Faculty Bibliography

OBJECTIVE: Cholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. APPROACH AND RESULTS: Here, we identify oxysterol-binding protein-like 6 (OSBPL6) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum-targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. CONCLUSIONS: These studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

Neurotrophins and glucocorticoids are robust synaptic modifiers, and deregulation of their activities is a risk factor for developing stress-related disorders. Low levels of brain-derived neurotrophic factor (BDNF) increase the desensitization of glucocorticoid receptors (GR) and vulnerability to stress, whereas higher levels of BDNF facilitate GR-mediated signaling and the response to antidepressants. However, the molecular mechanism underlying neurotrophic-priming of GR function is poorly understood. Here we provide evidence that activation of a TrkB-MAPK pathway, when paired with the deactivation of a GR-protein phosphatase 5 pathway, resulted in sustained GR phosphorylation at BDNF-sensitive sites that is essential for the transcription of neuronal plasticity genes. Genetic strategies that disrupted GR phosphorylation or TrkB signaling in vivo impaired the neuroplasticity to chronic stress and the effects of the antidepressant fluoxetine. Our findings reveal that the coordinated actions of BDNF and glucocorticoids promote neuronal plasticity and that disruption in either pathway could set the stage for the development of stress-induced psychiatric diseases.

The outcome for pediatric ALL patients that relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the MAPK pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrated that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of MEK1/2 target ERK in matched diagnosis and relapse primary samples and observed increased pERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Altogether, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents, and is an attractive target for prevention and/or treatment of relapsed disease.

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by the Liver X Receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRalpha serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRalpha at S198 is non-phosphorylated. In bone marrow derived-macrophages (BMDMs) we also observe induction of CCR7 by ligands that promote non-phosphorylated LXRalpha S198 and this is lost in LXR deficient BMDMs. LXRalpha occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing non-phosphorylated (RAW-LXRalphaS198A) compared to phosphorylated LXRalpha (RAW-LXRalphaWT). Expression profiling from ligand treated RAW-LXRalphaS198A compared to RAW-LXRalphaWT cells revealed induction of cell migratory and anti-inflammatory genes, and repression of pro-inflammatory genes. Modeling of LXRalpha S198 in non-phosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with sites for protein interaction. Therefore, gene transcription is regulated by LXRalpha S198 phosphorylation including anti-atherogenic genes like CCR7.

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

Uterine carcinosarcoma (UCS) is a rare type of cancer and accounts for 5% of uterine malignancies. However, UCS patients suffer a high prevalence of chemo-resistance and a very poor prognosis compared to uterine cancer patients. URI is a chaperone with functions in transcription. We analyzed the somatic URI1 copy number variation in 57 post-menopausal non-metastatic UCS patients in comparison to 363 uterine corpus endometrial carcinomas. URI1 amplification was detected in 40% (23/57) of primary UCS and 5.5% (20/363) of uterine carcinomas. UCS patients with URI1 amplification exhibited 13% (3/23) tumor-free survival compared to 41% (14/34) in the absence of URI amplification (P=0.023). URI1 amplification (OR=6.54, P=0.027), weight (OR=1.068, P=0.024), hypertension (OR=3.35, P=0.044), and tumor stage (OR=2.358, P=0.018) associated with poor survival. Patients treated with hormone replacement therapy (OR=15.87, P=0.011) displayed enhanced overall survival. Combined radiation and chemotherapy improved patient survival (median survival=2043 days) compared to single (median survival=597 days) or no treatment (median survival=317 days, P=0.0016). Importantly, patients with URI1 amplification had poor response to adjuvant treatment compared to control group (P=0.013). Tumors with URI1 amplification displayed decreased transcription of genes encoding tumor suppressor and apoptotic regulators and increased expression of genes regulating oncogenesis, survival and metastasis. Overexpression of URI1 in a cultured cell model induced ATM expression and resistance to cisplatin. Our findings suggest that high prevalence in UCS may associate with poor prognosis and worse response to adjuvant treatment.

The Androgen Receptor (AR) is a major therapeutic target in prostate cancer pharmacology. Progression of prostate cancer has been linked to elevated expression of AR in malignant tissue, suggesting that AR plays a central role in prostate cancer cell biology. Potent therapeutic agents can be precisely crafted to specifically target AR, potentially averting systemic toxicities associated with non-specific chemotherapies. In this review, we describe various strategies to generate steroid conjugates that can selectively engage AR with high potency. Analogies to recent developments in non-steroidal conjugates targeting AR are also evaluated. Particular focus is placed on potential applications in AR pharmacology. The review culminates with a description of future prospects for targeting AR.

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one out of five patients will relapse and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor (NCoR) complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes, and ultimately decreased glucocorticoid signaling caused by increased levels of NCoR1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1 depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1 depleted cells. Altogether, our data indicates that loss of TBL1XR1 is a novel driver of glucocorticoid-resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.