Since the dawn of whole-genome sequencing in cancers, it well-defined that AML cells carry the lowest mutation number as compared with other malignant cells[2]. This characteristic may be in part due to the genome-wide hypermethylation, which occurs in many AML patients, a feature that could guard leukemic cells against genome instability but simultaneously increased the chance of point mutation in CpG islands and dampened the expression of TSGs[28]. On the contrary, genome-wide hypomethylation is a marker of a dozen types of solid tumors, which is accompanied by genome instability and consequently a higher mutation rate per TSGs[29]. However, disfavoring cancer cells, general hypomethylation could attenuate the net suppressive effect of epigenetic on TSGs[29]. In this study, to shed light on the molecular mechanisms of p16INK4A and p53 suppression in AML patients, we investigated the expression of a panel of important epigenetic regulators and compared their expression with the control group.
Epigenetic could inactivate or silent the gene expression. It has been corroborated that the role of epigenetic in the silencing of TSGs in cancer cells is at least as effective as gene mutations[30]. DNA methylation is even thought to be the primary mechanism of silencing TSGs in patients with myelodysplastic syndrome (MDS) and AML[30]. We previously showed that p16INK4A expression significantly reduced in AML patients and its expression pattern had a tight association with the age of the patients. Our recent findings also showed that there was a remarkable and widespread decrease in the expression level of p53 by the mean of 12.5-fold in AML patients as compared with healthy control. DNA methylation is a possible mechanism in the suppression of p16INK4A and p53 genes. In this study, we demonstrated for the first time that akin to that seen in p16INK4A, p53 expression was also significantly reduced with advancing age in AML patients. Declining p53 function in the aging process has been suggested as a possible mechanism for the development of cancer in older populations[31]. Conversely, an augmented activation of many of TSGs, including p53, p21, p16, p27, and p15 have previously been documented in the cells isolated from normal tissues of elder individuals[32]. This feature confers cellular senescence, a process acting as a strict physiological antitumor response to counteract the oncogenic insults in cells with an accumulated oncogenic mutation [32]. Indeed, a recent study demonstrates that non-mutational p53 dysfunction may exist in roughly all AML patients[6]. Overall, the reduction of p53 protein and overexpression of their negative regulators named MDM2/4 have been previously attributed to p53 aberrancy in AML[6].
Our results showed a strong positive correlation with a prominent statistical significance between p16INK4A and p53 expressions in the control group, but not in the AML patients. A study on normal fibroblasts and osteoblasts of mouse organs indicates p53 enforced expression could repress p16INK4A gene expression in normal condition, but not in response to noxious stimuli. The authors have proposed an indirect mechanism for p16INK4A suppression mediated by Ets1 transcription factor activity [33]. Another study showed that p16INK4A up-regulation after exposure of the cells to ultraviolet light is required for efficient p53 over-expression [34]. So, it could be concluded that same to similarities that exist between p16INK4A and p53 functions, their expressions also could changes in parallel in response to stimulant environmental factors such as genotoxic stresses. The absence of p16INK4A and p53 correlation among AML patients coupled with the downregulation of these genes provokes further investigation to determine if AML cells properly respond to genotoxic stimuli through induction of p16INK4A and p53.
With this background, we were avid for looking for the plausible mechanisms behind the suppression of these critical TSGs in the AML patients with a special focus on histones modifiers. In the present study, of the five epigenetic regulators, four genes represented a grossly altered expression in AML patients. For the epigenetic reader, analyzing the expression level of UHRF2 showed that there was a 20-fold decrease in the expression of this gene as compared to the control group, suggestive of the tumor suppressor property of this gene in the patients. We did not find any significant correlation between UHRF2 expression and TSGs. UHRF2 repression in AML patients is in keeping with this fact that the UHRF2 coding region located at 9p24 is subjected to deletion in a variety of malignancies[35]. Moreover, Lu and colleagues have been indicated that the protein level of UHRF2 significantly reduced or mislocalized in diverse human cancers, in particular AML[36]. In sharp contrast, there are many studies in explanation of the oncogenic role of UHRF2 and its clinical relevance. UHRF2 was uncovered as a breast cancer promoter by suppression of p16, p21, and p27, through inducing DNA methylation and histone modification[37]. Furthermore, UHRF2 overexpression was attributed to poor prognosis in colon cancer, likely through transcriptional induction of ERK1/2 axis [38]. Regarding the various function of UHRF2 in the regulation of cell cycle, epigenetic and apoptosis, it seems that UHRF2 serves either as a tumor promoter or a tumor suppressor in a context-dependent manner[38]. According to the role of UHRF2 in the degradation of DNMTs and genome-wide hypomethylation[39], the down-regulation of this gene in AML patients could be defensible by the evidence of DNA hypermethylation and elevation of DNMTs in patients[40]. The absence of correlation between UHRF2 and TSGs could be explained in part by the impressive reduction of mRNA, which may attenuate their function in AML patients. In keeping with the previous studies, we found a significant negative correlation between the expression of UHRF1 and p53 in AML patients. UHRF1 is frequently activated in various malignancies and plays various oncogenic roles particularly by repression of TSGs[41].
Noteworthy, both p16INK4A and p53 gene expressions have been demonstrated to be influenced by histones modifiers [42, 43]. In this study, we found that two epigenetic writers named PRDM16 and SUV39H1 had significant high transcriptional expressions in 50% and 36% of AML patients respectively. PRDM16 was previously detected to drive a prognostically unfavorable inflammatory signature and also to regulate the expression of cytokines and chemokines, including VEGF, HGF, and TNF in AML patients[44]. Overexpression of PRDM16 has been demonstrated in approximately 30% of AML patients, which was associated with poor prognosis[44]. A great deal of evidence is already in hand that explores the suppressive role of SUV39H1 on TSGs in the AML patients[45]. The synoptic conclusion from these studies could be that SUV39H1 is required for the formation of AML largely through transcriptional repression of p15, p21 and E-cadherin[45]. Moreover, the results of their study also declared that SUV39H1 inhibition could induce differentiation and apoptosis in AML cells by restoring the expression of epigenetically silenced genes[45].
In corroboration with the previous studies[46], our results demonstrated that SUV39H1, which acts as an important H3K9 methyltransferase, adversely correlated with the expression of p16INK4A and p53 gens. PRDM16 overexpression coupled with p53 inactivation could be enough for the development of AML[21]. Interestingly, we found that SUV39H1 may have repressive effects on p53 expression, so, further studies are required to uncover whether parallel overexpression of PRDM16 and SUV39H1 could create similar results.
The mounting body of evidence has shown that both KDM3C and KDM2B have a crucial role in the maintenance and the self-renewal of AML cells through epigenetic regulation of a wide array of genes[19, 24]. Notably, the results of the qRT‐PCR analysis revealed a significant upregulation of KDM3C in AML patients, while the expression level of KDM2B in AML patients was similar to the control group. Moreover, we found that the expression level of KDM3C was higher in M3 patients in comparison with other subtypes of AML. Previous reports have suggested that via regulation of IL-3/RAS/JAK pathway and also through cooperation with the HOXA9/MEIS1 axis, KDM3C could exert pro-oncogenic function in AML[47]. On the other hand, in contrast to Oncomine databases, which assigned an elevated expression of KDM2B in acute leukemias, we found a normal expression for this gene in the AML patients[19]. An elevated level of this gene was also attributed to the advanced stages of pancreatic ductal adenocarcinoma [48]. KDM2B has been reported to have a remarkable suppressive effect for p15ink4a and modestly for p16ink4a and p19arf in a murine model of AML[19]. In keeping with this study, we found that KDM2B expression was negatively correlated with both p16INK4A and p53 expressions. The lack of correlation between KDM3C and TSGs in our study could be explained by this fact that KDM3C is mainly involved in erasing the suppressive marks, such as H3K9me3, particularly from promoters of genes involved in metabolic pathways.
We demonstrated that the epigenetic regulators were remarkably perturbed at the level of expression in the majority of AML patients. Distorted expression of some genes was correlated with repression of "guardians of the genome" namely p16INK4A and p53. We also delineated that the expression pattern of some genes is dependent on the age, and maybe involved in a greater tendency of AML occurrence toward advanced ages. Prospectively, our introduced panel consisting of epigenetic reader, writer and eraser enzymes would be promising to recruit by further investigation in the terms of prognosis determination, risk stratification, and monitoring in AML patients.