Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells.

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

Correction: ß-Agonists Selectively Modulate Proinflammatory Gene Expression in Skeletal Muscle Cells via Non-Canonical Nuclear Crosstalk Mechanisms.

[This corrects the article DOI: 10.1371/journal.pone.0090649.].

Effect of combining glucocorticoids with Compound A on glucocorticoid receptor responsiveness in lymphoid malignancies.

Glucocorticoids (GCs) are a cornerstone in the treatment of lymphoid malignancies such as multiple myeloma (MM) and acute lymphoblastic leukemia (ALL). Yet, prolonged GC use is hampered by deleterious GC-related side effects and the emergence of GC resistance. To tackle and overcome these GC-related problems, the applicability of selective glucocorticoid receptor agonists and modulators was studied, in search of fewer side-effects and at least equal therapeutic efficacy as classic GCs. Compound A (CpdA) is a prototypical example of such a selective glucocorticoid receptor modulator and does not support GR-mediated transactivation. Here, we examined whether the combination of CpdA with the classic GC dexamethasone (Dex) may improve GC responsiveness of MM and ALL cell lines. We find that the combination of Dex and CpdA does not substantially enhance GC-mediated cell killing. In line, several apoptosis hallmarks, such as caspase 3/7 activity, PARP cleavage and the levels of cleaved-caspase 3 remain unchanged upon combining Dex with CpdA. Moreover, we monitor no additional inhibition of cell proliferation and the homologous downregulation of GR is not counteracted by the combination of Dex and CpdA. In addition, CpdA is unable to modulate Dex-liganded GR transactivation and transrepression, yet, Dex-mediated transrepression is also aberrant in these lymphoid cell lines. Together, transrepression-favoring compounds, alone or combined with GCs, do not seem a valid strategy in the treatment of lymphoid malignancies.

Coregulator profiling of the glucocorticoid receptor in lymphoid malignancies.

Coregulators cooperate with nuclear receptors, such as the glucocorticoid receptor (GR), to enhance or repress transcription. These regulatory proteins are implicated in cancer, yet, their role in lymphoid malignancies, including multiple myeloma (MM) and acute lymphoblastic leukemia (ALL), is largely unknown. Here, we report the use and extension of the microarray assay for real-time nuclear receptor coregulator interactions (MARCoNI) technology to detect coregulator associations with endogenous GR in cell lysates. We use MARCoNI to determine the GR coregulator profile of glucocorticoid-sensitive (MM and ALL) and glucocorticoid-resistant (ALL) cells, and identify common and unique coregulators for different cell line comparisons. Overall, we identify SRC-1/2/3, PGC-1α, RIP140 and DAX-1 as the strongest interacting coregulators of GR in MM and ALL cells and show that the interaction strength does not correlate with GR protein levels. Lastly, as a step towards patient samples, we determine the GR coregulator profile of peripheral blood mononuclear cells. We profile the interactions between GR and coregulators in MM and ALL cells and suggest to further explore the GR coregulator profile in hematological patient samples.

ß-agonists selectively modulate proinflammatory gene expression in skeletal muscle cells via non-canonical nuclear crosstalk mechanisms.

The proinflammatory cytokine Tumour Necrosis Factor (TNF)-α is implicated in a variety of skeletal muscle pathologies. Here, we have investigated how in vitro cotreatment of skeletal muscle C2C12 cells with ß-agonists modulates the TNF-α-induced inflammatory program. We observed that C2C12 myotubes express functional TNF receptor 1 (TNF-R1) and ß2-adrenoreceptors (ß2-ARs). TNF-α activated the canonical Nuclear Factor-κB (NF-κB) pathway and Mitogen-Activated Protein Kinases (MAPKs), culminating in potent induction of NF-κB-dependent proinflammatory genes. Cotreatment with the ß-agonist isoproterenol potentiated the expression of inflammatory mediators, including Interleukin-6 (IL-6) and several chemokines. The enhanced production of chemotactic factors upon TNF-α/isoproterenol cotreatment was also suggested by the results from migrational analysis. Whereas we could not explain our observations by cytoplasmic crosstalk, we found that TNF-R1-and ß2-AR-induced signalling cascades cooperate in the nucleus. Using the IL-6 promoter as a model, we demonstrated that TNF-α/isoproterenol cotreatment provoked phosphorylation of histone H3 at serine 10, concomitant with enhanced promoter accessibility and recruitment of the NF-κB p65 subunit, cAMP-response element-binding protein (CREB), CREB-binding protein (CBP) and RNA polymerase II. In summary, we show that ß-agonists potentiate TNF-α action, via nuclear crosstalk, that promotes chromatin relaxation at selected gene promoters. Our data warrant further study into the mode of action of ß-agonists and urge for caution in their use as therapeutic agents for muscular disorders.

Hunting for serine 276-phosphorylated p65.

The transcription factor nuclear factor kappaB (NF-kappaB) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF-kappaB, regulating its transactivation ability, have been described. Especially phosphorylation of the NF-kappaB subunit p65 has been investigated in depth and several commercial phosphospecific antibodies, targeting selected p65 residues, are available. One of the p65 residues, that is subject to phosphorylation by protein kinase A (PKA) as well as by mitogen-stimulated kinase-1 (MSK-1), is the serine at position 276. Here, we have performed a detailed analysis of the performance of the most commonly used commercial anti-P-p65 Ser276 antibodies. Our findings indicate that at least three widely used anti-P-p65 Ser276 antibodies do not detect p65 in vivo via Western Blot, but instead crossreact with PKA-regulated proteins. As PKA is one of the main kinases responsible for phosphorylation of p65 at Ser276, this observation warrants cautious interpretation of data generated using the tested antibodies.

Hyperactivated NF-{kappa}B and AP-1 transcription factors promote highly accessible chromatin and constitutive transcription across the interleukin-6 gene promoter in metastatic breast cancer cells.

Interleukin-6 (IL-6), involved in cancer-related inflammation, acts as an autocrine and paracrine growth factor, which promotes angiogenesis, metastasis, and subversion of immunity, and changes the response to hormones and to chemotherapeutics. We explored transcription mechanisms involved in differential IL-6 gene expression in breast cancer cells with different metastatic properties. In weakly metastatic MCF7 cells, histone H3 K9 methylation, HP1 binding, and weak recruitment of AP-1 Fra-1/c-Jun, NF-kappaB p65 transcription factors, and coactivators is indicative of low chromatin accessibility and gene transcription at the IL-6 gene promoter. In highly metastatic MDA-MB231 cells, strong DNase, MNase, and restriction enzyme accessibility, as well potent constitutive transcription of the IL-6 gene promoter, coincide with increased H3 S10 K14 phosphoacetylation and promoter enrichment of AP-1 Fra-1/c-Jun and NF-kappaB p65 transcription factors and MSK1, CBP/p300, Brg1, and Ezh2 cofactors. Complementation, silencing, and kinase inhibitor experiments further demonstrate involvement of AP-1 Fra-1/c-Jun and NF-kappaB p65/RelB members, but not of the alpha estrogen receptor in promoting chromatin accessibility and transcription across the IL-6 gene promoter in metastatic breast cancer cells. Finally, the natural withanolide Withaferin A was found to repress IL-6 gene transcription in metastatic breast cancer cells upon dual inhibition of NF-kappaB and AP-1 Fra-1 transcription factors and silencing of IL-6 promoter chromatin accessibility.