Table_1_The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein.docx

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.

缺氧诱导转录因子（Hypoxia-inducible transcription factors, HIFs）可驱动血管生成与癌细胞增殖，促成侵袭性肿瘤表型。HIF-α蛋白的水平与活性，由HIF羟化酶在翻译后水平进行调控。经羟基化修饰的HIF-α可被希佩尔-林道（von Hippel Lindau, VHL）抑癌蛋白识别，并被靶向降解。HIF羟化酶属于铁离子与2-氧戊二酸依赖性双加氧酶家族，该家族酶类需以抗坏血酸作为活性辅因子。透明细胞肾细胞癌（clear cell renal cell carcinoma, ccRCC）携带VHL基因的突变，而乳头状肾细胞癌（papillary RCC, pRCC）则携带有功能性的VHL蛋白。肾细胞癌中存在的这类天然VHL变异体，使得我们能够在临床样本中验证“抗坏血酸可通过充当HIF羟化酶的辅因子，调控HIF-α水平”这一假说。我们对73例VHL缺陷型透明细胞肾细胞癌与41例VHL功能正常型乳头状肾细胞癌的人体肿瘤组织，检测了抗坏血酸、HIF-1α、HIF-2α蛋白，以及HIF下游靶标BNIP3、CA9、cyclin D1、GLUT1与VEGF的表达水平，并将上述靶标整合以计算HIF通路评分。相较于配对的肾皮质组织，透明细胞肾细胞癌与乳头状肾细胞癌组织中的HIF水平与抗坏血酸水平均有所升高。在乳头状肾细胞癌组织中，随着抗坏血酸水平升高，HIF-1活性与整体HIF通路激活评分均呈下降趋势（斯皮尔曼相关系数r=-0.38，p<0.05；r=-0.35，p<0.05），而这一关联在透明细胞肾细胞癌组织中并未显现。体外机制研究显示，抗坏血酸可影响携带功能性VHL的透明细胞肾细胞癌细胞的HIF-1活性，但对VHL缺陷的透明细胞肾细胞癌细胞无此调控作用。我们的研究结果表明，缺乏功能性VHL的透明细胞肾细胞癌，无法对抗坏血酸介导的HIF通路调控产生应答。这与在乳头状肾细胞癌及其他携带功能性VHL的肿瘤中观察到的抗坏血酸水平与HIF通路的关联相悖。上述结果支持：在具备功能性缺氧应答通路的癌症类型中，抗坏血酸可作为HIF活性与肿瘤侵袭性的调控因子。