13 research outputs found
Genetic and functional characterisation of the LIMD1 promoter and gene product: from lung cancer to the hypoxic response
No full textLIM domain containing protein 1 (LIMD1) is a tumour suppressor located at 3p21.3, a region that harbours multiple tumour suppressor genes and is commonly subject to homozygous deletions and loss of heterozygosity in many cancers. The mechanism of LIMD1 tumour suppressive activities are not fully elucidated, however to date it has been shown to bind to the retinoblastoma protein (pRb) and repress E2F driven transcription as well as being a critical component of miRNA mediated gene silencing. Recent work has also identified LIMD1 as a possible negative regulator of hypoxia inducible factor α (HIF1α) and the hypoxic response. In lung cancer, LIMD1 protein expression is down regulated in up to 79% of tumours when compared to normal tissue with gene deletion and loss of heterozygosity accounting for 32 and 12% respectively, leaving 30% of tumours with unexplained mechanism of LIMD1 protein loss.
In an aim to identify other possible mechanisms of LIMD1 loss, scrutinisation of the LIMD1 promoter identified a CpG Island in the 5’ promoter region, within which a small region was found to be critical for transcriptional activation. This region was methylated in the non-LIMD1 expressing MDA-MB435 cell line, but became hypomethylated and LIMD1 expressed following treatment with the DNA methylation inhibitor 5-Aza-2’-deoxycytidine. In primary lung tumours, analysis of genomic DNA also identified increased methylation of this region as well as a reduction in LIMD1 mRNA levels when compared to matched normal lung tissue. Furthermore, in silico analysis identified a conserved binding motif for the Ets transcription factor PU.1. Experimentally PU.1 was verified as binding to the LIMD1 promoter with siRNA mediated depletion of PU.1 significantly reducing endogenous LIMD1 protein levels, thus identifying two possible novel mechanisms of LIMD1 silencing. Transcription of LIMD1, like that of other HIF1α regulatory proteins, was enhanced when cells were exposed to hypoxia (1% O2), facilitated by HIF1α binding a hypoxic responsive element (HRE) within the promoter. At the molecular level, in vivo LIMD1 forms an endogenous complex with proline hydroxylase 2 (PHD2) and the von Hippel-Lindau (VHL) protein, with LIMD1 loss decreasing the efficiency of HIF1α degradation and impeding the resultant cellular adaptation to chronic hypoxia.
In summary these studies identified epigenetic silencing of LIMD1 as a possible explanation for LIMD1 protein loss in transformed cells. Furthermore, LIMD1 transcription was identified as being activated by PU.1 and enhanced by HIF1α, and a revised, LIMD1 integrated, model of HIF1α regulation is proposed
Genetic and functional characterisation of the LIMD1 promoter and gene product : from lung cancer to the hypoxic response
No full textLIM domain containing protein 1 (LIMD1) is a tumour suppressor located at 3p21.3, a region that harbours multiple tumour suppressor genes and is commonly subject to homozygous deletions and loss of heterozygosity in many cancers. The mechanism of LIMD1 tumour suppressive activities are not fully elucidated, however to date it has been shown to bind to the retinoblastoma protein (pRb) and repress E2F driven transcription as well as being a critical component of miRNA mediated gene silencing. Recent work has also identified LIMD1 as a possible negative regulator of hypoxia inducible factor α (HIF1α) and the hypoxic response. In lung cancer, LIMD1 protein expression is down regulated in up to 79% of tumours when compared to normal tissue with gene deletion and loss of heterozygosity accounting for 32 and 12% respectively, leaving 30% of tumours with unexplained mechanism of LIMD1 protein loss. In an aim to identify other possible mechanisms of LIMD1 loss, scrutinisation of the LIMD1 promoter identified a CpG Island in the 5’ promoter region, within which a small region was found to be critical for transcriptional activation. This region was methylated in the non-LIMD1 expressing MDA-MB435 cell line, but became hypomethylated and LIMD1 expressed following treatment with the DNA methylation inhibitor 5-Aza-2’-deoxycytidine. In primary lung tumours, analysis of genomic DNA also identified increased methylation of this region as well as a reduction in LIMD1 mRNA levels when compared to matched normal lung tissue. Furthermore, in silico analysis identified a conserved binding motif for the Ets transcription factor PU.1. Experimentally PU.1 was verified as binding to the LIMD1 promoter with siRNA mediated depletion of PU.1 significantly reducing endogenous LIMD1 protein levels, thus identifying two possible novel mechanisms of LIMD1 silencing. Transcription of LIMD1, like that of other HIF1α regulatory proteins, was enhanced when cells were exposed to hypoxia (1% O2), facilitated by HIF1α binding a hypoxic responsive element (HRE) within the promoter. At the molecular level, in vivo LIMD1 forms an endogenous complex with proline hydroxylase 2 (PHD2) and the von Hippel-Lindau (VHL) protein, with LIMD1 loss decreasing the efficiency of HIF1α degradation and impeding the resultant cellular adaptation to chronic hypoxia. In summary these studies identified epigenetic silencing of LIMD1 as a possible explanation for LIMD1 protein loss in transformed cells. Furthermore, LIMD1 transcription was identified as being activated by PU.1 and enhanced by HIF1α, and a revised, LIMD1 integrated, model of HIF1α regulation is proposed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Abstract A31: Targeting APC loss using synthetic lethality in Colorectal Cancer
No full textAbstract
Background: Mutations in the tumor suppressor gene Adenomatous Polyposis Coli (APC) are found in 80% of sporadic colorectal cancer (CRC) tumors and are also responsible for the inherited form of CRC, Familial Adenomatous Polyposis (FAP). APC mutations typically occur early in the development of CRC, followed by mutations in KRAS, SMAD2/3/4 and TP53. The majority of mutations in APC occur in a region known as the mutation cluster region (MCR). This region is responsible for negatively regulating levels of B-catenin and therefore the level of Wnt signaling activation. In APC mutated tumors, B-catenin is not tightly regulated resulting in hyperactivation of the Wnt signaling pathway leading to uncontrolled cellular proliferation, differentiation, migration and apoptosis.
Aim: We aim to use the concept of synthetic lethality to identify novel therapeutic targets for the treatment of APC mutated CRC.
Results: In order to identify novel therapeutic targets for the treatment of APC mutated CRC, we have generated an in vitro model of APC mutant CRC using a lentiviral CRISPR-cas9 system. Using the APC wildtype colorectal carcinoma cell line RKO, we targeted the cells with a guide RNA targeting the start of the final exon of APC, which covers 80% of the coding region. These cell lines allow us to identify targets, which are synthetically lethal with the APC mutation. To identify novel potential drug targets, we have used two parallel screening approaches. Firstly, we have screened our cell lines with a library of siRNA silencing over 700 kinases. Typically kinases are easy to target pharmaceutically, with many kinase inhibitors already clinically available. Upon analysis, we have identified seven genes which show synthetic lethality with the APC mutation, whilst not harming the control wildtype APC cells. In parallel, we have screened over 1000 FDA-approved compounds to identify drugs which cause increased selective lethality in APC mutant cells. From screening our FDA-approved compounds, we have identified a number of compounds which display synthetic lethality with the APC mutation.
Conclusions: We have identified seven genes as potential therapeutic targets and a number of FDA-approved compounds, which could potentially be new selective therapies for 80% of CRC patients. Currently we are validating these findings and investigating the mechanism of synthetic lethality with APC mutation. To further validate our findings we are also exploring whether these results extend to other CRC cell lines with different mutational backgrounds, this will help us access how many patients may benefit from our novel therapeutic targets.
Acknowledgments: We would like to thank Bowel and Cancer Research and The Rosetree Trust for co-funding this project.
Citation Format: Hannah Shailes, Gemma Bridge, Daniel Foxler, Tyson V. Sharp, Andrew Silver, Sarah A. Martin. Targeting APC loss using synthetic lethality in Colorectal Cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A31.</jats:p
LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing
No full textLIMD1 is induced by and required for LMP1 signaling, and protects EBV-transformed cells from DNA damage-induced cell death.
No full textLIMD1 (LIM domain-containing protein 1) is considered as a tumor suppressor, being deregulated in many cancers to include hematological malignancies; however, very little is known about the underlying mechanisms of its deregulation and its roles in carcinogenesis. Epstein-Barr Virus (EBV) is associated with a panel of malignancies of lymphocytic and epithelial origin. Using high throughput expression profiling, we have previously identified LIMD1 as a common marker associated with the oncogenic transcription factor IRF4 in EBV-related lymphomas and other hematological malignancies. In this study, we have identified potential conserved IRF4- and NFκB-binding motifs in the LIMD1 gene promoter, and both are demonstrated functional by promoter-reporter assays. We further show that LIMD1 is partially upregulated by EBV latent membrane protein 1 (LMP1) via IRF4 and NFκB in EBV latency. As to its role in the setting of EBV latent infection, we show that LIMD1 interacts with TRAF6, a crucial mediator of LMP1 signal transduction. Importantly, LIMD1 depletion impairs LMP1 signaling and functions, potentiates ionomycin-induced DNA damage and apoptosis, and inhibits p62-mediated selective autophagy. Taken together, these results show that LIMD1 is upregulated in EBV latency and plays an oncogenic role rather than that of a tumor suppressor. Our findings have identified LIMD1 as a novel player in EBV latency and oncogenesis, and open a novel research avenue, in which LIMD1 and p62 play crucial roles in linking DNA damage response (DDR), apoptosis, and autophagy and their potential interplay during viral oncogenesis.This work was supported by NIH grants to SN
(1R15DE027314) and ZQY/JPM (R01DK093526;
R01AI114748; R15AG050456), an VA Merit Review
Award to ZQY/JPM (INFA-016-15S), and BBSRC, MRC
and CRUK grants (BB/L027755/1; MR/N009185/1;
CRUK-A12733 respectively) to TVS, and in part by the
NIH grant C06RR0306551. This publication is the result
of work supported with resources and the use of facilities
at the James H. Quillen Veterans Affairs Medical Cente
PU.1 is a major transcriptional activator of the tumour suppressor gene LIMD1
No full textAbstractLIMD1 is a tumour suppressor gene (TSG) down regulated in ∼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1
Human feeder cell line for derivation and culture of hESc/hiPSc
No full textWe have generated a human feeder cell line from early second trimester Placental Stromal Fibroblasts (ihPSF) stably over-expressing the polycomb protein BMI-1. These feeder cells retain the ability to maintain human Embryonic Stem cells (hESc) over long-term culture whereas hTERT or BMI-1/hTERT immortalised feeder cell lines do not. ihPSFs were able to support the derivation of a new hESc line in near xenofree (free of non-human animal components) conditions and support continued culture of newly derived hESc and human induced Pluripotent Stem (hiPS) cell lines in complete xenofree conditions necessary for clinical use.</p
The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity.
No full textThere are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators
LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing
No full textIn recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex
