Quantitative profiling of cochlear synaptosomal proteins in cisplatin-induced synaptic dysfunction.

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction.

MicroRNA dysregulation in the heart and lung of infants with bronchopulmonary dysplasia.

BACKGROUND AND OBJECTIVES: Bronchopulmonary dysplasia (BPD) is a serious complication of preterm birth, resulting in significant morbidity and mortality. Recent studies have suggested that microRNA (miRNA) dysregulation is involved in the pathogenesis of BPD and may serve as biomarkers for early detection. We conducted a directed search for dysregulated miRNAs in lung and heart autopsy samples of infants with histologic BPD. METHODS: We used archived lung and heart samples from BPD (13 lung, 6 heart) and control (24 lung, 5 heart) subjects. To measure miRNA expression, RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue specimens then reverse-transcribed, labeled, and hybridized to miRNA microarrays. The microarrays were scanned, and data were quantile normalized. Statistical analysis with a moderated t-test and control of the false discovery rate (5%) was used to compare normalized miRNA expression values between clinical categories. RESULTS: With our set of 48 samples, 43 miRNAs had a significant difference in expression comparing BPD to non-BPD controls. Among the most statistically significant miRNAs, miR-378b, miRNA-184, miRNA-3667-5p, miRNA-3976, miRNA-4646-5p, and miRNA-7846-3p were all consistently upregulated in both the heart and lung tissues of BPD subjects. The cellular pathway predicted to be most affected by these miRNAs is the Hippo signaling pathway. CONCLUSIONS: This study identifies miRNAs that are similarly dysregulated in postmortem lung and heart samples in subjects with histologic BPD. These miRNAs may contribute to the pathogenesis of BPD, have potential as biomarkers, and may provide insight to novel approaches for diagnosis and treatment.

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia.

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

Exosomes in Epilepsy of Tuberous Sclerosis Complex: Carriers of Pro-Inflammatory MicroRNAs.

Exosomes are a class of small, secreted extracellular vesicles (EV) that have recently gained considerable attention for their role in normal cellular function, disease processes and potential as biomarkers. Exosomes serve as intercellular messengers and carry molecular cargo that can alter gene expression and the phenotype of recipient cells. Here, we investigated alterations of microRNA cargo in exosomes secreted by epileptogenic tissue in tuberous sclerosis complex (TSC), a multi-system genetic disorder that includes brain lesions known as tubers. Approximately 90% of TSC patients suffer from seizures that originate from tubers, and ~60% are resistant to antiseizure drugs. It is unknown why some tubers cause seizures while others do not, and the molecular basis of drug-resistant epilepsy is not well understood. It is believed that neuroinflammation is involved, and characterization of this mechanism may be key to disrupting the "vicious cycle" between seizures, neuroinflammation, and increased seizure susceptibility. We isolated exosomes from epileptogenic and non-epileptogenic TSC tubers, and we identified differences in their microRNA cargo using small RNA-seq. We identified 12 microRNAs (including miR-142-3p, miR-223-3p and miR-21-5p) that are significantly increased in epileptogenic tubers and contain nucleic acid motifs that activate toll-like receptors (TLR7/8), initiating a neuroinflammatory cascade. Exosomes from epileptogenic tissue caused induction of key pathways in cultured cells, including innate immune signaling (TLR), inflammatory response and key signaling nodes SQSTM1 (p62) and CDKN1A (p21). Genes induced in vitro were also significantly upregulated in epileptogenic tissue. These results provide new evidence on the role of exosomes and non-coding RNA cargo in the neuroinflammatory cascade of epilepsy and may help advance the development of novel biomarkers and therapeutic approaches for the treatment of drug-resistant epilepsy.

Levels of plasma glycan-binding auto-IgG biomarkers improve the accuracy of prostate cancer diagnosis.

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥ 2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment.

Monozygotic twins with neuroblastoma MS have a similar molecular profile: a case of twin-to-twin metastasis.

Fetoplacental neuroblastoma metastasis has been postulated as a mechanism accounting for concordant cases where one twin develops a primary tumour and the second twin manifests the disease without an identifiable primary site. These tumours may originate and spread concomitantly due to the same genetic background shared by monozygotic twins. This study investigated the molecular profile of stage MS neuroblastoma presenting concomitantly in monozygotic twins. Comparative genomic hybridisation (aCGH) was done for each of the twin liver tumour and peripheral blood samples at diagnosis. Comparison of copy-number variation (CNV) regions revealed a set of CNVs that were common to both tumour specimens and not apparent in the blood. The CNV signature in both twins' tumours was highly similar, suggesting a common clonal origin. Additional findings included large deletion of chromosome 10 and amplification of chromosome 17. Notably, both liver samples had amplification of a short region involving DEIN (chromosome 4q34.1). Similar CNVs strongly support a common clonal origin and metastatic spread from one twin to the other. DEIN is a long-coding RNA (IncRNA) that has been found highly expressed in stage MS neuroblastoma and is likely involved in biological processes such as cell migration and metastasis.

Exposure of Larval Zebrafish to the Insecticide Propoxur Induced Developmental Delays that Correlate with Behavioral Abnormalities and Altered Expression of hspb9 and hspb11.

Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed.

TLR7 activation in epilepsy of tuberous sclerosis complex.

BACKGROUND: Neuroinflammation and toll-like receptors (TLR) of the innate immune system have been implicated in epilepsy. We previously reported high levels of microRNAs miR-142-3p and miR-223-3p in epileptogenic brain tissue resected for the treatment of intractable epilepsy in children with tuberous sclerosis complex (TSC). As miR-142-3p has recently been reported to be a ligand and activator of TLR7, a detector of exogenous and endogenous single-stranded RNA, we evaluated TLR7 expression and downstream IL23A activation in surgically resected TSC brain tissue. METHODS: Gene expression analysis was performed on cortical tissue obtained from surgery of TSC children with pharmacoresistent epilepsy. Expression of TLRs 2, 4 and 7 was measured using NanoString nCounter assays. Real-time quantitative PCR was used to confirm TLR7 expression and compare TLR7 activation, indicated by IL-23A levels, to levels of miR-142-3p. Protein markers characteristic for TLR7 activation were assessed using data from our existing quantitative proteomics dataset of TSC tissue. Capillary electrophoresis Western blots were used to confirm TLR7 protein expression in a subset of samples. RESULTS: TLR7 transcript expression was present in all TSC specimens. The signaling competent form of TLR7 protein was detected in the membrane fraction of each sample tested. Downstream activation of TLR7 was found in epileptogenic lesions having elevated neuroinflammation indicated by clinical neuroimaging. TLR7 activity was significantly associated with tissue levels of miR-142-3p. CONCLUSION: TLR7 activation by microRNAs may contribute to the neuroinflammatory cascade in epilepsy in TSC. Further characterization of this mechanism may enable the combined of use of neuroimaging and TLR7 inhibitors in a personalized approach towards the treatment of intractable epilepsy.

The extrema of circulating miR-17 are identified as biomarkers for aggressive prostate cancer.

MicroRNAs (miRNAs) constitute short non-coding RNAs that can post-transcriptionally modulate the expression of many oncogenes and tumor suppressor genes engaged in key cellular processes. Deregulated serum miRNA signatures have been detected in various solid cancers including prostate cancer, suggesting that circulating miRNAs could function as non-invasive biomarkers of tumor emergence and progression. To determine whether serum miRNA expression levels are different between patients with aggressive and non-aggressive prostate cancer, we analyzed a panel of miRNAs from the blood of African American (AA) prostate cancer patients using a new recursive partitioning method that allows hypothesis testing of each split. We observed that both extrema of circulating miR-17, i.e. upregulation and downregulation, are associated with aggressive prostate cancer. A similar effect was observed in tumor samples from a separate dataset representing a different population of prostate cancer patients and in AA prostate cancer samples from the TCGA. The dual effect is consistent with the contradictory findings on the role of miR-17 in prostate cancer progression, whereby it controls important oncogenic and tumor-suppressive genes.

Global Signaling Profiling in a Human Model of Tumorigenic Progression Indicates a Role for Alternative RNA Splicing in Cellular Reprogramming.

Intracellular signaling is controlled to a large extent by the phosphorylation status of proteins. To determine how human breast cells can be reprogrammed during tumorigenic progression, we profiled cell lines in the MCF10A lineage by phosphoproteomic analyses. A large cluster of proteins involved in RNA splicing were hypophosphorylated as cells progressed to a hyperplastic state, and then hyperphosphorylated after progression to a fully metastatic phenotype. A comprehensive transcriptomic approach was used to determine whether alterations in splicing factor phosphorylation status would be reflected in changes in mRNA splicing. Results indicated that the degree of mRNA splicing trended with the degree of tumorigenicity of the 4 cell lines tested. That is, highly metastatic cell cultures had the greatest number of genes with splice variants, and these genes had greater fluctuations in expression intensities. Genes with high splicing indices were mapped against gene ontology terms to determine whether they have known roles in cancer. This group showed highly significant associations for angiogenesis, cytokine-mediated signaling, cell migration, programmed cell death and epithelial cell differentiation. In summary, data from global profiling of a human model of breast cancer development suggest that therapeutics should be developed which target signaling pathways that regulate RNA splicing.

A study of circulating microRNAs identifies a new potential biomarker panel to distinguish aggressive prostate cancer.

Prostate cancer is one of the most common cancers in men worldwide. Currently available diagnostic and prognostic tools for this disease, such as prostate specific antigen, suffer from lack of specificity and sensitivity, resulting in over- and misdiagnosis. Hence, there is an urgent need for clinically relevant biomarkers capable of distinguishing between aggressive and nonaggressive forms of prostate cancer to aid in stratification, management and therapeutic decisions. To address this unmet need, we investigated the patterns of expression of a panel of 68 plasma-derived microRNAs (miRNAs) in a cohort of African American (AA) and European American (EA) prostate cancer patients (n = 114). miRNA qPCR results were analyzed using in-depth statistical methods, and a bioinformatics analysis was conducted to identify potential targets of the differentially expressed miRNAs. Our data demonstrate that a new previously unreported circulating miRNA signature consisting of a combination of interacting miRNAs (miR-17/miR-192) and an independent miRNA (miR-181a) are capable of segregating aggressive and nonaggressive prostate cancer in both AA and EA patients. The interacting miRNAs outperformed independent miRNAs in identifying aggressiveness. Our results suggest that these circulating miRNAs may constitute novel biomarkers of prostate cancer aggressiveness in both races and warrant further investigation.

A nutrigenetic approach for investigating the chemopreventive effects of black raspberries during the development of preneoplastic esophagi in rats.

BACKGROUND: Large epidemiological studies have shown that diets high in fruits reduce the risk of esophageal squamous cell carcinoma (ESCC). OBJECTIVE: The current study investigated the effects of black raspberries (BRBs) on gene expression during the development of preneoplastic esophagi in rats. METHODS: Using a post-initiation protocol, F344 rats were injected with N-nitrosomethylbenzylamine (NMBA) and then fed either a control diet or 5% BRBs. At weeks 9, 15, and 35, we euthanized subgroups of the rats and collected preneoplastic esophagi to isolate RNA samples for DNA microarray. RESULTS: Along the development of NMBA-induced preneoplastic esophagi, NMBA injections led to differential expression of 1181 genes comparing to control rats, and dietary BRBs modulated 428 genes in esophagi from NMBA-treated rats. There are 137 common genes between 1181 and 428 gene sets, and BRBs significantly reversed the expression of 133 genes. These genes are associated with multiple gene oncology functions. BRBs induced an 8.8-fold gene enrichment on the pathway of inflammatory response and regulated 10 genes involved in this pathway. Among them, BRBs significantly reversed the expression of pro-inflammatory cytokines, such as CCL2, S100A8, and IL19. CONCLUSIONS: BRBs exhibit strong anti-inflammatory effects against NMBA-induced rat esophageal tumorigenesis.

Neuroinflammatory Nexus of Pediatric Epilepsy.

A rapidly growing body of evidence supports the premise that neuroinflammation plays an important role in initiating and sustaining seizures in a range of pediatric epilepsies. Clinical and experimental evidence indicate that neuroinflammation is both an outcome and a contributor to seizures. In this manner, seizures that arise from an initial insult (e.g. infection, trauma, genetic mutation) contribute to an inflammatory response that subsequently promotes recurrent seizures. This cyclical relationship between seizures and neuroinflammation has been described as a 'vicious cycle.' Studies of human tissue resected for surgical treatment of refractory epilepsy have reported activated inflammatory and immune signaling pathways, while animal models have been used to demonstrate that key inflammatory mediators lead to increased seizure susceptibility. Further characterization of the molecular mechanisms involved in this cycle may ultimately enable the development of new therapeutic approaches for the treatment of epilepsy. In this brief review we focus on key inflammatory mediators that have become prominent in recent literature of epilepsy, including newly characterized microRNAs and their potential role in neuroinflammatory signaling.

A distinct microRNA expression profile is associated with α[11C]-methyl-L-tryptophan (AMT) PET uptake in epileptogenic cortical tubers resected from patients with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in various organs and arises due to mutations in the TSC1 or TSC2 genes. TSC mutations lead to a range of neurological manifestations including epilepsy, cognitive impairment, autism spectrum disorders (ASD), and brain lesions that include cortical tubers. There is evidence that seizures arise at or near cortical tubers, but it is unknown why some tubers are epileptogenic while others are not. We have previously reported increased tryptophan metabolism measured with α[11C]-methyl-l-tryptophan (AMT) positron emission tomography (PET) in epileptogenic tubers in approximately two-thirds of patients with tuberous sclerosis and intractable epilepsy. However, the underlying mechanisms leading to seizure onset in TSC remain poorly characterized. MicroRNAs are enriched in the brain and play important roles in neurodevelopment and brain function. Recent reports have shown aberrant microRNA expression in epilepsy and TSC. In this study, we performed microRNA expression profiling in brain specimens obtained from TSC patients undergoing epilepsy surgery for intractable epilepsy. Typically, in these resections several non-seizure onset tubers are resected together with the seizure-onset tubers because of their proximity. We directly compared seizure onset tubers, with and without increased tryptophan metabolism measured with PET, and non-onset tubers to assess the role of microRNAs in epileptogenesis associated with these lesions. Whether a particular tuber was epileptogenic or non-epileptogenic was determined with intracranial electrocorticography, and tryptophan metabolism was measured with AMT PET. We identified a set of five microRNAs (miR-142-3p, 142-5p, 223-3p, 200b-3p and 32-5p) that collectively distinguish among the three primary groups of tubers: non-onset/AMT-cold (NC), onset/AMT-cold (OC), and onset/AMT-hot (OH). These microRNAs were significantly upregulated in OH tubers compared to the other two groups, and microRNA expression was most significantly associated with AMT-PET uptake. The microRNAs target a group of genes enriched for synaptic signaling and epilepsy risk, including SLC12A5, SYT1, GRIN2A, GRIN2B, KCNB1, SCN2A, TSC1, and MEF2C. We confirmed the interaction between miR-32-5p and SLC12A5 using a luciferase reporter assay. Our findings provide a new avenue for subsequent mechanistic studies of tuber epileptogenesis in TSC.

Black raspberries demethylate Sfrp4, a WNT pathway antagonist, in rat esophageal squamous cell papilloma.

Aberrant methylation of DNA is a common event in the development of cancers, including squamous cell carcinoma (SCC) of the human esophagus. In the present study, we determined: (a) whether aberrant DNA methylation also occurs in the development of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus, a model of human esophageal SCC; and (b) if so, whether dietary black raspberries (BRBs) are capable of preventing this aberrant DNA methylation. A diet containing 5% BRBs inhibited the development of NMBA-induced tumors in the rat esophagus. This inhibition was associated with reduced mRNA levels of the DNA methyltransferases, Dnmt1 and Dnmt3b, in both dysplastic lesions and in papillomas of the esophagus. In addition, promoter methylation of Sfrp4, a WNT pathway antagonist, was significantly reduced by the berry diet, and this was associated with decreased nuclear localization of ß-CATENIN and reduced expression of c-MYC protein in NMBA-treated esophagi. Decreased promoter methylation of Sfrp4 correlated with decreased expression of Dmnt3b and, ultimately, with increased Sfrp4 mRNA expression. This suggests that epigenetic alterations in NMBA-induced rat esophageal tumorigenesis recapitulate epigenetic events in human esophageal SCC, and that BRBs could be useful in preventing the aberrant DNA methylation involved in the development of human esophageal SCC. © 2015 Wiley Periodicals, Inc.

Gene Signature of High White Blood Cell Count in B-Precursor Acute Lymphoblastic Leukemia.

In this study we sought to identify genetic factors associated with the presenting white blood cell (WBC) count in B-precursor acute lymphoblastic leukemia (BP-ALL). Using ETV6-RUNX1-positive BP-ALL patient samples, a homogeneous subtype, we identified 16 differentially expressed genes based on the presenting WBC count (< 50,000/cumm vs > 50,000). We further confirmed that IL1R1, BCAR3, KCNH2, PIR, and ZDHHC23 were differentially expressed in a larger cohort of ETV6-RUNX1-negative BP-ALL patient samples. Statistical analysis demonstrated that expression levels of these genes could accurately categorize high and low WBC count subjects using two independent patient sets, representing positive and negative ETV6-RUNX1 cases. Further studies in leukemia cell line models will better delineate the role of these genes in regulating the white blood cell count and potentially identify new therapeutic targets.

Differential Regulation of Gene Expression by Cholesterol Biosynthesis Inhibitors That Reduce (Pravastatin) or Enhance (Squalestatin 1) Nonsterol Isoprenoid Levels in Primary Cultured Mouse and Rat Hepatocytes.

Squalene synthase inhibitors (SSIs), such as squalestatin 1 (SQ1), reduce cholesterol biosynthesis but cause the accumulation of isoprenoids derived from farnesyl pyrophosphate (FPP), which can modulate the activity of nuclear receptors, including the constitutive androstane receptor (CAR), farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs). In comparison, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (e.g., pravastatin) inhibit production of both cholesterol and nonsterol isoprenoids. To characterize the effects of isoprenoids on hepatocellular physiology, microarrays were used to compare orthologous gene expression from primary cultured mouse and rat hepatocytes that were treated with either SQ1 or pravastatin. Compared with controls, 47 orthologs were affected by both inhibitors, 90 were affected only by SQ1, and 51 were unique to pravastatin treatment (P < 0.05, ≥1.5-fold change). When the effects of SQ1 and pravastatin were compared directly, 162 orthologs were found to be differentially coregulated between the two treatments. Genes involved in cholesterol and unsaturated fatty acid biosynthesis were up-regulated by both inhibitors, consistent with cholesterol depletion; however, the extent of induction was greater in rat than in mouse hepatocytes. SQ1 induced several orthologs associated with microsomal, peroxisomal, and mitochondrial fatty acid oxidation and repressed orthologs involved in cell cycle regulation. By comparison, pravastatin repressed the expression of orthologs involved in retinol and xenobiotic metabolism. Several of the metabolic genes altered by isoprenoids were inducible by a PPARα agonist, whereas cytochrome P450 isoform 2B was inducible by activators of CAR. Our findings indicate that SSIs uniquely influence cellular lipid metabolism and cell cycle regulation, probably due to FPP catabolism through the farnesol pathway.

AP-2ß Is a Downstream Effector of PITX2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development.

PURPOSE: The homeodomain transcription factor, PITX2, is at the apex of a genetic pathway required for corneal development, but the critical effector genes regulated by the PITX2 remain unknown. The purpose of this study was to discover and validate PITX2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea. METHODS: Microarrays were used to compare gene expression in corneas isolated from temporal Pitx2 knockout embryos and control littermates. Quantitative RT-PCR and immunohistochemistry was used to further validate Tfap2b expression differences in Pitx2 knockout versus control corneas. In situ hybridization and protein immunohistochemistry were used to assay eyes of a Tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea, blood vessel endothelium, or lymphatic vessel endothelium. RESULTS: We show that PITX2 is required for the expression of Tfap2b, encoding the AP-2ß transcription factor, in the neural crest during corneal development. Markers of differentiated corneal epithelium and stroma are expressed in the absence of AP-2ß. In contrast, markers of differentiated corneal endothelium are not expressed in the absence of AP-2ß. Endomucin+ blood vessels are present throughout the developing corneal stroma in the absence of AP-2ß, whereas LYVE1+ lymphatic vessels are not found. CONCLUSIONS: The AP-2ß transcription factor is an important effector of PITX2 function during corneal development, required for differentiation of corneal endothelium and establishment of angiogenic privilege. Unlike PITX2, AP-2ß is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis, nor establishment of lymphangiogenic privilege. Therefore, additional PITX2-dependent factors likely regulate these latter processes during embryonic development. These results extend our understanding of the genetic mechanisms regulating cornea development.

Amplification of WHSC1L1 regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over-expression in breast cancer.

The 8p11-p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B-type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate that over-expression of WHSC1L1 is linked to over-expression of ERα in SUM-44 breast cancer cells and in primary human breast cancers. Knock-down of WHSC1L1, particularly WHSC1L1-short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock-down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP-Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 motifs under estrogen-free conditions and regulated expression of estrogen-responsive genes. Short-term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen-free conditions. Finally, knock-down of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions, which was restored upon exposure to estradiol. These results indicate the SUM-44 cells are a good model of a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over-express WHSC1L1, and over-express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.