Abstract
To the Editor:
Today more and more studies are illustrating the involvement of epigenetic alterations in cancer biology and therapeutics (Dzobo, 2019). Previously, it has been shown that the increased expression of Jumonji domain-containing protein 3 (JMJD3) demethylase was able to remove histone 3 lysine 27 trimethylated (H3K27me3) methylation in prostate cancer cell lines and human tumor biopsies (Daures et al., 2016). As a result of the latter research, four candidate genes (MGMT, TRA2A, RPS6KA2, and U2AF1) have been identified as a putative biosignature for prostate cancer. These genes are involved in different cellular processes such as DNA damage repair, mRNA splicing, or in the mitogen-activated protein kinase 6 (MAPK) regulatory pathway, thus having biological plausibility as potential markers.
In addition, after determination of the half maximal inhibitory concentration (IC50), it was shown that the inhibitor of JMJD3, ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoate (GSK-J4), was able to modulate the expression of these candidate genes in the DU-145, PC-3, and LNCaP cell lines (Daures et al., 2018).
Thus, to provide further evidence on the involvement of JMJD3 demethylase in prostate tumor progression, and particularly on MGMT, TRA2A, RPS6KA2, and U2AF1 candidate gene expressions, an analysis of the H3K27me3 enrichment was performed by chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) after inhibition of JMJD3 by GSK-J4 in prostate cancer cell lines. Subsequently, a reverse transcription (RT)-qPCR was conducted after inhibition of JMJD3 by small interfering RNA (siRNA) or GSK-J4 inhibitor to demonstrate its demethylase activity on the expression of studied genes. In addition, the inhibition of JMJD3 by siRNA was validated by Western blotting.
The analysis of H3K27me3 (Fig. 1A) showed an increase in H3K27me3 on the MYOGLOBIN and GAPDH control genes after treatment with GSK-J4 for the DU-145 and LNCaP cell lines, but no variation was observed in PC-3 cell line. In contrast, the enrichment of the marker on the genes of interest (MGMT, TRA2A, RPS6KA2, and U2AF1) tended to increase in different cell lines. Also, the accumulation of JMJD3 on target genes did not vary for DU-145 and PC-3 cell lines, but there was an increased trend for LNCaP cell line. These results collectively suggest that GSK-J4 treatment inhibits the activity of JMJD3 leading to an increase in the enrichment of H3K27me3 on genes.
Effects of JMJD3 inhibition on H3K27me3 enrichment and gene expression in prostate cancer cell lines.
Inhibition of JMJD3 by siRNA was chosen to show a decrease in JMJD3 protein accumulation after Western blotting analysis (Fig. 1B). Figure 1C shows gene expression by RT-qPCR analysis after inhibition of JMJD3 by siRNA and chemical inhibition by GSK-J4. Inhibition of JMJD3 by GSK-J4 exhibited nonsignificant decrease in JMJD3 expression in the three cell lines. Conversely, the inhibition of JMJD3 by siRNA led to a significant decrease of JMJD3 expression in DU-145 and PC-3 cell lines. Consecutively, a significant decrease in the TRA2A gene expression was noted with GSK-J4 in DU-145 and LNCaP cell lines. RPS6KA2 and U2AF1 exhibited a significant decrease with JMJD3 siRNA in PC-3 cell line.
mRNA analysis confirmed that the enrichment in H3K27me3 on target genes, shown in Figure 1A, led to an inhibition of MGMT, TRA2A, RPS6KA2, and U2AF1 expression, affirming H3K27me3 as a repressive marker.
Epigenetic drug mechanisms are increasingly studied today (Dzobo, 2019). Indeed, the low accumulation of JMJD3 on different genes (Fig. 1A) and the slight decrease in the expression of JMJD3 (Fig. 1C) after inhibition by GSK-J4 showed the efficacy of an epigenetic drug approach on the activity of JMJD3. The use of siJMJD3 resulted in a similar expression profile of the target genes.
The androgen receptor (AR) status seems to influence the accumulation of JMJD3 on candidate genes. Indeed, AR- cell lines (DU-145 and PC-3) showed no increase in JMJD3 after inhibition, whereas AR+ cell line (LNCaP) showed a tendency to accumulate JMJD3. This result was also demonstrated at the transcriptional level of JMJD3 between AR- and AR+ cell lines (Daures et al., 2016).
MGMT gene, involved in DNA repair, showed overexpression after inhibition by GSK-J4 in the LNCaP cell line. Presumably, this gene expression is dependent on the AR status. Indeed, a study showed a significant difference in MGMT gene methylation between AR+ and AR- cells in a prostate cancer model (Mishra et al., 2010).
Upregulation of genes such as U2AF1 and TRA2A that are involved in splicing pathway appears to be important as studies are showing the link between splicing and tumor progression. The decrease in gene expression by inhibition of JMJD3 needs to be highlighted. It has already been shown that demethylase JMJD1 is a coactivator of AR in prostate cancer and that overexpression of a number of factors involved in splicing would lead to a wide variety of gene functions in carcinogenesis (Takayama, 2019).
Furthermore, inhibition of RPS6KA2 has already been shown to decrease proliferation in LNCaP and PC-3 cell lines (Yu et al., 2015). In this context, the inhibition of an upstream actor of RPS6KA2 could be a new therapeutic target for JMJD3.
In conclusion, these data and analysis emphasize the link between JMJD3 and the regulation of MGMT, TRA2A, RPS6KA2, and U2AF1 genes in prostate cancer cell lines. Accumulation of H3K27me3 on specific genes leads to a decrease in their expression, making H3K27me3 a repressive epigenetic marker. JMJD3 is regulated in vitro by siRNA or GSK-J4, making JMJD3 a potential therapeutic target. In the future, it would be interesting to see the effect of JMJD3 inhibition on MGMT, TRA2A, U2AF1, and RPS6KA2 genes in vivo.
Footnotes
Author Disclosure Statement
The authors declare that no conflicting financial interests exist.
Funding Information
This study was supported by grants from the French Ligue Régional Contre le Cancer—Comité du Puy-de-Dôme and from ARTP (Association pour la Recherche sur les tumeurs de la Prostate).