分泌型拮抗剂（Frzb/SFRP3）和Bevacizumab联合用药对晚期前列腺癌的疗效及机制的研究

Anti-vascular endothelial growth factor (VEGF) therapy with Bevacizumab is currently the most promising anti-angiogenesis approach for treatment of metastatic cancers including metastatic prostate cancer (PCa). However, emerging animal and clinical evidences indicate that a host response to anti-VEGF therapy may lead to treatment failure and more invasive, metastatic cancers. This could be because the tumor switches from the bevacizumab targeted pathway to other pathways that support tumor growth, invasion and metastasis. Neuropilin (NRP) 1 is a co-receptor for both VEGF receptors and c-Met and play an important role in tumor growth and metastasis. Our preliminary data have demonstrated that bevacizumab increases expression of NRP1 and c-Met and their interaction leading to enhanced PCa cell migration and invasion. This result therefore led us to hypothesize that bevacizumab, while blocking VEGF and VEGF receptors, may activate co-receptor NRP1 and c-Met mediated signaling leading to cancer cell migration, invasion and metastasis. In addition, c-Met is a known Wnt target gene and the promoter sequence of NRP1 contains five predicted TCF4/LEF1 binding motifs. We have also shown that bevacizumab increases expression of Wnt transcriptional factor TCF4 and its target genes (c-Myc and survivin). Furthermore, anti-VEGF therapies cause hypoxia. Under hypoxia conditions, epithelial cancer cells acquire migratory and invasive capacity through (1) activation of Wnt signaling and transcriptional factors (Twist and snail) leading to epithelial to mesenchymal transition (EMT) and (2) activation of the HGF/c-Met pathway. It is possible that bevacizumab may activate Wnt signaling and then induce c-Met and NRP2 expression and EMT. Therefore, the focus of this application is to examine whether a rationally designed approach for using secreted Wnt antagonists can improve anti-VEGF therapy. We have published data that a secreted Wnt antagonist (Frzb/SFRP3) can reverse EMT and reduce PCa tumor growth and cellular invasiveness. We have also shown that Frzb/SFRP3 can down-regulate expression of NRP1 and c-Met. Therefore, we hypothesize that Frzb/SFRP3 may be an ideal candidate agent for preventing the pro-invasive effects of anti-VEGF therapy via targeting Wnt signaling mediated EMT and expression of NRP1 and c-Met. The proposal is designed to test this hypothesis and investigate the efficacy of Frzb/SFRP3 and Bevacizumab combined therapy for castration-resistant PCa.

Bevacizumab是作用于血管内皮细胞生长因子（VEGF）的抗肿瘤血管生成的一线靶向药物，但临床上大多数肿瘤（特别是晚期前列腺癌）患者容易对其产生抗药性。因为Bevacizumab阻断了VEGF-A与VEGF辅助受体- - 神经纤毛蛋白1（Neuropilin 1, NRP1）的结合,从而增加了NRP1与癌基因蛋白c-Met结合,促进肿瘤细胞的生存和转移，所以产生了抗药性。本课题组前期研究结果表明：分泌型WNT拮抗剂可以下调c-Met和NRP1的表达，从而降低机体对Bevacizumab的抗药性。本项目将构建PC3原位前列腺癌裸鼠模型；并通过模型测试研究Bevacizumab和分泌型WNT拮抗剂（Frab/sFRP3）重组蛋白联合使用对肿瘤的生长和转移的抑制作用，深入研究分泌型WNT拮抗剂的抗癌作用机制，寻找一种治疗晚期前列腺癌症新方法。

本课题组前期设计、合成了20多种新的查尔酮化合物，并利用一些肿瘤细胞株（如EC109, SKNSH, HepG2, MGC803)进行了初步的活性测定，发现H72和S17对前列腺癌细胞PC-3和胃癌细胞MGC-803较为敏感,并且对正常的胃上皮细胞GES-1毒性作用较小。我们的实验结果表明，H72处理MGC-803和PC-3、S17处理MGC-803后，可诱导产生活性氧，导致caspase9/3和线粒体膜电位的变化，并且H72和S17可通过上调DR5、DR4及BimEL, 下调Bid, Bcl-XL和XIAP蛋白诱导癌细胞的凋亡。另外，活性氧抑制剂N-acetyl cysteine (NAC)较好的逆转了H72和S17的细胞凋亡作用及一些蛋白的变化，在利用裸鼠的体内实验结果显示，H72和S17在不影响体重变化的情况下，明显地抑制了肿瘤的生长。这些结果表明，H72和S17在体内外效果良好、作用机制明确，可作为新的查尔酮衍生物进一步探讨和研究，以上结果也为开发抗前列腺癌和抗胃癌新药提供理论依据。