Medicine & Health

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Now showing 1 - 10 of 29
  • (2013) Apte, Minoti; Yang, Lu; Phillips, Phoebe; Xu, Zhihong; Kaplan, Warren; Cowley, Mark
    Journal Article
    Activated pancreatic stellate cells (PSCs) are responsible for the fibrotic matrix of chronic pancreatitis and pancreatic cancer. In vitro protocols examining PSC biology have usually involved PSCs cultured on plastic, a non-physiological surface. However, PSCs cultured on physiological matrices e.g. MatrigelTM (normal basement membrane) and collagen (fibrotic pancreas), may have distinctly different behaviours compared to cells cultured on plastic. Therefore, we aimed to i) compare PSC gene expression after culture on plastic, MatrigelTM and collagen I; ii) validate the gene array data for transgelin, the most highly dysregulated gene in PSCs grown on activating versus non-activating matrices, at mRNA and protein levels; iii) examine the role of transgelin in PSC function; and iv) assess transgelin expression in human chronic pancreatitis sections. Culture of PSCs on different matrices significantly affected their gene expression pattern. 146, 619 and 432 genes respectively were differentially expressed (p < 0.001) in PSCs cultured on collagen I vs MatrigelTM, MatrigelTM vs plastic and collagen I vs plastic. The highest fold change (12.5 fold upregulation) in gene expression in cells on collagen I vs MatrigelTM, was observed for transgelin (an actin stress fibre associated protein). Transgelin was significantly increased in activated PSCs versus quiescent PSCs. Silencing transgelin expression decreased PSC proliferation and also reduced platelet derived growth factor (PDGF)-induced PSC migration. Notably, transgelin was highly expressed in chronic pancreatitis in stromal areas and peri-acinar spaces but was absent in acinar cells. These findings suggest that transgelin is a potentially useful target protein to modulate PSC function so as to ameliorate pancreatic fibrosis.

  • (2019) Voli, Florida
    Cancer immune evasion is recognised as a central hallmark of tumour development. One mechanism that cancer cells use to protect themselves from anti-tumour immune responses is the over-expression of Programmed Death Ligand 1 (PD-L1). The immune checkpoint protein Programmed Death receptor 1 (PD-1) expressed by lymphocytes negatively regulates T-cells effector functions against target cells, including tumour cells. Several therapeutic monoclonal antibodies targeting PD-L1/PD-1 have been approved by the FDA for adult melanoma and lung cancer. However, their efficacy is limited by acquired resistance and immune-related adverse events in many patients. Additionally, the regulation of PD-L1 expression on tumour cells is still poorly understood. Copper transporter 1 (CTR-1) and copper levels are elevated in tumours and the use of copper targeting agents is currently under intense investigations. It has been also reported that copper plays a major role in the immune system, but its activity is unclear. In this study, we demonstrated for the first time that copper plays a key role in the expression of PD-L1 in cancer cells. Tissue microarrays from neuroblastoma and glioblastoma patients showed a significant correlation between CTR-1 and PD-L1 expression (p<0.0001 and p=0.006 respectively). Deep analysis of the TCGA database showed that CTR-1 and PD-L1 association also occurs across many cancer types characterised by high copper levels, but not in the corresponding normal tissues. In vitro experiments showed that copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells. Consistently, Dextran-Catechin (DC) and TEPA, copper-lowering drugs, were able to down-regulate PD-L1 expression in cancer cells, both post-transcriptionally by inhibiting EGFR-phosphorylation and promoting ubiquitin-mediated PD-L1 degradation and transcriptionally by reducing STAT signalling pathway. In vivo studies showed that copper-lowering drugs slowed tumour growth and improved mice survival, by down-regulating PD-L1 expression, which in turn caused a significantly increase in number of tumour-infiltrating CD8+ T cells and natural killer cells. This study reveals an important role for copper in regulating PD-L1 expression and it raises the potential of repurposing copper-lowering drugs to enhance immune checkpoint blockade. This novel therapeutic approach could target a wide spectrum of copper-dependent cancers.

  • (2018) Sia, Chang Shyan
    Acute lymphoblastic leukaemia (ALL) is a highly heterogeneous disease which can be divided into various subtypes based on cytogenetics and recurrent chromosomal alterations. By associating clinical outcomes, recurrent mutations can be used as markers in risk stratification. Among other factors, risk-directed treatments based on specific genomic alterations have contributed significantly to the high overall cure rate of ALL. However, patients classified as high-risk are still in need of better treatment strategies. One such subtype of high-risk ALL is termed the Philadelphia-like (Ph-like) ALL. Results from two clinical trials conducted by the Children’s Oncology Group have uniformly reported the relatively poor outcome of Ph-like patients compared to non-Ph-like. Hence, the main aim of this study was to identify novel effective treatments for paediatric Ph-like ALL. The main strategy used in this study was by targeting the cytokine receptor-like factor 2 (CRLF2) signalling axis. CRLF2 signalling is important in Ph-like ALL as up to 50% patients overexpress this receptor tyrosine kinase as a result of recurrent genomic alterations. In children, CRLF2 overexpression is correlated to poor outcome in the high-risk patients, but not the standard-risk, further suggesting the potential therapeutic value of CRLF2 in high-risk ALL. While clinically approved agents targeting CRLF2 do not exist, small molecule inhibitors can be used to target signalling kinases downstream of CRLF2. Through a phosphoproteomic approach, downstream tyrosine kinases activated by CRLF2 and its ligand were identified in this study. Subsequently, a rationally designed combination treatment of the tyrosine kinase inhibitors BMS-754807 and ponatinib was tested against patient-derived xenograft (PDX) models of Ph-like ALL and showed remarkable in vitro efficacy. The combination was further validated in preclinical models of Ph-like ALL using immunodeficient mice, albeit with disappointing in vivo efficacy results. In addition, investigations were carried out to determine the mechanism of sensitivity of Ph-like ALL cells to the SMAC mimetic birinapant, by studying epigenetic regulation of tumour necrosis factor receptor 1 (TNFR1) expression. Collectively, multifaceted approaches were utilised in this study for the overall aim of improving the clinical outcomes of children with Ph-like ALL.

  • (2018) Karsa, Mawar
    Despite remarkable improvements being made in the treatment of childhood acute lymphoblastic leukaemia (ALL), prognosis remains dismal for a certain subgroups of high-risk patients including infants with leukaemia harbouring rearrangement of the Mixed Lineage Leukaemia (MLL) gene, thus warranting development of more effective therapeutics. The approach of drug repurposing, whereby an approved drug may be applied to target a disease other than that for which it was originally intended, is one that is gaining popularity due to the potential to avoid the rising cost and lengthy process of the traditional drug discovery pathway. To identify novel candidates for high-risk leukaemia, a library of approved drugs and pharmacologically active compounds was screened against ALL cell lines with or without MLL gene rearrangement, using a cell-based viability assay. The screen identified two MLL-selective bioactive compounds. The purinergic P2Y receptor agonist and guanylate cyclase inhibitor, 2-chloroadenosine triphosphate, showed in vitro efficacy against MLL-r ALL patient-derived xenografts (PDX) whereby sensitivity was associated with decreased expression of several P2Y receptors including P2RY14, which was additionally found to be differentially expressed in MLL-r vs MLL-wt paediatric ALL patients. The second MLL-selective candidate, SID7969543 which targets Steroidogenic Factor-1 (SF-1), showed activity against a subset of MLL-r and CALM-AF10 leukaemia cell lines and synergized with etoposide and cytarabine in vitro. A subsequent secondary screen aimed at selecting more potent compounds identified two FDA-approved drugs, auranofin and disulfiram, which revealed a common ROS-mediated mechanism in potently inhibiting the viability of high-risk leukaemia cell lines and PDX in vitro. Preclinical testing of drug combinations revealed the potential of combined treatment with auranofin and cytarabine in delaying leukaemia growth in an aggressive MLL-r ALL PDX mouse model. Auranofin also demonstrated synergy with disulfiram in vitro which could be promising for future studies. In conclusion, this work identified MLL-selective compounds that uncovered potential new targetable pathways in MLL-r leukaemia for further investigation and possible future therapeutic exploitation. The two FDA-approved drugs identified highlighted the therapeutic potential of targeting the ROS pathway for high-risk leukaemia and demonstrated promising clinical utility for these patient subgroups.

  • (2009) Gan, Pei Pei
    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

  • (2014) Sun, Yuting
    N-Myc induces neuroblastoma by regulating the expression of target genes and proteins, and N-Myc protein is degraded by Fbxw7 and NEDD4 and stabilized by Aurora A. The class IIa histone deacetylase HDAC5 suppresses gene transcription, and blocks myoblast and leukaemia cell differentiation. While histone H3 lysine 4 (H3K4) trimethylation at target gene promoters is a pre-requisite for Myc-induced transcriptional activation, WDR5, as a histone H3K4 methyltransferase presenter, is required for H3K4 methylation and transcriptional activation mediated by a histone H3K4 methyltransferase complex. Here, I investigated the roles of HDAC5 and WDR5 in N-Myc overexpressing neuroblastoma. I have found that N-Myc upregulates HDAC5 protein expression, and that HDAC5 represses NEDD4 gene expression, increases Aurora A gene expression and consequently upregulates N-Myc protein expression in neuroblastoma cells. HDAC5 and N-Myc commonly repress the expression of a subset of genes by forming a protein complex, while HDAC5 and the class III HDAC SIRT2 commonly, but independently, repress the expression of another subset of genes. Moreover, HDAC5 blocks cell differentiation and induces cell proliferation. I have also demonstrated that N-Myc upregulates WDR5 gene expression by binding to the WDR5 gene promoter. Conversely, WDR5 forms a protein complex with N-Myc at the gene promoter of N-Myc target genes, leading to histone H3K4 trimethylation and transcriptional activation of N-Myc target genes, including MDM2 and CCNE1. Importantly, WDR5 reduces the expression of wild type, but not mutant, p53 protein expression in MYCN oncogene-amplified neuroblastoma cells through reducing MDM2 expression, induces neuroblastoma cell proliferation in p53 dependent/independent fashions, and enhances neuroblastoma cell survival in a p53-dependent manner. In a publicly available large cohort of 476 neuroblastoma patients, high levels of WDR5 gene expression in tumours correlate with poor patient survival independent of MYCN amplification and disease stage. In addition, treatment with WDR5 inhibitors blocks the formation of N-Myc and WDR5 protein complex, reduces the expression of N-Myc and WDR5 target genes, and impedes neuroblastoma cell proliferation. In conclusion, these data identify HDAC5 and WDR5 as novel co-factors in N-Myc oncogenesis, and provide the critical evidence for the potential utilization of WDR5 inhibitors for the therapy of neuroblastoma.

  • (2014) Sutton, Selina
    The incidence of cutaneous malignant melanoma is rapidly increasing. Melanoma is an extremely aggressive malignancy making up only 4% of total skin cancers, but it is responsible for 80% of skin cancer deaths. The clinical benefit of BRAF-MEK-inhibitor therapy in BRAF mutant melanoma remains temporary, and the mechanisms of metastasis with advanced disease are incompletely understood. The tripartite motif (TRIM) family members have been implicated in the pathogenesis of multiple cancers functioning as both oncogenes and tumour suppressors. Intriguingly, in the histological progression of squamous cell carcinoma (SCC) from normal skin, TRlM16 was significantly reduced in vivo and overexpression of TRlM16 reduced SCC cell migration in vitro. Furthermore, TRlM16 has demonstrated some of the features of a tumour suppressor protein in neuroblastoma through down-regulation of protein binding partners: cytoplasmic vimentin and nuclear E2F1. Taken together, these data suggest that TRIM16 acts to repress cancer cell replication and migration. However, the role of TRIM16 in melanoma is presently unknown. We developed a keratinocyte-specific TRlM16 knockout mouse model for the evaluation of TRIM16 in the development of SCC. Surprisingly, these mice demonstrated large cutaneous melanocytic lesions, indicating a possible role in melanoma. We then used human melanoma cell lines and primary tissues to show that low TRIM16 expression associates with enhanced cell migration in vitro, metastatic disease in vivo, and poor prognosis in a large cohort of melanoma patients with lymph node metastasis. TRIM16 protein expression is increased with vemurafenib BRAF inhibitor treatment in vitro and is partially required for drug mechanism of action. ln addition, TRlM16 protein expression is increased by BRAF inhibitor treatment in vivo. Taken together, these data indicated that repression of TRIM16 expression enhanced replication and migration of melanoma cells, associated with poor prognosis and thus represents as a potential therapeutic target. Furthermore, we identified a novel small molecule, compound 012, which unexpectedly increased BRAF inhibitor potency in BRAF wild-type mutant melanoma cells, an effect partially dependent on TRIM16. This establishes a basis for investigation of the combination of efficacious small molecules synergistic with vemurafenib, and may open an important novel treatment option for BRAF wild-type patients.

  • (2016) Yi, Hangyu
    Acute myeloid leukaemia (AML) is a deadly form of leukaemia resulting in the highest number of leukaemia-associated deaths. The high mortality rate is due to frequent relapse caused by the persistence of drug-resistant leukaemic stem cells (LSCs). We have previously demonstrated an essential role for beta-catenin signalling in regulating LSCs in AML. Leucine-rich repeat containing G protein-coupled receptor 4 (Lgr4) has recently been identified as the receptor for R-spondin (Rspo) proteins to activate beta-catenin. This study showed that Rspo2/Rspo3 cooperating with Wnt3a potently potentiated beta-catenin activation in haematopoietic stem cell (HSC)-derived pre-LSCs. Overexpression of Lgr4 augmented activation of Wnt/beta-catenin signalling and promoted leukaemogenesis in vivo. Inhibition of Lgr4 reduced beta-catenin activity, completely abolished Rspo3/Wnt3a/beta-catenin signalling and prevented leukaemia development. A microarray experiment of 104 AML patient samples showed that high Lgr4 expression was associated with poor outcomes of AML patients. Altogether, these findings provide strong evidence demonstrating Lgr4 to be a critical regulator of beta-catenin signalling in AML. Gene expression profiling identified Rgs1 (regulator of G protein signalling 1) to be a major component of Lgr4 signalling. Rgs1 has been shown to bind directly to G protein subunits Gaq and Gai (Moratz et al., J Immunol, 2000), and treatment of pre-LSCs with Gaq and Gai inhibitors indicated Gaq to be a key downstream effector of Lgr4/Rgs1 signalling. Further functional studies showed that Gaq knockdown reduced beta-catenin expression, attenuated the effect of Wnt3a/Rspo3-potentiated beta-catenin activation and impaired LSC self-renewal, recapitulating the role of Lgr4 in LSC regulation. These data support the view that Gaq is an integral component of Lgr4 signalling in LSCs. Gene expression analysis also showed that Lgr4 knockdown increased expression of Gadd45a (growth arrest and DNA damage-inducible gene) and repressed several mitochondrial associated genes. Functional studies showed that Gadd45a deletion significantly increased in vivo LSC self-renewal and enhanced AML progression. Blockade of Lgr4 signalling inhibited mitochondrial energy metabolism, on which LSCs rely for survival. Collectively, this study has identified a novel Wnt3a/Rspo2/Rspo3-Lgr4-Gaq-beta-catenin signalling pathway governing LSCs and interference with components of this pathway may represent a promising therapeutic approach for eradicating LSCs in AML.

  • (2016) Wong, Matthew
    N-Myc induces neuroblastoma by regulating the expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisites for Myc-induced transcriptional activation. DOT1L is the only known histone methyltransferase that catalyses mono-methylation (me), di-methylation (me2) and tri-methylation (me3) at the histone H3K79 position, which have been linked to gene transcriptional activation. JMJD6 is a bi-functional arginine demethylase and lysyl-hydroxylase. The JMJD6 gene is located on the chromosome 17q25 position. 17q21-gter gain has been identified as the most frequent chromosome alternation in neuroblastoma and an indicator of poor patient prognostic. Here, I investigated the roles of DOT1L and JMJD6 in N-Myc over-expressing neuroblastoma. I found that N-Myc up-regulated DOT1L mRNA and protein expression, by binding to an E-box at the DOT1L gene promoter. Knocking-down DOT1L reduced the mRNA and protein expression of the N-Myc target genes, ODC1 and E2F2. DOT1L and N-Myc formed a protein complex, and knocking-down DOT1L reduced histone H3K79me2 and N-Myc protein binding at the promoters of the N-Myc target genes ODC1 and E2F2, and reduced neuroblastoma cell proliferation in vitro and tumour progression in neuroblastoma-bearing mice. In a publicly available microarray gene expression dataset, high levels of DOT1L gene expression in tumours correlated with high levels of MYCN gene expression and poor patient survival independent of MYCN amplification, age at diagnosis and disease stage. I have also demonstrated that JMJD6 up-regulated both N-Myc and c-Myc in neuroblastoma cell lines. Conversely N-Myc and c-Myc did not affect JMJD6 mRNA or protein expression. Knocking down JMJD6 reduced neuroblastoma cell proliferation in vitro and tumour progression in neuroblastoma-bearing mice. JMJD6 gene expression correlated with MYCN gene expression in human neuroblastoma tissue microarray gene expression datasets. High DOT1L gene expression was also a prognostic factor for poor neuroblastoma patient outcome. In conclusion, these data identify DOT1L as a novel co-factor in N-Myc oncogenesis, and provide critical evidence for the potential utilization of DOT1L inhibitors for the therapy of MYCN amplified neuroblastoma. JMJD6 up-regulates N-Myc and c-Myc gene expression and JMJD6 gene gain is a potential mechanism for 17q21-qter gain driven neuroblastoma tumourigenesis.

  • (2015) Leung, Halina
    Acute myeloid leukaemia (AML) is a heterogeneous blood disease with high relapse rates. A small population of highly drug resistant leukaemic stem cells (LSC) persists after chemotherapy, causing disease relapse. This emphasises the need to develop novel LSC-targeted therapies. While increasing evidence has emerged in recent years highlighting the role of epigenetic regulators in cancer development and maintenance, knowledge in epigenetic regulation of LSC is limited. This study investigated the role of two novel histone demethylases, Jmjd1c and Jmjd5, in regulating LSC, as well as the effectiveness of currently available epigenetic agents on leukaemogenesis. This study has shown that Jmjd1c is required for AML development and maintenance. Experiments involving enforced expression of Jmjd1c in haematopoietic stem cells (HSC) suggested that Jmjd1c has the capacity to enhance stem cell proliferation. Jmjd1c overexpression also promoted proliferation of HSC transduced with oncogenes, Hoxa9/Meis1a, and accelerated leukaemia development in mice. Gene expression profiling identified Jmjd1c to be a critical regulator of metabolic pathways such as glycolysis. These novel findings support the importance of targeting Jmjd1c. This study also provided the first evidence of Jmjd5 as a potential tumour suppressor in AML. Overexpression of Jmjd5 significantly impaired LSC proliferation and prolonged mouse survival. Gene expression profiling and functional studies suggested that Jmjd5 negatively regulated a LSC self-renewal pathway driven by G protein-coupled receptor 84 (Gpr84). Given the partially shared downstream signalling of Jmjd5 and Gpr84, it is likely that Jmjd5 negatively regulates LSC function, at least in part, by inhibiting Gpr84 signalling. Jmjd1c and Jmjd5 have previously been shown to regulate histone methylation. Although global H3K9me2 levels were not altered by Jmjd1c, Jmjd5 reduced global H3K36me2 and H3K27me3 levels. Reduction in H3K27me3 could also be achieved by DZNep-induced Ezh2 inhibition at low doses, which altered the gene expression profile of DZNep-treated AML cells. In summary, both Jmjd1c and Jmjd5 were found to be crucial in the proliferation and maintenance of AML cells, suggesting that targeting aberrant methylation mediated by these epigenetic regulators may provide a promising approach in AML therapy.