Research areas pursued by MMI Clinical & Translational Research Scholars include inflammatory bowel disease, perinatal health, hepatitis C infection, drug screening technologies, medical devices, diabetic kidney disease, rheumatoid arthritis and cancers of the prostate, breast, colon and lung.
The impact of brain disorders on the human population is significant, with neuropsychiatric illness, and in particular mood and psychotic disorders, contributing to over 25% of the 800 billion euro annual healthcare costs of neurological disorders across Europe. To combat this socio-economic challenge, novel therapeutic strategies are required that can identify sequences of events that underlie the processes of long-term synaptic function during post-natal development, in adult life and in the pathogenesis of neural diseases. Detecting this relationship between genes, synapse development and functional circuit maturation in disease models of neuronal development requires the analysis of multiple parameters in order to evaluate neurobiological activity, determine molecular biomarkers of the disease and identify novel therapeutic targets. However, current gold-standard techniques used to study the CNS have limitations that pose unique challenges to furthering our understanding of functional CNS development.
In order to bridge this gap, researchers require enhanced tools to study functional CNS function, both structurally and physiologically, in order to identify mechanisms that lead to devastating and debilitating disorders. The recent advancement in nanotechnologies for biomedical applications has seen the emergence of nanoscience as a key enabling technology for delivering a translational bridge between basic and clinical research. In particular, in vitro neuronal networks grown on nanoengineered devices can be used to investigate circuit physiology, with a particular focus on understanding the morphological and functional properties of neuronal development. This study presents the development of a nano-engineered biosensor platform designed for the growth, interaction and visualisation of neuronal network activity. The development of such systems may provide a unique opportunity for a multimodal platforms for modelling neuronal development and studying the cellular and molecular properties of neurodevelopmental disorders.
Macrophage subsets in tissue sites such as the peritoneal cavity are extremely heterogeneous in their phenotype and functions. These peritoneal macrophage subsets can be of dual origin: monocyte-derived or tissue-resident embryo-derived. Transcription factors are key to every step of the generation of both monocyte-derived and tissue-resident embryo-derived macrophages. Rorα is a transcription factor that has a role in a number of different processes within the body including cell development, inflammation and circadian rhythms. The role that Rorα has in the regulation of macrophage development, in particular in the development of monocyte-derived macrophages or tissue-resident macrophages in the peritoneal cavity was investigated. Rorαsg/sg mice contain a naturally occurring mutation in Rorα and were used as to investigate Rorα function in this thesis.
Results showed that Rorαsg/sg mice had decreased levels of blood monocyte-derived peritoneal macrophages and also decreased levels of Ly6CLow blood monocytes compared to controls. Levels of Ly6CHigh bone marrow monocytes were increased in Rorαsg/sg mice compared to controls. Rorαfl/sg LysMCre mice were generated to test if these observations were due to Rorα acting intrinsically in macrophages. In Rorαfl/sg LysMCre mice Rorα is deleted only in myeloid cells as a result of Cre-Lox recombination. Results in Rorαfl/sg LysMCre mice appear to suggest that the alterations in peritoneal macrophages seen in Rorαsg/sg mice are due to Rorα acting extrinsically of myeloid cells as opposed to acting intrinsically.
Rorα has been shown to have a role in inflammatory responses. It is presented here that Rorαsg/sg mice are less susceptible to LPS-induced endotoxemia compared to controls. The response to LPS in Rorαsg/sg mice was characterised by decreased serum levels of the pro-inflammatory cytokine IL-1β compared to controls. Peritoneal macrophages from Rorαsg/sg mice also produced decreased levels of IL-1β compared to control macrophages. Results also show that in WT mice the blood monocyte-derived macrophage peritoneal population, decreased in Rorαsg/sg mice, produces pro-inflammatory cytokines in response to LPS stimulation. Similar to Rorαsg/sg mice, Rorαfl/sg LysMCre mice are less susceptible to LPS-induced endotoxemia compared to controls indicating that the decrease in the blood monocyte-derived peritoneal macrophage population is not linked to decreased susceptibility to LPS-induced endotoxemia in Rorαfl/sg LysMCre mice.
Rorα is a molecular component of the circadian clock. Recent work by others has indicated that the levels of certain immune cells have a diurnal pattern. Levels of the blood monocyte-derived peritoneal macrophage population and tissue-resident peritoneal macrophage population were analysed over 24 hours. Results show that both populations displayed a diurnal rhythm with levels of both being highest during the active phase. It was also found that peritoneal macrophages from WT mice express Rorα, along with Bmal1 in a diurnal manner.
Human blood monocytes are currently classified into three subsets: CD14++CD16- “Classical”, CD14++CD16+ “Intermediate” and CD14+CD16++ “Non-Classical”. Distinct functional differences between Classical and Non-Classical have been described but the role of Intermediate monocytes is less clear. In profiling monocytes from healthy adults by multi-colour flow cytometry, we observed that Intermediate monocytes exhibit dichotomous surface expression of the Class II major histocompatibility protein HLA-DR, with separate sub-populations expressing mid- (DRmid) and high-levels (DRhi). Further profiling of cell surface markers demonstrated that, compared to the DRhi subset, DRmid Intermediate monocytes express higher levels of CCR2 and CD62L, and lower levels of CD45, CX3CR1, LFA-1, VLA-4 and Mac-1, indicating heterogeneity for multiple functionally-relevant proteins.
We assessed how the newly described Intermediate sub-populations interact with and migrate through endothelium in in vitro assays. Results indicated both the DRmid and DRhi subsets are highly adherent to resting and activated primary human aortic endothelium, with adherence of the DRmid subset being partially mediated by CD11a. Both sub-populations exhibited poor CCL2-induced transmigration in contrast to the highly migratory CCR2+ Classical monocytes, despite the fact that DRmid and DRhi subset expressing CCR2+ and CCR2int phenotypes respectively. Further experiments revealed reduced intracellular calcium release and filamentous actin polymerisation, suggesting early termination of the CCL2-CCR2 signal. Chemokine receptors are G-protein coupled receptors (GPCRs), and GPCR signalling may be regulated by Regulator of G-Protein Signalling (RGS) proteins. We quantified mRNA levels of RGS1, 2, 12 and 18 in the monocyte subsets. Interestingly, elevated RGS1 was detected in the newly-described Intermediate monocyte subpopulations. RGS1 has been implicated as a negative regulator of CCR2 signalling in monocytes. Therefore, the results are consistent with a role for RGS1 up-regulation in the blunted CCL2-induced signalling and migration of the DRmid and DRhi intermediate monocytes. Overall, the results of this project add novel details to current knowledge regarding human Intermediate monocytes provide further evidence for heterogeneity within this monocyte subset and indicate that changes in the intracellular regulation of chemokine receptor signalling may contribute to DRmid intermediate monocyte expansion in the circulation during inflammatory disorders.
Lung cancer is the most common form of cancer-related death in the world, accounting for approximately 19.4% of all cancer mortalities. Currently platinum-based therapies are the gold-standard of care for non-small cell lung cancer (NSCLC), however the prognosis of advanced NSCLC remains bleak. As such there is a drive to identify novel therapeutics that can combat the disease more effectively. Widespread epigenetic alterations are observed in NSCLC, such as aberrant DNA methylation and histone acetylation patterns. The reversible nature of these modifications makes them an attractive prospect as novel therapeutic targets.
Lymphocyte subpopulations were enumerated in blood and bronchial lavage (BAL) samples from NSCLC patients and control subjects. Invariant natural killer T (iNKT) cells were found to be significantly depleted from both tissues. iNKT cells can recognize and kill cancer cells in a CD1d-dependent manner, however, we found that NSCLC cell lines do not express CD1d. We hypothesized that CD1d expression in NSCLC is epigenetically regulated and that increasing CD1d expression in NSCLC would increase their susceptibility to iNKT cell lysis. NSCLC cell lines were treated with a panel of DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) and quantitative polymerase chain reaction amplification of reverse transcribed mRNA and flow cytometry were performed to detect changes in CD1d expression. Both DNMTi and HDACi significantly induced CD1d expression in NSCLC cell lines, and this increase in CD1d expression was associated with increased susceptibility to iNKT cell-mediated cytolytic degranulation.
These results indicate that epigenetic targeting therapies can up-regulate CD1d expression in NSCLC, and that over-expression of CD1d leads to increased susceptibility to iNKT cell cytotoxicity. These results indicate that epigenetic manipulation of CD1d is a viable therapeutic option in combatting NSCLC.
Oesophageal and rectal adenocarcinoma are treated with neoadjuvant chemoradiotherapy in order to reduce tumour size prior to surgery, however only 10-30% of patients have a complete pathological response and there are no clear biomarkers to indicate which patients will respond. Radioresistant tumours have altered metabolism and high rates of angiogenesis and DNA repair, biological processes which are closely interlinked.
We tested the ability of 11B, our novel anti-angiogenic agent, and its structural analogues CC11, ZHCl and CC8 to affect these biological processes. We used an isogenic model of oesophageal adenocarcinoma: OE33P (radiosensitive) and OE33R (radioresistant) cells and a radioresistant colorectal cancer cell line, HT29-LUC. We also used ex vivo explant and in vivo oesophageal and rectal adenocarcinoma treatment-naïve tissue.
Firstly, we investigated if our novel drugs affected cancer cell growth and radiosensitivity in vitro. By BrdU assay, 11B and ZCHl inhibited proliferation of OE33P cells, and ZHCl inhibited proliferation of HT29-LUC cells. By clonogenic assay, 11B, CC11, ZHCl and CC8 reduced survival of OE33P and HT29-LUC cells, while CC11, ZHCl and CC8 reduced survival in OE33R cells. CC8 improved radiation response in OE33P, OE33R and HT29-LUC cells by clonogenic assay. ZHCl also improved radiation response in OE33R cells.
Having identified radiosensitising abilities with our novel drugs, we examined the effects of the drugs on energy metabolism, which is often altered in radioresistant cancers. Energy metabolism was assessed in vitro by Seahorse Biosciences technology. Oxidative phosphorylation rates were higher in OE33R compared to OE33P cells. CC11 and ZHCl reduced oxidative phosphorylation in OE33P cells and all four drugs reduced oxidative phosphorylation in OE33R and HT29-LUC cells. ZHCl also reduced aerobic glycolysis in OE33P cells. ATP levels were reduced in both OE33P and OE33R cells treated with 11B.
Radioresistance can also be due to increased rates of DNA repair. Therefore, we examined expression of DNA repair genes associated with radioresistance following treatment with our novel drugs in vitro. 11B reduced PARP1 expression in OE33P cells and MMS19 and PARP1 expression in OE33R cells. MLH1 expression was reduced in OE33P cells treated with CC11, ZHCl and CC8; OE33R cells treated with 11B, CC11, ZHCl and CC8 and HT29-LUC cells treated with CC8. In OE33P cells, pre-treatment with ZHCl increased levels of DNA damage, assessed by γH2AX assay, at 20 minutes, 6 hours and 24 hours post-irradiation. At 6 hours post irradiation, 11B also increased levels of DNA damage.
CLU is a radiation-inducible, DNA repair gene that encodes clusterin. We found that expression of CLU was higher in OE33R than OE33P cells and higher in oesophageal adenocarcinoma treatment-naïve in vivo tissue from poor radiation responders compared to good responders. CLU expression also correlated with expression of MMS19, SMUG1, PARP1 and MLH1. Clusterin secretion from treatment-naïve ex vivo tissue correlated with secretions of angiogenic mediators bFGF, VEGF, PAI-, sICAM-1 and sVCAM-1 and inflammatory mediators IL-2 and MMP-9.CLU knockdown reduced ANG-2 secretion in OE33P cells and reduced secretion of ANG-2, bFGF and IL-10 in OE33R cells. CLU knockdown also increased secretion of MCP-1 in OE33R cells.
Both excessive angiogenesis and inflammation have been linked to radioresistance. We found that there were higher secreted levels of angiogenic mediators PAI-1, sICAM-1, sVCAM-1 and ANG-1, and inflammatory mediators GROα, IL-6 and MCP-1 in oesophageal adenocarcinoma treatment-naïve ex vivo tissue from patients with a poor response to radiation compared to those with a good response; however no differences were seen with rectal adenocarcinoma tissue. Treatment with 2Gy radiation reduced ANG-1 secretion from oesophageal tissue and reduced MCP-1 secretions from rectal tissue. Treatment with 11B did not affect secretions of angiogenic or inflammatory mediators in either tumour tissue.
In a preliminary study, we tested the effects of gold nanoparticles in vitro, which may act as radiosensitisers. AuNP2 coupled with 11B reduced cell number and OXPHOS, and increased aerobic glycolysis and mitochondrial membrane potential of OE33P and OE33R cells. There were no effects on radiosensitivity.
In conclusion, we have identified a number of novel anti-angiogenic drugs that alter energy metabolism, DNA repair gene expression and response to radiation. We have also identified secretions of angiogenic and inflammatory mediators than can segregate oesophageal adenocarcinoma neoadjuvant radiation responders and non-responders. This is patentable information that could be used to develop a customised multiplex to identify responders, greatly improving quality of life and care of these patients. Overall, through investigation of metabolism, angiogenesis and DNA repair in oesophageal and rectal adenocarcinoma, we have demonstrated for the first time that differential expression patterns associated with all of these biological processes are linked with a radioresistant phenotype.
Interferon-α (IFN-α) is a pro-inflammatory cytokine used for the treatment of Hepatitis B and C as well as various malignancies. However, therapy is often associated with neuropsychiatric side effects, including depression and anxiety (Schaefer et al., 2002, Raison et al., 2005), as well as the development of painful symptoms (Shakoor et al., 2010, Nogueira et al., 2012, Capuron et al., 2002). Several preclinical studies which have employed IFN-α for the investigation of inflammation-induced depression have yielded varying results and to date, no study has evaluated the effect of repeated IFN-α treatment on nociceptive behaviour. Therefore, this thesis sought to devise an experimental protocol that would mimic the clinical situation and provide a robust and reproducible model of IFN-α-induced depression and hyperalgesia. The results demonstrated that repeated administration of a high, but not low, dose of IFN-α resulted in depressive-like behaviour and hyperalgesia to a noxious inflammatory stimulus in mice. Establishment of this model of IFN-α-induced depression and pain subsequently allowed for the evaluation of possible neurobiological substrates underpinning concurrent changes in emotional and nociceptive responding. Given the recognised role for the endocannabinoid system in emotional and nociceptive responding, we chose to examine if changes existed in this system following repeated administration of IFN-α. The data revealed no change in the levels of the endocannabinoids or expression of the receptors or enzymes responsible for metabolism of the endocannabinoids between saline and IFN-α-treated mice. However, in the presence of a noxious stimulus, levels of the endocannabinoids, AEA and 2-AG, were increased in the PAG and RVM, key components of the descending pain pathway in IFN-α-treated animals. In comparison, formalin administration increased levels of 2-AG, AEA and related N-acylethanolamines at the site of injury in saline-, but not IFN-α-treated animals. Thus, IFN-α-treated mice may have an inability to mobilise endocannabinoids at the site of injury in response to a noxious stimulus, an effect which may underlie the hyperalgesia observed. As such, the remaining studies evaluated the effect of enhancing endocannabinoid tone either at the site of injury (paw) or globally, on formalin-evoked nociceptive behaviour. Inhibition of FAAH, the primary enzyme responsible for the metabolism of AEA and N-acylethanolamines, or inhibition of MAGL, the 2-AG-metabolising enzyme, at the level of the paw, attenuated IFN-α-induced hyperalgesia, without altering formalin-evoked nociceptive behaviour in saline-treated animals. These data provide further support for an impaired ability of IFN-α-treated mice to engage the peripheral endocannabinoid system in response to a noxious stimulus, an effect which underlies the hyperalgesia observed in these animals. In comparison, systemic MAGL, but not FAAH, inhibition attenuated formalin-evoked nociceptive behaviour in both saline- and IFN-α-treated animals, indicating that enhancing 2-AG tone can elicit analgesic effects in the presence and absence of a depressive-like state. Taken together, this thesis has demonstrated for the first time that it is possible to establish a preclinical mouse model of IFN-α-induced depression and pain behaviour, and highlight a role for the endocannabinoid system in mediating and modulating IFN-α-related hyperalgesia.
The current mainstay treatment for advanced castration-resistant prostate cancer (CRPC) is the taxane chemotherapeutic drug, docetaxel. However in addition to causing a number of severe side effects, docetaxel therapy provides only a modest survival advantage of approximately two months in comparison to other treatment strategies, due to the emergence of docetaxel resistance.
Docetaxel resistance can develop in CRPC through a number of mechanisms, including decreased drug accumulation due to p-glycoprotein, activation of pro-survival signalling pathways and through the emergence of drug-resistant subpopulations within the tumour which possess a cancer stem cell (CSC) morphology. Using previously generated in vitro models of docetaxel resistance in the PC-3, DU145 and 22RV1 human prostate cancer cell lines, which are significantly resistant to docetaxel, we explored epithelial-mesenchymal transition (EMT) as a mechanism of docetaxel resistance. EMT is a process in which epithelial cells undergo a developmental switch, to acquire a mesenchymal phenotype to enable an enhanced migratory capacity and invasiveness; with loss of the epithelial marker E-cadherin considered a hallmark of EMT. Recent studies have highlighted a role for EMT in prostate cancer progression and metastasis and most recently as a mechanism mediating docetaxel resistance. In this PhD thesis, EMT was functionally characterised in docetaxel-resistant prostate cancer sub-lines, through an increased invasive capacity, cell colony scattering and matrixmetalloproteinase-1 production. In addition, docetaxel-resistant cells displayed an increased susceptibility to the CSC inhibitor salinomycin, thereby providing evidence of a link between EMT and the emergence of chemo-resistant CSCs in docetaxel-resistant prostate cancer. These functional changes were associated with increased expression of the EMT drivers ZEB1 and ZEB2 along with an increased expression of βIII-tubulin, a tubulin isotype linked to taxane resistance and tumour aggressiveness. Through siRNA manipulation of ZEB1, ZEB2 and βIII-tubulin, we established ZEB1, through its transcriptional repression of E-cadherin to be a key driver of both EMT and docetaxel resistance in docetaxel-resistant prostate cancer cells. This was clinically validated, with ZEB1 tumour expression found to be significantly higher in localised prostate cancer treated with docetaxel. In addition, we identified ZEB1 to be a driver of multi-drug resistance, with manipulation of ZEB1 expression reversing cross-resistance to both docetaxel and cisplatin-mediated cell death. The role of epithelial plasticity in mediating the progression and metastasis of prostate cancer is becoming increasingly appreciated; a phenomenon in which tumour cells simultaneously acquire both epithelial and mesenchymal features to aid in their metastatic progression. Further analysis of the in vitro models of docetaxel resistance identified a differential susceptibility of the PC-3 D12 and DU145 R docetaxel-resistant sub-lines to docetaxel, cisplatin and TRAIL-mediated apoptosis; with cross-resistance of the PC-3 D12 docetaxel-resistant sub-line associated with mesenchymal, single-cell migration, increased cell colony scattering and a marked loss in E-cadherin protein expression. In addition the PC-3 D12 docetaxel-resistant sub-line displayed a significant susceptibility to the CSC inhibitor salinomycin. The DU145 R docetaxel-resistant sub-line however was exclusively resistant to docetaxel and displayed a more ‘partial EMT’ morphology, as demonstrated by both an epithelial colonisation phenotype and a concomitant expression of both E-cadherin and EMT drivers ZEB1 and ZEB2. In addition, this partial EMT phenotype was associated with a reduced susceptibility to the CSC-inhibitor salinomycin; thereby emphasising the clinical relevance of epithelial plasticity in docetaxel-resistant CRPC.
In conclusion, this thesis investigated the role of ZEB1, ZEB2 and βIII-tubulin in mediating docetaxel-resistant prostate cancer and provides strong in vitro and clinical evidence for a key role of ZEB1, through its transcriptional repression of E-cadherin to be a key driver of both EMT and docetaxel resistance in docetaxel-resistant prostate cancer. In addition, we provide evidence in in vitro models of docetaxel-resistant prostate cancer of an acquisition of a partial EMT phenotype to be associated with a differential response to chemotherapy and in turn highlight the complexity of clinical management of advanced docetaxel-resistant prostate cancer.
Hepatitis C viral [HCV] infection is a leading cause of liver disease and infects 170 million people worldwide. Recently, new direct acting anti-virals (DAAs) which target the specific HCV proteins have been approved. Treatment efficacy with DAAs for genotype 1 patients is close to 100% . However, genotype 3 infection is more difficult to treat . While there is increasing success with the approved DAAs, host based therapies are an alternative model of treatment. Advantages to host based therapy include, low probability of the development of resistance and pan-genotypic antiviral activity. For patients who are not suitable for DAA therapy for example, genotype 3 non-responders to DAA and HIV-HCV co-infected patients, host based therapies are an alternative model of treatment . In an attempt to identify new targets for possible host based therapies, we sought to investigate interactions between HCV proteins and cellular proteins involved in autophagy and lipid metabolism.
Currently, there is a very restricted pool of replicon systems available for in vitro investigation of HCV infection, moreover, those that are available are primarily restricted to genotypes 1 and 2. We sought to develop an infection model using patient derived human serum containing HCV and human hepatocytes, Huh7 cells. Using the model, we have shown intracellular expression of incoming HCV RNA (5′ UTR region and region spanning the E1/E2 glycoproteins), expression of the HCV structural protein, core, and the non-structural protein, NS5B. We have also demonstrated a cellular response to HCV infection; the stimulation of INF β and MxA in HCV infected cells and a change in distribution of ADRP. These data suggests this model can be used to analyse the early stage of HCV infection.
HCV utilises the autophagy pathway to both establish infection and to complete its life cycle . Using sdHCV infection and a sub-genomic HCV replicon expressing cell line, we analysed two distinct points of the HCV life cycle; HCV entry and HCV RNA replication. Reports suggest that HCV utilises early stage autophagic membranes during initial HCV protein translation and during early stage HCV RNA replication . We sought to investigate HCV interaction with the early stage autophagy protein ATG5. We found that although ATG5 mRNA is unchanged in sdHCV infected cells and HCV replicon cells, protein expression of ATG5 is significantly upregulated in sdHCV infected and HCV replicon expressing cells relative to untreated cells. These data indicated HCV controls the post-transcriptional regulation of ATG5. We used the upstream open reading frame (uORF) region to examine the regulation of ATG5 . Several plasmids were constructed to analyse uORF and complete 5′ UTR regulation of main open reading frame (mORF). In untreated Huh7 cells and cells infected with sdHCV, the 5′ UTR of ATG5 caused an upregulation in the mORF suggesting the presence of translational promoter elements. Importantly, the 5′ UTR mediated upregulation was not evident in Huh7.5 cells containing HCV replicon or Huh7.5 cells which have been cured of the replicon. These data suggest HCV RNA replication either directly or indirectly causes post-transcriptional regulation of the early autophagy protein, ATG5 in a 5′ UTR and uORF independent manner.
HCV infection leads to an increase in SREBP controlled genes e.g. HMG-CoA Reductase, cholesterol, LDL and fatty acid synthesis [12-15]. Further knowledge of HCV interaction with lipid metabolism needs to be acquired to elucidate the mechanism by which HCV disrupts the lipid metabolism of hepatocytes. We hypothesised that HCV infection causes the activation of SREBP pathway by interacting directly or indirectly with proteins involved in the initiation of the pathway. We sought to determine if HCV interacts with SCAP or INSIG to cause overstimulation of lipogenic genes. Lipid metabolism is a complex divergent pathway. Lipid droplets (LDs) and HMG-CoA reductase are products of the divergent pathways. We confirmed a change in LD distribution and HMG-CoA reductase activity as a result of HCV RNA replication. Significantly, we show SCAP protein expression was also altered during HCV RNA replication. Furthermore, the data suggests that HCV core protein interacts with SCAP.
Lung cancer is the leading cause of cancer-related death in Ireland and worldwide. Despite advances in anti-cancer therapies, the overall 5-year survival for non-small cell lung cancer (NSCLC) remains poor (<15%). As such, it is critical that new strategies are explored to overcome this cancer type. In the absence of specific treatable mutations such as EGFR and EML4-ALK, cisplatin-based doublet chemotherapy remains the gold standard treatment for the majority of NSCLC patients. These patients are therefore subject to significant toxicities and in many instances, without therapeutic gain. While understanding the mode of action of cisplatin is desirable in refining therapeutic approaches that may further enhance the anti-tumour activity of this drug, cisplatin continues to pose a number of significant challenges in the clinical setting. The selection of pre-existing resistant cells and the acquisition of resistance in tumour cells during the course of treatment with chemotherapy undermines its curative potential and has become a significant clinical challenge in the treatment of NSCLC patients. Since the discovery of cancer stem cells (CSCs) in haematopoietic cancers and other solid tumours, little is known to date regarding the biology of lung cancer stem cells. The existence of cancer stem cells within the tumour cell population may explain the ineffectiveness of current treatments in eradicating tumour cells. While conventional cytotoxic therapies may target the majority of cancer cells, residual lung cancer stem cells can regenerate a cancer cell population resulting in tumour relapse in patients following chemotherapy. As such, there is an increasing need to identify and develop new therapeutic targets for specifically eradicating this specific cell population.
The overall aim of this study was to investigate the role of CSCs as the root of cisplatin resistance in NSCLC and to identify targetable entities to overcome such resistance. For the most part, a panel of isogenic parental (PT) and corresponding cisplatin resistant (CisR) NSCLC cell lines (SKMES-1, H460, MOR, H1299, A549) were used throughout this study as an in vitro model of chemoresistance. An initial model of CSCs was generated using a well-documented CSC-selective medium which promoted the growth of stem-like tumourspheres in the H460 and MOR CisR sublines, their corresponding PT cell lines did not thrive or colonise within the media. While the marker profile of lung cancer stem cells remains to be fully explored, some commonly used isolation strategies that have been used to date include the cell surface markers, CD133, CD44, EpCAM and the intracellular enzyme, aldehyde dehydrogenase 1 (ALDH1). While the above markers were extensively examined, in this study, ALDH1 was identified as a promising marker in cisplatin resistant NSCLC. ALDH1-positive subpopulations were identified and isolated from CisR sublines of all NSCLC subtypes (large cell carcinoma, adenocarcinoma, squamous cell carcinoma). Putative CSCs were characterised based on their resistance to cisplatin and their ability to self-renew and asymmetrically divide. Furthermore, gene expression profiling of the human embryonic stem cell markers (otherwise known as Yamanaka factors), Nanog, Oct-4, Sox-2, Klf-4 and cMyc, was examined in both models of putative CSCs, tumourspheres selected for in CSC selective media, and the ALDH1-positive and negative subpopulations from each CisR subline.
Based on the identification of an increased stem-like ALDH1-positive subset within the CisR sublines, specific inhibition of ALDH1 was employed using the ALDH1 inhibitors, DEAB and Disulfiram (Antabuse). Disulfiram has been widely used as an effective treatment of alcoholism for over 60years. Its primary pharmacological action is the inhibition of ALDH, which is responsible for converting acetaldehyde to acetate in the metabolism of alcohol. It is a potent inhibitor of the ALDH1 isoform. The development of novel anti-cancer drugs against various malignant tumours is both time-consuming and expensive and involves pre-clinical and clinical testing. Therefore, finding new uses for existing drugs with known safety profiles, otherwise known as “repurposing” is of great economic benefit. As Disulfiram is an FDA-approved drug, it represents an important drug for testing the proof of principle of ALDH1 inhibitors as CSC targeting agents in cisplatin resistant NSCLC. More importantly, Disulfiram has been safely used in cancer patients in combination with cisplatin and vinorelbine and shown beneficial effects on overall survival. When the CisR NSCLC sublines were treated with Disulfiram, in combination with cisplatin, the resistant cells were re-sensitised to the cytotoxic effects of cisplatin, as demonstrated by their decreased proliferative and clonogenic survival and increased apoptosis. Similar results were observed when DEAB was used in combination with cisplatin. Several ALDH isoforms including ALDH1 function in the metabolism of Vitamin A to retinoic acid cell signalling via retinoic acid production by oxidation of all-trans-retinal and 9-cis-retinal. This function in particular has been linked to the stemness characteristics of CSCs via the modulatory capacity of retinoic acid to induce differentiation. ALDH1 as a CSC marker and a catalytic enzyme of Vitamin A metabolism has highlighted the retinoic acid pathway as an exploitable signalling pathway. Treatment of the resistant cells with Vitamin A (retinol) or all-trans retinoic acid (ATRA) in combination with cisplatin re-sensitised the cells to cisplatin-induced apoptosis and decreased clonogenic and proliferative propensities compared to cisplatin alone. Our data also show that combination treatments using the cancer stemness inhibitor, BBI608, currently in clinical development, reversed cisplatin resistance in NSCLC cells, highlighting the potential of targeting cancer stemness as a novel approach to developing the next generation of cancer therapeutics.
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Altered miRNA expression can result in significant effects on the regulation of genes implicated in critical survival pathways that play a role in cell survival and tumour response to cytotoxic therapies. In order to identify a signature of cisplatin resistance in NSCLC, microRNA profiling of our panel of CisR and PT NSCLC cell lines was carried out using 7th generation in-situ hybridisation arrays (Exiqon). A preliminary 10-miR signature associated with resistance was identified. Subsequent validation by qPCR confirmed a panel of 5 miRNAs (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p and miR-4286) that were significantly differentially expressed in the CisR sublines relative to their PT counterparts, highlighting a role for these miRNAs in cisplatin resistant NSCLC. In order to delineate a role for these miRNAs in this resistance phenotype, miRNA expression was genetically manipulated using Pre-miRs (miR-4286) and miRNA inhibitors (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p) after which time various in vitro functional assays were assessed. Expression analysis of the 5-miR signature was examined in a cohort of matched normal lung and tumour tissues from NSCLC patients of different histological subtypes (adenocarcinoma and squamous cell carcinoma). The 5-miR signature was successfully able to distinguish between cancerous and normal tissue and between squamous cell carcinoma and adenocarcinoma. Furthermore, serum expression of miR-4286 was able to identify squamous cell carcinoma from both normal and adenocarcinoma sera.
Based on these findings, together with the data relating to CSCs in cisplatin resistant NSCLC, the final part of this project investigated the link between our microRNAs of interest and CSCs identified in the earlier part of the study. Tumourspheres derived from CSC-selective growth medium exhibited increased expression of the 5-miR panel relative to similarly treated PT cells and the CisR sublines from which they were derived. Of the microRNAs studied, miR-34a-5p was significantly up-regulated in the ALDH1-positive subpopulations compared to their corresponding ALDH1-negative counterparts and the CisR sublines from which they were isolated, highlighting a regulatory role of miR-34a-5p within the CSC phenotype in NSCLC.
Cells possess checkpoint pathways which are important for maintaining genome stability and preventing cancer. These signalling pathways include the ATR and CHK1 kinases which are activated in response to DNA damage or replication stress. Activation of CHK1 by ATR requires the mediator protein Claspin. Claspin also regulates the rate of fork progression during DNA replication.
The levels of Claspin are cell cycle regulated and importantly Claspin is stabilised during S-phase. A growing number of ubiquitin ligases and of deubiquitylating enzymes (DUBs) have been shown to affect Claspin stability, however how they cooperate in regulating Claspin levels remains unclear.
In this thesis, I have characterised an affinity tagged overexpression system allowing for the induction of Claspin. I extensively optimised a purification protocol for this protein and using, SILAC, a quantitative proteomic approach, I identified several proteins that differentially co-purify with Claspin compared to control cells. Among these, I found a novel DUB, USP9X. I have confirmed by reciprocal immunoprecipitation experiments that it binds to Claspin Using siRNA depletion and pharmacological inhibition strategies, I found that USP9X controls Claspin stability in an S-phase specific manner. I have also demonstrated that USP9X protects proteasome-dependent Claspin degradation. Using a DNA fiber spreading approach I have established that USP9X contributes to DNA replication fork stability. I have also established that USP9X contributes to the intra S-phase replication checkpoint response and the ability of a replication fork to recover following replication stress. Further still I have demonstrated that the depletion of USP9X leads to the spontaneous accumulation of DNA damage; however this damage can be significantly rescued with the simultaneous overexpression of Claspin.
Therefore we propose that USP9X, through its role in the regulation of Claspin, is a novel player involved in the maintenance of genomic stability and in the DNA replication stress response pathway.
Toll-like receptor 2 (TLR2) is a membrane bound pathogen recognition receptor responsible for the recognition of microbial stimuli and endogenous danger or stress signals. In this thesis we examine the effect of TLR2 activation on pro-inflammatory mechanisms in RA including (i) cell migration/invasion, (ii) mitochondrial dysfunction and metabolism and (iii) inflammasome activity.
Pam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants. β1-integrin expression was significantly higher in RA synovial tissue compared to controls. Pam3CSK4 specifically induced β1-integrin binding in RASFC, with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamics through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 from RA synovial explants. Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.
Previous work from our group demonstrated mitochondrial dysfunction in RA. In this study, we demonstrate that PAM3CSK4 significantly increased mitochondrial DNA mutations, ROS and 4HNE, inhibited mitochondrial membrane potential, altered mitochondrial ultrastructure and expression of key mitochondrial genes involved in anti-apoptotic pathways. To assess if TLR2-induced mitochondrial dysfunction was paralleled by altered metabolism, we demonstrated increased expression of PKM2, ATP5B and Glut1 in RA synovium compared to OA, paralleled by a decrease in key glucose metabolism genes, increased ATP levels and induced nuclear translocation of PKM2 in RASFC in response to Pam3CSK4. Using the glycolytic inhibitor 3PO, we showed a decrease in TLR2-induced cytokine and chemokine secretion, migration, invasion, angiogenic tube formation and signalling pathways, demonstrating a key role for TLR2 in metabolism.
Finally, we examined the expression of the NLRP3 inflammasome in the RA joint. Using in vivo, in vitro and ex vivo models, we demonstrated that NLRP3, caspase-1, IL-1β and IL-18 are highly expressed and active in the RA synovium. RA ex vivo explant conditioned media and synovial fluid induced NLRP3 in healthy PBMC. Pam3CSK4 further potentiated NLRP3 activity, increasing NLRP3, caspase-1, IL-1β and IL-18 in RA ex vivo explants. Finally, specific blockade of NLRP3 using novel inhibitor MCC950 inhibited inflammasome components and pro-inflammatory mediators IL-6 and IL-8 in RA ex vivo explants.
In conclusion, TLR2 activation regulates pro-inflammatory mechanisms in the RA joint. Together this data provides sufficient evidence to consider the development of a targeted therapy for the control of inflammation by the inhibition of TLR2-mediated pathways and/or metabolic mechanisms.
The work described in this thesis focuses on the development of an innovative bioimpedance device for the detection of breast cancer using electrical impedance as the detection method. The ability for clinicians to detect and treat cancerous lesions as early as possible results in improved patient outcomes and can reduce the severity of the treatment the patient has to undergo. Therefore, new technology and devices are continually required to improve the specificity and sensitivity of the accepted detection methods. The gold standard for breast cancer detection is digital x-ray mammography but it has some significant downsides associated with it. The development of an adjunct technology to aid in the detection of breast cancers could represent a significant patient and economic benefit. In this project silicon substrates were pattern with two gold microelectrodes that allowed electrical impedance measurements to be recorded from intact tissue structures. These probes were tested and characterised using a range of in vitro and ex vivo experiments. The end application of this novel sensor device was in a first-in-human clinical trial. The initial results of this study showed that the silicon impedance device was capable of differentiating between normal and abnormal (benign and cancerous) breast tissue. The mean separation between the two tissue types 5,594 Ω with p = 0.001. The cancer type and grade at the site of the probe recordings was confirmed histologically and correlated with the electrical impedance measurements to determine if the different subtypes of cancer could each be differentiated. The results presented in this thesis showed that the novel impedance device demonstrated excellent electrochemical recording potential; was biocompatible with the growth of cultured cell lines and was capable of differentiating between intact biological tissues. The results outlined in this thesis demonstrate the potential feasibility of using electrical impedance for the differentiation of biological tissue samples. The novelty of this thesis is in the development of a new method of tissue determination with an application in breast cancer detection.
Monocytes, recently classified as CD14++CD16- (classical), CD14++CD16+ (intermediate) and CD14dimCD16++ (non-classical) are considered to play a role in the pathogenesis of inflammatory bowel diseases (IBD). Anti-tumor necrosis factor (TNF)-α monoclonal antibodies (mAb) such as infliximab (IFX) dampen inflammation in diseases such as IBD and may in part exert their therapeutic effects via actions on cells that synthesize TNFα such as monocytes. Although clinically effective in many patients, anti-TNFα mAb do not work in some, and lose efficacy in others. A more detailed understanding of the actions of IFX on blood monocytes may provide important insights into the mechanism of action of anti-TNFα mAb and mechanisms of drug resistance. We therefore studied the acute effects of the anti-TNFα mAb IFX on blood monocytes and other mononuclear cells in a cohort of IBD patients.
Multi-colour flow cytometry was used to analyse freshly isolated peripheral blood mononuclear cells (PBMCs) from Healthy Controls (HC), Crohn’s Disease (CD) and Ulcerative colitis (UC) patients not receiving IFX and CD and UC patients before (trough) and immediately after (peak) IFX infusion. An array of cytokines and chemokines produced by stimulated PBMCs was assessed at trough and peak IFX drug levels. Monocyte apoptosis (cleaved caspase-3) and lipopolysaccharide (LPS) stimulated TNFα and interleukin (IL)-12 production by intracellular staining of the trough and peak IFX samples were also examined.
Compared with the age matched HC group, CD and UC patients had a more pro-inflammatory monocyte and lymphocyte phenotype. CD patients had increased numbers of intermediate monocytes and elevated expression of the inflammatory markers CD86 and CD163. The UC patients had increased total monocyte and granulocyte numbers as well as an expansion of the CD4+ T cells and the CD19+ B cells. IFX caused a prompt reduction in the classical and intermediate subsets in both the CD and UC groups. The CD4+ T cells were significantly reduced in CD patients only. Expression levels of the inflammatory surface markers were not altered with this drug. Quantification of IFX drug levels in CD patients revealed a negative correlation between total monocyte and classical monocyte numbers with increasing levels of IFX.
We could find no evidence of IFX-induction of apoptosis to explain the reduction in the number of the circulating PBMCs. The remaining cells secreted less of certain cytokines and chemokines following in-vitro stimulation. The ex vivo production of TNFα and IL-12 by monocytes following LPS stimulation was blunted in whole blood obtained from patients at the post-IFX dosing timepoint, compared to pre-dose. However, this blunting was not observed when isolated monocytes were stimulated.
These results show that IBD is associated with a pro-inflammatory monocyte and lymphocyte phenotype when compared to HC. IFX therapy causes a reduction in the number of these circulating monocytes, lymphocytes and respective subsets. This reduction in cell number is not due to apoptosis. The remaining monocytes are dysfunctional in their ability to produce cytokines when stimulated in a whole blood assay but not when isolated cells were used. This observation suggests that ‘reverse signalling’ seems to require the cells to be in contact with other cells and when isolated the blunting effect of IFX is diminished.
Overall, monocytes seem to be a key therapeutic target of IFX. The monocytopenic and functional inhibitory actions of IFX on those cells may underlie its clinical efficacy. Future work will be needed to establish if resistance to IFX or other anti-TNFα mAb is mediated at the level of those cells.
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive form of idiopathic interstitial pneumonia, the cause of which is unknown. The current understanding of pathogenesis involves a model of abnormal wound healing in response to multiple injuries in the lung microenvironment. The CXC chemokine receptor 3 (CXCR3) receptor is 7-transmebrane G-protein coupled receptor that plays a role in limiting fibrosis following lung injury. Interleukin 13 (IL)-13 is a pro-inflammatory cytokine that mediates the development of fibrosis and signals via STAT6; it can induce expression of and bind to its own receptor IL-13Rα2. This receptor is thought to function as a non-signalling decoy receptor. Previous work in our lab has shown the anti-fibrotic effects of IL-13Rα2 in vivo and the requirement of CXCR3 for IL-13 mediated regulation of IL-13Rα2 in murine fibroblasts. This study hypothesises that CXCR3 is an anti-fibrotic receptor that is negatively regulated by IL-13 and may be positively regulated by PPARγ. This study demonstrates the expression of CXCR3 on cultured pulmonary fibroblasts from both mouse and human lung. The CXCR3 ligands; CXCL9, CXCL10 and CXCL11 significantly upregulate the expression of IL-13Rα2 by NIH3T3 fibroblasts. Ectopic CXCR3 overexpression in this cell line was shown to alter fibroblast phenotype, function and signalling capabilities. These results suggest that CXCR3 potentiates the expression of IL-13Rα2 and modulates an anti-fibrotic phenotype in fibroblasts. The regulation of CXCR3 by IL-13 on murine fibroblasts was shown to be promoter independent and cell type specific in human fibroblasts. CXCR3 -/- fibroblasts exhibit enhanced fibrotic capabilities in vitro while CXCR3-/- mice have an increased fibrotic response to IL-13 induced lung injury in vivo. The PPARγ agonist rosiglitazone regulates CXCR3 expression in primary mouse and human lung fibroblasts in a PPARγ dependent manner. Rosiglitazone alters the myofibroblast phenotype be significantly decreasing α-SMA gene expression. IPF fibroblasts have significantly higher CXCR3 gene expression than normal fibroblasts. IL-13 negatively regulates the expression of CXCR3 in normal fibroblasts and upregulates CXCR3 expression in IPF fibroblasts. Differential signalling pathways are activated in response to IL-13 in normal and IPF fibroblasts. Downregulation of STAT6 was observed in normal fibroblasts while SMAD3, TYK2, CEBPB, SP1, STAM1 and TYK2 were downregulated in the IPF fibroblast. A common STAT5B pathway was downregulated in both normal and IPF fibroblasts. Immunohistochemistry shows IL-13Rα2 and CXCR3 are expressed by fibroblasts within the fibroblastic focus, on the epithelium and by individual fibroblasts. Fibroblasts stained intensely for CXCR3 and IL-13Rα2 with weaker staining for PPARγ.
Upregulation of two anti-fibrotic receptors CXCR3 and IL-13Rα2 in IPF may representative a protective mechanism that could be harnessed therapeutically while PPARγ agonists may represent a protective therapy. Further elucidation of the signalling pathways involved may lead to novel therapeutic targets in pulmonary fibrosis.
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- January 26, 2016
- October 31, 2013
- August 3, 2012