JMJD3 Is a Potential Tumor Suppressor Gene That Cooperates with TP53 in Chromosome 17p13

Introduction:

Hemizygous deletion of 17p13 (17p13(del)) has been identified in up to 30% of multiple myeloma (MM) patients and is a powerful prognostic marker. We have previously identified that TP53 demonstrates haploinsufficiency, which could render an aggressive nature in the 17p13(del) MM cells (Teoh et al., Leukemia, 2014). Importantly, there are various other genes that are also located within the minimal deleted region of 17p13. Therefore, we postulated that beyond TP53 deletion alone, the adverse effects conferred by 17p13(del) may be due to the concomitant loss of other potential tumor suppressor genes. Herein, we sought to identify a novel gene that can co-operate with TP53 in mediating growth and proliferation of MM cells and found KDM6B/JMJD3, which is a histone demethylase, to be of particular interest. The role of JMJD3 in cancer has remained controversial, not to mention, unknown in MM.

Methods:

We utilised publicly available MM patient datasets and various human myeloma cell lines (HMCLs) of differential p53 status as our study models. We created a dynamic system to elucidate the potential role of JMJD3 by performing a single knockdown of either p53 or JMJD3 as well as double knockdown of both p53 and JMJD3 in an isogenic HMCL by using the lentivirus transduction mechanism. The former approach was taken so as to enable us to study the independent tumor suppressive role of JMJD3. These cell lines were subjected to selection agents (puromycin) and stable cell lines were established within three to four weeks. Functional assays (CTG, colony formation and cell cycle) were done on these stable HMCLs to investigate the phenotypes upon loss of expression of JMJD3 or/and p53.

Results:

Comprehensive gene mapping on patient datasets has unveiled 33 genes to be residing within the minimally deleted 17p13 (0.7Mbp). Besides TP53, we identified JMJD3 to be our gene of interest. We interrogated the impact of 17p13(del) on JMJD3 expression and found that loss of the chromosomal copy number led to a substantial reduction in JMJD3 expression in both MM patients and HMCLs, denoting a possible biological significance of JMJD3 in this cytogenetic aberration. Consistently, our functional analyses then revealed JMJD3 to possess potential tumor suppressor properties. Even in the presence of WT-p53, single knockdown of JMJD3 promoted a more proliferative cellular state, suggesting that independently of TP53, loss of JMJD3 alone was already able to increase MM tumorigenicity. Nevertheless, concurrent knockdown of both p53 and JMJD3 could actually lead to a significantly higher cellular growth and proliferation. These results could also be replicated when JMJD3 was depleted in p53-deficient cells, thus, proving that concomitant loss of both genes had synergistic oncogenic effects to the MM cells. Interestingly, we observed that JMJD3 expression was not associated with the global histone methylation status. Protein localization assay demonstrated that majority of JMJD3 is located within the cytoplasm, hence, its tumor suppressive properties in these HMCLs are deemed to be demethylase-independent. Additionally, we identified positive p53-JMJD3 protein interactions, hinting the possibility of both proteins being a cofactor for each other.

Conclusions:

Beyond the classical belief, our preliminary data indicates that 17p13(del) is actually so much more than just TP53 itself. Although JMJD3 could exert tumor suppressor properties independently of p53, our results underline the importance of combined effects of dose reduction of two tumor suppressor genes within 17p13. Our study is important in shedding some lights to the doom and gloom of the disease biology of the high-risk patients with 17p13(del).