食管鳞癌（esophageal squamous cell carcinoma, ESCC）作为一种高发的消化道 恶性肿瘤，具有病程发展迅速及预后不良的特点，对于中晚期ESCC患者而言，其术后五年存活率大约只有15%至25%。由于缺乏有效的靶向药物，开发新的药物和治疗策略是ESCC研究的重点。

前期的研究结果显示，联合应用PLK1抑制剂BI 6727和AURKA抑制剂TC-S 7010 可对ESCC细胞在体内外的恶性增殖产生协同抑制作用，但相关分子机制尚未阐明。在前期研究的基础上，我们在本研究中进一步分析发现，与单药治疗组相比， 同时使用两种药物进行联合治疗可诱导裸鼠移植瘤组织细胞产生更为显著的有丝分裂阻滞和细胞凋亡，该结果与体外实验一致。与对照组和单药治疗组相比，经过 21 天的治疗，联合用药组肿瘤细胞呈现出明显的治疗反应，表现为角化增多、退变明显，Ki67阳性细胞率明显降低，TUNEL阳性细胞率明显升高。

分子水平的研究结果显示，ESCC细胞经PLK1特异性siRNA和抑制剂处理之后，p-AURKA 蛋白表达水平下调，经AURKA特异性siRNA和抑制剂处理之后， p-PLK1 蛋白表达水平下调，提示 PLK1 和 AURKA 之间存在交互调控关系，因此同时抑制PLK1和AURKA有可能产生协同的抗肿瘤效应。其后，RNA-seq数据分析结果初步提示，BI 6727和TC-S 7010联合应用可能通过影响ESCC细胞中YAP 和c-Myc的表达发挥对ESCC细胞增殖的协同抑制作用。进一步的研究发现，与单 独用药相比，联合用药可以更为明显地下调ESCC细胞中YAP和c-Myc的蛋白水平。由于高浓度或低浓度单药处理均不影响ESCC细胞中YAP和c-Myc的mRNA 水平，因此我们进一步检测了高浓度单药对YAP和c-Myc蛋白水平的影响及分子 机制，发现 PLK1 可通过泛素-蛋白酶体途径调控 YAP 和 c-Myc 的蛋白丰度，而 AURKA 可通过泛素-蛋白酶体途径调控c-Myc 的蛋白水平并通过自噬-溶酶体途径调控YAP的蛋白丰度。进而，Co-IP结合Western blot检测结果提示，PLK1可通过阻断E3连接酶β-TrCP与YAP蛋白的结合，抑制泛素-蛋白酶体途径介导的YAP蛋白的降解，从而增强ESCC细胞中的YAP蛋白稳定性。此外，我们还通过RNA-seq 数据分析及 qRT-PCR 验证发现，联合用药处理可抑制 ESCC 细胞中 MGST1 的 mRNA水平。

上述结果初步提示联合靶向 PLK1 与 AURKA 可能通过抑制 YAP、c-Myc 和 MGST1 的mRNA水平，以及YAP和c-Myc蛋白的丰度，对ESCC细胞的恶性增殖产生协同抑制作用，联合靶向抑制PLK1和AURKA可能成为一个新的、候选的 ESCC 治疗策略。

大量研究显示，YAP的表达水平升高或活性增强可导致细胞增殖和凋亡之间的平衡失调，导致肿瘤发生。目前已在结直肠癌、肝细胞癌、肺癌等多种人类恶性肿瘤中发现YAP过表达，且有研究发现YAP的异常活化与肿瘤干细胞的干性维持、化疗及靶向治疗抗性密切相关，提示其可能是ESCC 的潜在治疗靶点。作为YAP-TEAD 的小分子抑制剂，GNE-7883 可对包括结直肠癌在内的多种恶性肿瘤细胞的增殖能力产生显著抑制作用，但目前尚无其对ESCC细胞影响的研究。

本研究中，我们以DMSO为对照，使用不同浓度的GNE-7883处理ESCC细胞 KYSE410 和KYSE510细胞，检测细胞增殖活力、集落形成能力、迁移能力及凋亡变化，并用实时荧光定量逆转录PCR（qRT-PCR）和Western blot 分析相关分子的表达变化。结果显示，与对照组比，较高浓度（50和100 μmol/L）的GNE-7883可显著抑制KYSE410和KYSE510细胞的增殖活力（P < 0.05）及集落形成能力（P < 0.05），并可在血清饥饿条件下诱导KYSE410和KYSE510细胞发生显著凋亡（P < 0.05）。同时，在血清饥饿条件下，低浓度（8和16 μmol/L）的 GNE-7883可显著抑制KYSE410和KYSE510细胞的迁移能力（P < 0.01）。 分子水平检测结果显示，较高浓度GNE-7883处理后，ESCC细胞中与增殖相关的CTGF、CYR61、c-Myc、p ERK、p-AKT和p-S6蛋白水平明显降低。在血清饥饿条件下，较高浓度GNE-7883 处理后，ESCC细胞中凋亡标志分子Cleaved PARP、Cleaved Caspase-3的水平明显升高，同时伴随CTGF、CYR61、Bcl-2、Bcl-xL、c-Myc、p-ERK、p-AKT、p-S6蛋白水平的降低。同时，在血清饥饿条件下，低浓度GNE-7883处理后，ESCC细胞中 CTGF、CYR61、MMP14及p-ERK蛋白水平明显下调。 

综上，我们的研究发现GNE-7883可显著抑制ESCC细胞的增殖、迁移及存活能力，并可明显下调CTGF、CYR61、c-Myc、Bcl-2、Bcl-xL 和 MMP14 的表达及 MAPK/ERK和AKT/mTOR信号通路的活性，其有可能成为ESCC治疗的候选药物。

Esophageal squamous cell carcinoma (ESCC) is a common digestive system tumor with rapid progression and poor prognosis. The five-year survival rate of ESCC patients after surgery for patients in the middle and advanced stages is only 15% tp 25%. At present, there are no effective targeted therapeutic drugs in clinical practice. Therefore, searching for effective targeted therapeutic drugs and treatment strategies remains the focus of ESCC research.

Our previous study showed that the combined application of PLK1 small molecule inhibitor BI 6727 and AURKA small molecule inhibitor TC-S 7010 yielded synergistic antitumor activity against ESCC cells in vitro and in vivo, but the underlying molecular mechanism has not been elucidated. On the basis of our earlier findings, in this study, we further analyzed and found that compared with the monotherapy group, the combination of two drugs strikingly enhanced mitotic arrest and apoptosis in KYSE450 cell-derived xenograft tumors in nude mice, which was consistent with the the results of the in vitro experiments. Compared with the control group and the monotherapy group, after 21 days of treatment, the tumor cells in the combination therapy group exhibited obvious therapeutic responses, showing an increase in keratinization, obvious degeneration, a distinctly reduction in the rate of Ki67 positive cells, and an apparent increase in the rate of TUNEL positive cells.

At the molecular level, our current results showed that p-AURK A protein levels were markedly down-regulated in the ESCC cells transfected with PLK1 specific siRNA or treated with PLK1 small molecular inhibitor, and vice versa, suggesting an interactive regulatory relationship between PLK1 and AURKA. Therefore, concurrent inhibition of  PLK1 and AURKA may produce a synergistic anti-tumor effect. Subsequently, RNA-seq data analysis initially suggested that the combined treatmen combination therapy may exert a synergistic inhibitory effect on ESCC cell proliferation by downregulating the expression of YAP and c-Myc genes in ESCC cells. Additionally, the protein levels of YAP and c-Myc were also evidently reduced upon combined treatment. Since the mRNA expression levels of YAP and c-Myc were not affected by neither high nor low concentration of any individual inhibitor in ESCC cells, we further examined the effect of high concentrations of single inhibitors on YAP and c-Myc protein levels and relavate molecular mechanism. The corresponding results suggested that PLK1 can increase the protein abundances of YAP and c-Myc by suppressing the ubiquitin-proteasome pathway, while AURKA can increase the protein levels of c-Myc and YAP through inhibiting the ubiquitin-proteasome pathway and the autophagy-lysosomal pathway, respectively. Furthermore, the results of Co-IP combined with Western blot detection suggest that PLK1 could inhibit the degradation of YAP protein mediated by the ubiquitin-proteasomsome pathway through blocking the binding of E3 liagase β-TrCP to YAP protein, thereby enhancing the stability of YAP protein in ESCC cells. In addittion, through RNA-seq data analysis and qRT-PCR verification, we found that combined treatment with BI 2767 and TC-S 7010 also effectively reduced the mRNA level of MGST1 in ESCC cells as well.

In summary, our findings suggest that concurrent targeting of PLK1 and mTOR yields a synergistic inhibitory effect on the proliferation potential of ESCC cells, at least partially due to a more pronounced reduced the mRNA levels of YAP, c-Myc and MGST1, as well as the protein abundance of YAP and c-Myc, and simultaneous targeting of PLK1 and AURKA may serve as a new and promising therapeutic strategy for patients with ESCC. 

Previous studies have shown that YAP overexpression or heightened activity can imbalance cell proliferation and apoptosis, triggering cancer. YAP overexpression is found in colorectal, hepatocellular, and lung cancers, indicating its key role in tumor development. Its abnormal activation is also linked to cancer stem cell maintenance and drug resistance. Thus, YAP is a promising cancer therapy target. Our prior work showed YAP overexpression boosts esophageal squamous cell carcinoma (ESCC) cell proliferation in vitro and in vivo, suggesting its ESCC treatment potential.

GNE-7883, a YAP-TEAD - targeting small - molecule inhibitor, works by blocking YAP-TEAD binding. It has been proven to significantly suppress diverse malignant tumor cell proliferation in preclinical studies, yet its impact on ESCC cells remains unexplored in literature.

This experimental series employed two esophageal squamous cell carcinoma (ESCC) models (KYSE410 and KYSE510), wherein cells were exposed to GNE-7883 treatment, with dimethyl sulfoxide (DMSO) serving as the vehicle control group. Cell proliferation, migration, and apoptosis were assessed. Molecular expression changes were analyzed by qPCR and Western Blot. Compared to controls, high concentrations of GNE-7883 (50 and 100 μmol/L) significantly suppressed proliferation (P < 0.001) and colony formation (P < 0.001) in both cell lines, while inducing marked apoptosis under serum-starvation conditions (P < 0.05). Low concentrations (8 and 16 μmol/L) notably inhibited serum starvation-induced cell migration (P < 0.01). Mechanistically, high-dose GNE-7883 downregulated proliferation-associated proteins (CTGF, CYR61, c-Myc, p-ERK, p-AKT, p-S6) and elevated apoptotic markers (Cleaved PARP, Cleaved Caspase-3) while reducing anti-apoptotic proteins (Bcl-2, Bcl-xL) and pathway activators (p-ERK, p-AKT, p-S6).Low-dose treatment under serum starvation suppressed CTGF, CYR61, MMP14, and p ERK levels.

Overall, the study's findings indicated that GNE-7883 strongly inhibits proliferation, migration, and viability of ESCC cells. It downregulates the expression of YAP-TEAD downstream targets (CTGF, CYR61, c-Myc) as well as Bcl-2, Bcl-xL, and MMP14, while suppressing the activity of MAPK/ERK and AKT/mTOR signaling pathways. These findings suggest its potential as a therapeutic candidate for ESCC.