Deletion of the glucocorticoid receptor chaperone FKBP51 prevents glucocorticoid-induced skin atrophy

FKBP51 (FK506-binding protein 51) is a known co-chaperone and regulator of the glucocorticoid receptor (GR), which usually attenuates its activity. FKBP51 is one of the major GR target genes in skin, but its role in clinical effects of glucocorticoids is not known. Here, we used FKBP51 knockout (KO) mice to determine FKBP51's role in the major adverse effect of topical glucocorticoids, skin atrophy. Unexpectedly, we found that all skin compartments (epidermis, dermis, dermal adipose and CD34+ stem cells) in FKBP51 KO animals were much more resistant to glucocorticoid-induced hypoplasia. Furthermore, despite the absence of inhibitory FKBP51, the basal level of expression and glucocorticoid activation of GR target genes were not increased in FKBP51 KO skin or CRISPR/Cas9-edited FKBP51 KO HaCaT human keratinocytes. FKBP51 is known to negatively regulate Akt and mTOR. We found a significant increase in AktSer473 and mTORSer2448 phosphorylation and downstream pro-growth signaling in FKBP51-deficient keratinocytes in vivo and in vitro. As Akt/mTOR-GR crosstalk is usually negative in skin, our results suggest that Akt/mTOR activation could be responsible for the lack of increased GR function and resistance of FKBP51 KO mice to the steroid-induced skin atrophy

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc

Androgen Receptor Drives Cellular Senescence

The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor

Asymmetric expression of connexins between luminal epithelial- and myoepithelial- cells is essential for contractile function of the mammary gland

AbstractIntercellular communication is essential for glandular functions and tissue homeostasis. Gap junctions couple cells homotypically and heterotypically and co-ordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and the distribution of some Cx subtypes in the heterotypic gap junctions is not symmetrical; in the murine mammary gland, Cx26, Cx30 and Cx32 are expressed only in the luminal epithelial cells and Cx43 is expressed only in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy and throughout lactation suggesting essential roles for these proteins in the functional secretory activity of the gland. Transgenic (Tg) mice over-expressing Cx26 driven by keratin 5 promoter had an unexpected mammary phenotype: the mothers were unable to feed their pups to weaning age leading to litter starvation and demise in early to mid-lactation. The mammary gland of K5-Cx26 female mice developed normally and produced normal levels of milk protein, suggesting a defect in delivery rather than milk production. Because the mammary gland of K5-Cx26 mothers contained excessive milk, we hypothesized that the defect may be in an inability to eject the milk. Using ex vivo three-dimensional mammary organoid cultures, we showed that tissues isolated from wild-type FVB females contracted upon treatment with oxytocin, whereas, organoids from Tg mice failed to do so. Unexpectedly, we found that ectopic expression of Cx26 in myoepithelial cells altered the expression of endogenous Cx43 resulting in impaired gap junction communication, demonstrated by defective dye coupling in mammary epithelial cells of Tg mice. Inhibition of gap junction communication or knock-down of Cx43 in organoids from wild-type mice impaired contraction in response to oxytocin, recapitulating the observations from the mammary glands of Tg mice. We conclude that Cx26 acts as a trans-dominant negative for Cx43 function in myoepithelial cells, highlighting the importance of cell type-specific expression of Cxs for optimal contractile function of the mammary myoepithelium

Discovery of Compound A : a selective activator of the glucocorticoid receptor with anti-inflammatory and anti-cancer activity

CITATION: Lesovaya, E. et al. 2015. Discovery of Compound A : a selective activator of the glucocorticoid receptor with anti-inflammatory and anti-cancer activity. Oncotarget, 6(1):30730-30744, doi:10.18632/oncotarget.5078.The original publication is available at www.impactjournals.com/oncotargetGlucocorticoids are among the most effective anti-inflammatory drugs, and are widely used for cancer therapy. Unfortunately, chronic treatment with glucocorticoids results in multiple side effects. Thus, there was an intensive search for selective glucocorticoid receptor (GR) activators (SEGRA), which retain therapeutic potential of glucocorticoids, but with fewer adverse effects. GR regulates gene expression by transactivation (TA), by binding as homodimer to gene promoters, or transrepression (TR), via diverse mechanisms including negative interaction between monomeric GR and other transcription factors. It is well accepted that metabolic and atrophogenic effects of glucocorticoids are mediated by GR TA. Here we summarized the results of extensive international collaboration that led to discovery and characterization of Compound A (CpdA), a unique SEGRA with a proven “dissociating” GR ligand profile, preventing GR dimerization and shifting GR activity towards TR both in vitro and in vivo. We outlined here the unusual story of compound’s discovery, and presented a comprehensive overview of CpdA ligand properties, its anti-inflammatory effects in numerous animal models of inflammation and autoimmune diseases, as well as its anti-cancer effects. Finally, we presented mechanistic analysis of CpdA and glucocorticoid effects in skin, muscle, bone, and regulation of glucose and fat metabolism to explain decreased CpdA side effects compared to glucocorticoids. Overall, the results obtained by our and other laboratories underline translational potential of CpdA and its derivatives for treatment of inflammation, autoimmune diseases and cancer.http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=5078Publisher's versio

Molecular mechanism of the AR-induced senescence.

<p>This figure summarizes our findings. (<b>A</b>) mTOR activity in PC3-AR cells. Cells were treated with Dox alone (D) or in combination with DHT. Cell extracts were collected at days 3 and 5 of treatment and analyzed by Western blot for phosphorylated mTOR and p70S6K. Tubulin and total p70S6K served as loading controls. (<b>B</b>) Schematic representation of pathways leading to AR-indiced senescence: AR triggers two parallel pathways, necessary to ensure senescence and possibly engaged in a cross-talk: (1) Activated AR directly binds p21 promoter and thus causes protein expression and accumulation. P21 attenuates the levels of TAp63-α, increasing the number of PML nuclear bodies and causing senescence (2) AR enhances ROS production thus reducing phospho-Rb levels, while active Rb binds and sequesters E2F, causing senescence. Elevated p21 levels may additionally decrease phospho-Rb by blocking Cdks. Conversely, transcriptionally inactive Rb/E2F/HDAC complexes may be sequestered in PML bodies to maintain irreversible growth arrest/senescence. These pathways cumulatively contribute to quiescence, which progresses to senescence due to constitutive high mTOR activity.</p

AR-dependent tumor suppression was associated with increased cellular senescence.

<p>AR increased cellular senescence in vivo. (A) Experimental design. Male athymic mice (nu/nu, 5 mice/group) were injected s.c. with vector control (PC3-V) or PC3-AR cells and received Dox with drinking water, where indicated, to induce AR expression. Flutamide was given to counteract endogenous testosterone. (B) Mean tumor volumes and standard deviations are shown at 45-day time point. Note lower volume of PC3-AR tumors in male mice treated with Dox, and the lack of the decrease when Fl was added to the treatment. (C) Sections of frozen tumors collected in the experiments above were subjected to SA-βGal assay to detect senescence. Note increased βGal positivity (blue) in PC3-AR tumors treated with Dox and of LNCaP tumors treated with DHT.</p

p63 expression is blocked by P21 and contributes to AR-dependent senescence.

<p>This figure illustrates the link between p21 and p63 n AR-driven senescence. (<b>A</b>) PC3-AR cells were transfected with non-target (NT), p21 or p63 siRNA. Dox ± DHT were added to the media 24 hours post-transfection. Whole cell lysates were collected after 3 days and p63 expression measured by Western blot. Arrows point to TAp63-α and ΔNp63; * indicates non-specific band. Note the lack of p63 downregulation in p21 knock-down cells. (<b>B</b>) RWPE-1 cells were transfected with pLVX-AR or control pLVX and treated with vehicle EtOH or DHT. TAp63-α and ΔNp63 were measured as in (A). (<b>C</b>) PC3-AR cells were transfected with p21 or non-target (NT) siRNA. The cells were placed in Dox, ± DHT. RNA was isolated 3 days post-transfection. P63 message was measured by real-time RT-PCR. Note an approximately seven-fold reduction upon DHT treatment and increased expression after p21 knock-down. (<b>D</b>) In situ immunofluorescence of PC3-AR cells with pan-p63 antibody. Note pronounced p63 nuclear staining in Dox-treated cells, the lack of nuclear staining after 3 days in DHT and restored nuclear staining in the presence of Fl (top). Below: P63 nuclear localization (green pseudocolor), nuclei are counterstained with DAPI (red pseudocolor); colocalization appears in yellow. (<b>E</b>) PC3-AR cells were transfected with p21, p63 or control siRNA, cultured 48 hours in Dox ± DHT and stained with p63 antibodies. Note similar, diffuse p63 localization with the nuclear presence (filled arrows) in Dox-treated cells, the lack of nuclear localization in the presence of DHT (empty arrows), which is lost after p21 knock-down (filled arrows). P63 knock-down results in a weak, residual cytoplasmic staining. (<b>F, G</b>) Parental PC3 cells lacking AR expression were transfected with NT and p63 siRNA, cultured 5 days and senescence measured by SA-βGal assay. Representative images (left) and quantitative analysis (right) are shown. * indicates P<0.05.</p

p21 is regulated by AR and required for cellular senescence.

<p>AR-dependent senescence required increased p21. (<b>A</b>) PC3-AR cells were treated with Dox, DHT and Fl, at indicated combinations; cell extracts were analyzed by Western blot for p21, and ppRb. Tubulin was used as a loading control (A). Note the decrease in Rb phosphorylation and p21 increase in DHT-treated cells at every time point. (<b>B</b>) RWPE-1 cells, non-transfected or transfected with pLVX-AR or pLVX vector were treated with vehicle EtOH and DHT with or without Fl. Cell extracts were analyzed for AR, p21 and GAPDH to assess loading. (<b>C</b>) AR binding to p21 promoter: ChIP was performed with AR antibodies and AR-bound DNA amplified with the primers for the promoter region adjacent to the putative ARE element within p21 promoter (top) using real-time PCR. Each sample was run in triplicates, normalized per input DNA, and fold change in occupancy was calculated as FC = 2 ^{(−ΔΔCT [exp-con])}. Note increased AR binding to the p21 promoter in the presence of DHT and the reversal by flutamide (Fl, concentration shown in µM). The results of three independent experiments are pulled together (P<0.04). (<b>D</b>) PC3-AR cells were transfected with p21 or non-target control siRNA. Cell extracts were collected after 48 hours and analyzed by Western blot for p21, phospho-Rb (ppRb) and tubulin (loading control). Untransfected cells (-) are shown for comparison. (<b>E, F</b>) The transfectants were allowed 48 hours to recover and treated 5 days with Dox, DHT and Fl, as indicated; senescence was measured by SA-βGal assay. Note a significantly lower senescence levels after p21 knock-down (F, P<0.006). SA-βGal positive cells were quantified as above. Means and S.D. were calculated for three independent experiments. Representative images are shown (<b>F</b>).</p