Tandem histone methyltransferase upregulation defines a unique aggressive prostate cancer phenotype.

BACKGROUND: Histone modifications alter transcriptional gene function and participate in cancer progression. Enhancer-of-Zeste-Homologue-2 (EZH2) and Nuclear-Receptor-Binding-SET-domain2 (NSD2) methylate H3K27 and H3K36, respectively, to regulate transcription. Given the therapeutic interest in these enzymes, we investigated expression and coregulation in hormone-sensitive (HS) and castrate-resistant (CR) prostate cancer (PC). METHODS: EZH2 and NSD2 levels were quantified using VECTRA analysis in HS and CRPC tissue microarrays (n = 105 + 66). Expression data from The Cancer Genome Atlas (n = 498), Memorial Sloan Kettering Cancer Center (n = 240), and Stand Up to Cancer/Prostate Cancer Foundation (n = 444) cBioportal datasets were queried, and associations between EZH2 and NSD2 and clinicopathologic variables determined. RESULTS: Tumour expression of NSD2, but not EZH2, increased in CRPC (p = 0.05, 0.09). Epithelial nuclei co-expressing NSD2 and EZH2 increased in CRPC compared to HSPC (69 vs 42%, p = 0.02), and in metastatic tissue relative to benign (55 vs 35%, p = 0.02). cBioportal analysis revealed collinear NSD2/EZH2 expression (Spearman = 0.57, 0.58, 0.58, all p < 0.001). NSD2/EZH2 co-expression significantly associates with clinicopathologic characteristics including grade group, stage and seminal vesicle involvement. On univariate and multivariate analysis tumours co-expressing NSD2 and EZH2 conferred increased risk of recurrence (hazard ratio: 2.6, 95% confidence inerval: 1.2-5.4, p = 0.01). Kaplan-Meier analysis revealed reduced progression-free-survival of NSD2 and EZH2 co-expression patients in datasets (p < 0.001, 0.002). CONCLUSIONS: Increased EZH2/NSD2 co-expression is overrepresented in CRPC, metastases and associates with shorter disease-free survival in PC patients. Coregulation of these two histone methyltransferases is a biomarker for aggressive PC and licenses them as therapeutic targets.

Dysregulation of Sirtuin 2 (SIRT2) and histone H3K18 acetylation pathways associates with adverse prostate cancer outcomes.

BACKGROUND: Histones undergo extensive post-translational modifications and this epigenetic regulation plays an important role in modulating transcriptional programs capable of driving cancer progression. Acetylation of histone H3K18, associated with gene activation, is enhanced by P300 and opposed by the deacetylase Sirtuin2 (SIRT2). As these enzymes represent an important target for cancer therapy, we sought to determine whether the underlying genes are altered during prostate cancer (PCa) progression. METHODS: Tissue microarrays generated from 71 radical prostatectomy patients were initially immunostained for H3K18Ac, P300 and SIRT2. Protein levels were quantified using VECTRA automation and correlated with clinicopathologic parameters. The Cancer Genome Atlas (TGCA, n = 499) and Gene Expression Omnibus (n = 504) databases were queried for expression, genomic and clinical data. Statistics were performed using SPSSv23. RESULTS: Nuclear histone H3K18Ac staining increases in primary cancer (p = 0.05) and further in metastases (p < 0.01) compared to benign on tissue arrays. P300 protein expression increases in cancer (p = 0.04) and metastases (p < 0.001). A progressive decrease in nuclear SIRT2 staining occurs comparing benign to cancer or metastases (p = 0.04 and p = 0.03 respectively). Decreased SIRT2 correlates with higher grade cancer (p = 0.02). Time to Prostate Specific Antigen (PSA) recurrence is shorter in patients exhibiting high compared to low H3K18Ac expression (350 vs. 1542 days respectively, P = 0.03). In GEO, SIRT2 mRNA levels are lower in primary and metastatic tumors (p = 0.01 and 0.001, respectively). TGCA analysis demonstrates SIRT2 deletion in 6% and increasing clinical stage, positive margins and lower PSA recurrence-free survival in patients with SIRT2 loss/deletion (p = 0.01, 0.04 and 0.04  respectively). In this dataset, a correlation between decreasing SIRT2 and increasing P300 mRNA expression occurs in tumor samples (R = -0.46). CONCLUSIONS: In multiple datasets, decreases in SIRT2 expression portend worse clinicopathologic outcomes. Alterations in SIRT2-H3K18Ac suggest altered P300 activity and identify a subset of tumors that could benefit from histone deacetylation inhibition.

S100A12 in vascular smooth muscle accelerates vascular calcification in apolipoprotein E-null mice by activating an osteogenic gene regulatory program.

OBJECTIVE: The proinflammatory cytokine S100A12 is associated with coronary atherosclerotic plaque rupture. We previously generated transgenic mice with vascular smooth muscle-targeted expression of human S100A12 and found that these mice developed aortic aneurysmal dilation of the thoracic aorta. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in atherosclerosis-prone apolipoprotein E (ApoE)-null mice would accelerate atherosclerosis. METHODS AND RESULTS: ApoE-null mice with or without the S100A12 transgene were analyzed. We found a 1.4-fold increase in atherosclerotic plaque size and more specifically a large increase in calcified plaque area (45% versus 7% of innominate artery plaques and 18% versus 10% of aortic root plaques) in S100A12/ApoE-null mice compared with wild-type/ApoE-null littermates. Expression of bone morphogenic protein and other osteoblastic genes was increased in aorta and cultured vascular smooth muscle, and importantly, these changes in gene expression preceded the development of vascular calcification in S100A12/ApoE-null mice. Accelerated atherosclerosis and vascular calcification were mediated, at least in part, by oxidative stress because inhibition of NADPH oxidase attenuated S100A12-mediated osteogenesis in cultured vascular smooth muscle cells. S100A12 transgenic mice in the wild-type background (ApoE+/+) showed minimal vascular calcification, suggesting that S100A12 requires a proinflammatory/proatherosclerotic environment to induce osteoblastic differentiation and vascular calcification. CONCLUSIONS: Vascular smooth muscle S100A12 accelerates atherosclerosis and augments atherosclerosis-triggered osteogenesis, reminiscent of features associated with plaque instability.

Proper modulation of AHR signaling is necessary for establishing neural connectivity and oligodendrocyte precursor cell development in the embryonic zebrafish brain.

2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD) is a persistent global pollutant that exhibits a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. Epidemiological studies have associated AHR agonist exposure with multiple human neuropathologies. Consistent with the human data, research studies using laboratory models have linked pollutant-induced AHR activation to disruptions in learning and memory as well as motor impairments. Our understanding of endogenous AHR functions in brain development is limited and, correspondingly, scientists are still determining which cell types and brain regions are sensitive to AHR modulation. To identify novel phenotypes resulting from pollutant-induced AHR activation and ahr2 loss of function, we utilized the optically transparent zebrafish model. Early embryonic TCDD exposure impaired embryonic brain morphogenesis, resulted in ventriculomegaly, and disrupted neural connectivity in the optic tectum, habenula, cerebellum, and olfactory bulb. Altered neural network formation was accompanied by reduced expression of synaptic vesicle 2. Loss of ahr2 function also impaired nascent network development, but did not affect gross brain or ventricular morphology. To determine whether neural AHR activation was sufficient to disrupt connectivity, we used the Gal4/UAS system to express a constitutively active AHR specifically in differentiated neurons and observed disruptions only in the cerebellum; thus, suggesting that the phenotypes resulting from global AHR activation likely involve multiple cell types. Consistent with this hypothesis, we found that TCDD exposure reduced the number of oligodendrocyte precursor cells and their derivatives. Together, our findings indicate that proper modulation of AHR signaling is necessary for the growth and maturation of the embryonic zebrafish brain.

Vascular remodeling and arterial calcification are directly mediated by S100A12 (EN-RAGE) in chronic kidney disease.

BACKGROUND: The proinflammatory cytokine S100A12 (also known as EN-RAGE) is associated with cardiovascular morbidity and mortality in hemodialysis patients. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in nonatherosclerosis-prone C57BL/6J mice on normal rodent chow diet, but exposed to the metabolic changes of chronic kidney disease (CKD), would develop vascular disease resembling that observed in patients with CKD. METHODS: CKD was induced in S100A12 transgenic mice and wild-type littermate mice not expressing human S100A12 by surgical ligation of the ureters. The aorta was analyzed after 7 weeks of elevated BUN (blood urea nitrogen), and cultured aortic smooth muscle cells were studied. RESULTS: We found enhanced vascular medial calcification in S100A12tg mice subjected to CKD. Vascular calcification was mediated, at least in part, by activation of the receptor for S100A12, RAGE (receptor for advanced glycation endproducts), and by enhanced oxidative stress, since inhibition of NADPH-oxidase Nox1 and limited access of S100A12 to RAGE attenuated the calcification and gene expression of osteoblastic genes in cultured vascular smooth muscle cells. CONCLUSION: S100A12 augments CKD-triggered osteogenesis in murine vasculature, reminiscent of features associated with enhanced vascular calcification in patients with chronic and end-stage kidney disease.

Epigenetic field alterations in non-tumor prostate tissues detect prostate cancer in urine.

Prostate cancer (PC) development involves epigenetic DNA methylation changes that occur in the tumor. However, distinct DNA methylation changes have been previously found to encompass a widespread cancer field defect involving normal prostate tissue. In the current study, we analyzed a series of DNA methylation field markers to determine if they predict the presence of PC in urine. Urine samples were collected from patients undergoing prostate biopsy with biopsy-proven PC (90), and without PC (77). From the urine pellet, methylated DNA was quantified across several previously identified CpG island regions near the caveolin 1 (CAV1), even-skipped homeobox 1 (EVX1), fibroblast growth factor 1 (FGF1), natural cytotoxicity triggering receptor 2 (NCR2) and phospholipase A and acyltransferase 3 (PLA2G16) genes using bisulfite pyrosequencing. Univariate and multivariate analyses were performed. Urine cell pellets show significant increases in methylation in four of the markers from patients with PC compared to those without PC including EVX1 12.2 vs. 7.7%, CAV1 15.7 vs. 10.36%, FGF1 12.0 vs. 7.1%, and PLA2G16 12.2 vs. 8.3% [all P<0.01]. Area under the ROC Curve (AUCs) were generated for EXV1 (0.74, Odds ratios (OR) 1.09; 95% confidence intervals (CI) 0.94-1.25, CAV1 (0.72, OR 1.18; 95% CI 1.09-1.28) and PLA2G16 (0.76, OR 1.35; 95% CI 1.199-1.51). In combination, a two-marker assay performs better than prostate specific antigen (PSA), AUC 0.77 vs. PSA AUC of 0.6 (P = 0.01) with the lowest error. In addition, FGF1 distinguished between grade group 1 (GG1) and higher grade cancers (P<0.03). In conclusion, applying methylation of field defect loci to urine samples provides a novel approach to distinguish patients with and without cancer.

A mechanism linking perinatal 2,3,7,8 tetrachlorodibenzo-p-dioxin exposure to lower urinary tract dysfunction in adulthood.

Benign prostatic hyperplasia/lower urinary tract dysfunction (LUTD) affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging paradigms about when this disease process begins. We delivered a single dose of a widespread environmental contaminant present in the serum of most Americans [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 1 µg/kg], and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues, and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.

Synthetic Lethal Metabolic Targeting of Androgen-Deprived Prostate Cancer Cells with Metformin.

The initiation of androgen-deprivation therapy (ADT) induces susceptibilities in prostate cancer cells that make them vulnerable to synergistic treatment and enhanced cell death. Senescence results in cell-cycle arrest, but cells remain viable. In this study, we investigated the mechanisms by which prostate cancer cells undergo senescence in response to ADT, and determined whether an FDA-approved antidiabetic drug metformin has a synergistic effect with ADT in prostate cancer both in vitro and in vivo Our results show that longer term exposure to ADT induced senescence associated with p16INK4a and/or p27kip2 induction. The activation of PI3K/AKT and inactivation of AMPK in senescent cells resulted in mTORC1 activation. In addition, the antiapoptotic protein XIAP expression was increased in response to ADT. The addition of metformin following ADT induced apoptosis, attenuated mTOR activation, reduced senescent cell number in vitro, and inhibited tumor growth in prostate cancer patient-derived xenograft models. This study suggests that combining ADT and metformin may be a feasible therapeutic approach to remove persistent prostate cancer cells after ADT.

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers.

BACKGROUND: The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. RESULTS: Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. CONCLUSION: These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.

Sox9 in mouse urogenital sinus epithelium mediates elongation of prostatic buds and expression of genes involved in epithelial cell migration.

Previous studies identified Sox9 as a critical mediator of prostate development but the precise stage when Sox9 acts had not been determined. A genetic approach was used to delete Sox9 from mouse urogenital sinus epithelium (UGE) prior to prostate specification. All prostatic bud types (anterior, dorsolateral and ventral) were stunted in Sox9 conditional knockouts (cKOs) even though the number of prostatic buds did not differ from that of controls. We concluded that Sox9 is required for prostatic bud elongation and compared control male, control female, Sox9 cKO male and Sox9 cKO female UGE transcriptomes to identify potential molecular mediators. We identified 702 sex-dependent and 95 Sox9-dependent genes. Thirty-one genes were expressed in both a sex- and Sox9-dependent pattern. A comparison of Sox9 cKO female vs control female UGE transcriptomes revealed 74 Sox9-dependent genes, some of which also function in cell migration. SOX9 regulates, directly or indirectly, a largely different profile of genes in male and female UGE. Eighty-three percent of Sox9-dependent genes in male UGE were not Sox9-dependent in female UGE. Only 16 genes were Sox9-dependent in the UGE of both sexes and seven had cell migration functions. These results support the notion that Sox9 promotes cell migration activities needed for prostate ductal elongation.

Combination therapy with androgen deprivation for hormone sensitive prostate cancer: A new frontier.

Androgen deprivation therapy (ADT) has been the standard of care for the last 75 years in metastatic hormone sensitive prostate cancer (PCa). However, this approach is rarely curative. Recent clinical trials have demonstrated that ADT combined with other agents, notably docetaxel and abiraterone, lead to improved survival. The mechanisms surrounding this improved cancer outcomes are incompletely defined. The response of cancer cells to ADT includes apoptosis and cell death, but a significant fraction remains viable. Our laboratory has demonstrated both in vitro and in vivo that cellular senescence occurs in a subset of these cells. Cellular senescence is a phenotype characterized by cell cycle arrest, senescence-associated ß-galactosidase (SA-ß-gal), and a hypermetabolic state. Positive features of cellular senescence include growth arrest and immune stimulation, although persistence may release cytokines and growth factors that are detrimental. Senescent tumor cells generate a catabolic state with increased glycolysis, protein turnover and other metabolic changes that represent targets for drugs, like metformin, to be applied in a synthetic lethal approach. This review examines the response to ADT and the putative role of cellular senescence as a biomarker and therapeutic target in this context.

sox9b is required in cardiomyocytes for cardiac morphogenesis and function.

The high mobility group transcription factor SOX9 is expressed in stem cells, progenitor cells, and differentiated cell-types in developing and mature organs. Exposure to a variety of toxicants including dioxin, di(2-ethylhexyl) phthalate, 6:2 chlorinated polyfluorinated ether sulfonate, and chlorpyrifos results in the downregulation of tetrapod Sox9 and/or zebrafish sox9b. Disruption of Sox9/sox9b function through environmental exposures or genetic mutations produce a wide range of phenotypes and adversely affect organ development and health. We generated a dominant-negative sox9b (dnsox9b) to inhibit sox9b target gene expression and used the Gal4/UAS system to drive dnsox9b specifically in cardiomyocytes. Cardiomyocyte-specific inhibition of sox9b function resulted in a decrease in ventricular cardiomyocytes, an increase in atrial cardiomyocytes, hypoplastic endothelial cushions, and impaired epicardial development, ultimately culminating in heart failure. Cardiomyocyte-specific dnsox9b expression significantly reduced end diastolic volume, which corresponded with a decrease in stroke volume, ejection fraction, and cardiac output. Further analysis of isolated cardiac tissue by RT-qPCR revealed cardiomyocyte-specific inhibition of sox9b function significantly decreased the expression of the critical cardiac development genes nkx2.5, nkx2.7, and myl7, as well as c-fos, an immediate early gene necessary for cardiomyocyte progenitor differentiation. Together our studies indicate sox9b transcriptional regulation is necessary for cardiomyocyte development and function.

Beneficial effects of quinoline-3-carboxamide (ABR-215757) on atherosclerotic plaque morphology in S100A12 transgenic ApoE null mice.

OBJECTIVE: There is an emerging widespread interest in the role of damage-associated molecular pattern molecules (DAMP) S100A8, S100A9 and S100A12 in cardiovascular and other diseases. In this study we tested the efficacy of ABR-215757, a S100 protein binding immuno-modulatory compound to stabilize atherosclerosis in transgenic ApoE null mice that express the human pro-inflammatory S100A12 protein within the smooth muscle cell (SM22α-S100A12). METHODS: Twelve-week old S100A12 transgenic/ApoE(-/-) and WT/ApoE(-/-) mice were treated with ABR-21575 for 5 weeks and were analyzed 4 month later. RESULTS: Surface plasmon resonance analysis demonstrated that S100A12 interacts with ABR-215757 in a zinc dependent manner in vitro. In vivo, ABR-215757 administration reduced features of advanced plaque morphology resulting in smaller necrotic cores, diminished intimal and medial vascular calcification, and reduced amount of infiltrating inflammatory cells. ABR-215757 normalized aortic expression of RAGE protein and normalized experimentally-induced delayed hypersensitivity. The effect of ABR-215757 was more prominent in ApoE(-/-) mice expressing S100A12 than in ApoE(-/-) animals lacking expression of human S100A12 protein. CONCLUSION: Our data suggest that S100A12 is important for progression of atherosclerosis and can be targeted by the small molecule ABR-215757. The specific binding of quinoline-3-carboxamides to S100A12 attenuates S100A12-mediated features of accelerated murine atherosclerosis.

S100A12 expression in thoracic aortic aneurysm is associated with increased risk of dissection and perioperative complications.

OBJECTIVES: The purpose of this study was to determine the relevance of S100A12 expression to human thoracic aortic aneurysms and type A thoracic aortic aneurysm dissection and to study mechanisms of S100A12-mediated dysfunction of aortic smooth muscle cells. BACKGROUND: Transgenic expression of proinflammatory S100A12 protein in murine aortic smooth muscle causes thoracic aneurysm in genetically modified mice. METHODS: Immunohistochemistry of aortic tissue (n = 50) for S100A12, myeloperoxidase, and caspase 3 was examined and S100A12-mediated pathways were studied in cultured primary aortic smooth muscle cells. RESULTS: We found S100A12 protein expressed in all cases of acute thoracic aortic aneurysm dissection and in approximately 25% of clinically stable thoracic aortic aneurysm cases. S100A12 tissue expression was associated with increased length of stay in patients undergoing elective surgical repair for thoracic aortic aneurysm, despite similar preoperative risk as determined by European System for Cardiac Operative Risk Evaluation. Reduction of S100A12 expression in human aortic smooth muscle cells using small hairpin RNA attenuates gene and protein expression of many inflammatory- and apoptosis-regulating factors. Moreover, genetic ablation of the receptor for S100A12, receptor for advanced glycation end products (RAGE), in murine aortic smooth muscle cells abolished cytokine-augmented activation of caspase 3 and smooth muscle cell apoptosis in S100A12-expressing cells. CONCLUSIONS: S100A12 is enriched in human thoracic aortic aneurysms and dissections. Reduction of S100A12 or genetic ablation of its cell surface receptor, the receptor for advanced glycation end products (RAGE), in aortic smooth muscle resulted in decreased activation of caspase 3 and in reduced apoptosis. By establishing a link between S100A12 expression and apoptosis of aortic smooth muscle cells, this study identifies novel S100A12 signaling pathways and indicates that S100A12 may be a useful molecular marker and possible target for treatment for human aortic diseases.