GSKJ4, an H3K27me3 demethylase inhibitor, effectively suppresses the breast cancer stem cells
Graphical abstract
Introduction
In the past twenty years, a significant trend towards a worldwide reduction in mortality from breast cancer has been observed. This trend has been largely attributed to improved early detection methods and the development of improved treatment strategies. However, a substantial fraction of breast cancer patients still relapse and even die after five years. Current opinion proposes that this failure is largely due to the existence of breast cancer stem cells (BCSCs), which have unlimited proliferative potential and are responsible for drug resistance and disease relapse [1], [2]. Therefore, therapeutic strategies specifically targeting BCSCs are warranted.
Cancer stem cells (CSCs), which are found in most solid tumours, share with normal stem cells several properties, such as self-renewal, a better ability to repair DNA and resistance to apoptosis and hypoxia. It is conceivable that several key regulatory pathways involved in the regulation of self-renewal and differentiation of stem cells, such as WNT [3], Notch [4] and Sonic Hedgehog [5], are also frequently deregulated in CSCs. In addition, more and more studies have revealed that epigenetic mechanisms play a vital role in the maintenance of stemness in normal stem cells [6], [7], and the influence of epigenetic modifications on self-renewal and the pluripotency of CSCs is gaining a great deal of attention as well [8], [9], [10].
As a critical factor in epigenetic modification, histone methylation is mediated by methyltransferases that catalyse the mono-, di-, or tri-methylation of specific lysine residues [11] and is involved in many biological processes. Of note, four lysine residues (K4, K9, K27, K36) in the conserved N-terminal tail of the histone are primary targets of specific histone methyltransferases and demethylases. Furthermore, methylation (mono-, di-, and tri-methylation) on trimethylated lysine 27 of histone H3 (H3K27me3) induces transcriptional repression, and thereby is associated with controlling gene expression patterns [12]. By virtue of its influence on chromatin configuration, which could modulate the accessibility of transcription factors and the transcriptional activity of nearby genes, H3K27me3 is found to be involved in the normal development and disease [13], [14], [15], especially in several cancer types, such as breast [16], ovarian [17], and prostate cancer [18]. Notably, for breast cancer, Wei et al. found that the loss of H3K27me3 tended to be associated with a poor prognosis [11]. Another study also confirmed the positive correlation between H3K27me3 and luminal A breast cancer after adjusting for reproductive and lifestyle breast cancer risk factors [19]. These results suggest that H3K27me3 participates in breast cancer carcinogenesis, however, the underlying mechanisms for its involvement remain unknown.
Interestingly, a rapidly growing body of research has revealed that the stemness of both normal stem cells and CSCs is also maintained by the epigenetic marker H3K27me3. For example, Hawkins et al. evaluated the H3K27me3 levels in human embryonic stem (ES) cells with several types of lineage-committed cells [20]. As a result, high H3K27me3 levels in all lineage-committed cells were observed when compared with ES cells. Moreover, a recent study also demonstrated that increased H3K27me3 levels were associated with the differentiation of CSCs in ovarian cancer [21]. As CSCs have much in common with normal stem cells and as the loss of H3K27me3 is closely related with breast cancer and CSCs, we hypothesize that global changes in the H3K27me3 levels might play a key role in stemness maintenance of BCSCs. Hence, drugs targeting the abundance of H3K27me3 might help to eliminate BCSCs.
GSKJ4 is a novel, selective inhibitor of the jumonji family of histone demethylases JMJD3 and UTX, which are the H3K27me2/3-specific demethylases that catalyse the demethylation of H3K27me2/3 [22], [23]. To test our hypothesis, GSKJ4 was employed to evaluate the effects of H3K27me3 demethylation inhibition on the phenotypes and biological functions of BCSCs. The tumourigenicity of the breast cancer cell line after GSKJ4 treatment was also analysed in vivo. The aim of the present study was to evaluate whether the pharmacologic inhibition of H3K27me3 demethylation could specifically target CSCs of breast cancer and to explore the possible molecular mechanisms for this process.
Section snippets
Cell culture
Human breast cancer cell lines MCF7 and MDA-MB-231 were purchased from the American Type Culture Collection (ATCC) and were maintained in medium as indicated by ATCC's instructions. The cell lines were cultured in a humidified atmosphere with 5% carbon dioxide at 37 °C and were subcultured until 90% confluency was reached.
Reagents
Rabbit monoclonal anti-H3K27me3 (CAT: 14034S), anti-UTX (CAT: 33510S), anti-NANOG (CAT: 4903S), anti-SOX2 (CAT: 3579S), anti-OCT4 (CAT: 2750S) and anti-GAPDH (CAT: 5174S) were
A decreased global H3K27me3 level is observed in BCSCs
As a reliable method to enrich CSCs from breast cancer cells in vitro [26], the mammosphere culture platform was successfully established previously [24], [27]. In the present study, an elevated expression of stemness markers including NANOG, SOX2 and OCT4 was found in the mammospheres (Fig. 1A-B). Of note, we found a decreased global H3K27me3 level in the mammosphere-derived BCSCs when compared with adherent breast cancer cells (Fig. 1A). Taken together, these results support the possibility
Discussion
For decades, mounting evidence has suggested that BCSCs are responsible for cancer progression, metastasis and recurrence [30], [31], [32], [33]. Conventional therapies, though effective at killing the majority of cancer cells, often eventually fail due to not eliminating the subpopulation of CSCs, leading in turn to the development and recurrence of cancers [34]. Therefore, it is critical to find ways to eliminate CSCs thoroughly.
To this end, accumulating preclinical studies and clinical
Conclusions
Collectively, our results suggest that H3K27me3 plays a negative role in modulating the behaviours of BCSCs and provide strong supports for the hypothesis that GSKJ4 is a prospective drug target. Mechanistically, this effect results from JMJD3 and UTX inactivation. Thus, our findings suggest that epigenetic modifications, including histone methylation, hold the potential to eliminate BCSCs. The specificity and off-target effects remains to be evaluated in the future.
Conflict of interest
All authors declare no conflict of interest and are in agreement with the publication of this manuscript.
Acknowledgements
This work was supported by Key R&D Project of Jiangsu Province (BE2016677), the Youth Science Foundation of Nanchang Province (20171BAB215045) and Suzhou Science and Technology Project (SS201508).
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Ningning Yan and Liang Xu contributed equally to this work.