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A thermostable drug suppresses a pERK-FosB/ΔFosB-VCAM-1 axis, endothelial activation, angiogenesis, and retinal permeability., Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months’ storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

Smooth muscle cells (SMC) in blood vessels are normally growth quiescent and transcriptionally inactive. Our objective was to understand promoter usage and dynamics in SMC acutely exposed to a prototypic growth factor or pro-inflammatory cytokine. Using cap analysis gene expression (FANTOM5 project) we report differences in promoter dynamics for immediate-early genes (IEG) and other genes when SMC are exposed to fibroblast growth factor-2 or interleukin-1β. Of the 1871 promoters responding to FGF2 or IL-1β considerably more responded to FGF2 (68.4%) than IL-1β (18.5%) and 13.2% responded to both. Expression clustering reveals sets of genes induced, repressed or unchanged. Among IEG responding rapidly to FGF2 or IL-1β were FOS, FOSB and EGR-1, which mediates human SMC migration. Motif activity response analysis (MARA) indicates most transcription factor binding motifs in response to FGF2 were associated with a sharp induction at 1 h, whereas in response to IL-1β, most motifs were associated with a biphasic change peaking generally later. MARA revealed motifs for FOS_FOS{B,L1}_JUN{B,D} and EGR-1..3 in the cluster peaking 1 h after FGF2 exposure whereas these motifs were in clusters peaking 1 h or later in response to IL-1β. Our findings interrogating CAGE data demonstrate important differences in promoter usage and dynamics in SMC exposed to FGF2 or IL-1β.

Uncaging promoter and enhancer dynamics In order to understand cellular differentiation, it is important to understand the timing of the regulation of gene expression. Arner et al. used cap analysis of gene expression (CAGE) to analyze gene enhancer and promoter activities in a number of human and mouse cell types. The RNA of enhancers was transcribed first, followed by that of transcription factors, and finally by genes that are not transcription factors. Science , this issue p. 1010

Immediate-early genes are those that are rapidly induced in response to a cellular stimulus in the absence of protein synthesis and can influence the biology and pathobiology of the cell. These span transcription factors, cytokines, growth factors, enzymes, secreted factors, cytoskeletal proteins, transporters and anti-apoptotic proteins that are attractive targets for the control of pathologic angiogenesis. This chapter focuses on immediate-early genes and specifically their regulation of processes underpinning angiogenesis.

Background and Aim: MicroRNAs (miRNAs or miRs) are an abundant class of short (17 to 25 nt) non-coding single-stranded RNAs that are significant controllers of cellular gene expression. Mature miRNA is created from long primary genomic transcripts (pri-miRNA), which are processed in the nucleus. The resulting 60-80-nucleotide precursor miRNAs (pre-miRNAs) are transferred to the cytoplasm and are treated to produce a short RNA duplex. One strand of the duplex is degraded, the remaining single-stranded miRNA molecule associates with members of the Argonaute protein family, to form the RNA-induced silencing complex (RISC). RISC plus the guide miRNA bind to the 3′untranslated region (3′-UTR) of the targeted mRNA and induce gene down-regulation by mRNA cleavage or translational repression. Serum stimulation of cancer cells has been shown to prompt positive regulatory transcription factors, such as c-Jun, c-Fos and Egr-1, as well as increasing transcriptional repressors such as YY1, NAB2 and GCF2. These transcription factors play critical parts in the early stimulation of cancer disease phenotypes and are prime targets for clarifying disease pathways and as therapeutic candidates. The identification of microRNAs regulating the mRNAs of transcription factors will improve our understanding of the mechanism of disease gene stimulation and subsequent pathway initiation leading to cancer disease phenotypes. MicroRNAs that ablate disease phenotypes are possible pharmaceutical therapies. Our aim was to identify and profile microRNAs that interact with the mRNA of transcription factors such as c-Jun and c-Fos. Results: We measured the expresion of c-Jun and c-Fos at the mRNA and protein levels (time course 0 to 6 hrs) in MM200 (human melanoma). We measured the expression of two microRNAs, miR155 and miR125b, which have been reported as playing a role in regulating c-Jun and c-Fos in Dermal Fibroblasts and melanoma cells (Song, Liu et al. Cellular Physiology and Biochemistry 2012 and Kappelmann, Kuphal et al. Oncogene 2013). We show that miR-155 and miR-125b expression is repressed following serum activation in these two cell lines (HEK293 and MM200). Loss of function and gain-of-function studies in these cancer cell lines, by supressing and overexpressing miR155/125b were performed. Subsquently, a target 3′UTR vs microRNA study was conducted that demonstrated that the miR-155 and miR-125b have the ability to recognize and repress c-Jun and c-Fos. Moreover, phenotypic studies such as migration, proliferation, cell cycle were performed. Conclusions: miR-155 and miR-125b are able to regulate c-Jun and c-Fos mRNAs and to inhibit their expression at the protein level. Citation Format: Ahmad M.N Alhendi, Leonel Prado-Lourenço, Noel Whitaker. Targeting c-Jun and c-Fos using microRNA's has a potential in contesting melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2015-2114

Melanoma is one the most commonly diagnosed cancers in Australia and while treatment is generally successful if the tumor is identified early, once the disease begins to spread the prognosis is considerably worse. The recent approval of new targeted therapies (Vemurafenib, Dabrafenib and Trametinib) has improved progression-free survival for patients carrying the common V600- B-Raf mutations. However even when these inhibitors are used in combination many tumors still develop resistance and furthermore there is a lack of treatment options available for patients who do not carry the mutated form of B-Raf. Therefore there is a pressing need for new therapeutics to address this deficit. The AP-1 transcription factor family has been shown to play an important role in numerous aspects of melanoma tumorgenesis including cellular proliferation and survival, making these transcription factors attractive targets for new treatments. Therefore, a high throughput screening was performed on several libraries of chemical compounds to identify those capable of inhibiting AP-1 directed transcription in vitro. Two lead compounds (X and BT2) were identified for further testing. From these a number of analogues were produced to modify their activity. Those that showed the most improvement above the leads against several melanoma lines were selected for further testing. The in vitro activity of these compounds was characterised in detail against two human melanoma cell lines, one wild type for B-Raf and one carrying the common V600E mutation. In 2D culture both compounds inhibit melanoma cell proliferation; induce apoptosis and cell cycle arrest and inhibit clonogenic capacity. These compounds have little cytotoxicity (LDH) and retain their activity for at least a month under a variety of environmental conditions. Furthermore in 3D spheroid assays the compounds show similar levels of activity. In vivo proof-of-concept testing is underway to assess the compounds activity in immunocompetent (xenograft) and immunocompromised (allograft) model. In parallel, bioinformatics analysis of mass spectrometry, kinome and microarray data is underway to identify the exact mechanism of action and the specificity of these inhibitors. In summary, the identified compounds show promising anti-melanoma activity in vitro. They inhibit human melanoma growth and clonogenic capacity and induce apoptosis and cell cycle arrest in 2D culture at submicromolar concentrations, comparable to the clinically approved drug Tramatenib. They show little cytotoxicity and also inhibit growth in a 3D spheroid model at submicromolar concentrations. The AP-1 transcription family, in particular c-Jun, have been shown to be important players in the development of melanoma. As such, these compounds may prove to be effective treatments for melanoma irrespective of B-Raf status and warrant further investigation both in vitro and in vivo. Citation Format: Kylie M. Taylor, Ahmad MN Alhendi, Gary Halliday, Lionel Prado-Lourenço. Novel transcriptional inhibitors for the inhibition of melanoma growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2015-1647