Abstract 5134: Cross-talk of EZH2/PRC2 and Gata5/Foxa2/miR-200 regulates EMT in lung cancer metastasis

Abstract Introduction: Lung cancer is the most lethal type of cancer. Part of this lethality is due to late diagnosis when more than 60% of cases present distant metastases at bones, brain and liver, leading to an overall survival of less than 5%. Thus, investigating the biology of metastatic process is mandatory as a public health policy. One molecular mechanism involved in lung cancer progression is the repression of miR-200 family of miRNAs that regulate the epithelial-mesenchymal transition (EMT). Our previous study showed that miR-200 deletion accelerated the formation of lung adenocarcinoma and progression in KP transgenic mice model. Objective: Investigate the transcriptional regulation of miR-200 cluster and its impact in lung cancer metastasis. Methods: For in silico analysis of promoter region, we used the FANTOM 5, genome browser and transcription factor (TF) prediction tools. We cloned several fragments of miR-200c/141 and miR-200b/miR-200a/miR-429 into the luciferase reporter plasmid pGL4-20, and transfected into a panel of murine lung cancer cell lines (metastatic: 889PF, 404M1 and KPT4-LM and non-metastatic: 368T1, 394T4 and KPT4). Gene expression analysis of TFs and miR-200s was performed for comparisons among lung cancer lines, including. For overexpression experiments, we cloned the coding sequence of TFs (Gata5 and Foxa2) into MSCV-puro plasmid and transfected into metastastic lines 889PF and 404M1. Modulation of Ezh2 was performed with CRISPR/Cas9 in 889PF cells. Results: we defined the minimal promoter region of miR-200c/141 and miR-200b/miR-200a/miR-429 as the fragments -1246 to 0bp and-4363 to -4027bp from transcription start site, respectively. In silico search for TFs revealed a list of several TFs related to lung biology, such as Nkx2-1, Gata5 and Foxa2. Gene expression analysis showed that reduced levels of miR-200s expression in metastatic lines (889PF and 404M1) inversely correlated with TFs Gata5 and Foxa2 levels. The reactivation of Gata5 and Foxa2 in the metastatic lines reverted the mesenchymal phenotype to an epithelial phenotype while restored miR-200s expression and blocked EMT factors. Further investigation of Gata5 and Foxa2 genes promoter region revealed a common signature for polycomb gene Ezh2/PRC2. Overexpression of Ezh2 in non-metastatic cancer lines reduced TFs and miR-200 expression. Moreover, KPT4-LM, another metastatic line, showed activation of EMT and reduction of miR-200s and TFs, while Ezh2 and PRC2 components were upregulated. Ezh2 gene editing in 889PF induced epithelial phenotype in some clones. Conclusion: mesenchymal phenotype is controlled by the loop between EZH2/PRC2 and TFs Gata5-Foxa2/miR-200s. Moreover, Gata5 and Foxa2 are pioneer TFs that can restore epithelial phenotype in lung cancer metastatic cell lines. Citation Format: Cesar Seigi Fuziwara, Guilherme A. Góis, Pedro L. Oliveira, Joice M. Menezes, Bin Xue, Lin He, Edna T. Kimura. Cross-talk of EZH2/PRC2 and Gata5/Foxa2/miR-200 regulates EMT in lung cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5134.