靶向CREB1-TGFβ2-ERK信号通路的欧前胡素在食管癌侵袭转移中的作用及机制研究

Esophageal cancer is the sixth most common cause of cancer-related deaths in the world，and China alone accounts for approximately half of all new esophageal cancer cases per year, with esophageal squamous cell carcinoma (ESCC) being the predominant histologic subtype. The disease carries a dismal prognosis particularly for patients who present with metastatic esophageal cancer. Development of novel nontoxic agents that can prevent the invasion and metastasis of cancer cells are therefore essential to tackle this highly aggressive disease. Natural products from plants are receiving increasing attention for their pharmacological effects in treatment and prevention of cancer because of their potential biological activity and low toxicity....A high-throughput screen in a compound library comprising of 1514 nature products identified imperatorin, a bioactive ingredient extracted from Angelica, as a novel inhibitor of cancer cell invasion. Neither the function of imperatorin in cancer invasion/metastasis nor the bioactivity of imperatorin in ESCC has been documented previously. The Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) quantitative proteomics and bioinformatics analysis suggested that transforming growth factor beta 2 (TGFβ2) may play an important role in the biological functions of imperatorin, which was confirmed by the decreases in expression and secretion of TGFβ2 in imperatorin-treated ESCC cells. Our preliminary data from tumor microarray, chamber invasion assay, proteomics and bioinformatics analysis, Western blot, qRT-PCR, ELISA, molecular docking and dynamics, and Biacore experiments showed that expression level of TGFβ2 is significantly higher in ESCC tumor tissue compared with adjacent normal tissues, and expression of TGFβ2 in ESCC is negatively associated with patient survival and metastasis; in addition, expression level of TGFβ2 was found to be further increased in metastatic ESCC in lymph nodes; and TGFβ2 induced epithelial-mesenchymal transition (EMT) and invasion of ESCC cells. Mechanistically, imperatorin could directly bind to CREB1, one of the upstream transcription factors of TGFβ2, interfere the binding of CREB1 to promoter region of TGFβ2 and inactivate transcription and expression of TGFβ2, therefore blocking ERK signaling pathway and suppressing ESCC cell invasion and metastasis. More importantly, addition of exogenous TGFβ2 could significantly abrogated the inhibitory effect of imperatorin on ESCC cell invasion. ...We therefore hypothesize that imperatorin can suppress tumor metastasis in ESCC through inhibition of CREB1-TGFβ2-ERK regulatory axis, and TGFβ2 has its clinical significance in diagnosis and prognosis as novel noninvasive biomarker. This regulatory mechanism has not been reported previously. In the proposed study, we will perform experiments with the following aims:.1..To evaluate the inhibitory effect of imperatorin on ESCC cell invasion and metastasis.2..To study the clinicopathological significance of TGFβ2 expression in ESCC, and its important role in mediating the bioactivity of imperatorin in cancer .3..To investigate the molecular mechanisms how imperatorin regulates TGFβ2 transcription through directly binding to CREB1...The outcome of this project will facilitate the identification of effective biomarker in cancer diagnosis and prognosis, and provide useful preclinical data for development of novel therapeutic strategies for this lethal disease.

肿瘤转移是癌症治疗失败的主要原因，缺乏有效的诊断预后和治疗方案。天然产物可预防和治疗疾病，来源丰富、副作用小。我们采用由1514个天然产物组成的化合物库，经过细胞侵袭实验高通量筛选率先发现，白芷提取物欧前胡素可明显抑制食管癌细胞侵袭，无细胞毒性。目前，欧前胡素的抗肿瘤侵袭转移作用及分子机制未知。蛋白质组学结合生物信息学结果提示欧前胡素可通过影响TGFβ2信号通路行使作用。经过一系列分子动力学、分子生物学、临床病理分析等前期实验，我们提出假说：欧前胡素与CREB1蛋白直接结合，通过干扰CREB1和TGFβ2启动子区域的结合，转录下调TGFβ2阻断ERK通路，从而抑制食管癌侵袭转移。TGFβ2在肿瘤组织中高表达，可作为食管癌诊断预后的标志物。我们拟从生物学功能、临床意义、分子调控机制三方面深入研究欧前胡素对食管癌侵袭转移的抑制作用。本项目将为食管癌无创诊断预后分子标志物和用药选择提供新线索。

食管癌是人类高发的癌症之一，具有进展快、预后差的特点，其五年生存率仅为15%左右。筛选发现一批毒副作用小的抗癌药物，且针对肿瘤个体的基因组/蛋白组图谱差异进行相应的个性化用药选择，对分秒必争的临床肿瘤治疗来说，就显得更为重要。肿瘤细胞本身和肿瘤微环境中基质细胞都可分泌大量的细胞因子包括transforming growth factor beta (TGFβ)、vascular endothelial growth factor (VEGF))等，这些细胞因子可通过酪氨酸蛋白激酶受体促进肿瘤细胞的侵袭以及肿瘤组织的血管生成。然而，关于TGFβ2能否诱导食管癌的侵袭转移仍然未知，其在食管癌中的表达水平及临床病理意义也未见报道。在此研究中，我们结合临床病理数据，分析TGF-β2在临床样本中的表达，首次指出TGF-β2在食管癌肿瘤组织中明显高表达, 并且与患者生存率显著负相关。这一发现将为食管癌患者的药物选择、以及靶向治疗提供新的靶点。此文从生物学功能、分子机制和潜在临床意义三个方面深入剖析TGF-β2在食管癌转移中的重要角色。在研究抑制肿瘤转移药物方面， 科学界一直缺乏合理的筛选模型。在此课题中，研究人员创新地运用高通量药物筛选平台，以小分子药物库为基础，筛选能显著同时抑制食管癌侵袭的和TGF-β信号通路的小分子药物。结合多种细胞体内体外模型指出，欧前胡素有效地抑制食管癌的侵袭和转移。其蛋白质组学结合生物信息学结果提示欧前胡素可证实影响TGFβ2信号通路行使作用。经过一系列分子动力学、分子生物学、临床病理分析等前期实验， 指出欧前胡素通过与CREB1 蛋白直接结合，抑制CREB1 磷酸化及其对TGFβ2的转录调控，进而降低TGFβ2 表达、阻断ERK信号通路，发挥对食管癌侵袭转移的抑制作用。课题组从生物学功能、临床意义、分子调控机制三方面深入研究欧前胡素对食管癌侵袭转移的抑制作用，此课题为食管癌无创诊断预后分子标志物和用药选择提供新线索。