The Histone Lysine Demethylase KDM4C Promotes Cell Proliferation And Modulates Metabolic Reprogramming In Cancer Cells

Epigenetic refers to the study of heritable changes in gene expression that are independent of changes in DNA sequences. Mechanisms of epigenetic regulation include DNA and histone modifications, as well as histone variants and non-coding RNAs. Overwhelming evidence indicates that epigenetic dysregulation is an initiating force in tumorigenesis by promoting the selection of cancer-associated phenotypes. It is well established that cancer rewire their cellular metabolism to meet the biosynthetic challenge of growth and proliferation. The metabolic reprogramming changed not only cell fates, but also their cellular metabolic pathway. Aerobic glycolysis reroutes metabolites from energy-producing catabolic pathways to biosynthetic pathways for the production of amino acids, nucleotides, and lipids, thereby providing the anabolic needs of cell growth and proliferation. Our previous study showed that KDM4 C has a key role in cell fates and metabolic reprogramming. Moreover, the KDM4 C gene is amplified or overexpressed in various cancer types, including lymphoma, breast cancer, esophageal squamous cell carcinoma, lung sarcomatoid carcinoma, and medulloblastoma. The study of KDM4 C is conducive to define the molecular mechanism for epigenetic regulation of cell fate and metabolic reprogramming, and provide a theoretical support for cancer treatment. In this report, we present evidence for the cooperation between the histone H3 demethylase KDM4 C and the transcription factor ATF4 in control of amino acid metabolism and cell cycle. These results shed that new light on the biological function of KDM4 C and its role as a metabolic oncogene in cancer pathogenesis and progression. The main results were as follows:1. KDM4 C is essential for the expression of the seine pathway genes and cancer cell proliferationWe silenced the expression of KDM4 C by small hairpin RNA(sh RNA), there were highly effective in up-regulation of H3K9me3 levels, but showed no significant effecct on the expression of other KDM4 family members. We found that knock down of KDM4 C in several cancer cell lines results in a significant reduction in cell proliferation and colony formation, which are consistent with the previous data(Cloose et al., Kim et al.). Ch IP-q PCR analysis in B(E)2C cell line revealed significant association of KDM4 C with the promoters of the serine biosynthetic pathway. These are consistent with the recent chromatin immunoprecipitation and sequencing(Ch IP-seq) study of KDM4 C binding sites(Pedersen et al., 2014). These results suggest that KDM4 C is required for maintaining the serine pathway genes in an epigenetically actve state. Together, KDM4 C is essential for the expression of the serine pathway genes and cancer cell proliferation.2. Ectopic KDM4 C expression transcriptionally activates the serine pathway to promote cancer cell proliferationAs noted earlier, the KDM4 C gene is amplified in various human cancers. We investigated the effect of KDM4 C over-expression using an inducible system because of demethylase. We found that induction of KDM4 C in cell lines led to significantly decreased H3K9me3 levels, significantly increased H3K9me1 levels and no significant changes in H3K9me2 levels. Futhermore, KDM4 C induction in several cancer cell lines markedly enhanced cell proliferation and tumorigenicity. The reason of these results may be that ectopic expression of KDM4 C alone was sufficient to transcriptionally activate the serine pathway genes. Therefore, we investigated the m RNA and protein levels following KDM4 C induction. We found that there are significant increases in the m RNA and protein levels of the pathway enzymes. Moreover, Ch IP-q PCR assays revealed significantly higher levels of KDM4 C associated with the promoters of PHGDH and PSAT1 following KDM4 C induction. Together, ectopic KDM4 C expression transcriptionally activates the serine pathway to promote cancer cell proliferation.3. KDM4 C has a central role in transcriptional activation of amino acid biosynthesis and transportFrom the Microarray analysis, KDM4 C play a key role in transcriptional activation of amino acid biosynthesis and transport. We confirmed the results of our microarray data analyses by RT-q PCR, such as ASNS, ASS1, CTH, GOT1, GPT2. These enzymes are involved in the biosynthesis of alanine, arginine, asparagine, aspartic acid, cysteine, and glutamic acid. We also confirmed by RT-q PCR that KDM4 C induction resulted in a significant increase in m RNA expression of 7 amino acid transporter genes, including SLC1A4, SLC1A5, SLC3A2, SLC6A9, SLC7A1, SLC7A5 å'Œ SLC7A11. These transporters are involved in the transport of all amino acids except aspartic acid, asparagine, and proline. It should be pointed out that data from the recent KDM4 C Ch IP-seq study indicates that KDM4 C binds to the promoters of most of these genes(Pedersen et al., 2014). Together, these data suggest a major role of KDM4 C in transcriptional activation of genes involved in amino acid biosynthesis and transport. Moreover, GC-MS analysis revealed marked elevation of the intracellular levels of 15 amino acids following KDM4 C induction, accompanied by a significant increase in the kinase activity of m TORC1, which promotes cell growth and proliferation by stimulating macromolecule biosynthesis and cell-cycle progression. Collectively, these findings reveal a central role of KDM4 C in promoting amino acid biosynthesis and transport for cancer cell proliferation.4. KDM4 C requires ATF4 for transcriptional activation of the serine pathwayActivating transcription factor 4(ATF4) has a key role in the amino acid deprivation response. We found that in all the cell lines examined, knockdown of KDM4 C expression led to a marked reduction in ATF4 expression at both m RNA and protein levels, indicating an essential role of KDM4 C in maintaining the basal level of ATF4 expression. It has been shown recently that KDM4 C specifically binds to the ATF4 promoter(Pedersen et al., 2014). These results demonstrate that ATF4 is a direct downstream target gene of KDM4 C. We next investigated whether KDM4 C has a physiological function in transcriptional control of ATF4 expression. It is known that ATF4 expression is markedly increased following amino acid deprivation, as a result of both transactivation of its gene and stabilization of its protein. We found that serine deprivation significantly increased the level of KDM4 C associated with the ATF4 promoter, which was accompanied by a significant decrease in the level of H3K9me3 at the promoter. Importantly, knockdown of KDM4 C expression by sh RNA completely abrogated the induction of ATF4 by serine deprivation. These findings reveal an essential role of KDM4 C both in the maintenance of the basal level of ATF4 expression and in the transcriptional upregulation of ATF4 in response to serine deprivation. Previous studies have shown that PHGDH and PSAT1 are direct transcriptional targets of ATF4, raising the possibility that ATF4 may function downstream of KDM4 C in transcriptional activation of the serine pathway genes. To test the hypothesis, we silenced ATF4 expression using sh ATF4 constructs. Knockdown of ATF4 expression completely abolished the ability of KDM4 C to promote cell proliferation and to upregulate the expression of the serine pathway genes. Interestingly, ATF4 knockdown also resulted in a marked reduction in the level of KDM4 C at the promoter, leading to a significant increase in H3K9 me levels at the promoter. Moreover, we observed that ATF4 could be co-imminoprecipitated with KDM4 C in under the condition of KDM4C overexpression or serine deprivation. Together, these findings demonstrate that KDM4 C interacts with and depends on ATF4 for targeting to the promoters of the serine pathway genes for their transcriptional induction in response to serine deprivation.