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Background Improvement of patient with cancer outcome can be achieved by identifying novel mechanisms that underlie cancer pathogenesis. GTP cyclohydrolase (GTPCH) is the rate-limiting enzyme for neopterin and tetrahydrobiopterin (BH4) synthesis. Elevated neopterin in patients associates strongly with cancer progression, while excessive BH4 favours tumour cell proliferation. We have recently found that GTPCH transforms normal fibroblasts and induces tumour angiogenesis; a similar role has been reported by another group in human colon and melanoma tumour xenografts. However, the mechanisms of GTPCH-driven tumour formation remain largely unknown. Method Immunohistochemistry (IHC) was used to investigate GTPCH expression in a variety of tumour tissue microarray (TMA). Gene microarray data were analyzed to correlate GTPCH elevation with prognosis of breast cancer patients. Cell cultures and mouse xenografts were models used to evaluate molecular effects of GTPCH expression in breast tumour cells. Results In screening TMA with IHC, GTPCH was found commonly highly expressed in breast, colon, lung and endometrium carcinomas and lymphomas. Analysis of a series of gene array data and interrogation of Oncomine showed a significant correlation of high GCH1 with worse prognosis and lower recurrence-free survival, particularly in the estrogen receptor (ER) or progesterone receptor (PR)-negative breast cancer in > 3,000 patients. In cell cultures, GTPCH transformed normal MCF10A epithelial cells and increased breast cancer BT474 malignancy; they displayed with an epithelial to mesenchymal transition phenotype. GTPCH increased MDA-MB231 cell proliferation and scratch wound healing, and upregulated RTK Tie2-associated Akt/ERK phosphorylation and spontaneously downregulated retinoblastoma tumour suppressor protein. In mouse xenotransplantation, GTPCH significantly increased tumour growth, angiogenesis and perfusion, and glycolytic metabolism pathway induction. These effects were attenuated by the GTPCH inhibitor. Conclusion Our study demonstrates a novel pathway by which GTPCH potentiates tumour growth, supporting further development of small molecule inhibitors and combination therapies most effective for clinical translation.

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.

Background: Hypoxia imaging is a promising tool for targeted therapy but the links between imaging features and underlying molecular characteristics of the tumour have not been investigated. The aim of this study was to compare hypoxia biomarkers and gene expression in oropharyngeal squamous cell carcinoma (OPSCC) diagnostic biopsies with hypoxia imaged with 64Cu-ATSM PET/CT. Methods: 64Cu-ATSM imaging, molecular and clinical data were obtained for patients. Primary tumour SUVmax, tumour to muscle ratio (TMR) and hypoxic volume were tested for association with reported hypoxia gene signatures in diagnostic biopsies. A putative gene signature for hypoxia in OPSCCs (hypoxic volume-associated gene signature, HVS) was derived. Results: Hypoxic volume was significantly associated with a reported hypoxia gene signature (rho=0.57, P=0.045), but SUVmax and TMR were not. Immunohistochemical staining with the hypoxia marker carbonic anhydrase 9 (CA9) was associated with a gene expression hypoxia response (rho=0.63, P=0.01). 16 genes were positively and 5 genes negatively associated with hypoxic volume (adjusted P

In cultured clear-cell renal carcinoma (CCRCC) 786-0 cells transfected with HIF1alpha (HIF-1+), HIF-2alpha (HIF-2+), or empty vector (EV), no significant differences were observed in the growth rates in vitro, but when grown in vivo as xenografts HIF-2alpha significantly increased, and HIF-1alpha significantly decreased growth rates, compared to EV tumors. Factors associated with proliferation were increased and factors associated with cell death were decreased in HIF-2+ tumors. Metabolite profiles showed higher glucose and lower lactate and alanine levels in the HIF-2+ tumors whilst immunostaining demonstrated higher pyruvate dehydrogenase and lower pyruvate dehydrogenase kinase 1, compared to control tumors. Taken together, these results suggest that overexpression of HIF-2alpha in CCRCC 786-0 tumors regulated growth both by maintaining a low level of glycolysis and by allowing more mitochondrial metabolism and tolerance to ROS induced DNA damage. The growth profiles observed may be mediated by adaptive changes to a more oxidative phenotype.

OBJECTIVE: To develop a relational database (Cancer Research Uro-Oncology Database, CRUD) to enable automatic data collection on all urological malignancies within our region, as there is increasing emphasis on good data collection for surgical patients with cancer, and numerous overlapping systems that are amassing data on the same patients. METHODS: Links have been established between pathological databases, multidisciplinary team data-collection systems and patient-survival monitoring facilities, providing accurate pathology, treatment and survival data on all of uro-oncology patients. We are also developing individual modules for the oncological surgeons within our unit that are compatible with the British Association of Urological Surgeons (Section of Oncology), and have plans to connect to the Medical and Clinical Oncology data systems in the future. RESULTS: Pre-existing protocols for fresh tissue, plasma and urine collection have been incorporated within CRUD via a tissue-tracking system, to comply with the Human Tissue Act 2004, and link these samples to accurate clinical, pathological and survival data. Many research and audit projects have already used these data, including the construction of a 274-case tissue microarray for renal cell carcinoma, microRNA hybridization arrays and analysis of 900 nephrectomy cases from the past three decades. CONCLUSIONS: Our work over the past 3 years in Oxford has established numerous links with organizations collecting data on our uro-oncological patients, and we are now able to collect this excellent combined data on all of these patients, in an automated manner.

AIMS: Angiogenesis in solid tumour pathology is well established but less is known about its role in haematological malignancies. Our study investigated the immunohistochemical expression of a variety of angiogenic and hypoxic factors and microvessel densities on 110 cases of high- and low-grade non-Hodgkin's lymphomas and reactive lymphoid tissues. methods and results: Expression of vascular endothelial growth factor (VEGF) was present in 82 (96%) of the non-Hodgkin's cases and 35 (100%) of the reactive lymphoid tissue cases. Both hypoxia inducible factors 1 alpha and 2 alpha (HIF 1 alpha, 2 alpha) were weakly expressed in the majority of high- and low-grade lymphomas. Carbonic anhydrase IX (CA IX), a HIF-inducible membrane-bound enzyme, expression was not abundant with membranous staining being present in seven (8%) of the lymphoma cases and none of the reactive cases. Thymidine phosphorylase (TP) was distributed amongst macrophages and follicular dendritic cells but was not present in the neoplastic population. The vasculature was stained using CD34 which gave rise to a distinct vascular, predominantly paracortical network present in low-grade lymphomas and reactive lymphoid tissue but which was lost in high-grade lymphomas. CONCLUSION: Our results suggest that non-Hodgkin's lymphomas may be less angiogenic and hypoxically driven than most solid tumours, which has implications for possible future therapies.

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Worldwide, breast cancer is the most frequently diagnosed cancer in women and the leading cause of cancer death among women. The concentrations of bio-metals are crucial for the homeostasis of human health and are being shown to have significantly different concentrations when comparing human cancer tissue and normal tissue. This is the first study that correlates the findings of the differences in the levels of certain elements between individual tumours, to the clinical prognostic factors such as oestrogen receptor (ER) status, lymph node status, tumour size, grade, menopause status, human epidermal growth factor receptor 2 status, epidermal growth factor receptor status, relapsed status and survival status. Micro probe synchrotron radiation X-ray fluorescence techniques have been used to determine the localization and the relative concentrations of Zn, Cu, Fe and Ca in 128 formalin-fixed paraffin-embedded invasive ductal breast cancer (IDC) samples and normal surrounding breast tissue. The statistical analysis reveals a significant increase in the levels of Ca, Fe, Cu and Zn concentrations by 85%, 20%, 23% and 117%, respectively, in IDC tissue when compared to the normal breast tissue. Our study shows that increased relative expressions of Zn, Fe and Ca are all associated with ER positive breast cancers and also indicates that the imbalance in iron concentration (deficiency) should be viewed as an important risk factor that is associated with aggressive features of the cancer. Characterisation of the difference of bio-metals in tumour to normal regions will help in selecting treatment for breast cancer with novel agents that chelate iron or zinc. © 2013 John Wiley and Sons, Ltd.

AIMS: Human AP endonuclease 1 (HAP1) plays a major role in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA by catalysing hydrolytic cleavage of the phosphodiester backbone 5' to the site. HAP1 is also known to be a potent reduction-oxidation (redox) factor, regulating the binding activity of a number of transcription factors. The purpose of the present study was to examine the expression of HAP-1 in a wide range of human tissues. METHODS AND RESULTS: Using a recently developed specific rabbit polyclonal antibody, we performed immunohistochemistry on paraffin-embedded tissue material. Nuclear staining was detected in crypt cells of the small and large intestine, epithelial cells of breast ducts, basal cells of the skin, alveolar cells of the lung, lymphocytes of the marginal zone of the spleen, in the surface epithelium and stromal cells of the ovary and the transitional epithelium of the bladder. Unexpectedly for a presumed nuclear protein, the staining pattern in some cell populations was mainly cytoplasmic (e.g. superficial cells of gastrointestinal tract, Langerhans cells, Leydig cells and spermatocytes, epithelium of the prostate glands), or both cytoplasmic and nuclear (e.g. epithelial cells of thymus, follicular thyroid cells, parietal cells of the stomach, glandular epithelial cells of the cervix, epithelial cells of exocrine pancreas). CONCLUSION: This differential expression in a wide spectrum of cells is indicative of a potential multifunctional action of HAP1, not necessarily restricted to a role in the nucleus. Histopathology