Dietary consumption of tea and the risk of prostate cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial

Tea contains polyphenols such as flavonoids, anthocyanidins, flavanols and phenolic acids which in laboratory studies have reported to promote antioxidant enzyme formation, reduces excess inflammation, slow cancer cell proliferation and promote apoptosis. Evidence from epidemiological studies, on the effect of tea consumption on CaP incidence has been conflicting. We analysed data from 25 097 men within the intervention arm of the 155000 participant Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial screening trial. Histologically confirmed cases of prostate cancer were reported in 3,088 men (12.3%) during the median 11.5 year follow up. Tea consumption was assessed with a food frequency questionnaire. Baseline characteristics were compared between groups using Chi-square and Kruskal-Wallis tests. Cox regression models were used to assess associations between tea intake and CaP incidence. There was no statistical difference between the risk of CaP between men who never drank tea to those who drank tea at any quantity. Amongst tea drinkers, those in the highest third of consumption group had a small but significantly lower risk compared to those in the lowest third (11.2% v 13.2% HR 1.16; 95% CI 1.05-1.29, p=0.004). This pattern persisted with adjustments for demographics and lifestyle. In conclusion, among tea drinkers, there was a small positive association between drinking tea and a reduced risk of prostate cancer. It does not support starting to drink tea, if men previously did not, to reduce the risk. Further research is needed to establish whether tea is justified for future prospective nutritional intervention studies investigating CaP prevention

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

[This corrects the article DOI: 10.3389/fonc.2018.00388.]

Medical treatment of renal cancer: new horizons.

Renal cell carcinoma (RCC) makes up 2-3% of adult cancers. The introduction of tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin inhibitors in the mid-2000s radically changed the management of RCC. These targeted treatments superseded immunotherapy with interleukin-2 and interferon. The pendulum now appears to be shifting back towards immunotherapy, with the evidence of prolonged overall survival of patients with metastatic RCC on treatment with the anti-programmed cell death 1 ligand monoclonal antibody, nivolumab. Clinical prognostic criteria aid prediction of relapse risk for resected localised disease. Unfortunately, for patients at high risk of relapse, no adjuvant treatment has yet shown benefit, although further trials are yet to report. Clinical prognostic models also have a role in the management of advanced disease; now there is a pressing need for predictive biomarkers to direct therapy. Treatment selection for metastatic disease is currently based on histology, prognostic group and patient preference based on side effect profile. In this article, we review the current medical and surgical management of localised, oligometastatic and advanced RCC, including side effect management and the evidence base for management of poor-risk and non-clear cell disease. We discuss recent results from clinical trials and how these are likely to shape future practice and a renaissance of immunotherapy for renal cell cancer

Medical treatment of renal cancer: new horizons.

Renal cell carcinoma (RCC) makes up 2–3% of adult cancers. The introduction of tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin inhibitors in the mid-2000s radically changed the management of RCC. These targeted treatments superseded immunotherapy with interleukin-2 and interferon. The pendulum now appears to be shifting back towards immunotherapy, with the evidence of prolonged overall survival of patients with metastatic RCC on treatment with the anti-programmed cell death 1 ligand monoclonal antibody, nivolumab. Clinical prognostic criteria aid prediction of relapse risk for resected localised disease. Unfortunately, for patients at high risk of relapse, no adjuvant treatment has yet shown benefit, although further trials are yet to report. Clinical prognostic models also have a role in the management of advanced disease; now there is a pressing need for predictive biomarkers to direct therapy. Treatment selection for metastatic disease is currently based on histology, prognostic group and patient preference based on side effect profile. In this article, we review the current medical and surgical management of localised, oligometastatic and advanced RCC, including side effect management and the evidence base for management of poor-risk and non-clear cell disease. We discuss recent results from clinical trials and how these are likely to shape future practice and a renaissance of immunotherapy for renal cell cancer

Investigating the role of the UDP-galactose transporter SLC35A2 in the regulation of HIF signalling

Background: The hypoxia inducible factor (HIF) family of dimeric transcription factors play a vital role in the cellular response to low oxygen (hypoxia). HIF activation leads to the transcriptional upregulation of a range of genes that are involved in diverse physiological and pathophysiological processes. In malignant tumours increased HIF-α expression, and HIF activation are commonly observed, and are associated with poorer prognosis and disease progression. HIF activity is controlled by a number of different mechanisms, and the identification of molecular regulators of HIF is of intense interest. The Golgi UDP-galactose transporter SLC35A2 was identified by the Ashcroft group as a novel regulator of HIF-α, and CHO cells that had lost SLC35A2 exhibited elevated HIF-α protein in normoxia. It is known that the absence of SLC35A2 in the Golgi leads to wide-ranging glycosylation defects. The underlying mechanisms linking SLC35A2 to HIF-α protein regulation were not known. This thesis aims to further characterise the role effect of SLC35A2 loss on HIF signalling and tumour cell behaviour, in CHO cells and a human cell system, specifically by exploring the hypotheses that these cells may harbour an autophagy defect, or differences in overall UDP-sugar content, in particular O-GlcNAc. Methods and Results: I used parental and Slc35a2 mutant CHO cells, SLC35A2 knockout (KO) and wild type (WT) suspension HeLa (sHeLa) cells, and a panel of cancer cells lines, including 786O, RCC4 renal carcinoma cells which exhibit constitutive HIF activation due to loss of VHL function. Confirming previous work from the Ashcroft group, I showed that Slc35a2 mutant (M6.19) CHO cells exhibit elevated normoxic HIF-1α protein levels and evidence of a glycosylation defect. Expanding on these findings, I found that SLC35A2 loss led to elevated normoxic HIF-α (HIF-1α and HIF-2α) protein and *HIFA* mRNΑ, and elevated expression of HIF-α target genes (*GLUT1*, *VEGF*). Using global RNAseq analysis of SLC35A2 KO and WT sHeLa cells, I found that SLC35A2 loss was associated with highly upregulated expression of a range of genes. Further exploration of the observed glycosylation defect in Slc35a2 mutant CHO cells, showed that SLC35A2 KO sHeLa cells also exhibited altered mobility of GLUT1 protein, which I found was consistent with treatment of WT sHeLa cells with tunicamycin, a protein *N*-glycosylation inhibitor. UDP-sugar analysis by HPLC of Slc35a2 mutant CHO and SLC35A2 KO sHeLa cells indicated increased levels of UDP-GlcNAc compared to their WT counterparts. Furthermore, I found similar patterns of increased UDP-GlcNAc levels in SLC35A2 KO compared to WT sHeLa cells in normoxia and hypoxia, while stable reconstitution of Slc35a2 in Slc35a2 mutant (M6.19) CHO cells reduced levels of UDP-GlcNAc and rescued CMP-sialic acid levels. Consistent with my findings and the involvement of UDP-GlcNAc in glutamine metabolism, I found that Slc35a2 mutant (M6.19) CHO cells were significantly more sensitive to glutamine withdrawal compared to parental (C4.5) CHO cells. Slc35a2 mutant (M6.19) and SLC35A2 KO sHeLa cells exhibited a glycosylation defect of LAMP2A, a protein involved in HIF-1α lysosomal degradation and critical for chaperone mediated autophagy. SLC35A2 mutant (M6.19) CHO cells were significantly more sensitive to the inhibition of proliferation by the inhibitors of autophagy bafilomycin and 3-methyladenine, and showed elevated levels of the autophagy marker LC3B. Finally, from analyses of the TCGA-KIRC database I found *SCL35A2* expression was higher in patients with non-mutant VHL versus mutant VHL in renal cancers. In support of these findings, I showed that basal SLC35A2 protein and mRNA levels were higher in patient-derived 786O renal carcinoma cells reconstituted with wild type VHL (786O-VHL) compared to matched 786-O empty vector control (786O-EV) cells. I also found that high *SLC35A2* expression is associated with poor prognosis in patients with *VHL* non-mutant ccRCC. Conclusion: Taken together, my thesis identifies SLC35A2 as a regulator of HIF-α and metabolism, potentially through its role in regulating UDP-sugars, and reveals a possible novel role for SLC35A2 in lysosomal processing and autophagy. *SLC35A2* expression in renal cell carcinoma is associated with VHL status, which may provide a new route for dysregulation of HIF, altered metabolism, and changes in lysosomal processing and autophagy

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.TB was funded by a British Heart Foundation (BHF) (FS/09/051) and METOXIA FP7 (HEALTH454 F2-2009-222741) awards to MA. JMS was funded by a Medical Research Council (MRC) Doctoral Training and Sackler award (RG70550) to MA. LWT was funded by MRC grants (MR/K002201/1, MR/K002201/2) to MA. CE was funded by a Cancer Research UK (CRUK) award (C7358/A19442) to MA. YLC was supported by the CR-UK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grant C1060/A10334, and CRUK grant C1060/A16464. LAM was funded by a CRUK PhD Studentship (RG91141) award to MA. BG was funded by a CRUK Clinical Research Training Fellowship (RG85993) award to MA. SV and SES were funded by MRC grant: Medical Research Council (MC_UU_12022/7)