第一部分：食管鳞癌（Esophageal squamous cell carcinoma，ESCC）是我国高发的恶性肿瘤之一，其预后差，死亡率高，多年来中晚期患者术后的五年生存率一直徘徊在15%-25%。目前临床上缺乏可有效治疗ESCC的药物，因此迫切需要研发新的有效药物及治疗策略。

我们的前期研究结果提示Polo-like kinase1（PLK1）是ESCC中重要的功能基因及潜在的治疗靶点。由于单独使用PLK1的靶向药物在大多数治疗恶性肿瘤的临床实验中并未取得令人满意的疗效，因此我们对PLK1相关的联合靶向治疗策略进行了探索。在本研究中，我们通过筛选含有346种小分子药物的抑制剂分子库，发现PLK1的小分子抑制剂BI 6727与蛋白激酶Aurora-A的抑制剂TC-S 7010联合应用对ESCC细胞的增殖活力具有显著的协同抑制作用。对公共基因表达数据库GEO中的数据集GSE23400进行分析发现，ESCC组织中PLK1与Aurora-A的mRNA表达量均显著高于癌旁正常组织（n = 53, P < 0.0001），并且PLK1与Aurora-A的mRNA表达水平在ESCC中呈显著正相关（n=53，P < 0.001，r =0.5052）。其后，我们检测了两种药物联用对ESCC细胞恶性表型的影响，发现BI 6727与TC-S 7010联合应用可协同抑制ESCC细胞系KYSE450和KYSE510的增殖活力和集落形成能力，并且可协同促进这两种细胞发生G2/M周期阻滞并诱发细胞凋亡（P值均小于0.05）。类似的，BI 6727与Aurora-A的另一种抑制剂MLN 8237联用，也显示出了对KYSE450和KYSE510细胞的增殖和存活具有协同的抑制作用。分子水平的检测结果显示，与单药处理相比，联合用药可明显增强细胞周期（Cyclin B1和p-Histone H3）和凋亡相关分子标志（Cleaved-Caspase 3和Cleaved-PARP）的表达变化；而且，也可进一步抑制YAP、c-MYC、Bcl-2和Bcl-xL等多个与细胞增殖及存活相关分子的表达。进而，我们构建了KYSE450细胞的裸鼠移植瘤模型进行抑瘤实验，结果显示BI 6727与TC-S 7010联合治疗对移植瘤生长的抑制率为82.7%。与单药治疗组相比，二者联用表现出协同的抑瘤作用（Deviation 值为0.25）。

综上所述，联合使用PLK1的小分子抑制剂BI 6727与Aurora-A的抑制剂TC-S 7010或MLN 8237均可协同抑制ESCC细胞的恶性增殖和存活能力，提示联合靶向抑制PLK1和Aurora-A可能是一种新的、潜在的ESCC 治疗策略。

第二部分：

既往研究表明，Wnt/β-catenin信号通路的异常激活与食管鳞癌（Esophageal squamous cell carcinoma，ESCC）的发生、发展及预后密切相关, 提示其可能是ESCC治疗的潜在靶点。ICG-001是Wnt/β-catenin信号通路的一种小分子抑制剂，已有研究证实ICG-001可显著抑制多种恶性肿瘤细胞在体内外的增殖能力，但ICG-001在ESCC中的作用目前尚无文献报道。

在本研究中，我们使用浓度为50 和100 μmol/L的小分子抑制剂ICG-001处理ESCC细胞系KYSE410和KYSE450，以溶剂DMSO处理细胞作为对照，进行细胞增殖活力、集落形成能力、细胞周期分布及凋亡检测。研究结果显示，ICG-001处理可显著抑制ESCC细胞系KYSE410和KYSE450的体外增殖能力和集落形成能力，并可诱导细胞发生G0/G1期阻滞（P < 0.01）。Western blot检测结果显示，ICG-001处理后细胞中细胞周期蛋白依赖性激酶CDK4、CDK6的表达明显下调，细胞周期的负调控因子p27 Kip1显著上调；b-catenin/TCF下游靶分子SKP2和Survivin的表达水平明显下调，而且可与b-catenin结合发生转录调控作用的转录因子TCF4的表达量也明显降低（P值均小于0.01）。同时，qRT-PCR分析结果显示，在ICG-001处理的ESCC细胞中SKP2、Survivin和TCF4的mRNA表达水平显著降低，而p27 Kip1 的mRNA表达水平显著上调（P值均小于0.01）。

综上所述，本研究结果提示ICG-001可能通过影响β-catenin/TCF的功能从而抑制下游靶分子SKP2、Survivin和TCF4的表达并上调p27 Kip1的表达，诱导细胞发生G0/G1期阻滞进而抑制ESCC细胞的增殖。同时本研究结果提示ICG-001是一个潜在的ESCC治疗药物，有可能单独或联合其他治疗手段一起提高临床疗效。

第一部分：

Esophageal squamous cell carcinoma（ESCC）is one of the most common malignant tumors in China, with poor prognosis and high mortality. For many years, the five-year survival rate for advanced patients has hovered around 15% to 25%. At present, there is a lack of effective drugs for the treatment of ESCC, so it is urgent to develop new effective drugs and therapeutic strategies.

Our previous research results suggest that Polo-likekinase1 (PLK1) is an important functional gene and a potential therapeutic target in ESCC. Since the efficacy of targeting PLK1 alone in the treatment of malignant tumors have not achieved satisfactory in most clinical trials, we explored PLK1-inhibition-based combination therapy strategies. In this study, we screened a compound library containing 346 small molecular inhibitors and found that the combination of small molecular inhibitor BI 6727 of PLK1 and inhibitor TC-S 7010 of protein kinase Aurora-A had a significant synergistic inhibitory effect on the proliferation of ESCC cells. Based on the analysis of the dataset GSE23400 in the public gene expression database GEO, it was found that the mRNA expression levels of PLK1 and Aurora-A in ESCC tissues were significantly higher than those in adjacent normal tissues (n=53，P<0.0001). The mRNA expression levels of PLK1 and Aurora-A were positively correlated in ESCC (n=53, P<0.001, r=0.5052). Subsequently, we investigated the effects of the combination of the two drugs on the malignant phenotype of ESCC cells, and found that the combination of BI 6727 and TC-S 7010 synergistically inhibited the proliferative activity and colony formation ability of ESCC cell lines KYSE450 and KYSE510. In addition, combination treatment synergistically promoted G2/M cycle arrest and induced cell apoptosis in these two cell lines (both P values were less than 0.05). Similarly, BI 6727 in combination with MLN 8237, another inhibitor of Aurora-A, also showed a synergistic inhibitory effect on proliferation and survival of KYSE450 and KYSE510 cells. The results at the molecular level showed that compared with monotherapy, combination therapy significantly enhanced the expression of cell cycle (CyclinB1 and p-HistoneH3) and apoptosis-related molecular markers (Cleaved-Caspase3 and Cleaved-PARP). Additionally, the expression levels of other molecules related to cell proliferation and survival, including YAP, c-MYC, Bcl-2, Bcl-xL, were also markerdly reduced after combination therapy. Furthermore, we constructed a nude mouse transplanted tumor model with KYSE450 cells for tumor inhibition experiments, and the results showed that the inhibition rate of BI 6727 combined with TC-S 7010 on the growth of transplanted tumor was 82.7%. Compared with the monotherapy group, the combination therapy showed synergistic anti-tumor effect (Deviation value was 0.25).

In summary, the combination of small molecular inhibitor BI 6727 of PLK1 and inhibitor TC-S 7010 or MLN 8237 of Aurora-A synergistically inhibited the malignant proliferation and survival of ESCC cells, suggesting that the co-targeting of PLK1 and Aurora-A may be a promising therapeutic strategy for ESCC.

第二部分：

Previous studies have shown that abnormal activation of Wnt/β-catenin signaling pathway is closely related to the occurrence, development and prognosis of ESCC, suggesting that it may be a potential target for ESCC treatment. ICG-001 is a small molecule inhibitor of Wnt/β-catenin signaling pathway. The results of in vitro and in vivo studies have shown that ICG-001 can significantly inhibit the proliferation of various malignant tumor cells, but the effect of ICG-001 on ESCC cells has not been reported.

In this study, the ESCC cell lines KYSE410 and KYSE450 were treated with small molecule inhibitor ICG-001 of 50 and 100 μmol/L, and the cells treated with solvent DMSO were used as control to detect the cell proliferation viability, colony formation ability, cell cycle distribution and apoptosis. The results showed that ICG-001 treatment significantly inhibited the proliferation and colony formation ability of ESCC cell lines KYSE410 and KYSE450, and significantly induced G0/G1 phase arrest (P < 0.01). Correspondingly, Western blot results showed that the expression of cyclin-dependent kinase CDK4 and CDK6 was obviously decreased, while the negative regulator of cell cycle p27 Kip1 was markedly up-regulated upon ICG-001 treatment. The expression of downstream target molecules SKP2 and Survivin of β-catenin/TCF was significantly decreased, and the expression of transcription factor TCF4 which could bind to β-catenin was also significantly decreased (P < 0.01). At the same time, qRT-PCR analysis showed that the mRNA expression of SKP2, Survivin and TCF4 was significantly decreased, while the mRNA expression of p27 Kip1 was significantly up-regulated in ICG-001-treated ESCC cells (all P values were less than 0.01).

Collectively, the results of this study showed that ICG-001 significantly inhibited the proliferation viability and colony formation ability of ESCC cell line KYSE410 and KYSE450, and induced G0/G1 phase arrest, which may be attributed to the suppression of b-catenin/TCF4 transcriptional activity and the expression of its target molecules Survivin、SKP2 and TCF4，and the up-regulation of p27 Kip1 . Our findings suggest that ICG-001 may be a potential therapeutic drug for the treatment of ESCC, and it may improve the clinical efficacy alone or in combination with other therapeutic methods.