Supplementary Material for: Upregulation of RASSF1A in Colon Cancer by Suppression of Angiogenesis Signaling and Akt Activation

<b><i>Background/Aims:</i></b> Silencing of tumor suppressor genes (TSGs) and promotion of angiogenesis are associated with tumor development and metastasis. However, little is known if angiogenic molecules directly control TSGs and vice versa. <b><i>Methods:</i></b> A regulatory link between angiogenesis and down regulation of TSGs was evaluated using an anti-cancer agent, andrographolide (AGP) in cancer cells, mouse xenograft tissues and patient derived organoids through gene/protein expression, gene silencing, and immunohistochemical analyses. <b><i>Results:</i></b> AGP treatment demonstrated significant expression of RASSF1A and PTEN TSGs in colon cancer and other cancer cells, mouse tissues and organoids. Depletion of RASSF1A with siRNA limited cyclin D1 and BAX expression. SiRNA depletion of PTEN, upstream regulator of RASSF1A resulted in a 50% reduction in RASSF1A expression. Histopathological analysis of the AGP treated tumor sections showed significant reduction in vessel size, microvascular density and tumor mitotic index suggesting suppression of angiogenesis. This was corroborated by protein analysis demonstrating significant reductions in angiogenesis signaling pathway molecules VEGF₁₆₅, FOXM1, and pAkt, but significant elevation of the endogenous angiogenesis inhibitor Tsp-2. Treatment of cells with exogenous VEGF prevented the suppression of angiogenesis signaling by AGP, resulting in sustained expression of pAkt, an upstream down-regulator of RASSF1A. RASSF1A expression remained low in VEGF treated cells despite the addition of AGP. <b><i>Conclusion:</i></b> Our results demonstrate for the first time that AGP induces RASSF1A expression in colon cancer cells and is dependent on angiogenesis signaling events. Therefore, our research may facilitate novel therapeutic options for advanced colon cancer therapy.

**背景与目的：** 肿瘤抑制基因（tumor suppressor genes，TSGs）的沉默与血管生成的促进均与肿瘤发生发展及转移密切相关，但目前尚未明确血管生成分子能否直接调控肿瘤抑制基因，反之亦然。
**方法：** 本研究以抗癌成分穿心莲内酯（andrographolide，AGP）为工具，通过基因/蛋白表达检测、基因沉默技术及免疫组化分析，在癌细胞、小鼠异种移植组织与患者来源类器官中，评估了血管生成与肿瘤抑制基因下调之间的调控关联。
**结果：** 穿心莲内酯处理可显著上调结直肠癌及其他癌细胞、小鼠组织与类器官中RASSF1A与PTEN这两种肿瘤抑制基因的表达水平。利用小干扰RNA（siRNA）沉默RASSF1A后，cyclin D1与BAX的表达受到抑制；而作为RASSF1A上游调控因子的PTEN经小干扰RNA沉默后，可使RASSF1A的表达量降低50%。对经穿心莲内酯处理的肿瘤切片进行组织病理学分析，结果显示血管管径、微血管密度及肿瘤有丝分裂指数均显著降低，提示血管生成受到抑制。蛋白分析进一步验证了这一结论：血管生成信号通路分子VEGF₁₆₅、FOXM1与pAkt的表达量显著下调，而内源性血管生成抑制因子Tsp-2的表达量则显著升高。向细胞添加外源性VEGF可阻断穿心莲内酯对血管生成信号通路的抑制作用，使pAkt——RASSF1A的上游负调控因子——的表达维持稳定；即便添加穿心莲内酯，经VEGF处理的细胞中RASSF1A的表达量仍处于较低水平。
**结论：** 本研究首次证实，穿心莲内酯可诱导结直肠癌细胞中RASSF1A的表达，且该过程依赖于血管生成信号事件。因此，本研究可为晚期结直肠癌治疗提供全新的治疗策略。