Gene delivery for the treatment of prostate cancer

Prostate cancer is one of the most common cancers diagnosed in men. Whilst treatments for early-stage disease are largely effective, current therapies for metastatic prostate cancer, particularly for bone metastasis, offer only a few months increased lifespan at best. Hence new treatments are urgently required. Small interfering RNA (siRNA) has been investigated for the treatment of prostate cancer where it can ‘silence’ specific cancer-related genes. However the clinical application of siRNA-based gene therapy is limited due to the absence of an optimised gene delivery vector. The optimisation of such gene delivery vectors is routinely undertaken in vitro using 2D cell culture on plastic dishes which does not accurately simulate the in vivo bone cancer metastasis microenvironment. The goal of this thesis was to assess the potential of two different targeted delivery vectors (gold or modified β-cyclodextrin derivatives) to facilitate siRNA receptor-mediated uptake into prostate cancer cells. Furthermore, this project aimed to develop a more physiologically relevant 3D in vitro cell culture model, to mimic prostate cancer bone metastasis, which is suitable for evaluating the delivery of nanoparticulate gene therapeutics. In the first instance, cationic derivatives of gold and β-cyclodextrin were synthesized to complex anionic siRNA. The delivery vectors were targeted to prostate cancer cells using the anisamide ligand which has high affinity for the sigma receptor that is overexpressed by prostate cancer cells. The gold nanoparticle demonstrated high levels of uptake into prostate cancer PC3 cells and efficient gene silencing when transfection was performed in serum-free media. However, due to the absence of a poly(ethylene glycol) (PEG) stabilising group, the formulation was unsuitable for use in serum-containing conditions. Conversely, the modified β-cyclodextrin formulation demonstrated enhanced stability in the presence of serum due to the inclusion of a PEG chain onto which the anisamide ligand was conjugated. However, the maximum level of gene silencing efficacy from three different prostate cancer cell lines (DU145, VCaP and PC3 cells) was 30 %, suggesting that further optimisation of the formulation would be required prior to application in vivo. In order to develop a more physiologically-relevant in vitro model of prostate cancer bone metastasis, prostate cancer cells (PC3 and LNCaP cells) were cultured in 3D on collagenbased scaffolds engineered to mimic the bone microenvironment. While the model was suitable for assessing nanoparticle-mediated gene knockdown, prostate cancer cells demonstrated a phenotype with lower invasive potential when grown on the scaffolds relative to standard 2D cell culture. Hence, prostate cancer cells (PC3 and LNCaP cells) were subsequently co-cultured with bone osteoblast cells (hFOB 1.19 cells) to enhance the physiological relevance of the model. Co-cultures secreted elevated levels of the MMP9 enzyme, a marker of prostate cancer metastasis, relative to prostate cancer cell monocultures (2D and 3D) indicating enhanced physiological relevance of the model. Furthermore, the coculture model proved suitable for investigating nanoparticle-mediated gene silencing. In conclusion, the work outlined in this thesis identified two different sigma receptor-targeted gene delivery vectors with potential for the treatment of prostate cancer. In addition, a more physiologically relevant model of prostate cancer bone metastasis was developed with the capacity to help optimise gene delivery vectors for the treatment of prostate cancer.

Ethnicity and the prostate cancer experience: a qualitative metasynthesis

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Digoxin use after diagnosis of prostate cancer and survival: a population-based cohort study

PURPOSE:
Preclinical studies have shown that digoxin exerts anticancer effects on different cancer cell lines including prostate cancer. A recent observational study has shown that digoxin use was associated with a 25% reduction in prostate cancer risk. The aim of this study was to investigate whether digoxin use after diagnosis of prostate cancer was associated with decreased prostate cancer-specific mortality.
METHODS:
A cohort of 13 134 patients with prostate cancer newly diagnosed from 1998 to 2009 was identified from English cancer registries and linked to the UK Clinical Practice Research Datalink (to provide digoxin and other prescription records) and to the Office of National Statistics mortality data (to identify 2010 prostate cancer-specific deaths). Using time-dependent Cox regression models, unadjusted and adjusted hazard ratios (HR) and 95% confidence intervals (CIs) were calculated for the association between post-diagnostic exposure to digoxin and prostate cancer-specific mortality.
RESULTS:
Overall, 701 (5%) patients with prostate cancer used digoxin after diagnosis. Digoxin use was associated with an increase in prostate cancer-specific mortality before adjustment (HR = 1.59; 95% CI 1.32-1.91), but after adjustment for confounders, the association was attenuated (adjusted HR = 1.13; 95% CI 0.93-1.37) and there was no evidence of a dose response.
CONCLUSIONS:
In this large population-based prostate cancer cohort, there was no evidence of a reduction in prostate cancer-specific mortality with digoxin use after diagnosis.

DNA vaccination for prostate cancer: key concepts and considerations

While locally confined prostate cancer is associated with a low five year mortality rate, advanced or metastatic disease remains a major challenge for healthcare professionals to treat and is usually terminal. As such, there is a need for the development of new, efficacious therapies for prostate cancer. Immunotherapy represents a promising approach where the host's immune system is harnessed to mount an anti-tumour effect, and the licensing of the first prostate cancer specific immunotherapy in 2010 has opened the door for other immunotherapies to gain regulatory approval. Among these strategies DNA vaccines are an attractive option in terms of their ability to elicit a highly specific, potent and wide-sweeping immune response. Several DNA vaccines have been tested for prostate cancer and while they have demonstrated a good safety profile they have faced problems with low efficacy and immunogenicity compared to other immunotherapeutic approaches. This review focuses on the positive aspects of DNA vaccines for prostate cancer that have been assessed in preclinical and clinical trials thus far and examines the key considerations that must be employed to improve the efficacy and immunogenicity of these vaccines.

Specific humoral response of hosts with variable schistosomiasis susceptibility

The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers-the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

Ethnicity and the prostate cancer experience: a qualitative metasynthesis

Objectives: To summarise black and minority ethnic (BME) patients' and partners
experiences of prostate cancer (PCa) by examining the findings of existing qualitative studies
Methods:
We undertook a systematic metasynthesis of qualitative studies using a modified version of
Noblit and Hare's 'meta-ethnography' approach, with a 2000-2015 search of seven databases.
Results: Thirteen studies of men from US and UK BME groups were included. We explored
constructs with BME-specific features. Healthcare provider relationships, formation of a
spiritual alliance with God (which enhanced the participants’ feeling of empowerment and
ability to cope with the cancer) and living on for others (generally to increase cancer
awareness), often connected to spiritual regrowth, were the three constructs most commonly
reported. A magnified effect from erectile dysfunction was also common. Initially this
affected men’s disclosure to others about their cancer and their sexual problems, but
eventually men responded by shifting their conceptualisations of masculinity to sustain self
and social identities. There was also evidence of inequality resulting from financial
constraints and adversity that necessitated resilience in coping.
Conclusions: The prostate cancer experience of BME men and their partners is affected by a
complex intersection of ethnicity with other factors. Healthcare services should acknowledge
this. If providers recognise the men’s felt masculinities, social identities and spiritual beliefs
and their shifting nature, services could be improved, with community as well as individual
benefits. More studies are needed in diverse ethnic groups

HNF1B variants associate with promoter methylation and regulate gene networks activated in prostate and ovarian cancer

Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor.

Dual stimulation of antigen presenting cells using carbon nanotube-based vaccine delivery system for cancer immunotherapy

Although anti−cancer immuno−based combinatorial therapeutic approaches have shown promising results, efficient tumour eradication demands further intensification of anti−tumour immune response. With the emerging field of nanovaccinology, multi−walled carbon nanotubes (MWNTs) have manifested prominent potentials as tumour antigen nanocarriers. Nevertheless, the utilization of MWNTs in co−delivering antigen along with different types of immunoadjuvants to antigen presenting cells (APCs) has not been investigated yet. We hypothesized that harnessing MWNT for concurrent delivery of cytosine−phosphate−guanine oligodeoxynucleotide (CpG) and anti-CD40 Ig (αCD40), as immunoadjuvants, along with the model antigen ovalbumin (OVA) could potentiate immune response induced against OVA−expressing tumour cells. We initially investigated the effective method to co−deliver OVA and CpG using MWNT to the APC. Covalent conjugation of OVA and CpG prior to loading onto MWNTs markedly augmented the CpG−mediated adjuvanticity, as demonstrated by the significantly increased OVA−specific T cell responses in vitro and in C57BL/6 mice. αCD40 was then included as a second immunoadjuvant to further intensify the immune response. Immune response elicited in vitro and in vivo by OVA, CpG and αCD40 was significantly potentiated by their co−incorporation onto the MWNTs. Furthermore, MWNT remarkably improved the ability of co−loaded OVA, CpG and αCD40 in inhibiting the growth of OVA−expressing B16F10 melanoma cells in subcutaneous or lung pseudo−metastatic tumour models. Therefore, this study suggests that the utilization of MWNTs for the co−delivery of tumour−derived antigen, CpG and αCD40 could be a competent approach for efficient tumours eradication.

Regulatory T cells control strain specific resistance to Experimental Autoimmune Prostatitis

Susceptibility to autoimmune diseases results from the encounter of a complex and long evolved genetic context with a no less complex and changing environment. Major actors in maintaining health are regulatory T cells (Treg) that primarily dampen a large subset of autoreactive lymphocytes escaping thymic negative selection. Here, we directly asked whether Treg participate in defining susceptibility and resistance to Experimental Autoimmune Prostatitis (EAP). We analyzed three common laboratory strains of mice presenting with different susceptibility to autoimmune prostatitis upon immunization with prostate proteins. The NOD, the C57BL/6 and the BALB/c mice that can be classified along a disease score ranging from severe, mild and to undetectable, respectively. Upon mild and transient depletion of Treg at the induction phase of EAP, each model showed an increment along this score, most remarkably with the BALB/c mice switching from a resistant to a susceptible phenotype. We further show that disease associates with the upregulation of CXCR3 expression on effector T cells, a process requiring IFNγ. Together with recent advances on environmental factors affecting Treg, these findings provide a likely cellular and molecular explanation to the recent rise in autoimmune diseases incidence.

Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia.

Background: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent. Objective: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection. Patient and Methods: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene). Results: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result. Conclusion: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.

Development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to T cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/-recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts. This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.