CaV channels and cancer: canonical functions indicate benefits of repurposed drugs as cancer therapeutics

The importance of ion channels in the hallmarks of many cancers is increasingly recognised. This article reviews current knowledge of the expression of members of the voltage-gated calcium channel family (CaV) in cancer at the gene and protein level and discusses their potential functional roles. The ten members of the CaV channel family are classified according to expression of their pore-forming α-subunit; moreover, co-expression of accessory α2δ, β and γ confers a spectrum of biophysical characteristics including voltage dependence of activation and inactivation, current amplitude and activation/inactivation kinetics. CaV channels have traditionally been studied in excitable cells including neurones, smooth muscle, skeletal muscle and cardiac cells, and drugs targeting the channels are used in the treatment of hypertension and epilepsy. There is emerging evidence that several CaV channels are differentially expressed in cancer cells compared to their normal counterparts. Interestingly, a number of CaV channels also have non-canonical functions and are involved in transcriptional regulation of the expression of other proteins including potassium channels. Pharmacological studies show that CaV canonical function contributes to the fundamental biology of proliferation, cell-cycle progression and apoptosis. This raises the intriguing possibility that calcium channel blockers, approved for the treatment of other conditions, could be repurposed to treat particular cancers. Further research will reveal the full extent of both the canonical and non-canonical functions of CaV channels in cancer and whether calcium channel blockers are beneficial in cancer treatment.

Targeting treatment resistant cancer stem cells with FKBPL and its peptide therapeutics

FKBPL and its peptide derivatives have already demonstrated well-established inhibitory effects on cancer growth and CD44-dependent anti-angiogenic activity. Since cancer stem cells (CSCs) are CD44 positive, we wanted to explore if these therapeutics could specifically target CSCs in breast and ovarian cancer. In a tumoursphere assay, FKBPL stable overexpression or FKBPL-based peptide (AD-01, preclinical peptide or ALM201, clinical peptide candidate) treatment were highly effective at reducing the CSC population measured by inhibiting tumoursphere forming efficiency in breast and ovarian cancer cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- and ALDH+ cell subpopulations representative of CSCs, validated these results. The ability of AD-01 and ALM201 to inhibit the self-renewal capacity of CSCs was confirmed across three generations, eradicating CSC completely by the third generation (p<0.001). Furthermore, clonogenic assay demonstrated that FKBPL-based peptides mediated CSC differentiation, with a significant decrease in the number of CSCs or holoclones and an associated increase in differentiated cancer cells or meroclones/paraclones. In addition, AD-01 treatment in vitro and in vivo led to a significant reduction in the stem cell markers, Nanog, Sox2 and Oct4 protein and mRNA levels; whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in tumoursphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). Additionally, when AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in CSCs. Importantly, using gold standard in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, FKBPL-based peptides appear to have dual anti-angiogenic and anti-CSC activity which will be advantageous as this agent enters clinical trial.

An investigation of the molecular pharmacology of G protein-coupled receptor 35

G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling drugs on the market and there are a substantial number of GPCRs that are yet to be characterised; these could offer interest for therapeutic targeting. GPR35 is one such receptor that, as a result of gene knockout and genome wide association studies, has attracted interest through its association with cardiovascular and gastrointestinal disease. Elucidation of the basic physiological function of GPR35 has, however, been difficult due a paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an assay format suitable for the characterisation of newly identified ligands. Although GPR35 associates with both Gαi/o and Gα13 families of small heterotrimeric G proteins, the G protein-independent β-arrestin-2 recruitment format was found to be the most suited to drug screening efforts. Small molecule compound screening, carried out in conjunction with the Medical Research Council Technology, identified compound 1 as the most potent ligand of human GPR35 reported at that time. However, the lower efficacy and potency of compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated human and rat GPR35 with equal potency. This finding offered–for the first time–the opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to consider when translating rodent in vivo functional studies to those in man. Additionally, using molecular modelling and site directed mutagenesis studies, these newly identified compounds were used to aid characterisation of the ligand binding pockets of human and rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the basic biology of GPR35 and investigate its contribution to disease.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.