Faculty Bibliography

Fibrosis of the vocal folds poses a substantive clinical challenge potentially underlying the rapid proliferation of direct steroid injections into the upper airway. The variable clinical response to glucocorticoids (GCs) in the vocal folds is likely related to diversity inherent to GCs and both patient-specific, and upstream, cell-specific responses to GCs. Broadly, we hypothesize the disparity in clinical outcomes are due to undesirable effects of GCs on resident fibroblasts. Transcriptome analysis identified significant GC-mediated modulation of Hippo signaling, a known regulator of fibrotic gene expression. Subsequent analysis confirmed GC-mediated YAP activation, a transcriptional co-factor in the Hippo signaling pathway. YAP inhibition attenuated ACTA2 expression in GC-treated human vocal fold fibroblasts. Nuclear localization and phosphorylation at Ser211, however, was not affected by YAP inhibition, suggesting nuclear translocation of YAP is indirectly driven by GR. RNA-seq analysis confirmed the influence of GCs on Wnt signaling, and canonical Wnt signaling target genes were upregulated by GCs. These data implicate YAP and its downstream targets as putative mediators of a pro-fibrotic response to GCs. Therapeutic YAP inhibition may ultimately be clinically relevant and warrants further consideration.

Atherosclerosis and obesity share pathological features including inflammation mediated by innate and adaptive immune cells. LXRα plays a central role in the transcription of inflammatory and metabolic genes. LXRα is modulated by phosphorylation at serine 196 (LXRα pS196), however, the consequences of LXRα pS196 in hematopoietic cell precursors in atherosclerosis and obesity have not been investigated. To assess the importance of LXRα phosphorylation, bone marrow from LXRα WT and S196A mice was transplanted into Ldlr^-/- mice, which were fed a western diet prior to evaluation of atherosclerosis and obesity. Plaques from S196A mice showed reduced inflammatory monocyte recruitment, lipid accumulation, and macrophage proliferation. Expression profiling of CD68⁺ and T cells from S196A mouse plaques revealed downregulation of pro-inflammatory genes and in the case of CD68⁺ upregulation of mitochondrial genes characteristic of anti-inflammatory macrophages. Furthermore, S196A mice had lower body weight and less visceral adipose tissue; this was associated with transcriptional reprograming of the adipose tissue macrophages and T cells, and resolution of inflammation resulting in less fat accumulation within adipocytes. Thus, reducing LXRα pS196 in hematopoietic cells attenuates atherosclerosis and obesity by reprogramming the transcriptional activity of LXRα in macrophages and T cells to promote an anti-inflammatory phenotype.

Androgen deprivation therapy (ADT) is a mainstay of prostate cancer treatment, given the dependence of prostate cells on androgen and the androgen receptor (AR). However, tumors become ADT-resistant, and there is a need to understand the mechanism. One possible mechanism is the upregulation of AR co-regulators, although only a handful have been definitively linked to disease. We previously identified the Mediator subunit MED19 as an AR co-regulator, and reported that MED19 depletion inhibits AR transcriptional activity and growth of androgen-insensitive LNCaP-abl cells. Therefore, we proposed that MED19 upregulation would promote AR activity and drive androgen-independent growth. Here, we show that stable overexpression of MED19 in androgen-dependent LNCaP cells promotes growth under conditions of androgen deprivation. To delineate the mechanism, we determined the MED19 and AR transcriptomes and cistromes in control and MED19-overexpressing LNCaP cells. We also examined genome-wide H3K27 acetylation. MED19 overexpression selectively alters AR occupancy, H3K27 acetylation, and gene expression. Under conditions of androgen deprivation, genes regulated by MED19 correspond to genes regulated by ELK1, a transcription factor that binds the AR N-terminus to induce select AR target gene expression and proliferation, and genomic sites occupied by MED19 and AR are enriched for motifs associated with ELK1. Strikingly, MED19 upregulates expression of monoamine oxidase A (MAOA), a factor that promotes prostate cancer growth. MAOA depletion reduces androgen-independent growth. MED19 and AR occupy the MAOA promoter, with MED19 overexpression enhancing AR occupancy and H3K27 acetylation. Furthermore, MED19 overexpression increases ELK1 occupancy at the MAOA promoter, and ELK1 depletion reduces MAOA expression and androgen-independent growth. This suggests that MED19 cooperates with ELK1 to regulate AR occupancy and H3K27 acetylation at MAOA, upregulating its expression and driving androgen independence in prostate cancer cells. This study provides important insight into the mechanisms of prostate cancer cell growth under low androgen, and underscores the importance of the MED19-MAOA axis in this process.

PIM1 is a serine/threonine kinase that promotes and maintains prostate tumorigenesis. While PIM1 protein levels are elevated in prostate cancer relative to local disease, the mechanisms by which PIM1 contributes to oncogenesis have not been fully elucidated. Here, we performed a direct, unbiased chemical genetic screen to identify PIM1 substrates in prostate cancer cells. The PIM1 substrates we identified were involved in a variety of oncogenic processes, and included N-Myc Downstream-Regulated Gene 1 (NDRG1), which has reported roles in suppressing cancer cell invasion and metastasis. NDRG1 is phosphorylated by PIM1 at serine 330 (pS330), and the level of NDRG1 pS330 is associated higher grade prostate tumors. We have shown that PIM1 phosphorylation of NDRG1 at S330 reduced its stability, nuclear localization, and interaction with AR, resulting in enhanced cell migration and invasion.

Similar to the hypertrophic scar and keloids, the efficacy of glucorticoids (GC) for vocal fold injury is highly variable. We previously reported dexamethasone enhanced the pro-fibrotic effects of transforming growth factor (TGF)-β as a potential mechanism for inconsistent clinical outcomes. In the current study, we sought to determine the mechanism(s) whereby GCs influence the fibrotic response and mechanisms underlying these effects with an emphasis on TGF-β and nuclear receptor subfamily 4 group A member 1 (NR4A1) signaling. Human VF fibroblasts (HVOX) were treated with three commonly-employed GCs+ /-TGF-β1. Phosphorylation of the glucocorticoid receptor (GR:NR3C1) and activation of NR4A1 was analyzed by western blotting. Genes involved in the fibrotic response, including ACTA2, TGFBR1, and TGFBR2 were analyzed by qPCR. RNA-seq was performed to identify global changes in gene expression induced by dexamethasone. GCs enhanced phosphorylation of GR at Ser211 and TGF-β-induced ACTA2 expression. Dexamethasone upregulated TGFBR1, and TGFBR2 in the presence of TGF-β1 and increased active NR4A1. RNA-seq results confirmed numerous pathways, including TGF-β signaling, affected by dexamethasone. Synergistic pro-fibrotic effects of TGF-β were observed across GCs and appeared to be mediated, at least partially, via upregulation of TGF-β receptors. Dexamethasone exhibited diverse regulation of gene expression including NR4A1 upregulation consistent with the anti-fibrotic potential of GCs.

The DNA damage response (DDR) is necessary to maintain genome integrity and prevent the accumulation of oncogenic mutations. Consequently, proteins involved in the DDR often serve as tumor suppressors, carrying out the crucial task of keeping DNA fidelity intact. Mediator of DNA damage checkpoint 1 (MDC1) is a scaffold protein involved in the early steps of the DDR. MDC1 interacts directly with γ-H2AX, the phosphorylated form of H2AX, a commonly used marker for DNA damage. It then propagates the phosphorylation of H2AX by recruiting ATM kinase. While the function of MDC1 in the DDR has been reviewed previously, its role in cancer has not been reviewed, and numerous studies have recently identified a link between MDC1 and carcinogenesis. This includes MDC1 functioning as a tumor suppressor, with its loss serving as a biomarker for cancer and contributor to drug sensitivity. Studies also indicate that MDC1 operates outside of its traditional role in DDR, and functions as a co-regulator of nuclear receptor transcriptional activity, and that mutations in MDC1 are present in tumors and can also cause germline predisposition to cancer. This review will discuss reports that link MDC1 to cancer and identify MDC1 as an important player in tumor formation, progression, and treatment. We also discuss mechanisms by which MDC1 levels are regulated and how this contributes to tumor formation.

The diversity of actions of the glucocorticoid stress hormones among individuals and within organs, tissues and cells is shaped by age, gender, genetics, metabolism, and the quantity of exposure. However, such factors cannot explain the heterogeneity of responses in the brain within cells of the same lineage, or similar tissue environment, or in the same individual. Here, we argue that the stress response is continuously updated by synchronized neural activity on large-scale brain networks. This occurs at the molecular, cellular and behavioral levels by crosstalk communication between activity-dependent and glucocorticoid signaling pathways, which updates the diversity of responses based on prior experience. Such a Bayesian process determines adaptation to the demands of the body and external world. We propose a framework for understanding how the diversity of glucocorticoid actions throughout brain networks is essential for supporting optimal health, while its disruption may contribute to the pathophysiology of stress-related disorders, such as major depression, and resistance to therapeutic treatments.

Post-translational modifications, such as phosphorylation, are a powerful means by which the activity and function of nuclear receptors such as LXRα can be altered. However, despite the established importance of nuclear receptors in maintaining metabolic homeostasis, our understanding of how phosphorylation affects metabolic diseases is limited. The physiological consequences of LXRα phosphorylation have, until recently, only been studied in vitro or non-specifically in animal models by pharmacologically or genetically altering the enzymes enhancing or inhibiting these modifications. Here we review recent reports on the physiological consequences of modifying LXRα phosphorylation at serine 196 (S196) in cardiometabolic disease including non-alcoholic fatty liver disease (NAFLD), atherosclerosis and obesity. A unifying theme from these studies is that LXRα S196 phosphorylation rewires the LXR-modulated transcriptome, which in turn alters physiological response to environmental signals, and that this is largely distinct from the LXR-ligand-dependent action.

PURPOSE/OBJECTIVE:Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. METHODS:Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. RESULTS:< .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. CONCLUSION/CONCLUSIONS:These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Stress can either promote or impair learning and memory. Such opposing effects depend on whether synapses persist or decay after learning. Maintenance of new synapses formed at the time of learning upon neuronal network activation depends on the stress hormone-activated glucocorticoid receptor (GR) and neurotrophic factor release. Whether and how concurrent GR and neurotrophin signaling integrate to modulate synaptic plasticity and learning is not fully understood. Here, we show that deletion of the neurotrophin brain-derived neurotrophic factor (BDNF)-dependent GR-phosphorylation (PO₄) sites impairs long-term memory retention and maintenance of newly formed postsynaptic dendritic spines in the mouse cortex after motor skills training. Chronic stress and the BDNF polymorphism Val66Met disrupt the BDNF-dependent GR-PO₄ pathway necessary for preserving training-induced spines and previously acquired memories. Conversely, enrichment living promotes spine formation but fails to salvage training-related spines in mice lacking BDNF-dependent GR-PO₄ sites, suggesting it is essential for spine consolidation and memory retention. Mechanistically, spine maturation and persistence in the motor cortex depend on synaptic mobilization of the glutamate receptor subunit A1 (GluA1) mediated by GR-PO₄ Together, these findings indicate that regulation of GR-PO₄ via activity-dependent BDNF signaling is important for the formation and maintenance of learning-dependent synapses. They also define a signaling mechanism underlying these effects.