Mechanism of extracellular citrate uptake and metabolism in cancer; specificity and potential use of gluconate in cancer therapy

为了转移和增殖，癌细胞需要调整其代谢以适应对能量和蛋白质合成的增加需求。从氧化磷酸化到厌氧糖酵解的Warburg效应被认为是癌症的一个标志。有趣的是，柠檬酸盐作为脂肪酸合成的主要底物，在癌细胞代谢中起着核心作用，因此被广泛研究。癌细胞中柠檬酸盐的来源提出了一个特别有趣和重要的问题，因为众所周知，提供正常细胞代谢所必需的柠檬酸盐的线粒体在癌症中活性降低。另外，谷氨酰胺还原羧化已被认为是恶性细胞中柠檬酸盐的主要来源。我们最近发现，癌细胞出人意料地吸收细胞外的柠檬酸盐，并用它来支持它们的新陈代谢。从癌细胞中克隆了质膜柠檬酸转运蛋白（pmCiC），并确定其主要负责柠檬酸盐的导入。此外，我们的研究表明pmCiC在人体组织中表达，其表达主要局限于癌细胞，与肿瘤分期和转移潜能相关。重要的是，我们发现葡萄糖酸盐是一种不可逆的pmCiC抑制剂，并在中试实验中表明，体内用葡萄糖酸盐治疗实验性胰腺癌可显著降低肿瘤生长并显著改变肿瘤组织的代谢特征。在本项目中，我们将：(1)研究癌细胞对柠檬酸盐摄取和代谢的机制，(2)利用CRISPR-Cas9技术稳定沉默pmCiC，(3)确定葡萄糖酸盐对pmCiC的特异性，并测试葡萄糖酸盐对癌症和良性细胞的作用，(4)在临床前体内模型中通过稳定沉默或与葡萄糖酸盐一起抑制pmCiC对癌症发展的治疗作用，(5)更精确地测定pmCiC在正常细胞和癌细胞中的表达。(6)确定pmCiC的表达作为肿瘤进展/分期的标志。所提出的研究将进一步揭示这种支持癌细胞代谢的新机制，这种机制可能被用于治疗癌症。此外，测定人体组织中柠檬酸转运蛋白的表达将有助于确定其作为癌症进展分子标志物的潜力。

To metastasize and proliferate, cancer cells need to adjust their metabolism to the increased need for energy and protein synthesis. Warburg effect, the switch from oxidative phosphorylation to anaerobic glycolysis, is considered a hallmark of cancer. Interestingly, citrate as the primary substrate in fatty acid synthesis occupies a central role in cancer cell metabolism, and therefore has been studied extensively. The source of citrate in cancer cells presents a particularly interesting and important question because mitochondria which supply citrate necessary for normal cell metabolism are known to have reduced activity in cancer. Alternatively, glutamine reductive carboxylation has been suggested as the main source of citrate in malignant cells. We have recently discovered that cancer cells unexpectedly take up extracellular citrate and use it to support their metabolism. Plasma membrane citrate transporter (pmCiC) was cloned from cancer cells and determined to be responsible for citrate import. Moreover, our studies indicate that the pmCiC is expressed in human tissues and its expression is mainly restricted to cancer cells, correlating with their stage and metastatic potential. Importantly, we have found gluconate to be an irreversible inhibitor of the pmCiC and show in pilot experiments that treatment of experimental pancreatic cancer with gluconate in vivo results in significantly decreased cancer growth and markedly changes metabolic characteristics of tumor tissues.In the present project we will: (1) study the mechanisms of citrate uptake and metabolism by cancer cells, (2) stably silence pmCiC using CRISPR-Cas9 technique, (3) determine the specificity of gluconate towards pmCiC and test effects of gluconate on cancer versus benign cells, (4) test therapeutic effects of pmCiC inhibition through stable silencing or with gluconate on cancer development in a preclinical in vivo model, (5) more precisely determine pmCiC expression in normal versus cancer cells, and (6) determine pmCiC expression as a marker of tumor progression/stage.The proposed studies will further reveal this novel mechanism supporting cancer cell metabolism that can potentially be exploited therapeutically to treat cancer. Furthermore, determination of citrate transporter expression in human tissues will be useful in establishing its potential as a molecular marker of cancer progression.