The Rogosin Institute, 310 East 67th Street, New York, NY, 10065, USA.
Graduate School of Biomedical Sciences and Engineering, University of Maine, 168 College Ave, Orono, 04469, ME, USA.
BMC Cancer. 2021 Mar 24;21(1):312. doi: 10.1186/s12885-021-07957-8.
Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported.
Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database.
FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-O. In vivo, xenografted 786-O showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-O had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-O, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation.
We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.
叉头转录因子控制多种癌症类型的细胞生长。Foxd1 对于肾脏发育和线粒体代谢是必不可少的，但它在肾细胞癌（ccRCC）中的意义尚未被报道。
使用 TCGA 数据库中的转录组数据来关联 FOXD1 表达与患者生存。在 786-O 细胞系中敲除 FOXD1，并分析已知的靶标。通过生长速率和 Seahorse XF 代谢测定以及异种移植模型在体外观察和研究细胞生长减少的情况。通过流式细胞术和免疫印迹法确定细胞周期特征。使用 TUNEL 和 γH2AX 免疫染色来测量 DNA 损伤。通过使用 TCGA 数据库进行相关性分析来研究 FOXD1 途径与细胞周期进展的关联。
ccRCC 中 FOXD1 的表达水平与患者的生存呈负相关。在 786-O 细胞中敲除 FOXD1 会改变 FOXD1 靶标的表达，特别是涉及代谢（MICU1）和细胞周期进展的基因。代谢状态的研究表明线粒体代谢和糖酵解发生了显著变化，但能量产生没有净变化。体外生长速率测定显示 786-O 的生长速度显著降低。在体内，异种移植的 786-O 显示出肿瘤形成能力降低和肿瘤体积减小。细胞周期分析显示 786-O 的 G2/M 期延长。有丝分裂的研究表明 786-O 的组蛋白 H3 磷酸化不足，并且 DNA 损伤增加。与 TCGA 数据集中的 FOXD1 相关的基因与有丝分裂的几个方面相关，包括组蛋白 H3 磷酸化。
我们表明，FOXD1 通过控制组蛋白 H3 磷酸化来调节 ccRCC 细胞的细胞周期，并且 FOXD1 的表达控制肿瘤的形成和肿瘤的生长。转录组分析支持 FOXD1 在 ccRCC 患者肿瘤中的作用，并为 FOXD1 肿瘤表达与患者生存之间的反比关系提供了解释。我们的研究结果揭示了 FOXD1 在维持染色质稳定性和促进细胞周期进展中的重要作用，并提供了一个新的工具来研究 FOXD1 在 ccRCC 中的生物学功能。
