乳腺癌CAFs中GPER介导肿瘤谷氨酰胺代谢重塑的机制及作用研究

Cancer-associated fibroblasts (CAFs) from tumor microenvironment supply abundant metabolite substrates for breast cancer energy metabolism reprogramming (EMR), biologic therapy targeting CAFs represents a new strategy in cancer management. Previous studies have demonstrated that G protein-coupled estrogen receptor (GPER) mediated estrogenic effects, and the cross-talk between breast cancer cells and CAFs specific activated downstream cAMP/PKA/CREB signaling axis via inducing cytoplasmic GPER translocation in CAFs. However, the effects of stromal GPER on breast tumor metabolic phenotype and its regulated mechanism were still not clear. Thus, microarray technology was used to obtain potent metabolic-related target genes of estrogen/GPER signaling pathway in CAFs under the co-culturing condition. According to bioinformational analysis, it was hypothesized that estrogen might activate GPER to enhance the production and secretion of glutamine (Gln) by targeted up-regulating several key metabolic genes (such as GLUL), then providing enough substrate resources for Gln metabolism of breast cancer cells. In the present program, western blot et al would be performed to identify the molecular mechanism of GPER-induced tumor Gln metabolism reprogramming. Gene interference et al would be used to explore the effects of GPER-mediated EMR on tumor biological behaviour. The potential therapeutic effects of targeting inhibition of GPER in CAFs-triggerred tumor Gln metabolism reprogramming in CAFs would be evaluated by tumor formation in nude mice et al. Our study may hopefully provide theoretical and clinical evidence for stromal GPER-targeted treatment strategy.

肿瘤微环境CAFs供给大量代谢底物参与乳腺癌能量代谢重塑（EMR），靶向CAFs是治疗新策略。研究团队前期证实，GPER在CAFs中具有雌激素受体活性；肿瘤细胞诱导CAFs中GPER细胞浆转位并特异性介导cAMP/PKA/CREB信号轴活化。然而，间质GPER对乳腺癌代谢的影响及机制尚不清楚，故运用基因芯片技术筛选出共培养下CAFs中雌激素/GPER信号下游代谢靶基因。结合生物信息学分析，提出雌激素可能活化GPER靶向上调GLUL等代谢关键基因促进CAFs合成及分泌谷氨酰胺（Gln），为乳腺癌Gln代谢提供底物来源。项目拟采用免疫印迹等技术明确GPER诱导肿瘤Gln代谢重塑的分子机制；应用基因干扰等方法探讨GPER介导的EMR对肿瘤恶性行为的影响；通过裸鼠成瘤等手段明确靶向抑制CAFs中GPER介导的肿瘤Gln代谢重塑对缓解乳腺癌进展及多药耐受的作用，为靶向间质GPER提供理论与临床依据。

肿瘤相关成纤维细胞（CAFs）是乳腺癌微环境中最主要的间质成分之一，它可供给大量代谢底物参与肿瘤能量代谢重塑（EMR），靶向CAFs有望成为未来治疗新策略。研究团队前期证实G蛋白偶联雌激素受体（GPER）在CAFs中具有雌激素受体活性；乳腺癌细胞诱导CAFs中GPER发生细胞浆转位并特异性介导cAMP/PKA/CREB信号活化促进CAFs有氧糖酵解。然而，间质GPER对乳腺肿瘤代谢的影响及机制尚不明确，仍有待进一步探讨。. 项目组前期运用基因芯片技术筛选出共培养下CAFs中雌激素/GPER信号下游代谢靶基因，结合文献报道与生物信息学分析，提出雌激素活化GPER靶向上调谷氨酰胺合成酶（GLUL）等代谢关键基因促进CAFs合成及分泌谷氨酰胺（Gln），为乳腺癌Gln代谢提供底物来源。针对上述假设，本研究以乳腺癌细胞与CAFs为共培养体外模型，临床乳腺癌组织为研究对象，探讨CAFs中GPER对Gln合成与分泌的调控关系；应用GPER、GLUL基因干扰及相关信号通路的特异性抑制剂明确CAFs中GPER靶向调控Gln合成及分泌的分子机制；应用细胞生长/划痕、线粒体活性检测、干扰谷氨酰胺转运体（ASCT1）与谷氨酰胺分解酶（GLS1）表达等方法明确CAFs来源的Gln供给对肿瘤能量代谢及生物学行为的影响，以及特异性阻断微环境GPER介导的肿瘤Gln代谢的临床潜在价值和可行性。. 通过上述研究方法，本项目首次发现：乳腺癌组织中间质GPER与间质GLUL及间质LDHB的表达呈现正相关性，并与临床药物耐受显著相关；雌激素活化CAFs中GPER/cAMP/PKA/CREB信号通路上调关键代谢基因GLUL及乳酸脱氢酶B（LDHB）的表达，一方面可促进CAFs合成及分泌大量的Gln，另一方面CAFs创造独特的乳酸微环境使乳腺癌MDA-MB-231、BT549细胞高表达ASCT1及GLS1，这进一步提高癌细胞对CAFs源性Gln的吸收及利用，导致肿瘤Gln代谢增强、线粒体活性增加、合成代谢旺盛，最终增强癌细胞生长、侵袭、转移能力及临床化疗药物耐受，靶向干预上述CAFs中GPER介导的肿瘤Gln代谢可能有较好的临床应用前景。本研究以乳腺癌CAFs中新型雌激素受体GPER为切入点，从肿瘤Gln代谢重塑角度创新性探讨其介导乳腺癌进展及临床耐药的新机制，或为提高乳腺癌患者的预后指出新的方向。