PCa) invariably develop resistance to anti-androgen therapy and taxane-based chemotherapy. Glucocorticoid receptor (GR) has been implicated in PCa therapy resistance; however, the mechanisms underlying GR-mediated chemoresistance remain unclear. Lens epithelium-derived growth factor p75 (LEDGF/p75, also known as PSIP1 and DFS70) is a glucocorticoid-induced transcription co-activator implicated in cancer chemoresistance. We investigated the contribution of the GR-LEDGF/p75 axis to docetaxel (DTX)-resistance in PCa cells. GR silencing in DTX-sensitive and -resistant PCa cells decreased LEDGF/p75 expression, and GR upregulation in enzalutamide-resistant cells correlated with increased LEDGF/p75 expression. ChIP-sequencing revealed GR binding sites in the LEDGF/p75 promoter. STRING protein-protein interaction analysis indicated that GR and LEDGF/p75 belong to the same transcriptional network, and immunochemical studies demonstrated their co-immunoprecipitation and co-localization in DTX-resistant cells. The GR modulators exicorilant and relacorilant increased the sensitivity of chemoresistant PCa cells to DTX-induced cell death, and this effect was more pronounced upon LEDGF/p75 silencing. RNA-sequencing of DTX-resistant cells with GR or LEDGF/p75 knockdown revealed a transcriptomic overlap targeting signaling pathways associated with cell survival and proliferation, cancer, and therapy resistance. These studies implicate the GR-LEDGF/p75 axis in PCa therapy resistance and provide a pre-clinical rationale for developing novel therapeutic strategies for advanced PCa.

Glucocorticoid Receptor and ß-Catenin Interact in Prostate Cancer Cells and Their Co-Inhibition Attenuates Tumorsphere Formation, Stemness, and Docetaxel Resistance.

Therapy resistance hinders the efficacy of anti-androgen therapies and taxane-based chemotherapy for advanced prostate cancer (PCa). Glucocorticoid receptor (GR) signaling mediates resistance to androgen receptor signaling inhibitors (ARSI) and has also been recently implicated in PCa resistance to docetaxel (DTX), suggesting a role in therapy cross-resistance. Like GR, ß-catenin is upregulated in metastatic and therapy-resistant tumors and is a crucial regulator of cancer stemness and ARSI resistance. ß-catenin interacts with AR to promote PCa progression. Given the structural and functional similarities between AR and GR, we hypothesized that ß-catenin also interacts with GR to influence PCa stemness and chemoresistance. As expected, we observed that treatment with the glucocorticoid dexamethasone promotednuclear accumulation of GR and active ß-catenin in PCa cells. Co-immunoprecipitation studies showed that GR and ß-catenin interact in DTX-resistant and DTX-sensitive PCa cells. Pharmacological co-inhibition of GR and ß-catenin, using the GR modulator CORT-108297 and the selective ß-catenin inhibitor MSAB, enhanced cytotoxicity in DTX-resistant PCa cells grown in adherent and spheroid cultures and decreased CD44+/CD24- cell populations in tumorspheres. These results indicate that GR and ß-catenin influence cell survival, stemness, and tumorsphere formation in DTX-resistant cells. Their co-inhibition could be a promising therapeutic strategy to overcome PCa therapy cross-resistance.

The LEDGF/p75 Integrase Binding Domain Interactome Contributes to the Survival, Clonogenicity, and Tumorsphere Formation of Docetaxel-Resistant Prostate Cancer Cells.

Patients with prostate cancer (PCa) receiving docetaxel chemotherapy invariably develop chemoresistance. The transcription co-activator lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 and PSIP1, is upregulated in several human cancers, including PCa and promotes resistance to docetaxel and other drugs. The C-terminal region of LEDGF/p75 contains an integrase binding domain (IBD) that tethers nuclear proteins, including the HIV-1 integrase and transcription factors, to active chromatin to promote viral integration and transcription of cellular survival genes. Here, we investigated the contribution of the LEDGF/p75 IBD interactome to PCa chemoresistance. Quantitative immunoblotting revealed that LEDGF/p75 and its IBD-interacting partners are endogenously upregulated in docetaxel-resistant PCa cell lines compared to docetaxel-sensitive parental cells. Using specific human autoantibodies, we co-immunoprecipitated LEDGF/p75 with its endogenous IBD-interacting partners JPO2, menin, MLL, IWS1, ASK1, and PogZ, as well as transcription factors c-MYC and HRP2, in docetaxel-resistant cells, and confirmed their nuclear co-localization by confocal microscopy. Depletion of LEDGF/p75 and selected interacting partners robustly decreased the survival, clonogenicity, and tumorsphere formation capacity of docetaxel-resistant cells. These results implicate the LEDGF/p75 IBD interactome in PCa chemoresistance and could lead to novel therapeutic strategies targeting this protein complex for the treatment of docetaxel-resistant tumors.

Twenty years of research on the DFS70/LEDGF autoantibody-autoantigen system: many lessons learned but still many questions.

The discovery and initial characterization 20 years ago of antinuclear autoantibodies (ANAs) presenting a dense fine speckled (DFS) nuclear pattern with strong staining of mitotic chromosomes, detected by indirect immunofluorescence assay in HEp-2 cells (HEp-2 IIFA test), has transformed our view on ANAs. Traditionally, ANAs have been considered as reporters of abnormal immunological events associated with the onset and progression of systemic autoimmune rheumatic diseases (SARD), also called ANA-associated rheumatic diseases (AARD), as well as clinical biomarkers for the differential diagnosis of these diseases. However, based on our current knowledge, it is not apparent that autoantibodies presenting the DFS IIF pattern fall into these categories. These antibodies invariably target a chromatin-associated protein designated as dense fine speckled protein of 70 kD (DFS70), also known as lens epithelium-derived growth factor protein of 75 kD (LEDGF/p75) and PC4 and SFRS1 Interacting protein 1 (PSIP1). This multi-functional protein, hereafter referred to as DFS70/LEDGF, plays important roles in the formation of transcription complexes in active chromatin, transcriptional activation of specific genes, regulation of mRNA splicing, DNA repair, and cellular survival against stress. Due to its multiple functions, it has emerged as a key protein contributing to several human pathologies, including acquired immunodeficiency syndrome (AIDS), leukemia, cancer, ocular diseases, and Rett syndrome. Unlike other ANAs, "monospecific" anti-DFS70/LEDGF autoantibodies (only detectable ANA in serum) are not associated with SARD and have been detected in healthy individuals and some patients with non-SARD inflammatory conditions. These observations have led to the hypotheses that these antibodies could be considered as negative biomarkers of SARD and might even play a protective or beneficial role. In spite of 20 years of research on this autoantibody-autoantigen system, its biological and clinical significance still remains enigmatic. Here we review the current state of knowledge of this system, focusing on the lessons learned and posing emerging questions that await further scrutiny as we continue our quest to unravel its significance and potential clinical and therapeutic utility.

Psychosocial Stress, Glucocorticoid Signaling, and Prostate Cancer Health Disparities in African American Men.

Recent advances in our understanding of racial disparities in prostate cancer (PCa) incidence and mortality that disproportionately affect African American (AA) men have provided important insights into the psychosocial, socioeconomic, environmental, and molecular contributors. There is, however, limited mechanistic knowledge of how the interplay between these determinants influences prostate tumor aggressiveness in AA men and other men of African ancestry. Growing evidence indicates that chronic psychosocial stress in AA populations leads to sustained glucocorticoid signaling through the glucocorticoid receptor (GR), with negative physiological and pathological consequences. Compelling evidence indicates that treatment of castration-resistant prostate cancer (CRPC) with anti-androgen therapy activates GR signaling. This enhanced GR signaling bypasses androgen receptor (AR) signaling and transcriptionally activates both AR-target genes and GR-target genes, resulting in increased prostate tumor resistance to anti-androgen therapy, chemotherapy, and radiotherapy. Given its enhanced signaling in AA men, GR-together with specific genetic drivers-may promote CRPC progression and exacerbate tumor aggressiveness in this population, potentially contributing to PCa mortality disparities. Ongoing and future CRPC clinical trials that combine standard of care therapies with GR modulators should assess racial differences in therapy response and clinical outcomes in order to improve PCa health disparities that continue to exist for AA men.

Glucocorticoids Induce Stress Oncoproteins Associated with Therapy-Resistance in African American and European American Prostate Cancer Cells.

Glucocorticoid receptor (GR) is emerging as a key driver of prostate cancer (PCa) progression and therapy resistance in the absence of androgen receptor (AR) signaling. Acting as a bypass mechanism, GR activates AR-regulated genes, although GR-target genes contributing to PCa therapy resistance remain to be identified. Emerging evidence also shows that African American (AA) men, who disproportionately develop aggressive PCa, have hypersensitive GR signaling linked to cumulative stressful life events. Using racially diverse PCa cell lines (MDA-PCa-2b, 22Rv1, PC3, and DU145) we examined the effects of glucocorticoids on the expression of two stress oncoproteins associated with PCa therapy resistance, Clusterin (CLU) and Lens Epithelium-Derived Growth Factor p75 (LEDGF/p75). We observed that glucocorticoids upregulated LEDGF/p75 and CLU in PCa cells. Blockade of GR activation abolished this upregulation. We also detected increased GR transcript expression in AA PCa tissues, compared to European American (EA) tissues, using Oncomine microarray datasets. These results demonstrate that glucocorticoids upregulate the therapy resistance-associated oncoproteins LEDGF/p75 and CLU, and suggest that this effect may be enhanced in AA PCa. This study provides an initial framework for understanding the contribution of glucocorticoid signaling to PCa health disparities.

RNA sequencing reveals upregulation of a transcriptomic program associated with stemness in metastatic prostate cancer cells selected for taxane resistance.

Patients with metastatic castration-resistant prostate cancer (mCRPC) develop resistance to conventional therapies including docetaxel (DTX). Identifying molecular pathways underlying DTX resistance is critical for developing novel combinatorial therapies to prevent or reverse this resistance. To identify transcriptomic signatures associated with acquisition of chemoresistance we profiled gene expression in DTX-sensitive and -resistant mCRPC cells using RNA sequencing (RNA-seq). PC3 and DU145 cells were selected for DTX resistance and this phenotype was validated by immunoblotting using DTX resistance markers (e.g. clusterin, ABCB1/P-gp, and LEDGF/p75). Overlapping genes differentially regulated in the DTX-sensitive and -resistant cells were ranked by Gene Set Enrichment Analysis (GSEA) and validated to correlate transcript with protein expression. GSEA revealed that genes associated with cancer stem cells (CSC) (e.g., NES, TSPAN8, DPPP, DNAJC12, and MYC) were highly ranked and comprised 70% of the top 25 genes differentially upregulated in the DTX-resistant cells. Established markers of epithelial-to-mesenchymal transition (EMT) and CSCs were used to evaluate the stemness of adherent DTX-resistant cells (2D cultures) and tumorspheres (3D cultures). Increased formation and frequency of cells expressing CSC markers were detected in DTX-resistant cells. DU145-DR cells showed a 2-fold increase in tumorsphere formation and increased DTX resistance compared to DU145-DR 2D cultures. These results demonstrate the induction of a transcriptomic program associated with stemness in mCRPC cells selected for DTX resistance, and strengthen the emerging body of evidence implicating CSCs in this process. In addition, they provide additional candidate genes and molecular pathways for potential therapeutic targeting to overcome DTX resistance.