亚牛磺酸代谢及其调控KCNN4对胶质瘤侵袭性影响的机制研究

The most notorious feature of glioma is its invasive growth. Our previous study proved that hypotaurine content in gliomas associated with WHO grade. Isocitrate dehydrogenases (IDHs) mutations are frequently found in gliomas. Hypotaurine could stabilize hypoxia inducible factor –1α (HIF-1α) through the similar mechanism as that of 2-hydroxyglutarate which is generated by mutant IDHs. Both of the two metabolites could promote tumorigenesis. Unfortunately, the dominant origin and its action mechanisms of hypotaurine in promoting glioma invasion are still elusive. We had observed that hypotaurine facilitated expression of KCNN4. In this study, we intend to explore the major origin of hypotaurine in the malignant and benign astrocytes to disclose which metabolic pathway, the 2-aminoethanethiol dioxygenase (ADO) or cysteine dioxygenase (CDO) pathway, is crucial to their individual intracellular hypotaurine. The activity of the corresponding key metabolic pathway in patient glioma tissues will also be evaluated to ascertain its relationship to tumor grade. Because IDH mutations can be frequently found in gliomas, next, through tumor tissue hypotaurine quantitation and IDH mutation analysis, we try to explain the relationship between hypotaurine metabolism and IDH mutations in view of metabolites. Hypotaurine and the mutant IDH enzymatic product 2-hydroxyglutarate share similar tumorigenesis mechanism. It is still unknown if they can function competitively, cooperatively or independently within the same cell. Lastly, by using various genetically modified cells, glioma tissues and tumor burden nude mice, we want to test if KCNN4 expression is positively linked to intracellular hypotaurine in vivo and in vitro. Simultaneously, whether KCNN4 is regulated by HIF-1α directly or indirectly through vascular endothelial growth factor (VEGF) will also be probed to disclose the potential mechanism of invasion promoting effects from hypotaurine. Through the answering of the abovementioned questions, hopefully, we can provide a potential metabolite target that can be managed to arrest the pathological progress and improve the prognosis of glioma.

胶质瘤的危害主要源于其侵袭性生长。我们发现亚牛磺酸可促进胶质瘤细胞侵袭，它能与胶质瘤中常见的IDHs突变产生的2-羟基戊二酸以类似机制稳定HIF-1α，并显著上调KCNN4表达，但胶质瘤中亚牛磺酸的代谢及促侵袭机制尚未完全明确。本研究将通过对正常和瘤细胞内亚牛磺酸主要来源比较，揭示胶质瘤中亚牛磺酸主要受控于ADO还是CDO途径，并在各级实体瘤中验证相应途径活动程度与肿瘤级别关系；继而，通过实体瘤中亚牛磺酸的定量和相应IDHs突变分析，探讨两种作用机制雷同的促瘤因素，在代谢物水平上是互补、竞争还是独立发挥作用，明确亚牛磺酸代谢是否受IDHs突变影响；最后，借助各种工程细胞、实体瘤组织和裸鼠等的体内外试验，验证亚牛磺酸含量与KCNN4表达程度之间是否正相关，阐明亚牛磺酸是通过HIF-1α直接还是间接通过VEGF调控KCNN4表达、促进胶质瘤侵袭，以期为有效干预胶质瘤进程和改善预后筛选新靶点。

胶质瘤的危害主要源于其侵袭性生长。我们发现亚牛磺酸可促进胶质瘤细胞侵袭，它能与胶质瘤中常见的IDHs突变产生的2-羟基戊二酸以类似机制稳定HIF-1alpha，并显著上调KCNN4表达。本研究通过对正常和瘤细胞内亚牛磺酸主要来源比较，发现胶质瘤中亚牛磺酸来源主要受控于2-氨基乙硫醇双加氧酶（ADO）途径，其另一主要合成途径半胱胺加双氧酶（CDO）途径在胶质瘤中表达并不活跃。通过实体瘤中亚牛磺酸的定量和相应IDHs突变分析，发现ADO-亚牛磺酸途径是独立于IDHs突变以外的促瘤因素。.我们在上一NSFC资助的研究中发现亚牛磺酸含量在低、高级别胶质瘤中的差异最显著，其含量与胶质瘤恶性程度呈高度正相关。且亚牛磺酸含量与其合成关键酶ADO的表达呈正相关。研究证实少突胶质细胞瘤的染色体1p 和19q 共缺失致使侵袭能力减弱，我们猜想，亚牛磺酸促进胶质瘤侵袭作用，可能是直接或间接影响到染色体1p 和/或19q 上相关基因表达继而左右了胶质瘤细胞的侵袭性。为此，我们对利用iRNA干扰ADO基因的U251细胞系和对照细胞系进行了转录组分析发现，ADO基因能够上调19q13.2 位点上的KCNN4 基因表达。经再次利用CRISPR-Cas9构造的LN229细胞ADO基因敲除株与对照株分析，我们发现ADO/亚牛磺酸轴通过激活NF-kappaB信号通路进而促进趋化因子CCL20的分泌，从而维持胶质瘤干细胞的干性和促进增殖和侵袭。相反，通过CRISPR/Cas9技术敲除ADO，则会导致体内、体外实验中胶质瘤细胞增殖、侵袭受抑制，破坏胶质瘤干细胞自我更新的能力等。进一步试验发现抑制KCNN4的表达能够同样达到抑制NF-kappa B信号通路作用，同时抑制CCL20的分泌。抑制VEGF信号通路可抑制KCNN4的表达。上述过程的抑制作用均可通过补充亚牛磺酸得到不同程度挽救。抑制HIF-1alpha表达也同时使KCNN4受抑，但是如果敲除HIF-1alpha基因后，再抑制VEGF、KCNN4或NF-kappa B信号通路中的任何一个则亚牛磺酸几乎不发挥明显的挽救作用。因此我们认为，ADO/亚牛磺酸轴是通过HIF-1alpha、VEGF、KCNN4、NF-kappa B、CCL20分子的顺次作用，发挥促进胶质瘤作用。