The impact and mechanism of FOXA3 on the Hippo signaling pathway in carcinoma of esophagus
ZHANG Shan,TIAN Jing-jing,CHEN Ming
Abstract:
Objective To elucidate the impact and underlying mechanisms of FOXA3 on the Hippo/YAP signaling pathway in esophageal squamous cell carcinoma. Methods The esophageal cancer cell line ECA109 was selected for culture. Stable up-regulated FOXA3 and downregulated FOXA3 groups cell lines were constructed through lentiviral transfection. The ECA109 cell lines were divided into four groups: the up-regulated FOXA3 group, down-regulated FOXA3 group, and their respective control group. Cell growth capability was assessed using the Cell Counting Kit-8 (CCK-8) assay and colony formation assays. The expression levels of LATS2 and YAP1 in Hippo signaling pathway were evaluated via real-time PCR and Western blotting analysis in each group. Additionally, to establish xenograft models of esophageal squamous cell carcinoma, ECA109 cells with either up-regulated or down-regulated FOXA3 expression were respectively subcutaneously injected into the dorsal region of nude mice. After a 6-week inoculation period, tumors were excised and evaluated for volume and weight. Real-time PCR, Western blotting, and immunohistochemistry were employed to evaluate the expression levels of LATS2 and YAP1 in Hippo signaling pathway in tumor tissues from the FOXA3 up-regulated group, FOXA3 down-regulated group, and control group. Additionally, the expression levels of Hippo signaling pathway-related proteins LATS2 and YAP1 in tumor tissues from the up-regulated FOXA3 group, down-regulated FOXA3 group, and control group were assessed using real-time PCR, Western blotting analysis, and Immunohistochemical staining. Results In vitro experiments demonstrated that the proliferation rate of ECA109 cells with FOXA3 up-regulation was significantly slower compared to that of the control cells (P <0.05). Conversely, the proliferation rate of ECA109 cells with FOXA3 down-regulation was markedly faster than that of the control cells (P <0.05). The results of real-time PCR and Western blotting analysis demonstrated that LATS2 expression was significantly upregulated in cell lines with high FOXA3 expression (P <0.05), whereas it was markedly downregulated in cell lines with low FOXA3 expression (P <0.05). Additionally, YAP1 expression was reduced in cell lines with high FOXA3 expression (P <0.05), while it was increased in cell lines with low FOXA3 expression (P <0.05). In vivo experiments demonstrated that the tumor growth rate, volume, and weight in the FOXA3 up-regulation group were significantly reduced compared to the control group (P <0.05). Conversely, the tumor growth rate, volume, and weight in the FOXA3 down-regulation group were markedly increased relative to the control group (P <0.05). Real-time PCR, Western blotting, and Immunohistochemistry revealed that LATS2 expression was significantly increased in tumor tissues of the FOXA3 up-regulated group and significantly decreased in the FOXA3 down-regulated group. In contrast, YAP1 expression was significantly decreased in the FOXA3 up-regulated group and significantly increased in the FOXA3 down-regulated group. All differences were statistically significant(P <0.05). Additionally, cells in the FOXA3 up-regulated group exhibited a significantly faster proliferation rate compared to the control group and the FOXA3 down-regulated group(P <0.05). Conclusion FOXA3 exerts an inhibitory effect on the proliferation and growth of esophageal squamous cell carcinoma, potentially through modulation of the Hippo/YAP signaling pathway.
