甘油醛-3-磷酸脱氢酶细胞核定位缺失相关的乳腺癌三苯氧胺耐药的机制研究

About 60-75% human breast tumors express estrogen receptor (ER) α which is an estrogen-dependent transcriptional factor facilitating breast tumorigenesis and progression. Tamoxifen (Tam) is the most often prescribed antiestrogen for breast cancers, and Tam resistance (TR) remains one of the most critical obstacles for breast cancer management. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme and considered as a housekeeping gene. Its potential role in breast cancer pathogenesis is understudied. We have previously reported that GAPDH is upregulated in breast cancer mammosphere-derived cells when compared to the parental cells, indicated that GAPDH might be implied in breast cancer tumorigenesis and progression. The preliminary studies for this current proposal showed that the nuclear expression of GAPDH could independently predict better outcome for ERα-positive breast cancer patients and significantly correlated with the expression of progesterone receptor whose clinical role is to predict breast cancer responsiveness to tamoxifen, we also discovered the presence of nuclear localized GAPDH in parental MCF-7 cells but not in tamoxifen resistant MCF-7 cells (MCF-7 TR) , concomitantly, we found the reduced concentration of nitric oxide (NO) in MCF-7 TR when compared to parental MCF-7 cells. Further we determined that nitrosoglutathione（GSNO) , an endogenous bioavailable source of NO, could drive GAPDH translocate to the nucleus of MCF-7 TR cells while repressed the proliferation of MCF-7 TR cells much more profoundly than that of parental MCF-7 cells, indicated that deficiency of nuclear GAPDH might be causal for breast cancer resistance to tamoxifen. Since nuclear GAPDH could function as an ERα coactivator to induce the transcription of sodium coupled neutral amino acid transporter (SNAT2), we thus examined the expression of SNAT2 and unexpectedly detected an enhanced mRNA level in MCF-7 TR cells, in comparison to MCF-7 cells. This interesting finding promoted us to examine the expression of glutamine synthetase (GLUL) whose expression endowed glutamine-independency of MCF-7 cells, and discovered a decreased expression of GLUL. Eventually, through analyzing the public dataset, we found that either higher expression of SANT2 or lower expression of GLUL could predict worse outcome for tamoxifen treated breast cancers, indicated that enhanced glutamine uptake and subsequent glutaminolyis might be related to TR. These data enabled us to postulate that nuclear GAPDH deficiency related TR might be related to the transition of addicted nutrients for luminal breast cancers. In this proposal, we will extend our preliminary studies to define the mechanisms underlying nuclear GAPDH deficiency related TR..The Specific Aims are:.1..To determine whether nuclear deficiency of GAPDH is causal for TR and to elucidate the potential mechanism;.2..To examine whether nuclear GAPDH deficiency might be resulted from the transition of addicted metabolic patterns, and vice versa;.3..To test whether and how the yet to be defined transition of addicted metabolic metabolic patterns might confer breast cancer TR;.4..To explore whether targeting GAPDH to the nucleus or aiming at TR-related metabolism could reverse TR.

三苯氧胺耐药（TR）是乳腺癌临床最重要的问题之一，与代谢的关系却鲜见报道。我们前期工作发现甘油醛-3-磷酸脱氢酶（GAPDH）核定位缺失可独立指示雌激素受体（ERα）阳性乳癌患者预后不良，继而发现TR的乳腺癌细胞（MCF-7 TR）中也存在GAPDH核定位缺失，伴随一氧化氮（NO）浓度降低，MCF-7 TR细胞对可驱使其GAPDH转位入核的NO供体异常敏感。继续研究发现MCF-7 TR细胞中谷氨酰胺合成酶表达降低而一种谷氨酰胺转运蛋白SNAT2表达升高。进一步研究发现这两个有利于肿瘤细胞利用谷氨酰胺降解途径的变化与乳腺癌患者TR显著相关。提示与GAPDH核定位缺失相关的TR或与代谢途径的变化有关， 而靶向GAPDH入核的策略或可用于逆转TR。本研究将阐释GAPDH核定位缺失与乳腺癌代谢途径改变的关系及其导致TR的机制，并将探讨靶向GAPDH入核和靶向相关代谢的策略可否逆转TR。

三苯氧胺是乳腺癌临床上应用最为广泛的抗雌激素药物，其耐药（TR）是乳腺癌临床最重要的问题之一，与代谢的关系却鲜见报道。本项目主要研究甘油醛-3-磷酸脱氢酶（GAPDH）核定位缺失与乳腺癌代谢途径改变的关系及其导致TR的机制，并将探讨靶向GAPDH入核和靶向相关代谢的策略可否逆转TR。.1. GAPDH的的核定位缺失可以指示三苯氧胺耐药；.2. 对三苯氧胺耐药前后细胞模型的代谢组学分析显示，精氨酸水平的下降与三苯氧胺耐药有关；.3. 三苯氧胺耐药模型中一氧化氮（NO）的水平降低，而脂肪酸合成酶（FASN）的高表达可以指示三苯氧胺耐药；.4. 三苯氧胺耐药模型中FASN的亚硝基化减少，谷胱甘肽过氧化物酶4（GPX4）水平下降，铁死亡被抑制。