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    • Based on the differentiation and siRNA studies reported

    2018-11-06

    Based on the differentiation and siRNA studies reported here, the mechanistic regulation of FOXM1 towards pluripotency control and differentiation might differ between mouse and human ES cells. The discrepancy can be attributed to the different developmental status of human versus mouse ES cells (Nichols and Smith, 2009; Pera and Tam, 2010). On the other hand, despite being pluripotent the EC cells have adapted towards tumor growth via suppression of differentiation, and are distinctly different from ES cells (Sperger et al., 2003; Andrews et al., 2005). In EC cells there are additional copies of chromosome 12p (Andrews et al., 2005), where FOXM1 is located (Korver et al., 1997a). Given that FOXM1 is over-expressed in EC cells (Sperger et al., 2003), FOXM1 may regulate a different set of biological pathways (see below), explaining the discrepancy observed. Human ESCs undergo rapid proliferation with uniquely abbreviated cell cycles to maintain self-renewal and pluripotent capacity (Ruiz et al., 2011; Hindley and Philpott, 2013). The underlying molecular regulation is believed to be distinct from somatic cells (Ruiz et al., 2011; Hindley and Philpott, 2013). In this study, we demonstrated that FOXM1 knockdown led to impairment of hESC proliferation, mainly due to delayed progression through the G2/M phase of the sr9009 accompanied by chromosome abnormalities during mitosis. The G2/M delay with FOXM1 knockdown is consistent with the known function of FOXM1 reported in other mouse and human cell lines (Laoukili et al., 2005; Wang et al., 2005; Wonsey and Follettie, 2005). It is also reminiscent of the arrest in G2 phase and accumulation of cells in mitosis after CDK1 knockdown recently reported in hESCs (Neganova et al., 2014). The critical role of FOXM1 in mitosis in hESCs is supported by the enrichment of cell cycle genes (in particular M phase relevant genes) in the GO analysis of the potential FOXM1 target genes discovered in the ChIP-seq analysis. We confirmed that the known FOXM1 direct targets CDK1 and CCNB1 are also downstream targets of FOXM1 in hESCs based on their downregulation upon FOXM1 knockdown and association of FOXM1 with their promoters. In somatic cells, FOXM1 is phosphorylated and activated by the CDK1/CCNB1 complex at the S/G2 phase transition (Chen et al., 2009). FOXM1 might also work synergistically with CDK1 and CCNB1 in a positive feedback manner to promote timely progression through G2/M phase in hESCs. CDK1 depletion showed more obvious effect on hESC pluripotency (Neganova et al., 2014) than FOXM1 knockdown reported here. This might be attributed to the incomplete suppression of CDK1 function upon FOXM1 knockdown and there may also be compensatory regulation of CDK1 upon FOXM1 depletion. Human ESCs exhibit higher resistance to H2O2-induced stress compared to their differentiated counterparts (George et al., 2009) by having an efficient repair capacity with elevated expression of DNA repair genes (Maynard et al., 2008). Our study supports the involvement of FOXM1 in the defense mechanism of hESCs against oxidative stress. FOXM1 knockdown impaired recovery of hESCs following H2O2 treatment, as evidenced by elevated ROS level and reduced cell growth, indicating that FOXM1 depletion renders hESCs more vulnerable to oxidative stress. FOXM1 depletion was accompanied by downregulation of CAT but not SOD2 at both the RNA and protein levels, supporting a role for CAT in the defense mechanism. However, no significant binding of FOXM1 to the proximal promoter region of CAT (~10kb upstream) was detected by ChIP-seq and ChIP analysis (data not shown). It remains to be tested whether FOXM1 regulates CAT transcription by binding to sites far away or whether FOXM1 mediates its protective effect via other target genes. The significant overlap of potential FOXM1 targets between VAL-3 (reported here) and RPE cells points to a core set of targets regulated by FOXM1 in non-cancerous cells with healthy genomes. In fact, ZBTB33 and TP53 are the top two binding motifs coexisting with FOXM1-bound sequences in both data sets (Choudhary et al., 2015). Unexpectedly, the VAL-3 peak set overlaps with relatively less targets identified in the cancer cell line OE33. The overexpression of FOXM1 and thereby the deregulation of downstream targets in various cancer cells contributes to its principal regulatory role throughout tumorigenesis, including tumor initiation, invasion and metastasis (Wierstra, 2013a). The disparate FOXM1 chromatin binding profile may imply that FOXM1 orchestrates very different sets of downstream targets in non-cancerous cells versus cancer cells, which are subjected to regulation by very different signaling pathways. Thus, further investigation is warranted to differentiate the molecular mechanisms underscoring FOXM1 regulation in normal healthy cells and transformed cells to understand its regulatory roles in diverse biological processes, which is of therapeutic relevance for targeting cancer cells.