The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein

Hypoxia-inducible transcription factors (HIFs) drive angiogenesis and cancer cell growth, contributing to an aggressive tumor phenotype. HIF-α protein levels and activity are controlled at the post-translational level by HIF hydroxylases. Hydroxylated HIF-α is recognized by the von Hippel Lindau (VHL) tumor suppressor and targeted for degradation. The HIF hydroxylases are members of the iron and 2-oxoglutarate-dependent dioxygenases, which require ascorbate as cofactor for activity. Clear cell renal cell carcinomas (ccRCC) harbor mutations in the VHL gene, whereas papillary RCC (pRCC) have a functional VHL. These natural occurring VHL variants in RCC enable the testing, in clinical samples, of the hypothesis that ascorbate modulates HIF-α levels through its role as a cofactor for the HIF hydroxylases. We measured ascorbate, HIF-1α, and HIF-2α protein and HIF downstream targets BNIP3, CA9, cyclin D1, GLUT1, and VEGF (combined to generate the HIF pathway score) in VHL-defective ccRCC (n = 73) and VHL-proficient pRCC human tumor tissue (n = 41). HIF and ascorbate levels were increased in ccRCC and pRCC tumors compared to matched renal cortex. HIF-1 and total HIF pathway activation scores were decreased with higher ascorbate in pRCC tumors (Spearman r = −0.38, p < 0.05 and r = −0.35, p < 0.05). This was not evident for ccRCC tumors. In mechanistic studies in vitro, ascorbate influenced HIF-1 activity in VHL-proficient, but not VHL-defective ccRCC cells. Our results indicate that ccRCC, which lacks a functional VHL, does not respond to ascorbate-mediated modulation of the HIF response. This contrasts with the demonstrated association between ascorbate content and the HIF pathway observed in pRCC and other tumors with a functional VHL. The results support a role for ascorbate as a modulator of HIF activity and tumor aggression in cancer types with a functional hypoxic response

The role of ascorbate in controlling hypoxia factors in renal cell carcinoma

Clear cell RCC (ccRCC) is the most common and also most aggressive RCC type harbouring alterations in the VHL tumour suppressor gene leading to uncontrolled accumulation of pro-survival hypoxia inducible transcription factors (HIF-1/HIF-2). The second most common form, papillary RCC (pRCC), has a functional VHL protein with regulated HIF levels. Protein levels and activity of hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by enzymes belonging to the family of iron- and 2-oxoglutarate-dependent dioxygenases. Hydroxylation of HIF by proline hydroxylases (PHDs) targets the protein for degradation via the VHL-ubiquitin-ligase complex, and hydroxylation by factor inhibiting HIF (FIH) leads to its inactivation. Both PHD and FIH enzymes require ascorbate as cofactor, and absence of ascorbate leads to increased HIF activation. Previous data in endometrial, colorectal, breast and thyroid cancer patients has shown that higher tumour ascorbate levels are associated with lower HIF-1 levels and reduced markers of tumour aggression. To verify the role of ascorbate in cancer, ccRCC tumours were specifically selected as model of VHL mutation in comparison to VHL-proficient pRCCs. Humans acquire ascorbate from their diet. It is actively absorbed from the intestines via sodium-dependent vitamin C transporter 1 (SVCT1), carried in the blood and taken up into all cells of the body via SVCT2. SVCT1 is also responsible for ascorbate reuptake in the kidney. Information of the ascorbate transporters in cancer is very sparse. The aim of this thesis was to verify that ascorbate modulates HIF proteins by supporting the activity of the regulatory hydroxylases. Markers of HIF-1 and HIF-2 activation were analysed in banked tumour samples from kidney cancer patients with matched normal renal cortex and related to tissue ascorbate levels and clinico-pathological data. In addition, the influence of ascorbate on HIF pathway activity was monitored in VHL-proficient and VHL-defective ccRCC cell lines in vitro. Furthermore, efficiency of proline hydroxylation of HIF-1a was monitored in gene-modified VHL-defective ccRCC cells after ascorbate treatment. In addition, this thesis aimed to understand the relationship between SVCTs and ascorbate in clinical RCC samples and cell lines. In pRCC patients, HIF pathway activity was increased in ascorbate-deficient tumours, supporting previous data in other cancer types. However, the same association was not evident in ccRCC patients. Ascorbate content was not associated with patient survival in either cohort, while a higher HIF-2 pathway score was negatively associated with disease-free survival in patients with ccRCC. In vitro, ascorbate was able to dampen induction of HIF only in VHL-proficient cells. In VHL-defective cells, elevated levels of hydroxylated HIF-1a in response to increasing intracellular ascorbate was observed. In patient samples, protein levels of SVCT1 were increased compared to normal renal cortex tissue. Tumour samples also showed an increase in the (presumed) glycosylated form of SVCT2, with a decrease seen in non-glycosylated SVCT2. Although overall levels of ascorbate were higher in tumours compared to renal cortex, no association between transporter levels and ascorbate content was detected in individual patients. Protein expression and localisation was also not significantly affected by the ascorbate in vitro. This thesis was the first study to investigate the association between ascorbate and HIF-1/HIF-2 in RCC. Data from VHL-proficient pRCC provided evidence for a universal association between ascorbate and the HIF-pathway in cancer. The comparison with VHL-deficient ccRCC tumours supports the role of ascorbate as a modulator of tumour aggression, but only in cancer types with a functional hypoxic response which is important for the design of future clinical trials. We show mechanistic proof in living cells that ascorbate can modulate the hypoxic pathway in cancer cells by increasing HIF hydroxylase activity. The investigation into SVCTs demonstrates the complexities of ascorbate uptake mechanisms in RCC. The results from this thesis provide the first direct evidence for the requirement of ascorbate for effective HIF-hydroxylase activity in cancer cells and strongly support a role for ascorbate as a modulator of HIF activity in cancer types with a functional hypoxic response

The role of ascorbate in controlling hypoxia factors in renal cell carcinoma

Clear cell RCC (ccRCC) is the most common and also most aggressive RCC type harbouring alterations in the VHL tumour suppressor gene leading to uncontrolled accumulation of pro-survival hypoxia inducible transcription factors (HIF-1/HIF-2). The second most common form, papillary RCC (pRCC), has a functional VHL protein with regulated HIF levels. Protein levels and activity of hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by enzymes belonging to the family of iron- and 2-oxoglutarate-dependent dioxygenases. Hydroxylation of HIF by proline hydroxylases (PHDs) targets the protein for degradation via the VHL-ubiquitin-ligase complex, and hydroxylation by factor inhibiting HIF (FIH) leads to its inactivation. Both PHD and FIH enzymes require ascorbate as cofactor, and absence of ascorbate leads to increased HIF activation. Previous data in endometrial, colorectal, breast and thyroid cancer patients has shown that higher tumour ascorbate levels are associated with lower HIF-1 levels and reduced markers of tumour aggression. To verify the role of ascorbate in cancer, ccRCC tumours were specifically selected as model of VHL mutation in comparison to VHL-proficient pRCCs. Humans acquire ascorbate from their diet. It is actively absorbed from the intestines via sodium-dependent vitamin C transporter 1 (SVCT1), carried in the blood and taken up into all cells of the body via SVCT2. SVCT1 is also responsible for ascorbate reuptake in the kidney. Information of the ascorbate transporters in cancer is very sparse. The aim of this thesis was to verify that ascorbate modulates HIF proteins by supporting the activity of the regulatory hydroxylases. Markers of HIF-1 and HIF-2 activation were analysed in banked tumour samples from kidney cancer patients with matched normal renal cortex and related to tissue ascorbate levels and clinico-pathological data. In addition, the influence of ascorbate on HIF pathway activity was monitored in VHL-proficient and VHL-defective ccRCC cell lines in vitro. Furthermore, efficiency of proline hydroxylation of HIF-1a was monitored in gene-modified VHL-defective ccRCC cells after ascorbate treatment. In addition, this thesis aimed to understand the relationship between SVCTs and ascorbate in clinical RCC samples and cell lines. In pRCC patients, HIF pathway activity was increased in ascorbate-deficient tumours, supporting previous data in other cancer types. However, the same association was not evident in ccRCC patients. Ascorbate content was not associated with patient survival in either cohort, while a higher HIF-2 pathway score was negatively associated with disease-free survival in patients with ccRCC. In vitro, ascorbate was able to dampen induction of HIF only in VHL-proficient cells. In VHL-defective cells, elevated levels of hydroxylated HIF-1a in response to increasing intracellular ascorbate was observed. In patient samples, protein levels of SVCT1 were increased compared to normal renal cortex tissue. Tumour samples also showed an increase in the (presumed) glycosylated form of SVCT2, with a decrease seen in non-glycosylated SVCT2. Although overall levels of ascorbate were higher in tumours compared to renal cortex, no association between transporter levels and ascorbate content was detected in individual patients. Protein expression and localisation was also not significantly affected by the ascorbate in vitro. This thesis was the first study to investigate the association between ascorbate and HIF-1/HIF-2 in RCC. Data from VHL-proficient pRCC provided evidence for a universal association between ascorbate and the HIF-pathway in cancer. The comparison with VHL-deficient ccRCC tumours supports the role of ascorbate as a modulator of tumour aggression, but only in cancer types with a functional hypoxic response which is important for the design of future clinical trials. We show mechanistic proof in living cells that ascorbate can modulate the hypoxic pathway in cancer cells by increasing HIF hydroxylase activity. The investigation into SVCTs demonstrates the complexities of ascorbate uptake mechanisms in RCC. The results from this thesis provide the first direct evidence for the requirement of ascorbate for effective HIF-hydroxylase activity in cancer cells and strongly support a role for ascorbate as a modulator of HIF activity in cancer types with a functional hypoxic response

Vitamin C Transporters in Cancer: Current Understanding and Gaps in Knowledge

Sufficient uptake and whole body distribution of vitamin C (ascorbate) is essential for many biochemical processes, including some that are vital for tumor growth and spread. Uptake of ascorbate into cancer cells is modulated by availability, tumor blood flow, tissue diffusion parameters, and ascorbate transport proteins. Uptake into cells is mediated by two families of transport proteins, namely, the solute carrier gene family 23, consisting of sodium-dependent vitamin C transporters (SVCTs) 1 and 2, and the SLC2 family of glucose transporters (GLUTs). GLUTs transport the oxidized form of the vitamin, dehydroascorbate (DHA), which is present at negligible to low physiological levels. SVCT1 and 2 are capable of accumulating ascorbate against a concentration gradient from micromolar concentrations outside to millimolar levels inside of cells. Investigating the expression and regulation of SVCTs in cancer has only recently started to be included in studies focused on the role of ascorbate in tumor formation, progression, and response to therapy. This review gives an overview of the current, limited knowledge of ascorbate transport across membranes, as well as tissue distribution, gene expression, and the relevance of SVCTs in cancer. As tumor ascorbate accumulation may play a role in the anticancer activity of high dose ascorbate treatment, further research into ascorbate transport in cancer tissue is vital

Best practices on critical reagent characterization, qualification, and life cycle management for HCP immunoassays

The performance of immunoassays for the detection and quantification of host cell proteins (HCPs) in biopharmaceuticals depends on the quality of the critical assay reagents. Not only their preparation, but also a stringent life-cycle management, including reagent qualification, requalification and replacement, plays a crucial role in ensuring consistent and reliable results. To provide a cross-industry perspective on HCP reagent management, we conducted a survey on common practices among several pharmaceutical and biotech companies. Based on its outcome, as well as informed by a corresponding roundtable session (“Managing critical reagents over time”) at the BioPharmaceutical Emerging Best Practices Association (BEBPA) HCP conference in 2019, this work presents specific recommendations and proven concepts to support immunoassay reagent management for monitoring HCPs

Cancer of the uterus and treatment of stress incontinence: a pilot study

The purpose of this pilot study was to determine whether women with early-stage endometrial cancer could be screened for stress urinary incontinence (SUI) at their initial gynecologic oncology visit and referred to a urogynecologist for concurrent treatment of their endometrial cancer and SUI

A Profound Basic Characterization of eIFs in Gliomas: Identifying eIF3I and 4H as Potential Novel Target Candidates in Glioma Therapy

Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients’ overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I–IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients’ survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients