乳腺癌叶酸转运受体表达水平调节及其经核转录因子CREB调控的分子机制研究

Antifolate agent is one of the most widely used drugs in chemotherapy. Optimizing selection of targeted antifolate drugs is critical to individualized chemotherapy of breast carcinoma. Previous studies had demonstrated that uptake of folate and antifolate drugs were mainly processed by reduced folate carrier (RFC) and folate receptor (FR) into tumor cells.These two receptors affect cell proliferation and apoptosis by metabolic enzymes. The results of our preliminary experiments had demonstrated that over-expression of FR-α was significantly associated with decreased RFC in various breast cancer cell lines with different molecular subtypes. Both expression levels of RFC and FR-α were regulated by folic acid and estrogen / tamoxifen (TAM). At the same time, we also found that these unbalance expressions of RFC and FR-α was correlated with the nuclear transcription factor-CREB under stimulation with exogenous estrogen. The promoter region of RFC contains CRE and cAMP response element binding protein (CREB) could regulate the transcription of CRE. We had demonstrated the co-expression among CREB, FR and RFC in breast cancer based on the experimental results of breast cancer cell lines and the database of TCGA. This project aims to study the effect of CREB signaling pathway on cell proliferation after activating or inhibiting FR-α of breast cancer cell by folate and/or estrogen in vitro and animal model and also research the molecular regulation mechanism of CREB in transcription of RFC. To investigate the molecular mechanism of CREB on two folate transport receptors will benefit to antifolate targeted therapy of breast carcinoma.

合理选择抗叶酸代谢药物是实现乳腺癌患者个体化化疗迫切需要研究的方向。既往研究表明叶酸及抗叶酸代谢药物主要通过还原叶酸载体(reduced folate carrier，RFC)及叶酸受体（folate receptor，FR）转运入肿瘤细胞，通过代谢关键酶影响细胞的增殖和凋亡。我们前期实验首次发现各分子亚型乳腺癌细胞株均存在RFC表达下调和FR-α表达升高；两种叶酸受体表达失衡受到叶酸、雌激素/雌激素拮抗剂的调节。RFC基因启动子区域含有CRE，可受到cAMP应答元件结合蛋白（CREB）的调控。基于多种乳腺癌细胞株及TCGA肿瘤数据库的实验结果，我们证实了乳腺癌中CREB与FR和RFC的共表达关系。本项目拟通过体外实验和动物模型研究乳腺癌细胞FR-α激活或抑制后，以CREB为中心轴的信号传导通路对细胞增殖的影响，研究CREB对RFC的分子调控机制，为乳腺癌靶向叶酸受体提供理论基础。

乳腺癌是女性最常见的恶性肿瘤，也是危害妇女生命健康最主要的恶性肿瘤，目前发病率已超过肺癌，成为发病率最高的恶性肿瘤。研究证实乳腺癌是一组高度异质性的肿瘤，基于基因表达谱可分为5种分子亚型。不同分子亚型的乳腺癌具有不同的临床病理特征，对治疗的反应也有所不同。如何对乳腺癌患者进行更为精确的个体化治疗，选择最有效的治疗方式和药物是目前该领域的研究重点。.细胞生长代谢需要多种营养元素的共同作用，其中微量元素叶酸（folic acid, FA）在胚胎生长发育中起到重要作用。叶酸不能自身合成，必须完全依赖外源性供给和摄入。近年研究发现过多摄入叶酸不仅对机体无保护作用，而且具有肿瘤促进作用。深入研究叶酸转运相关调控通路，寻找相应肿瘤治疗靶向位点已成为目前国内外研究的热点。.FR-α作为最主要的叶酸受体亚型，在正常组织中低水平表达，在多种恶性肿瘤，如卵巢癌、宫颈癌、鼻咽癌中高表达，且表达水平与肿瘤组织学类型相关。叶酸另一受体RFC表达水平在恶性间皮瘤等少数肿瘤出现升高。目前，关于RFC和FR-α两种转运体的表达水平与乳腺癌的相关性研究较少。其表达水平在不同分子亚型乳腺癌中是否具有差异性，是否与乳腺癌细胞的恶性行为有关均有待研究。.本项目拟根据前期工作基础，进一步检测FR-α在浸润性乳腺癌肿瘤组织中的表达情况，观察RFC和FR-α在不同分子亚型的乳腺癌细胞株中的表达差异，了解不同叶酸浓度、雌激素及雌激素拮抗剂对乳腺癌细胞RFC和FR-α表达水平的影响，为深入研究浸润性乳腺癌叶酸转运相关受体在乳腺癌发展、增殖中的作用奠定基础。本项目发表SCI论文4篇，北大核心期刊一篇，多次国内、外学术交流，项目基本完成计划任务，达到预期目标。