Abstract
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.
Highlights
The hypoxia-inducible factors (HIFs) are heterodimeric prosurvival transcription factors consisting of oxygen-sensitive regulatory α subunits (HIF-1α, HIF-2α, or HIF-3α) and a stably expressed HIF-1β subunit [1, 2]
Most papillary RCC (pRCC) tumors were classified as type 2 (53%) vs. type 1 (34%); patients with type 2 have been reported to have a poorer prognosis than those with type 1 [20]
Our study in 114 renal cell carcinoma (RCC) tumors demonstrates an association between the HIF pathway and ascorbate in tumors that is dependent on the presence of a functional von Hippel-Lindau (VHL) tumor suppressor
Summary
The hypoxia-inducible factors (HIFs) are heterodimeric prosurvival transcription factors consisting of oxygen-sensitive regulatory α subunits (HIF-1α, HIF-2α, or HIF-3α) and a stably expressed HIF-1β subunit [1, 2]. Under physiological conditions HIF-α protein levels are tightly regulated by post-translational hydroxylation by enzymes belonging to the family of Fe(II) and 2-oxoglutaratedependent dioxygenases. These hydroxylases require oxygen, 2oxoglutarate, ascorbate, and iron for activity [3]. Inadequate supply of the hydroxylases with either of the two substrates oxygen or 2-oxoglutarate, as well as absence of the essential cofactors ascorbate or iron, impairs activity, leading to increased HIF stabilization and activation [4, 5]. We and others have demonstrated an inverse association between tumor ascorbate levels and HIF-1 activation in clinical samples from patients with colorectal, endometrial, and thyroid cancer [7,8,9]. Increased tumor ascorbate content was associated with significantly improved disease-free survival for colorectal cancer patients [8]
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