FOXC1 promotes HCC proliferation and metastasis by Upregulating DNMT3B to induce DNA Hypermethylation of CTH promoter

Abstract

Background Forkhead box C1 (FOXC1), as a member of the FOX family, is important for promote HCC invasion and metastasis. FOX family protein lays a pivotal role in metabolism. ROS is involved in tumor progression and is associated with the expression of lots of transcription factors. We next explored the mechanism underlying FOXC1 modulating the metabolism and ROS hemostasis in HCC. Methods We used amino acids arrays to verify which metabolism is involved in FOXC1-induced HCC. The kits were used to detect the ROS levels in HCC cells with over-expression or down-expression of FOXC1. After identified the downstream target genes and candidate pathway which regulated by FOXC1 during HCC progression in vitro and in vivo, we used western blot, immunohistochemistry, bisulfite genomic sequencing, methylation-specific PCR, chromatin immunoprecipitation analysis and luciferase reporter assays to explore the relationship of FOXC1 and downstream genes. Moreover, the correlation between FOXC1 and target genes and the correlation between target genes and the recurrence and overall survival were analyzed in two independent human HCC cohorts. Results Here, we reported that FOXC1 could inhibit the cysteine metabolism and increase reactive oxygen species (ROS) levels by regulating cysteine metabolism-related genes, cystathionine γ-lyase (CTH). Overexpression of CTH significantly suppressed FOXC1-induced HCC proliferation, invasion and metastasis, while the reduction in cell proliferation, invasion and metastasis caused by the inhibition of FOXC1 could be reversed by knockdown of CTH. Meanwhile, FOXC1 upregulated de novo DNA methylase 3B (DNMT3B) expression to induce DNA hypermethylation of CTH promoter, which resulted in low expression of CTH in HCC cells. Moreover, low levels of ROS induced by N-acetylcysteine (NAC) which is an antioxidant inhibited the cell proliferation, migration, and invasion abilities mediated by FOXC1 overexpression, whereas high levels of ROS induced by L-Buthionine-sulfoximine (BSO) rescued the suppression results mediated by FOXC1 knockdown. Our study demonstrated that the overexpression of FOXC1 that was induced by the ROS dependent on the extracellular regulated protein kinases 1 and 2 (ERK1/2)- phospho-ETS Transcription Factor 1 (p-ELK1) pathway. In human HCC tissues, FOXC1 expression was positively correlated with oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), p-ELK1 and DNMT3B expression, but negatively correlated with CTH expression. HCC patients with positive co-expression of 8-OHdG/FOXC1 or p-ELK1/FOXC1 or FOXC1/DNMT3B had the worst prognosis, whereas HCC patients who had positive FOXC1 and negative CTH expression exhibited the worst prognosis. Conclusion In a word, we clarify that the positive feedback loop of ROS-FOXC1-cysteine metabolism-ROS is important for promoting liver cancer proliferation and metastasis, and this pathway may provide a prospective clinical treatment approach for HCC.

The interaction of disulfiram and H2S metabolism in inhibition of aldehyde dehydrogenase activity and liver cancer cell growth

“…We previously reported that CSE expression was reduced in drug resistant liver cancer cells and supply of exogenous H2S could reverse drug resistance in these cells 81 . Other studies also found that overexpression of CSE in T cells significantly inhibited tumour growth in a mouse model of adoptive cell transfer, while silencing CSE gene transcription via FOXC1-mediated DNMT3B expression and DNA hyper-methylation promoted hepatocellular carcinoma survival 102,103 . This evidence demonstrated a key role of CSE/H2S system in regulating cancer growth and drug resistance possibly by targeting at ALDH.…”

Low‐level EFCAB1 promoted progress by upregulated DNMT3B and could be as a potential biomarker in lung adenocarcinoma

“…MiR‐29b inhibits the progression of cholangiocarcinoma by releasing the inhibition of CDKN2B expression mediated by DNMT3B 10 . FOXC1 induced CTH promoter DNA hypermethylation through upregulation of DNMT3B to promote proliferation and metastasis in primary hepatocellular carcinoma 11 . We found that the expression level of DNMT1 mRNA of PC9, A549, and HCC827 were higher than that of BEAS‐2B, while the expression level of A549 and HCC827 was lower than the expression levels of A549 and HCC827 were lower than those of BEAS‐2B.…”

Therapeutically Targeting Cancers That Overexpress FOXC1: A Transcriptional Driver of Cell Plasticity, Partial EMT, and Cancer Metastasis

“…This is evident in Table 2 based on the number of studies across a wide variety of cancers where this has been demonstrated to be the case. In addition to the clinical correlative studies outlined above, clear examples of the critical importance of FOXC1 in metastagenesis includes studies performed in breast cancer (77,85), lung cancer (57), HCC (54,143), colon cancer (49) and salivary gland cancer (144). In these studies, overexpression led to the development of an increased number of metastatic lesions in preclinical animal models.…”

“…FOXC1, which has been shown to play an important role in the development and progression of multiple malignancies, also plays a pivotal role in metabolism. Xia and colleagues reported that FOXC1 could inhibit the cysteine metabolism-related genes, cystathionine glyase (CTH) through upregulation of de novo DNA methylase 3B (DNMT3B) expression, which resulted in the decrease of cysteine levels and increase reactive oxygen species (ROS) levels (143). In human HCC cells FOXC1 was in turn upregulated by ROS-ERK1/2-p-ELK1 signaling axis.…”