Genomic insights into triple-negative and HER2-positive breast cancers using isogenic model systems

Introduction In general, genomic signatures of breast cancer subtypes have little or no overlap owing to the heterogeneous genetic backgrounds of study samples. Thus, obtaining a reliable signature in the context of isogenic nature of the cells has been challenging and the precise contribution of isogenic triple negative breast cancer (TNBC) versus non-TNBC remains poorly defined. Methods We established isogenic stable cell lines representing TNBC and Human Epidermal Growth Factor Receptor 2 positive (HER2+) breast cancers by introducing HER2 in TNBC cell lines MDA-MB-231 and MDA-MB-468. We examined protein level expression and functionality of the transfected receptor by treatment with an antagonist of HER2. Using microarray profiling, we obtained a comprehensive gene list of differentially expressed between TNBC and HER2+ clones. We identified and validated underlying isogenic components using qPCR and also compared results with expression data from patients with similar breast cancer subtypes. Results We identified 544 and 1087 statistically significant differentially expressed genes between isogenic TNBC and HER2+ samples in MDA-MB-231 and MDA-MB-468 backgrounds respectively and a shared signature of 49 genes. By comparing results from MDA-MB-231 and MDA-MB-468 backgrounds with two patient microarray datasets, we identified 17 and 22 common genes with same expression trend respectively. Additionally, we identified 56 and 78 genes from MDA-MB-231 and MDA-MB-468 comparisons respectively present in our published RNA-seq data. Conclusions Using our unique model system, we have identified an isogenic gene expression signature between TNBC and HER2+ breast cancer. A portion of our results was also verified in patient data samples, indicating an existence of isogenic element associated with HER2 status between genetically heterogeneous breast cancer samples. These findings may potentially contribute to the development of molecular platform that would be valuable for diagnostic and therapeutic decision for TNBC and in distinguishing it from HER2+ subtype

30S Beam Development and X-ray Bursts

Over the past three years, we have worked on developing a well-characterized 30S radioactive beam to be used in a future experiment aiming to directly measure the 30S(alpha,p) stellar reaction rate within the Gamow window of Type I X-ray bursts. The importance of the 30S(alpha,p) reaction to X-ray bursts is discussed. Given the astrophysical motivation, the successful results of and challenges involved in the production of a low-energy 30S beam are detailed. Finally, an overview of our future plans regarding this on-going project are presented.Comment: 7 pages, 2 figures, 5th European Summer School on Experimental Nuclear Astrophysics, Santa Tecla, Sicily, September 200

Two-Dimensional Molecular Patterning by Surface-Enhanced Zn-Porphyrin Coordination

In this contribution, we show how zinc-5,10,15,20-meso-tetradodecylporphyrins (Zn-TDPs) self-assemble into stable organized arrays on the surface of graphite, thus positioning their metal center at regular distances from each other, creating a molecular pattern, while retaining the possibility to coordinate additional ligands. We also demonstrate that Zn-TDPs coordinated to 3-nitropyridine display a higher tendency to be adsorbed at the surface of highly oriented pyrolytic graphite (HOPG) than noncoordinated ones. In order to investigate the two-dimensional (2D) self-assembly of coordinated Zn-TDPs, solutions with different relative concentrations of 3-nitropyridine and Zn-TDP were prepared and deposited on the surface of HOPG. STM measurements at the liquid-solid interface reveal that the ratio of coordinated Zn-TDPs over noncoordinated Zn-TDPs is higher at the n-tetradecane/HOPG interface than in n-tetradecane solution. This enhanced binding of the axial ligand at the liquid/solid interface is likely related to the fact that physisorbed Zn-TDPs are better binding sites for nitropyridines.

Glomerular-specific protein kinase C-β-induced insulin receptor substrate-1 dysfunction and insulin resistance in rat models of diabetes and obesity

Insulin resistance has been associated with the progression of chronic kidney disease in both diabetes and obesity. In order to determine the cellular mechanisms contributing to this, we characterized insulin signaling in renal tubules and glomeruli during diabetic and insulin-resistant states using streptozotocin-diabetic and Zucker fatty-insulin-resistant rats. Compared with nondiabetic and Zucker lean rats, the insulin-induced phosphorylation of insulin receptor substrate-1 (IRS1), Akt, endothelial nitric oxide synthase, and glycogen synthase kinase 3α were selectively inhibited in the glomeruli but not in the renal tubules of both respective models. Protein, but not mRNA levels of IRS1, was decreased only in the glomeruli of streptozotocin-diabetic rats likely due to increased ubiquitination. Treatment with the protein kinase C-β inhibitor, ruboxistaurin, enhanced insulin actions and elevated IRS1 expression. In glomerular endothelial cells, high glucose inhibited the phosphorylation of Akt, endothelial nitric oxide synthase, and glycogen synthase kinase 3α; decreased IRS1 protein expression and increased its association with ubiquitin. Overexpression of IRS1 or the addition of ruboxistaurin reversed the inhibitory effects of high glucose. Thus, loss of insulin's effect on endothelial nitric oxide synthase and glycogen synthase kinase 3α activation may contribute to the glomerulopathy observed in diabetes and obesity

Novel insights into breast cancer genetic variance through RNA sequencing

Using RNA sequencing of triple-negative breast cancer (TNBC), non-TBNC and HER2-positive breast cancer sub-types, here we report novel expressed variants, allelic prevalence and abundance, and coexpression with other variation, and splicing signatures. To reveal the most prevalent variant alleles, we overlaid our findings with cancer- and population-based datasets and validated a subset of novel variants of cancer-related genes: ESRP2, GBP1, TPP1, MAD2L1BP, GLUD2 and SLC30A8. As a proof-of-principle, we demonstrated that a rare substitution in the splicing coordinator ESRP2(R353Q) impairs its ability to bind to its substrate FGFR2 pre-mRNA. In addition, we describe novel SNPs and INDELs in cancer relevant genes with no prior reported association of point mutations with cancer, such as MTAP and MAGED1. For the first time, this study illustrates the power of RNA-sequencing in revealing the variation landscape of breast transcriptome and exemplifies analytical strategies to search regulatory interactions among cancer relevant molecules

Bidirectional autoregulatory mechanism of metastasis-associated protein 1-alternative reading frame pathway in oncogenesis

Although metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling and histone deacetylation complex, is widely up-regulated in human cancers and correlates with tumor metastasis, its regulatory mechanism and related signaling pathways remain unknown. Here, we report a previously unrecognized bidirectional autoregulatory loop between MTA1 and tumor suppressor alternative reading frame (ARF). MTA1 transactivates ARF transcription by recruiting the transcription factor c-Jun onto the ARF promoter in a p53-independent manner. ARF, in turn, negatively regulates MTA1 expression independently of p53 and c-Myc. In this context, ARF interacts with transcription factor specificity protein 1 (SP1) and promotes its proteasomal degradation by enhancing its interaction with proteasome subunit regulatory particle ATPase 6, thereby abrogating the ability of SP1 to stimulate MTA1 transcription. ARF also physically associates with MTA1 and affects its protein stability. Thus, MTA1-mediated activation of ARF and ARF-mediated functional inhibition of MTA1 represent a p53-independent bidirectional autoregulatory mechanism in which these two opposites act in concert to regulate cell homeostasis and oncogenesis, depending on the cellular context and the environment

Effects of Soil Types and Fertilizers on Growth, Yield, and Quality of Edible Amaranthus tricolor lines in Okinawa, Japan

Soil types and fertilizer regimes were evaluated on growth, yield, and quality of Amaranthus tricolor lines, IB (India Bengal), TW (Taiwan), BB (Bangladesh B), and BC (Bangladesh C) in developing management practices in Okinawa. Growth and yield of all amaranth lines were higher in gray soil (pH 8.4) than in dark red soil (pH 6.6) and red soil (pH 5.4). The combined NPK fertilizer resulted in highest growth parameters and yield of amaranths in all soils. Nitrogen fertilizer alone did not affect growth parameters and yield of amaranths in dark red and red soils. Growth parameters and yield increased similarly with the 30, 40, and 50 g m−2 of NPK fertilizer in BB line, and with the 20, 30, 40, and 50 g m−2 in BC line. Agronomic efficiency of NPK fertilizer at 50 g m−2 was not prominent on the amaranths, compared to the fertilizer at 40 g m−2. Amaranth lines had higher Na in dark red and red soils, while K and Mg in gray soil, Ca in gray and red soils, and Fe in dark red soil. The NPK fertilizer resulted in higher Na, Ca, Mg, and P in BB line in glasshouse. These minerals in BB line were not clearly affected, but in BC line were lower with NPK fertilizer at 20–50 g m−2 in field. These studies indicate that gray soil is best for amaranth cultivation and combined NPK fertilizer at 20–40 g m−2 is effective in gray soil in Okinawa for higher yield and minerals of amaranth

High-Throughput Screening of Australian Marine Organism Extracts for Bioactive Molecules Affecting the Cellular Storage of Neutral Lipids

Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 µM inhibiting LD formation and triglyceride accumulation by ∼50% in the presence of 50 µM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo