Lakjaya Buluwela

T:G Mismatch-specific Thymine-DNA Glycosylase Potentiates Transcription of Estrogen-regulated Genes through Direct Interaction with Estrogen Receptor α

Nuclear receptors (NR) classically regulate gene expression by stimulating transcription upon binding to their cognate ligands. It is now well established that NR-mediated transcriptional activation requires the recruitment of coregulator complexes, which facilitate recruitment of the basal transcription machinery through direct interactions with the basal transcription machinery and/or through chromatin remodeling. However, a number of recently described NR coactivators have been implicated in cross-talk with other nuclear processes including RNA splicing and DNA repair. T:G mismatch-specific thymine DNA glycosylase (TDG) is required for base excision repair of deaminated methylcytosine. Here we show that TDG is a coactivator for estrogen receptor α (ERα). We demonstrate that TDG interacts with ERα in vitro and in vivo and suggest a separate role for TDG to its established role in DNA repair. We show that this involves helix 12 of ERα. The region of interaction in TDG is mapped to a putative α-helical motif containing a motif distinct from but similar to the LXXLL motif that mediates interaction with NR. Together with recent reports linking TFIIH in regulating NR function, our findings provide new data to further support an important link between DNA repair proteins and nuclear receptor function.

miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.

Defining the mechanism of action and enzymatic selectivity of psammaplin A against its epigenetic targets.

Psammaplin A (11c) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that 11c functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC(50) 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for 11c functioning as a DNMT inhibitor.

ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases

The regulation of gene expression by estrogen receptor-α (ERα) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERα as bait in a yeast two-hybrid screen, we have identified a novel ERα-interacting protein, ZNF366, which is a potent corepressor of ERα activity. The interaction between ZNF366 and ERα has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERα corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen.

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein–protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.

HGF/SF in Mammary Epithelial Growth and Morphogenesis: In Vitro and In Vivo Models

HGF/SF is a multifunctional cytokine whichthrough binding to its cellular receptor, c-MET, canelicit mitogenic, morphogenic and motogenic responses intarget cells. Expression of HGF/SF and c-MET has been shown to be critical in earlyembryogenesis affecting development of many organs andtissues. The effects of HGF/SF4 on established human andmouse mammary cell lines have previously been reported. This review describes the source and targetsfor HGF/SF in both human and mouse mammary tissue anddiscusses the effects of HGF/SF on primary mouse andhuman mammary epithelial cells in vitro, detailing the individual response of the two epithelialsub-population of cells which comprise this organ.Additionally, the effects of HGF/SF overexpression onmouse mammary gland development in vivo, aresummarized.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-α and the transcriptional repressor PLZF

Estrogen receptor α (ERα) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERα plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERα and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERα is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERα expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERα is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

Retroviral infection of the FGF2 gene into MCF-7 cells induces branching morphogenesis, retards cell growth and suppresses tumorigenicity in nude mice.

FGF2 (basic fibroblast growth factor) is a multifunctional growth factor and exhibits diverse function in different cell types. In breast, loss of FGF2 expression is associated with malignant progression. In order to understand the role of FGF2 in maintenance of normal breast structures and control of cell growth, we restored FGF2 expression in the breast cancer cell line MCF-7. The FGF2 retrovirally infected MCF-7 cells (MCF-7.F2.5) not only expressed FGF2 in cytoplasm and nuclei, but also released FGF2 into culture medium both on plastic and in Matrigel conditions. The MCF-7.F2.5 cells formed branches in Matrigel and this effect was abolished by the addition of a neutralising anti-FGF2 antibody or function blocking antibodies to alpha2, alpha3 and beta1 integrins. Furthermore, MCF-7.F2.5 cells lost their ability for anchorage-independent growth in soft agar. When MCF-7 and MCF-7.F2.5 cells were injected into nude mice, there was a 1.6- to 3.2-fold reduction of tumour volume with MCF-7.F2.5 cells in comparison with the parental MCF-7 cells. MCF-7.F2.5 cells also demonstrated a reduction in oestrogen receptor-alpha (ERalpha) both in vitro and in vivo. Our results suggest that introduction of the FGF2 gene into MCF-7 cells altered the malignant tumour cells towards a more benign phenotype in vitro and in vivo.

Estrogen Receptors and Anti-Estrogen Therapies

The realisation that breast cancer growth is regulated by the hormone estrogen led to the development of tamoxifen as the first target-directed cancer drug. Clearly, there is considerable headway to be made in further elucidating estrogen receptor function, in particular in identifying the mechanisms underlying endocrine resistance. It is hoped that these studies will lead to the development of novel therapies aimed at overcoming endocrine resistance. Nevertheless, the success of anti-estrogen therapies in the treatment of breast cancer have paved the way for another first in cancer treatment, that of breast cancer prevention.

Abstract B166: Defining the mechanism of action and enzymatic selectivity of psammaplin A against its epigenetic targets.

Naturally isolated products continue to play a significant role in anticancer drug discovery. In line with our interest in small molecule compounds which effect epigenetic gene regulation, we are interested in the structural and biological properties of the natural product Psammaplin A. While it has been implicated as an inhibitor of numerous targets such as mycothiol-S-conjugate amidase, topoisomerase II and aminopeptidase N, studies by Crews and co-workers showed it to be an extremely potent inhibitor of both histone deacetylase (HDAC) and DNA methyltransferase (DNMT) enzymes. Our goal was to establish structure-activity relationships around psammaplin A, probing the molecular features responsible for the chemical biology of this natural product. We developed a number of versatile synthetic routes towards small compound libraries based on the psammaplin core [1]. We subsequently assessed the activity of these compounds against a selection of histone deacetylases, DNMT1, and in cell based assays. Analysis of the data revealed psammaplin A to be a natural prodrug; the central disulphide being reduced to a free thiol. Indeed, we showed the corresponding thiol not only to be a potent inhibitor of HDAC1 (IC50 0.001 μM), but to have remarkable selectivity against HDAC6 (IC50 0.360 μM), HDAC7 (IC50 17.4 μM) and HDAC8 (IC50 1.34 μM) in cell-free assays. Cell-based (MCF7) studies confirmed the HDAC targeting potential of psammaplin A and confirmed the selectivity observed in vitro. Curiously, our psammaplin A derivatives demonstrated no activity against DNMT1 in cell-free assays. Taken together with other reports, this data suggests psammaplin A may not be an inhibitor of DNMT1 as previously reported. In conclusion, we have explored the epigenetic activity of psammaplin A and have found it to have significant potency and selectivity against HDACs, whereas we find no evidence for DNMT1 activity. Reference: 1. “New synthetic strategies towards Psammaplin A, access to natural product analogues” Baud, M. G. J.; Leiser, T.; Meyer-Almes, F.-J.; Fuchter, M. J. Org. Biomol. Chem. 2011, 9, 659–662. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B166.