Prostate cancer is the most commonly diagnosed non-cutaneous cancer and one of the leading causes of cancer death for North American men. Whereas localized prostate cancer can be cured, there is currently no cure for metastatic prostate cancer. Here we report a novel approach that utilizes designed chimeric transcription activator-like effectors (dTALEs) to control prostate cancer metastasis. Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications. These manipulations markedly altered expression of endogenous CRMP4, a metastasis suppressor gene. Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis. CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression. This proof-of-concept study with dTALEs for locus-specific epigenomic manipulation validates the selected CpG methylation of CRMP4 gene as an independent biomarker for diagnosis and prognosis of prostate cancer metastasis and opens up a novel avenue for mechanistic research on cancer biology.

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt −377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming.

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Ketamine produces rapid and sustained antidepressant activity, but its mechanism of action remains uncertain. Williams & colleagues (2018) reported that naltrexone pretreatment attenuated ketamine’s effects in treatment resistant depressed patients. Naltrexone antagonizes both kappa (KOR) and mu opioid receptors (MORs). We hypothesized that ketamine requires acute KOR activation, leading to long‐lasting suppression of KOR signaling to exert its antidepressant effects. The following series of experiments demonstrated: 1) exposure to ketamine produces internalization of KORs; 2) the behavioral effects of ketamine in mice were prevented by prior KOR blockade; and 3) ketamine pretreatment prevented behavioral and physiological responses caused by subsequent KOR activation. KOR internalization was tested in HEK293 cells transfected with yPET‐OPRK1 plasmids following ketamine stimulation (2 μM). Approximately 30% of KORs were internalized, which was comparable to the internalization achieved with the endogenous KOR agonist dynorphin (40 μM). To demonstrate that activation of KORs by ketamine contributes to its protracted behavioral profile, C57BL/6J mice (8–12 weeks) were pretreated with naltrexone (1 mg/kg) or the selective KOR antagonist LY2444296 (3 mg/kg) 30 minutes prior to ketamine (10 mg/kg) and were tested in the forced swim test 24 h later. Reductions in immobility scores by ketamine were blocked by naltrexone and LY2444296 pretreatment, confirming the importance of KORs in initiating ketamine’s long‐lasting effects. Next, the physiological impact of ketamine on KOR activity was examined ex vivo in the lateral habenula (LHb), a reward‐related brain region. Bath application of the KOR agonist U50,488 (10 μM) robustly increased the number of action potentials in response to depolarization. The increase in LHb neuronal excitability was absent when ketamine (10 mg/kg) was administered to mice 24 h previously. In parallel, U50,488 (10 or 20 mg/kg) induced behavioral deficits on nest building, prepulse inhibition and antinociception on the hot plate were effectively prevented by ketamine (10 or 20 mg/kg) given 24 h earlier. Finally, LY2444296 (3 mg/kg) administered 30 min prior to ketamine (10 mg/kg) prevented ketamine‐induced suppression of U50,488‐induced alterations on nesting behavior 24 h later. This established that KOR occupation prior to ketamine prevented the initiation of ketamine’s long‐lasting behavioral effects. Collectively, these studies provide evidence supporting the hypothesis that activation of KORs by acute ketamine administration leads to enduring reductions of KOR signaling, which may contribute to the protracted clinical antidepressant effects of ketamine. Support or Funding Information This work was funded by US Public Health Service (USPHS) grant R01 MH105623.

Transdifferentiation of stem cells into insulin-producing cells for the treatment of diabetes have shown promising but inconsistent results. We examined the potential for attracting bone marrow stem cells (BMSCs) to the pancreas using a chemokine, stromal cell derived factor-1 (SDF-1). SDF-1 treatment markedly increased the number of GFP labeled BMSCs in the pancreas, but surprisingly, the majority was observed in liver. The liver cells had typical pancreatic endocrine cell gene expression including insulin I, insulin II, PDX-1, somatostatin and glucagon. Combined treatment with SDF-1 and BMSC transplant reduced hyperglycemia and prolonged the long-term survival of diabetic mice, and a sub group had complete normoglycemia (<150 mg/dl), restored blood insulin levels, and normal glucose tolerance. Our results suggest that SDF-1 could potentially be used to improve the homing of stem cells and β-cell regeneration. The mechanism appears to involve an increase in insulin producing cells mainly in the liver.