Immunotherapy for the treatment of poor prognosis cancers

Cancer is amongst the leading causes of death worldwide and the number one cause in the developed world. Every year there are close to 10 million cancer related deaths and this corresponds to hundreds of millions of euro in health care costs and lost productivity, placing a substantial drain on the economy. The efficacy of traditional treatment modalities for cancer therapy, such as surgery, radiotherapy and chemotherapy has plateaued, and while they are undoubtedly effective at prolonging patient lifespan, there is a high rate of adverse side effects and fatal reoccurrence. Currently, there is a huge amount of interest in the areas of cancer immunosurveillance and cancer immuno-editing, which explain some of the complex interactions between the host immune system and cancer. If left unchecked, cancerous malignancies have the ability to generate an immunosuppressive microenvironment, effectively shielding themselves from elimination and promoting tumour growth and progression. To overcome this, the potential of the immune system must be harnessed and the work undertaken in this thesis sought to contribute to this goal. Focus was placed on using novel therapies, combining tumour ablation with immune-modulating antibodies to maximise tumour elimination in an immune dependent manner, to overcome immunosuppression and promote immune activation. Chapter 2 focuses on the use of ECT as a method of tumour ablation and its effects on the immune system. ECT proved to be effective at inhibiting the tumour growth both in vitro and in vivo, and conferred significant survival advantages in both small and large animal models. More importantly, ECT proved to cause tumour death in an immune dependent manner, displaying the hallmarks of Immunogenic Cell Death, increases in immune cell infiltration and generating tumour-specific immune responses. Chapter 3 focuses on combining ECT with immune checkpoint blockade inhibitors; anti- CTLA-4 and anti-PD-1. Both combinations proved to be effective at inhibiting both primary and distal tumour growth, indicating the generation of tumour specific immune responses and prolonged animal survival. In addition, the treatments caused increases in the levels of certain intra-tumoural immune cell subsets and modulated the cytokine profile of treated animals in a way that was favourable overall. Chapter 4 focuses on the combining ECT with an anti-iCOS agonist antibody, capable of causing immune co-stimulation. This novel combinational therapy proved to be the most effective by far, with a high cure rate achieved across a number of different in vivo tumour models. Total regression was seen in both primary and distal tumours, as well as spontaneous metastases, with the tumour specific immune response generated conferring total protection to animals on tumour rechallenge. Overall the data presented here adds further insight into the area of cancer immunotherapy with some of the novel combinational therapies demonstrating substantial clinic potential.

Leptin modifies the prosecretory and prokinetic effects of the inflammatory cytokine interleukin-6 on colonic function in Sprague–Dawley rats

Leptin ameliorates the prosecretory and prokinetic effects of the pro-inflammatory cytokine interleukin-6 on rat colon. Leptin also suppresses the neurostimulatory effects of irritable bowel syndrome plasma, which has elevated concentrations of interleukin-6, on enteric neurons. This may indicate a regulatory role for leptin in immune-mediated bowel dysfunction. In addition to its role in regulating energy homeostasis, the adipokine leptin modifies gastrointestinal (GI) function. Indeed, leptin-resistant obese humans and leptin-deficient obese mice exhibit altered GI motility. In the functional GI disorder irritable bowel syndrome (IBS), circulating leptin concentrations are reported to differ from those of healthy control subjects. Additionally, IBS patients display altered cytokine profiles, including elevated circulating concentrations of the pro-inflammatory cytokine interleukin-6 (IL-6), which bears structural homology and similarities in intracellular signalling to leptin. This study aimed to investigate interactions between leptin and IL-6 in colonic neurons and their possible contribution to IBS pathophysiology. The functional effects of leptin and IL-6 on colonic contractility and absorptosecretory function were assessed in organ baths and Ussing chambers in Sprague–Dawley rat colon. Calcium imaging and immunohistochemical techniques were used to investigate the neural regulation of GI function by these signalling molecules. Our findings provide a neuromodulatory role for leptin in submucosal neurons, where it inhibited the stimulatory effects of IL-6. Functionally, this translated to suppression of IL-6-evoked potentiation of veratridine-induced secretory currents. Leptin also attenuated IL-6-induced colonic contractions, although it had little direct effect on myenteric neurons. Calcium responses evoked by IBS plasma in both myenteric and submucosal neurons were also suppressed by leptin, possibly through interactions with IL-6, which is elevated in IBS plasma. As leptin has the capacity to ameliorate the neurostimulatory effects of soluble mediators in IBS plasma and modulated IL-6-evoked changes in bowel function, leptin may have a role in immune-mediated bowel dysfunction in IBS patients.

Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Function of the PDLIM2 protein in oncogenic Wnt signalling pathways

Aberrant regulation of the Wnt signalling pathway is a recurrent theme in cancer biology. Hyper activation due to oncogenic mutations and paracrine activity has been found in both colon cancer and breast cancer, and continues to evolve as a central mechanism in oncogenesis. PDLIM2, a cytoskeletal PDZ protein, is an IGF-1 regulated gene that is highly expressed in cancer cell lines derived from metastatic tumours. Suppression of PDLIM2 inhibits polarized cell migration, reverses the Epithelial to Mesenchymal transition (EMT) phenotype, suppresses the transcription of β-catenin target genes, and regulates gene expression of key transcription factors in EMT. This thesis investigates the mechanism by which PDLIM2 contributes to the maintenance of Wnt signalling in cancer cells. Here we show that PDLIM2 is a critical regulator of the Wnt pathway by regulating β-catenin at the adherens juctions, as also its transcriptional activity by the interaction of PDLIM2 with TCF4 at the nucleus. Evaluation of PDLIM2 in macrophages and co-culture studies with cancer cells and fibroblasts showed the influence exerted on PDLIM2 by paracrine cues. Thus, PDLIM2 integrates cytoskeleton signalling with gene expression by modulating the Wnt signalling pathway and reconciling microenvironmental cues with signals in epithelial cells. Negative correlation of mRNA and protein levels in the triple negative breast cancer cell BT549 suggests that PDLIM2 is part of a more complex mechanism that involves transcription and posttranslational modifications. GST pulldown studies and subsequent mass spectrometry analysis showed that PDLIM2 interacts with 300 proteins, with a high biological function in protein biosynthesis and Ubiquitin/proteasome pathways, including 13 E3 ligases. Overall, these data suggest that PDLIM2 has two distinct functions depending of its location. Located at the cytoplasm mediates cytoskeletal re-arrangements, whereas at the nucleus PDLIM2 acts as a signal transduction adaptor protein mediating transcription and ubiquitination of key transcription factors in cancer development.

Therapeutically targeting mitochondrial redox signalling alleviates endothelial dysfunction in preeclampsia

Aberrant placentation generating placental oxidative stress is proposed to play a critical role in the pathophysiology of preeclampsia. Unfortunately, therapeutic trials of antioxidants have been uniformly disappointing. There is provisional evidence implicating mitochondrial dysfunction as a source of oxidative stress in preeclampsia. Here we provide evidence that mitochondrial reactive oxygen species mediates endothelial dysfunction and establish that directly targeting mitochondrial scavenging may provide a protective role. Human umbilical vein endothelial cells exposed to 3% plasma from women with pregnancies complicated by preeclampsia resulted in a significant decrease in mitochondrial function with a subsequent significant increase in mitochondrial superoxide generation compared to cells exposed to plasma from women with uncomplicated pregnancies. Real-time PCR analysis showed increased expression of inflammatory markers TNF-α, TLR-9 and ICAM-1 respectively in endothelial cells treated with preeclampsia plasma. MitoTempo is a mitochondrial-targeted antioxidant, pre-treatment of cells with MitoTempo protected against hydrogen peroxide-induced cell death. Furthermore MitoTempo significantly reduced mitochondrial superoxide production in cells exposed to preeclampsia plasma by normalising mitochondrial metabolism. MitoTempo significantly altered the inflammatory profile of plasma treated cells. These novel data support a functional role for mitochondrial redox signaling in modulating the pathogenesis of preeclampsia and identifies mitochondrial-targeted antioxidants as potential therapeutic candidates.

Steroid induced neuroprotection of damaged photoreceptor cells

Retinitis Pigmentosa (RP) is the name given to a group of hereditary diseases causing progressive and degenerative blindness. RP affects over 1 in 4000 individuals, making it the most prevalent inherited retinal disease worldwide, yet currently there is no cure. In 2011, our group released a paper detailing the protective effects of the synthetic progestin ‘Norgestrel’. A common component of the female oral contraceptive pill, Norgestrel was shown to protect against retinal cell death in two distinct mouse models of retinal degeneration: in the Balb/c light damage model and the Pde6brd10 (rd10) model. Little was known of the molecular workings of this compound however and thus this study aimed to elucidate the protective manner in which Norgestrel worked. To this aim, the 661W cone photoreceptor-like cell line and ex vivo retinal explanting was utilised. We found that Norgestrel induces a increase in neuroprotective basic fibroblast growth factor (bFGF) with subsequent downstream actions on the inhibition of glycogen synthase kinase 3β. Progesterone receptor expression was subsequently characterised in the C57 and rd10 retinas and in the 661W cell line. Norgestrel caused nuclear trafficking of progesterone receptor membrane complex one (PGRMC1) in 661W cells and thus Norgestrel was hypothesised to work primarily through the actions of PGRMC1. This trafficking was shown to be responsible for the critical upregulation of bFGF and PGRMC1- Norgestrel binding was proven to cause a neuroprotective bFGF-mediated increase in intracellular calcium. The protective properties of Norgestrel were further studied in the rd10 mouse model of retinitis pigmentosa. Using non-invasive diet supplementation (80mg/kg), we showed that Norgestrel gave significant retinal protection out to postnatal day 40 (P40). Overactive microglia have previously been shown to potentiate photoreceptor cell loss in the degenerating rd10 retina and thus we focussed on Norgestrel-mediated changes in photoreceptor-microglial crosstalk. Norgestrel acted to dampen pro-inflammatory microglial cell reactivity, decreasing chemokine (MCP1, MCP3, MIP-1α, MIP-1β) and subsequent damaging cytokine (TNFα, Il-1β) production. Critically, Norgestrel up-regulated photoreceptor-microglial, fractalkine-CX3CR1 signalling 1000-fold in the P20 rd10 mouse. Known to prevent microglial activation, we hypothesise that Norgestrel acts as a vital anti-inflammatory in the diseased retina, driving fractalkine-CX3CR1 signalling to delay retinal degeneration. This study stands to highlight some of the neuroprotective mechanisms utilised by Norgestrel in the prevention of photoreceptor cell death. We identify for the first time, not only a pro-survival pathway activated directly in photoreceptor cells, but also a Norgestreldriven mediation of an otherwise damaging microglial cell response. All taken, these results form the beginning of a case to bring Norgestrel to clinical trials, as a potential therapeutic for the treatment of RP.