High Levels Of Glucocorticoid Receptor Research Articles

Abstract P2-21-08: Digital spatial profiling of RNA and protein in the invasive lobular breast cancer microenvironment

Abstract Invasive lobular breast cancer (ILC) is an understudied subtype of breast cancer with late recurrence, metastasis to serosal surfaces, such as the peritoneum, and dismal long-term outcome. We previously reported better patient outcome in ER+ breast cancers with high glucocorticoid receptor (GR) expression (Pan et al 2011). Preliminary data suggests more favorable patient outlook in the ILC subtype when tumors express higher levels of GR. The dynamic interaction between a tumor and its microenvironment leads to phenotypic changes in stromal cells and the extracellular matrix to promote growth or invasion of malignant cells. ILC is histologically distinct from invasive ductal carcinoma and is characterized by discohesive tumor cells that grow as “Indian files” due to lack of the cell adhesion molecule E-cadherin. Because of these differences we expect the tumor microenvironment (TME) to be quite unique in ILC compared to other breast cancer subtypes. We hypothesized that differing levels of expression of nuclear receptors by tumor cells would impact cells residing within the stroma, presumably through paracrine signaling. Immunohistochemistry for 25 ILC biopsies revealed a wide variation in GR expression, ranging from completely negative to strongly positive. Our goal was to gain insight into how GR presence or absence in ILC cells might impact gene and protein expression in malignant cells and their TME. We were inspired to examine how crosstalk between the GR-positive or GR-negative cancer cells and their respective tumor microenvironment (TME) differentially impact stromal cell gene expression as well as the immune cell milieu. Bulk expression profiling is inadequate to examine molecular profiles of the tumor and stroma separately. Therefore, we used nanoString GeoMx® high-plex digital spatial profiling of RNA and protein expression in GR-positive and GR-negative primary ILC. We first performed spatial gene expression using the Whole Transcriptome Atlas (WTA) for six primary ILC, two of which were strongly positive for GR, two GR-negative and 2 tumors harboring both GR-positive and GR-negative regions. To examine the tumor cells and the stroma independently we segmented regions into panCK-positive and panCK-negative segments and analyzed those separately. Intriguing differences were observed between GR-positive and negative ILC, for the tumor cells as well as for the TME. Implementation of the Spatial Deconvolution script embedded within the GeoMx® software revealed striking differences in the abundance of immune cells, endothelial cells and fibroblasts in the stroma of GR-positive vs GR-negative ILC. Macrophages and other myeloid subsets were present in significantly higher numbers in the TME of GR-positive compared to GR-negative ILC. The contribution of B- and T-cell subsets as well as endothelial cells and fibroblasts were also notably different. To more precisely define the spatial distribution and abundance of various cell subtypes and their association with tumor cells, we are including protein DSP as an added layer to complement the WTA. We are also performing RNA and protein DSP for an additional ten GR-positive and ten GR-negative ILC. Thus, we will test our initial observations and expand our investigation to generate comprehensive molecular profiles of ILC tumors and stromal cells and acquire a deeper understanding of how GR expression/activation influences the crosstalk between ILC cells and their TME. We anticipate that differences in gene and protein expression will provide clues as to how GR activation affects proliferative and adhesive properties of ILC through modification of the TME. Citation Format: Lynda B. Bennett, Candace Frerich, Sunati Sahoo, Cheryl Lewis, Indu Raman, Min Xu, Guanchun Chen, Suzanne D. Conzen. Digital spatial profiling of RNA and protein in the invasive lobular breast cancer microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-08.

Glucocorticoid-induced expansion of classical monocytes contributes to bone loss

Classical monocytes are commonly involved in the innate inflammatory response and are the progenitors of osteoclasts. Excess endogenous glucocorticoids (GCs) can increase the levels of classical monocytes in blood and bone marrow. The role of this cell population in high-dose exogenous GC-induced osteoporosis (GIOP) remains to be elucidated. In this study, GIOP was established in rats and mice by daily methylprednisolone injection, and monocyte subsets were analyzed by flow cytometry. We demonstrated that classical monocytes accumulate in bone marrow during GIOP. Similarly, the monocyte proportion among bone marrow nucleated cells was also increased in patients with steroid treatment history. We sorted classical monocytes and analyzed their transcriptional profile in response to GCs by RNA sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that classical monocytes isolated from GC-treated rats exhibited osteoclast differentiation potential. Deletion of classical monocytes by clodronate liposome treatment prevented GIOP via inhibition of osteoclastogenesis and restoration of CD31HiendomucinHi vessels. Regarding the molecular mechanism, classical monocytes express high levels of glucocorticoid receptors. In vitro treatment with GCs increased both the percentage and absolute number of monocytes and promoted their proliferation. In summary, classical monocytes mediated GC-induced bone loss and are a potential target for therapeutic intervention in GIOP treatment.

Hippocampal interneurons are direct targets for circulating glucocorticoids.

The hippocampus has become a significant target of stress research in recent years because of its role in cognitive functioning, neuropathology, and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite the pervasive impact of stress on psychiatric and neurological disease, many of the circuit- and cell-dependent mechanisms giving rise to the limbic regulation of the stress response remain unknown. Hippocampal excitatory neurons generally express high levels of glucocorticoid receptors (GRs) and are therefore positioned to respond directly to serum glucocorticoids. These neurons are, in turn, regulated by neighboring interneurons, subtypes of which have been shown to respond to stress exposure. However, GR expression among hippocampal interneurons is not well characterized. To determine whether key interneuron populations are direct targets for glucocorticoid action, we used two transgenic mouse lines to label parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons. GR immunostaining of labeled interneurons was characterized within the dorsal and ventral dentate hilus, dentate cell body layer, and CA1 and CA3 stratum oriens and stratum pyramidale. While nearly all hippocampal SST+ interneurons expressed GR across all regions, GR labeling of PV+ interneurons showed considerable subregion variability. The percentage of PV+, GR+ cells was highest in the CA3 stratum pyramidale and lowest in the CA1 stratum oriens, with other regions showing intermediate levels of expression. Together, these findings indicate that, under baseline conditions, hippocampal SST+ interneurons are a ubiquitous glucocorticoid target, while only distinct populations of PV+ interneurons are direct targets. This anatomical diversity suggests functional differences in the regulation of stress-dependent hippocampal responses.

Variability in Behavioral Phenotypes after Forced Swimming-Induced Stress in Rats Is Associated with Expression of the Glucocorticoid Receptor, Nurr1, and IL-1β in the Hippocampus.

Individual differences in coping with stress may determine either a vulnerable or resilient phenotype. Therefore, it is important to better understand the biology underlying the behavioral phenotype. We assessed whether individual behavioral phenotype to acute stress is related with the hippocampal expression of glucocorticoid receptor (GR), Nurr1, interleukin-1 beta (IL-1β) or brain-derived neurotrophic factor (BDNF). Wistar male rats were exposed to forced swimming for 15 min and sacrificed at different times. Behavioral response was analyzed, and it was compared with the gene and protein expression of GR, Nurr1, IL-1β and BDNF in the hippocampus for each time point. Behavioral phenotyping showed a group with high immobility (vulnerable) while another had low immobility (resilient). No significant differences were found in the Nurr1, IL-1β and BDNF mRNA levels between resilient and vulnerable rats at different recovery times except for Nr3c1 (gene for GR). However, exposure to stress caused significantly higher levels of GR, Nurr1 and IL-1β proteins of vulnerable compared to resilient rats. This variability of behavioral phenotypes is associated with a differential molecular response to stress that involves GR, Nurr1, and IL-1β as mediators in coping with stress. This contributes to identifying biomarkers of susceptibility to stress.

A Growing Dilemma: Antenatal Corticosteroids and Long-Term Consequences.

A single course of synthetic antenatal corticosteroids is standard care for women considered to be at risk for preterm birth before 34 weeks of gestation. While the intended target is the fetal lung, the fetal brain contains remarkably high levels of glucocorticoid receptors in structures critical in the regulation of behavior and endocrine function. Negative programming signals may occur which can lead to permanent maladaptive changes and predispose the infant/child to an increased risk in physical, mental, and developmental disorders. Framed around these areas of concerns for physical, mental, and developmental disorders, this narrative review drew on studies (animal and clinical), evaluating the long-term effects of antenatal corticosteroids to present the case that a more targeted approach to the use of antenatal corticosteroids for the betterment of the fetus urgently needed. Studies raised concerns about the potential negative long-term consequences, especially for the exposed fetus who was born beyond the period of the greatest benefit from antenatal corticosteroids. The long-term consequences are more subtle in nature and usually manifest later in life, often beyond the scope of most clinical trials. Continued research is needed to identify sufficient safety data, both short term and long term. Caution in the use of antenatal corticosteroids should be exercised while additional work is undertaken to optimize dosing strategies and better identify women at risk of preterm birth prior to administration of antenatal corticosteroids. · A single-course ACS is a remarkable therapy with substantial benefits.. · There is a potential of long-term neurodevelopmental consequences in the ACS-exposed fetus.. · There is a need to improve dosing strategies and identification of appropriate at risk women..

Glucocorticoid receptor upregulation increases radioresistance and triggers androgen independence of prostate cancer.

Despite the overall success of radiotherapy, a significant number of patients develop radioresistance, which leads to local regional recurrence and distant metastasis. We studied whether repeated radiation treatment promotes androgen-independent survival of prostate cancer (PCa) cells and their metastatic potential. We also studied whether glucocorticoid receptor (GR) increase in radioresistant cells is associated with acquisition of these aggressive characteristics. Radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) PCa sublines were developed by repeated radiation treatments of parental cells. Levels and activations of androgen receptor (AR) and GR in radioresistant PCa cells and respective parental cells were investigated in quantitative real-time polymerase chain reaction/Western blot analyses and immunofluorescence staining. Androgen-independent survival of radioresistant cells was tested in in vitro cell growth assays and the castration-resistant survival of these cell-derived tumors were investigated in mouse xenografts. Higher GR levels, but lower AR levels were detected in radioresistant cells than in parental cells. Radiation-induced GR upregulation was associated with increased intracellular cyclic adenosine monophosphate. As a consequence of GR activation, LNCaPR18 cells survived well in an androgen-depleted culture condition while parental cells could not. Results of in vivo mouse studies showed survival of LNCaPR18 cell-derived tumors in castrated mice while parental cell-derived tumors regressed. The growth of LNCaPR18 cell-derived tumors in castrated mice was impaired when treated with the anti-GR agent mifepristone. In experiments with C4-2/C4-2R26 cell sets, GR activation in C4-2R26 cells increased their metastatic potential. GR activation in radioresistant cells mediates androgen independence and facilitates PCa progression.

Radiation-induced glucocorticoid receptor promotes CD44+ prostate cancer stem cell growth through activation of SGK1-Wnt/β-catenin signaling.

We observed cancer stem cell (CSC) population increase in radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) prostate cancer (PCa) cells compared with respective parental cells. Since the CD44 level increase was most significant in radioresistant PCa cells compared with parental cells among CSC markers tested, we isolated the CD44+ population from LNCaP/LNCaPR18 and C4-2/C4-2R26 cell sets via the immunomagnetic separation method and used them as CSC sources. We detected lower AR level, but higher glucocorticoid receptor (GR) level in CD44+ CSCs than CD44- non-CSCs. Higher GR level in CD44+ CSCs than CD44- cells was also detected when cells were isolated from mouse tumor tissues of LNCaPR18 cell and C4-2R26 cell-derived human xenografts and grown in culture. We then found blocking the GR signaling by adding the anti-GR agent mifepristone into the cell culture inhibited the CD44+ CSC growth while the addition of the anti-AR agent enzalutamide enhanced the CSC growth. In xenograft mouse studies in which tumors were developed from the injection of CD44+ CSCs of LNCaPR18 or C4-2R26 cell lines, retarded tumor growth in mifepristone-injected mice was observed compared with vehicle-treated mice. We next discovered the GR regulation of Wnt/β-catenin signaling pathway. We further found that the serum/glucocorticoid regulated kinase 1 (SGK1) is the GR downstream molecule that mediates Wnt/β-catenin signaling activation. Therefore, inhibition of either SGK1 or Wnt/β-catenin signaling impaired the in vitro CD44+ CSC growth. From these results, we suggest that blocking GR signaling or its downstream SGK1-Wnt/β-catenin signaling axis may suppress the radiation-induced CSC increase in PCa. KEY MESSAGES: Higher CSC population exists in radioresistant PCa cells than parental cells. Higher GR levels (and lower AR level) in CD44+ CSCs than CD44- non-CSCs. Use of anti-GR agent blocked the growth of CD44+ CSCs in in vitro/in vivo tests. GR downstream SGK1-Wnt/β-catenin signaling axis mediates the CSC increase. Targeting this signaling axis may enhance the radiotherapy efficacy in treating PCa.

SUN-008 The Glucocorticoid Receptor Is Essential For TGFβ and p38 MAPK Mediated Cancer Phenotypes in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is the most metastatic and deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. There is a critical need to identify molecular targets that could be exploited as new biomarkers of TNBC prognosis and for improving therapies. Although TNBC lacks estrogen and progesterone receptors, 15-40% of TNBC patients express the glucocorticoid receptor (GR). Women with TNBC that express high levels of GR have poor outcomes. We hypothesize that GR is a key mediator of advanced cancer phenotypes in TNBC. Specifically, we propose that GR acts as a “sensor” for stress signaling pathways commonly activated by soluble factors that are abundant within the tumor microenvironment (TME). Using TNBC models, we showed previously that GR is phosphorylated on Ser134 in response to cellular stress stimuli such as hypoxia. Herein, we show that pS134-GR is elevated in TNBC tumor tissue samples relative to non-TNBC tissues. In vitro studies in TNBC models demonstrate that GR Ser134 phosphorylation is promoted by cytokines (TGFbeta), and growth factors (HGF) and occurs in the absence of GR ligands such as Dexamethasone or cortisol. In response to stress signaling inputs, studies with kinase inhibitors confirmed that p38 MAPK is required for GR Ser134 (pS134-GR) phosphorylation. To evaluate the functional significance of pS134-GR, we created CRISPR models of MDA-MB-231 TNBC cells expressing either wt GR or phospho-mutant GR that cannot be phosphorylated at Ser134 (S134A). RNAseq studies were performed to identify pS134-GR target genes in TNBC models. Our transcriptome data demonstrated a requirement for pS134 GR in the expression of gene sets associated with TGFβ and p38 MAPK signaling. Pathway analysis revealed that pS134-GR target genes primarily regulate cancer cell migration. In vitro assays revealed that pS134-GR is essential for inducing cell migration and anchorage-independent growth in TNBC cells, even in the absence of exogenous GR ligands. Furthermore, using co-IP assays, we identified that upon phosphorylation at Ser134, GR interacts with the scaffolding protein 14-3-3zeta. Like pS134-GR, 14-3-3zeta is highly expressed in TNBC when compared to non-TNBC patients. We observed co-recruitment of both pS134-GR and 14-3-3zeta to known pS134-GR target genes (i.e. PTK6) in TNBC cells. These data prompted us to test the requirement for 14-3-3zeta in GR-mediated phenotypes. Short hairpin RNA knock-down experiments demonstrated that expression of 14-3-3zeta is required for serum and TGFbeta-induced TNBC cell migration. We conclude that the pS134-GR/14-3-3zeta complex is a key “sensor” of local stress signals within the TME (TGFbeta) and a potent mediator of cell migration in TNBC models. Further studies are aimed at exploring pS134 GR as a biomarker and therapeutic target in TNBC.

Antenatal Glucocorticoid Exposure Results in Sex-Specific and Transgenerational Changes in Prefrontal Cortex Gene Transcription that Relate to Behavioural Outcomes

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery to reduce respiratory distress syndrome in the newborn. The prefrontal cortex (PFC) is important in regulating stress responses and related behaviours and expresses high levels of glucocorticoid receptors (GR). Further, antenatal exposure to sGC results in a hyperactive phenotype in first generation (F1) juvenile male and female offspring, as well as F2 and F3 juvenile females from the paternal lineage. We hypothesized that multiple courses of antenatal sGC modify gene expression in the PFC, that these effects are sex-specific and maintained across multiple generations, and that the gene sets affected relate to modified locomotor activity. We performed RNA sequencing on PFC of F1 juvenile males and females, as well as F2 and F3 juvenile females from the paternal lineage and used regression modelling to relate gene expression and behavior. Antenatal sGC resulted in sex-specific and generation-specific changes in gene expression. Further, the expression of 4 genes (C9orf116, Calb1, Glra3, and Gpr52) explained 20–29% of the observed variability in locomotor activity. Antenatal exposure to sGC profoundly influences the developing PFC; effects are evident across multiple generations and may drive altered behavioural phenotypes.

Using Radiolabeled 3'-Deoxy-3'-18F-Fluorothymidine with PET to Monitor the Effect of Dexamethasone on Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality in the United States, and pemetrexed-based therapies are regularly used to treat nonsquamous NSCLC. Despite widespread use, pemetrexed has a modest effect on progression-free survival, with varying efficacy between individuals. Recent work has indicated that dexamethasone, given to prevent pemetrexed toxicity, is able to protect a subset of NSCLC cells from pemetrexed cytotoxicity by temporarily suppressing the S phase of the cell cycle. Therefore, dexamethasone might block treatment efficacy in a subpopulation of patients and might be contributing to the variable response to pemetrexed. Methods: Differences in retention of the experimental PET tracer 3'-deoxy-3'-fluorothymidine (FLT) were used to monitor S-phase suppression by dexamethasone in NSCLC cell models, animals with tumor xenografts, and patients with advanced cancer. Results: Significant reductions in tracer uptake were observed after 24 h of dexamethasone treatment in NSCLC cell lines and xenograft models expressing high levels of glucocorticoid receptor α, coincident with pemetrexed resistance visualized by attenuation of the flare effect associated with pemetrexed activity. Two of 4 patients imaged in a pilot study with 18F-FLT PET after dexamethasone treatment demonstrated reductions in tracer uptake from baseline, with a variable response between individual tumor lesions. Conclusion:18F-FLT PET represents a useful method for the noninvasive monitoring of dexamethasone-mediated S-phase suppression in NSCLC and might provide a way to individualize chemotherapy in patients receiving pemetrexed-based regimens.

Glucocorticoid receptor activation is associated with increased resistance to heat‐induced hyperthermia and injury

Anti-inflammatory mediators likely play a key role in maintaining thermal homeostasis and providing protection against heat stress. The aim of this study was to investigate the association between activation of the glucocorticoid receptor (GR) and resistance to heat-induced hyperthermia and injury. Effects of heat exposure on core body temperature, muscle GR phosphorylation status and subcellular expression were examined in control mice and thermal acclimation (TA)-exposed mice. In addition, effects of TA and corticosterone on C2C12 mouse myoblast viability and subcellular GR were assessed during heat exposure. Phosphorylated, nuclear and mitochondrial GR levels were significantly higher in the gastrocnemius muscles of mice with mild hyperthermia (tolerant), compared to mice with severe hyperthermia (intolerant) during a heat exposure test. Similar changes were found in mice after TA, compared to non-TA-exposed controls. Additional groups of TA and non-TA-exposed mice underwent a heat exposure test. TA mice presented a significantly lower hyperthermic response during heat exposure than non-TA-exposed control. C2C12 cells exposed to TA incubation had higher viability against heat shock and showed higher GR levels in their mitochondria and nuclei detected by Western blot analysis and fluorescence microscopy, compared to cells exposed to normal incubation. Furthermore, pre-incubation with 0.1 μM corticosterone increased C2C12 cell viability during heat exposure and mitochondrial and nuclear GR expression. The results of these in vivo and in vitro studies suggest that GR activation is associated with increased resistance against heat-induced hyperthermia and injury.

M4. Epigeneticly Related mRNA Levels in Lymphocytes of Schizophrenic and Nonpsychotic Controls

Background: Epigenetic dysregulation may be involved in the underlying molecular deficits in schizophrenia (SZ). Previous research by our group has show hypermethylation of GABAergic promoter gene in and increases in DNMT1 and DNMT3A in post mortem brain samples of patients with SZ. We have also shown that difference sin DNMT1 and other epigenetically related enzymes are also found in the lymphocytes of living patients with chronic schizophrenia (CSZ). We now report preliminary results on epigenetic related mRNA’s in lymphocytes on a larger sample of CSZ. Methods: CSZ (n = 29) and nonpsychotic controls (NPC) (n = 31) subjects had a blood sample (60–80 cc) drawn, and lymphocyte pellet extracted by Ficoll gradient procedure. qPCR assays were used to measure epigenetically related mRNA’s—DNMT1, DMNT3A, TET1, TET2, TET3, BDNF, NR3C (glucocorticoid receptor).We also assayed several immunological related mRNA (which appeared as strong hits from RNA sequence analysis) T-Cell Surface Glycoprotein CD4, CCR1 (C-C motif chemokine receptor 1), FPRL3 (Formyl Peptide Receptor). Assays were performed using Taqman probes with B-Actin as housekeeping gene. Patients were also evaluated with psychopathology with PANSS, for cognitive function with MATRICS, and for odor identification and discrimination with Sniff and Sticks smell test. Results: In this sample CSZ showed significantly higher DMNT3A (P = .048) than NPC Male CSZ showed significantly higher DNMT1 than NPC (P = .014), but the total sample there was a trend but no significant difference in DNMT1. Compared to NPC, CSZ subjects showed significantly higher levels of GABAergic enzymes measured by the GAD1 probe (which assays GAD67 and GAD25 mRNA) (P = .030), higher levels of glucocorticoid receptor measured by the NRC3 probe (P = .006), and significantly lower levels of FRPRL3(P = .039). Higher levels FPRL3 and CD4 were moderately correlated with PANSS positive symptoms in CSZ (FRPL3 r = +.43 P = .019, CD4 r = +.37 P = .054) and these correlations were slightly stronger in male CSZ. Higher DNMT1 and DNMT3A levels in lymphocytes of CSZ correlated negatively with scores n MATRICs battery (DNMT1—attention/vigilance—r = −.41, P = .031, working memory r = −.37, P = .048, composite score r = −.36, P = .063). Conclusion: CSZ demonstrate differences in epigenetically related mRNA’s in their lymphocytes. In CSZ higher levels of DNMT were related to poorer cognitive performance and higher levels of immunological related mRNA to greater positive symptom scores. Some potential differences to our previously published results may be related to differences in mRNA’s measured by the Taqman probes and earlier research with specifically generated sequence probes.

HPOB, an HDAC6 inhibitor, attenuates corticosterone-induced injury in rat adrenal pheochromocytoma PC12 cells by inhibiting mitochondrial GR translocation and the intrinsic apoptosis pathway

High levels of glucocorticoids (GCs) have been reported to damage normal hippocampal neurons, and such damage has been positively correlated with major depression (MD) and chronic stress. Our previous study showed that HDAC6 might be a potential target to regulate GC-induced glucocorticoid receptor (GR) translocation to the mitochondria and subsequent apoptosis. In the present study, we investigated the effect of HPOB, a selective HDAC6 inhibitor, on corticosterone (Cort)-induced apoptosis and explored the possible mechanism of action of HPOB in rat adrenal pheochromocytoma (PC12) cells, which possesses typical neuron features and expresses high levels of glucocorticoid receptors. We demonstrated that pre-treatment with HPOB remarkably reduced Cort-induced cytotoxicity and confirmed the anti-apoptotic effect of HPOB via the caspase-3 activity assay and H33342/PI and TUNEL double staining. Mechanistically, we demonstrated that HPOB reversed the Cort-induced elevation of GR levels in the mitochondria and blocked concomitant mitochondrial dysfunction and the intrinsic apoptosis pathway. Furthermore, HPOB was shown to attenuate expression of the multi-chaperone machinery (Hsp90-Hop-Hsp70) and cooperate with mitochondrial translocase of the outer/inner membrane (TOM/TIM) complex recruitment by triggering hyperacetylation of Hsps through HDAC6 inhibition. Considering all of these findings, the neuroprotective effect of HPOB demonstrated the crucial role of HDAC6 inhibition in reducing Cort-induced apoptosis in PC12 cells. The data further suggested that the anti-apoptotic activity of HDAC6 inhibition against the mitochondria-mediated impairment pathway might be mechanistically linked to the hyperacetylation of Hsps and consequent suppression of GR translocation to the mitochondria.

Phase I/II trial of X-396, a novel anaplastic lymphoma kinase (ALK) inhibitor, in patients with ALK+ non-small cell lung cancer (NSCLC)

We have previously demonstrated that in non-squamous non-small cell lung cancer (nsNSCLC) cells expressing relatively high levels of the glucocorticoid receptor (GR), dexamethasone (Dex) induces reversible G1 arrest that is virtually complete by 72h of Dex treatment. We have also shown that this effect of Dex protects the cells from the cytotoxicity of pemetrexed, a mainstay chemotherapy in advanced nsNSCLC that entails co-administration of Dex. Further, induction of G1 arrest by Dex was confirmed by FLT-PET imaging of tumor lesions in patients treated with Dex for 24h. Here we report the effects of long-term treatment of nsNSCLC cells with Dex. The nsNSCLC cell line models included A549 (GR), H292 (GR), H1650 (GR) and H1299 (GR) cells as well as clonal recombinant H1299 cells overexpressing GR. In only the GRhi cells, Dex caused an increase in p21 peaking on Day 7 and declining by Day 14. The cells retained proliferation potential on Day 3 as measured by colony formation. By Day 7 of Dex treatment, the GRhi cells but not the GRlo cells exhibited a senescence phenotype, marked by cytosolic beta-galactosidase activity and increases in p16 and p15 at the mRNA and protein levels as well as significant increases in cell size. The GR cells displayed a progressively decreasing ability to form colonies until 6 weeks of Dex treatment. The extent of this loss of proliferation potential was related to relative GR expression levels among the Dex-sensitive cells. When mice bearing xenografts of H1299 (GR) or isogenic recombinant H1299-GR (GR) cells were implanted with slow release Dex pellets, tumor growth was inhibited only in the H1299-GR cells. Evaluation of a tissue microarray as well as a cDNA array from clinical nsNSCLC tumors showed that about 20 percent of the tumors showed uniform expression of GR that were comparable to the levels required for Dex to induce senescence in vitro or to inhibit tumor growth in vivo. The results suggest that long-term administration of Dex could serve as an additional treatment option for a small cohort of lung adenocarcinoma patients that harbor uniform high levels of GR in their tumor lesions.

Long-term treatment with dexamethasone induces senescence and progressive loss of proliferation potential in lung adenocarcinoma cells expressing high levels of the glucocorticoid receptor

We have previously demonstrated that in non-squamous non-small cell lung cancer (nsNSCLC) cells expressing relatively high levels of the glucocorticoid receptor (GR), dexamethasone (Dex) induces reversible G1 arrest that is virtually complete by 72h of Dex treatment. We have also shown that this effect of Dex protects the cells from the cytotoxicity of pemetrexed, a mainstay chemotherapy in advanced nsNSCLC that entails co-administration of Dex. Further, induction of G1 arrest by Dex was confirmed by FLT-PET imaging of tumor lesions in patients treated with Dex for 24h. Here we report the effects of long-term treatment of nsNSCLC cells with Dex. The nsNSCLC cell line models included A549 (GR), H292 (GR), H1650 (GR) and H1299 (GR) cells as well as clonal recombinant H1299 cells overexpressing GR. In only the GRhi cells, Dex caused an increase in p21 peaking on Day 7 and declining by Day 14. The cells retained proliferation potential on Day 3 as measured by colony formation. By Day 7 of Dex treatment, the GRhi cells but not the GRlo cells exhibited a senescence phenotype, marked by cytosolic beta-galactosidase activity and increases in p16 and p15 at the mRNA and protein levels as well as significant increases in cell size. The GR cells displayed a progressively decreasing ability to form colonies until 6 weeks of Dex treatment. The extent of this loss of proliferation potential was related to relative GR expression levels among the Dex-sensitive cells. When mice bearing xenografts of H1299 (GR) or isogenic recombinant H1299-GR (GR) cells were implanted with slow release Dex pellets, tumor growth was inhibited only in the H1299-GR cells. Evaluation of a tissue microarray as well as a cDNA array from clinical nsNSCLC tumors showed that about 20 percent of the tumors showed uniform expression of GR that were comparable to the levels required for Dex to induce senescence in vitro or to inhibit tumor growth in vivo. The results suggest that long-term administration of Dex could serve as an additional treatment option for a small cohort of lung adenocarcinoma patients that harbor uniform high levels of GR in their tumor lesions.

Local glucocorticoid synthesis and regeneration in lymphoid organs: Neonatal and adult mice

Glucocorticoids are synthesized in the adrenal glands and circulate through the blood to coordinate organismal physiology. In early life, elevated glucocorticoids suppress growth and neural development, and altricial neonates avoid these deleterious effects by undergoing a stress hyporesponsive period during which circulating glucocorticoids are low. However, elevated glucocorticoids may be needed in other tissues, such as the thymus, for their critical role in production of immunocompetent T cells. Here, we show that lymphoid organs increase their local glucocorticoid levels via glucocorticoid production, and that production increases with age. Murine lymphoid organs (thymus, bone marrow, spleen) produced corticosterone in vitro, and this production was blocked by metyrapone, which inhibits both CYP11B1 synthesis of corticosterone from precursors (deoxycorticosterone) and 11beta-HSD1 regeneration of corticosterone from metabolites (dehydrocorticosterone). Gene expression and enzyme activity of 11beta-HSD1 were much higher than expression and activity of CYP11B1, which were extremely low or nondetectable. Adults showed higher 11beta-HSD1 expression and activity than neonates. These findings show that lymphoid organs produce glucocorticoids via regeneration, and that regeneration increases with age. Furthermore, regeneration occurs not only in the thymus, where glucocorticoids are necessary for T cell development, but also in bone marrow and spleen, the sites of B cell development. As developing B cells have high levels of glucocorticoid receptors, locally-produced glucocorticoids likely play a critical role in both T and B cell development.

Abstract B16: The pro-apoptotic effect of dexamethasone mediated by GILZ and Bim up-regulation is related to genetic heterogeneity of multiple myeloma.

Abstract Multiple myeloma (MM) is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. Briefly, MM that are hyperdiploid or have a t(11;14) translocation have a better prognosis than those with t(4;14) or t(14;16) translocations. Dexamethasone (Dex) is widely used in all phases of MM treatment as induction, consolidation or maintenance. However, the mechanism of Dex sensitivity still remains elusive. In the present study, we analyzed the ability of Dex to induce cell death by Apo-2.7 staining in 33 human myeloma cell lines (HMCLs) representative of the molecular subsets carrying t(11;14), t(4;14) or t(14;16) translocations, which deregulate CCND1, MMSET and c-MAF, respectively. The mechanisms controlling Dex-induced apoptosis were evaluated by analyzing glucocorticoid receptor (GR), glucocorticoid-induced leucin zipper (GILZ) and Bcl-2 protein family expression in the different MM molecular subtypes. Transient knock-down of glucocorticoid-responsive proteins were performed to define their role in the pro-apoptotic effect of Dex. We first demonstrated that direct pro-apoptotic effect of Dex was related to the genetic heterogeneity of MM, since sensitive cell lines were restricted to t(14;16) and t(4;14) subgroups. We next demonstrated by transcriptomic Affymetrix analysis that GR expression was heterogeneous among the different molecular subtypes of HMCLs. MAF subgroup significantly expressed higher levels of GR than all other subgroups (p=0.02). This result was also confirmed on 300 newly diagnosed MM patients (p<.0001). We also demonstrated that Dex sensitivity was associated with its ability to down-regulate MAF protein level and its well-known target CCND2. This result was in agreement with the fact that elevated levels of MAF were associated not only with t(14;16) translocation but also with MMSET over-expression, which is a characteristic of t(4;14) subgroup. We next demonstrated that Dex induced an up-regulation of GILZ, a glucocorticoid-responsive molecule, in dex-sensitive and insensitive HMCLs. Nevertheless, resistant HMCLs, which expressed low levels of GR, failed to up-regulate GILZ to the same extent than sensitive HMCLs, suggesting that GR levels could be an important limiting factor for GILZ up-regulation. Of note, GILZ silencing induced a strong reduction of Dex-induced cell death. These results suggest that transactivation has a pivotal role in the induction of cell death by Dex in myeloma cells. Dex also induced an up-regulation of Bim isoforms and a down-regulation of Bcl-xL in all sensitive cell lines. We further demonstrated that Bim up-regulation is required for the apoptotic program in response to Dex. Finally, the silencing of GILZ impaired the up-regulation of Bim and the down-regulation of Bcl-xL under Dex treatment. In conclusion, Dex exerted a direct anti-tumor effect on HMCLs of t(14;16) and t(4;14) molecular subgroups while t(11;14) subgroup was insensitive to it. This result suggested that conventional therapeutic approaches should be re-evaluated within molecular subgroups of MM patients to favor potential molecular subtype therapy based on rational preclinical data. Finally, while Dex interferes with multiple pathways, we begin to unravel its complex mechanism of action demonstrating that transactivation, inducing GILZ up-regulation, plays a pivotal role in Dex induced cell death through the regulation of the Bcl-2 protein network. Citation Format: Charlotte Kervoëlen, Emmanuelle Ménoret, Patricia Gomez-Bougie, Régis Bataille, Philippe Moreau, Catherine Pellat-Deceunynck, Martine Amiot. The pro-apoptotic effect of dexamethasone mediated by GILZ and Bim up-regulation is related to genetic heterogeneity of multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B16.

Modulation of c-Jun N-terminal kinase signaling and specific glucocorticoid receptor phosphorylation in the treatment of major depression

Glucocorticoid resistance is a common finding in major depressive disorder. Increased glucocorticoid receptor (GR) phosphorylation at serine 226 is associated with increased glucocorticoid resistance. Previously we have demonstrated that depressed patients exhibit higher levels of GR phosphorylated at serine 226 compared to healthy controls. The enzyme that is involved in this specific GR phosphorylation is c-Jun N-terminal kinase (JNK). We propose that modulation of glucocorticoid phosphorylation at serine 226, by targeting JNK signaling pathway, could be a potential strategy for antidepressant treatment. We base this assumption on the results of previous research that examined GR phosphorylation and JNK signaling in animal models and human studies. We also discuss the potential challenges in targeting JNK signaling pathway in depression.

Accumulation of cytoplasmic glucocorticoid receptor is related to elevation of FKBP5 in lymphocytes of depressed patients.

We have previously shown that patients with the major depressive disorder (MDD) exhibited elevated phosphorylation of the lymphocyte glucocorticoid receptor (GR) at serine 226 (S226). Here, we further analyse potential alterations of GR signalization in lymphocytes of MDD patients, i.e. the cytoplasmic/nuclear distribution of GR, levels of FK506-binding protein 5 (FKBP5) and glucocorticoid-induced leucine zipper (GILZ). The FKBP5 acts as an important regulator of GR activation, by decreasing ligand binding and impeding translocation of the receptor to the nucleus, while GILZ mediates glucocorticoid anti-inflammatory effects. Our result showed that the depressed patients had significantly higher GR levels in the cytoplasm compared to controls, which was accompanied by higher FKBP5 levels. Linear regression model demonstrated significantly higher correlation between FKBP5 and cytoplasmic GR than the presence of MDD itself or phosphorylation of nuclear GR at S226. There were no differences in the levels of GILZ isoforms. Therefore, the results suggest that accumulation of the GR in cytoplasm is related to the elevation of FKBP5, adding one more step in understanding altered GR signalling in lymphocytes, and potentially brain tissue, of MDD patients.

Abstract C28: Dexamethasone-induced apoptosis in multiple myeloma cells is restricted to t(14;16) and t(4;14) molecular subgroups due to a combined down-regulation of MAF and an up-regulation of Bim.

Abstract Multiple myeloma (MM) is heterogeneous with respect to its causative chromosomal abnormalities and the treatment response of patients. Briefly, MM patients with chromosomal hyperdiploidy or those carrying a t(11.14) translocation have a better prognosis than those with a t(4;14) or t(14;16) translocation. Dexamethasone (Dex) is widely used in all phases of the treatment of MM as induction, consolidation or maintenance. However, the mechanisms of sensitivity and resistance to Dex still remain elusive. In the present study, we analyzed the ability of Dex to induce cell death by Apo-2.7 staining in 33 human myeloma cell lines (HMCLs) representative of the molecular subsets i.e., carrying a t(11;14), t(4;14) or t(14;16) translocation, which deregulates the expression of CCND1, MMSET, and c-MAF respectively. The mechanisms controlling Dex-induced apoptosis were evaluated by analyzing the nuclear translocation of glucocorticoid receptor (GR), signalling pathways and modulation of Bcl-2 protein family in the different MM molecular subtypes. We first demonstrated by transcriptomic analysis (Affymetrix) that GR expression was heterogeneous among the different molecular subtypes of HMCLs and patients. Indeed, t(14;16) c-MAF subgroup significantly expressed higher levels of GR than all other subgroups in both HMCLs (Kruskall-Wallis p<.02) and primary MM cells from patients at diagnosis (n=309, Kruskall-Wallis p<.0001). We further demonstrated that the direct pro-apoptotic effect of Dex was related to the genetic heterogeneity of MM, since sensitive HMCLs were restricted to t(14;16) and t(4;14) subgroups. Dex-induced apoptosis was associated with caspase 6 and 3 activation. We next demonstrated that Dex sensitivity depended on its ability to induce the down-regulation of c-MAF protein, which led to a decrease in CCND2 expression, a well-known target gene of c-MAF. The down-regulation of c-MAF mainly occurred by the increase of its proteasomal degradation. Altogether, these results were in agreement with the elevated levels of c-MAF found not only in t(14;16) translocated primary cells and HMCLs but also in t(4;14) translocated cells and HMCLs. While Bim expression (all isoforms) was up-regulated in most of cell lines by Dex irrespectively of its capacity to induce apoptosis, we demonstrated by Bim silencing that its up-regulation was required for initiating Dex-induced apoptotic program. Of note, by a long-term culture in the presence of low doses of Dex, we generated a Dex-resistant t(14;16) which was characterized by a decreased expression of GR. Dex treatment of this Dex-resistant HMCL neither induced any down-regulation of c-MAF nor any up-regulation of Bim, showing that a minimal expression level of GR was the first limiting step to induce a death response. In the present study, we demonstrated that Dex exerted a direct anti-tumor effect on myeloma cells from t(14;16) and t(4;14) molecular subgroups by controlling c-MAF level while those belonging to the t(11;14) subgroup were insensitive to Dex. This result highlights the fact that conventional therapeutic approaches could be re-evaluated in concert with the definition of molecular subgroups of MM patients to favor molecular subtype-specific therapy based on rational preclinical data. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C28. Citation Format: Charlotte Kervoëlen, Emmanuelle Ménoret, Patricia Gomez-Bougie, Régis Bataille, Catherine Pellat-Deceunynck, Martine Amiot. Dexamethasone-induced apoptosis in multiple myeloma cells is restricted to t(14;16) and t(4;14) molecular subgroups due to a combined down-regulation of MAF and an up-regulation of Bim. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C28.