Martin Tenniswood

Background: The efficacy of epigenetic drugs, such as histone deacetylase inhibitors, is often diminished by poor aqueous solubility resulting in limited bioavailability and a low therapeutic index. To overcome the suboptimal therapeutic index, we have developed a biocompatible starch nanoparticle formulation of CG-1521, a histone deacetylase inhibitor in preclinical development for hard-to-treat breast cancers, which improves its bioavailability and half-life. Methods: The physicochemical parameters (size, zeta potential, morphology, loading, and release kinetics) of these nanoparticles (CG-NPs) have been optimized and their cytotoxic and apoptotic capacities measured in MCF-7 breast cancer cell line. The mechanism of action of the encapsulated drug was compared with the free drug at molecular level. Results: We show that encapsulation of CG-1521 substantially reduces the release rate of drug and provides a significantly enhanced cytotoxic ability of nanoparticles compared with equivalent dose of free CG-1521. CG-NPs induced cell cycle arrest and significant apoptosis in MCF-7 cells in vitro. The biological action of encapsulated drug has the similar impact with free drug on gene expression. Conclusion: The findings suggest that encapsulation of CG-1521 into starch nanoparticles can improve drug delivery of histone deacetylase inhibitors for breast cancer therapy without interfering with the mechanism of action of the drug.

The role of microRNAs (miRNA) in estrogen receptor (ER) signaling in the uterus and in endometrial cancer is not well understood. We therefore analyzed miRNA expression in uterine samples from a standard 3‐day uterotrophic assay using young female adult rats to identify E2‐regulated miRNAs. Microarray analysis identified 47 E2 down‐regulated miRNAs including miR‐30a, and 25 E2up‐regulated miRNAs including miR‐672, miR‐203, and miR‐146b. The strongly E2‐upregulated miR‐203 was selected for further analysis. miR‐203 was deleted in the rat endometrial adenocarcinoma cell line, RUCA‐I, using CRISPR/CAS9. Five clones devoid of miR‐203 expression were generated. Proliferation was reduced and G2‐arrest was observed in all miR‐203 deficient RUCA‐I clones. Transfection with a miR‐203‐3p mimic partially rescues this effect. Comparison of mRNA expression in three miR‐203 knockout clones to wild type RUCA‐I cells reveals 566 miR‐203‐upregulated and 592 miR‐203‐downregulated genes. 43 of the genes that are upregulated by miR‐203 knockout in vitro are downregulated in the uterus by E2. Of these Acer2, Zbtb20, Ptn, Rcbtb2, Mum1l1, Hmgn3, and Nfat5 possess one or more seed sequence matches in their 3′‐UTR that are predicted to be targets of miR‐203. These data demonstrate the importance of E2 regulated miRNAs in general, and miR‐203 in particular, for E2 regulated gene expression and physiological processes including proliferation and cell migration, in the uterus as well as in the etiology of endometrial carcinomas.

Background: Cross-talk between the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER) plays a major role in signaling processes in female reproductive organs. Objectives: We investigated the influence of the AHR ligand 3-methylcholanthrene (3-MC) on ER-mediated signaling in mammary gland tissue of ovariectomized (ovx) rats. Methods: After 14 days of hormonal decline, ovx rats were treated for 3 days with 4 μg/kg 17β-estradiol (E2), 15 mg/kg 8-prenylnaringenin (8-PN), 15 mg/kg 3-MC, or a combination of these compounds (E2 + 3-MC, 8-PN + 3-MC). Whole-mount preparations of the mammary gland were used to count terminal end buds (TEBs). Protein expression studies (immunohistochemistry, immunofluorescence), a cDNA microarray, pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the interaction between AHR- and ER-mediated signaling pathways. Results: E2 treatment increased the number of TEBs and the levels of Ki-67 protein and progesterone receptor (PR); this treatment also changed the expression of 325 genes by more than 1.5-fold. Although 3-MC treatment alone had marginal impact on gene or protein expression, when rats were co-treated with 3-MC and E2, 3-MC strongly inhibited E2-induced TEB development, protein synthesis, and the expression of nearly half of E2-induced genes. This inhibitory effect of 3-MC was partially mirrored when 8-PN was used as an ER ligand. The anti-estrogenicity of ligand-activated AHR was at least partly due to decreased protein levels of ERα in ductal epithelial cells. Conclusion: Our data show transcriptome-wide anti-estrogenic properties of ligand-activated AHR on ER-mediated processes in the mammary gland, thereby contributing an explanation for the chemopreventive and endocrine-disrupting potential of AHR ligands. Citation: Helle J, Bader MI, Keiler AM, Zierau O, Vollmer G, Chittur SV, Tenniswood M, Kretzschmar G. 2016. Cross-talk in the female rat mammary gland: influence of aryl hydrocarbon receptor on estrogen receptor signaling. Environ Health Perspect 124:601–610; http://dx.doi.org/10.1289/ehp.1509680

To identify chemical genetic interactions underlying the mechanism of action of histone deacetylase inhibitors (HDACi) a yeast deletion library was screened for hypersensitive deletion mutants that confer increased sensitivity to the HDACi, CG‐1521. The screen demonstrated that loss of GCN5 or deletion of components of the Gcn5 histone acetyltransferase (HAT) complex, SAGA, sensitizes yeast to CG‐1521‐induced cell death. Expression profiling after CG‐1521 treatment reveals increased expression of genes involved in metabolism and oxidative stress response, and oxidative stress response mutants are hypersensitive to CG‐1521 treatment. Accumulation of reactive oxygen species and increased cell death are enhanced in the gcn5Δ deletion mutant, and are abrogated by anti‐oxidants, indicating a central role of oxidative stress in CG‐1521‐induced cell death. In human cell lines, siRNA mediated knockdown of GCN5 or PCAF, or chemical inhibition of GCN5 enzymatic activity, increases the sensitivity to CG‐1521 and SAHA. These data suggest that the combination of HDAC and GCN5/PCAF inhibitors can be used for cancer treatment. J. Cell. Biochem. 116: 1982–1992, 2015.

Epidemiological data have demonstrated an inverse association between serum vitamin D3 levels, cancer incidence and related mortality. However, the effects of vitamin D on prostate cancer biology and its utility for prevention of prostate cancer progression are not as well defined. The data are often conflicting: some reports suggest that vitamin D3 induces apoptosis in androgen dependent prostate cancer cell lines, while others suggest that vitamin D3 only induces cell cycle arrest. Recent molecular studies have identified an extensive synergistic crosstalk between the vitamin D- and androgen-mediated mRNA and miRNA expression, adding an additional layer of post-transcriptional regulation to the known VDR- and AR-regulated gene activation. The Warburg effect, the inefficient metabolic pathway that converts glucose to lactate for rapid energy generation, is a phenomenon common to many different types of cancer. This process supports cell proliferation and promotes cancer progression via alteration of glucose, glutamine and lipid metabolism. Prostate cancer is a notable exception to this general process since the metabolic switch that occurs early during malignancy is the reverse of the Warburg effect. This “anti-Warburg effect” is due to the unique biology of normal prostate cells that harbor a truncated TCA cycle that is required to produce and secret citrate. In prostate cancer cells, the TCA cycle activity is restored and citrate oxidation is used to produce energy for cancer cell proliferation. 1,25(OH)2D3 and androgen together modulates the TCA cycle via transcriptional regulation of zinc transporters, suggesting that 1,25(OH)2D3 and androgen maintain normal prostate metabolism by blocking citrate oxidation. These data demonstrate the importance of androgens in the anti-proliferative effect of vitamin D in prostate cancer and highlight the importance of understanding the crosstalk between these two signaling pathways.

To develop new therapies for inflammatory breast cancer (IBC) we have compared the effects of two hydroxamic acid-based histone deacetylase (HDAC) inhibitors, CG-1521 and Trichostatin A (TSA) on the biology of two IBC cell lines: SUM149PT and SUM190PT. CG-1521 and TSA induce dose (0−10 µM) and time-dependent (0−96 h) increases in the proportion of cells undergoing cell cycle arrest and apoptosis in the presence or absence of 17β-estradiol. In SUM 149PT cells, both CG-1521 and TSA increase the levels of acetylated α-tubulin; however the morphological effects are different: CG-1521 blocks mitotic spindle formation and prevents abscission during cytokinesis while TSA results in an increase in cell size. In SUM190PT cells CG-1521 does not cause an increase in acetylated-α-tubulin and even though TSA significantly increases the levels of acetylated tubulin, neither inhibitor alters the morphology of the cells. Microarray analysis demonstrates that CG-1521 modulates the expression of 876 mRNAs and 63 miRNAs in ...