Epigenetic regulation significantly impacts critical biological processes such as genomic integrity, transcriptional regulation, and cell fate [24–26], garnering considerable attention in cancer biology [27]. Histone acetylation modification, as a form of epigenetics, can regulate various biological processes such as cell proliferation, differentiation and apoptosis. Dysregulation alters the normal expression and physiological functions of genes within cells, playing a role in the initiation and progression of tumors [28]. It has been reported that histone deacetylase inhibitors might represent a novel strategy for cancer treatment [29]. With advances in sequencing technology, the use of sequencing to seek new predictive markers and candidates for immune and targeted therapies is becoming one of the important treatment strategies for various cancers, including EWS [30]. Furthermore, research has already developed gene signature based on HARGs for predicting the prognosis of colorectal and identifying biomarkers for colorectal cancer and ovarian cancer [21, 22]. However, understanding of acetylation in EWS is still limited. These evidence provided a strong theoretical basis and preliminary groundwork for constructing a prognostic feature of HARGs in EWS patients. Furthermore, previous research has suggested that miR-22, which is typically suppressed by EWS-FLI1, may inhibit the growth of EWS cells by targeting the histone demethylase KDM3A, involved in H3K9me1/2 histone modifications [13].
This study systematically examined the expression of HARGs in EWS patients, and found that most HARGs are overexpressed in EWS samples and are associated with the prognoses of EWS patients. This suggests that these HARGs may be one of the factors influencing the development of EWS. To improve the efficiency of epigenetic therapy for EWS patients, this study constructed a HARGs signature related to EWS prognoses by performing Cox regression and LASSO regression. We discovered that the constructed signature exhibits specificity and sensitivity in predicting the prognosis of EWS patients, particularly in assessing the 1-year survival rate. The value of 1-year AUC in training and validation cohort are both higher than 0.85, showing that this prognostic signature may be good at predicting the short-term survival for EWS patients. Although the values of 2- and 3-year AUC in the validation cohort were relatively low, their values are close to 0.7, still implying the significance of HARGs in EWS prognoses. It is undeniable that many factors such as individual differences and tumor heterogeneity may affect the prognosis. In addition, the validation cohort was composed of two GEO datasets, and the process of combining two datasets might change the original gene expression, particularly when performing the normalization procedure. These situations may lead to the low value of AUC. However, the good performance of this HARGs prognostic signature cannot be overlooked. We believed that improved algorithms can assist us in achieving better results in the future.
For the GO enrichment analyses, it was noticed that neuropeptide Y receptor activity was enriched in both training and validation cohort. Neuropeptide Y receptor has five subtypes called Y1, Y2, Y4, Y5, and Y6, belonging to G protein-coupled receptor superfamily [31]. Y1 was highly expressed in EWS and it could potentially be a therapeutic target for EWS [32], while Y5 may promote the progression and bone metastasis for EWS [33]. In addition, Neuropeptide Y pathways seemed could be affected by histone acetylation [34]. However, the role of HARGs in the neuropeptide Y pathway in EWS is not yet clear. Our study suggested that HARGs might be associated with the neuropeptide Y pathway in EWS, which could aid in identifying new effective therapeutic targets for EWS treatment. This hypothesis requires further in-depth research to be substantiated. In the immune-related analyses, we found that APC co-stimulation and several immune checkpoints (CD276, TNFRSF9) were significantly different in two groups. Their scores or expression were relatively higher in the high-risk group, indicating that HARGs may have a certain impact on the immune microenvironment of EWS. However, this specific assertion requires further research for confirmation.
There are three roles in the process of histone acetylation, containing “writers”, “erasers” and “readers”. “Writers” are a group of enzymes that transfer chemical groups to histones, while “erasers” are also a group of enzymes responsible for removing these chemical groups, and “readers” are proteins which could recognize the modified histones [35]. In this study, the prognostic signature was composed of two protective factors (ATF2, OGA) and two risk factors (TAF4, HDAC2). ATF2 (Activating Transcription Factor 2) is a transcription factor with B-Zip-containing, and its encoded protein is a histone acetyltransferase (HAT) specifically acetylating histones H2B and H4 [36]. Previous studies have implicated ATF2 is closely associated with the development and progression of cancer. It has been reported that ATF2 promotes the occurrence and progression of melanoma and renal cell carcinoma, being identified as an oncogene [37–39]. Conversely, ATF2 is speculated to have a specific inhibitory effect on the tumor formation and metastasis of liver cancer [40]. In addition, ATF2 elicits tumor suppressor activity in mammary and skin tumors under a specific environment [36]. Similarly, OGA also plays a duplicated role in the development of tumors. OGA (O-GlcNAcase) contains a structural domain of histone acetyltransferase, which contributed to histone acetylation. OGA was found highly expressed in poorly differentiated laryngeal tumor cells, related to a poor prognosis [41]. OGA was considered as a potential target by stabilizing the activity of P53 pathway in ovarian carcinoma treatment [42]. Although the roles of two genes are different in the process of histone acetylation (ATF2: writers, OGA: erasers), high expression of these two genes may indicate a better prognosis for EWS patients, showing that ATF2 and OGA may be potentially therapeutic targets for EWS patients. TAF4 (TATA-Box Binding Protein Associated Factor 4) is a primary component of the TFIID complex, with a remarkably conserved molecular structure, maintaining stability and integrity of the TFIID complex [43]. TAF4 participates in a series of biological processes, including epigenetic modification and cell development. ATF4 inactivation alters the expression of matrix metalloproteases (CTGF and OPN), which are crucial to regulating metastasis [44]. Furthermore, the loss of ATF4 significantly changes cell adhesion and communication, further inducing the expression of oncogenic markers, and promoting malignant transformation to stimulate tumor formation [44]. Additionally, TAF4 might mediate the tumor invasion and metastasis in gastric cancer by co-expression with TWA1 [45]. These evidences indicated the oncogenic functions of ATF4. HDACs are not only involved in the deacetylation of chromatin proteins, but also take part in the deacetylation of non-histones by directly interacting with transcription factors like P53 and STAT3, influencing cell differentiation and cell cycle [46, 47]. As an “eraser” in histone acetylation, the expression of HDAC2 was highly detected in gastric cancer, and high expression of HDAC2 was associated with nodal spread, correlated with poor survival outcomes for gastric cancer patients [48]. Previous studies have shown that EWS patients with high expression of HDAC2 have a worse prognosis compared to those with low expression [49], which is consistent with our findings. Given the significant regulatory role of HDACs in cancer cells, and the fact that HDAC2 is an important part of a class I HDAC family, we decided to further explore the specific function of HDAC2 in the development of EWS. We noticed that the proliferation and migration abilities of EWS cells were inhibited after silencing HDAC2, indicating oncogenic functions of HDAC2 in EWS. Considering the considerable promise shown by HDAC inhibitors in cancer treatment [50], we believe that targeting HDAC2 may represent a novel and effective approach for treating EWS patients.
This study has certain limitations, which are related to our being the first to use HARGs to establish a prognostic gene model for EWS patients. These limitations are mainly reflected in the following aspects. On the one hand, both the construction and validation of the EWS prognostic model using HARGs are based on retrospective data from public databases. Due to the low incidence of EWS, limited research, and small sample size, the data sets are relatively small in scale. On the other hand, although the function of HDAC2 in EWS has been identified, the roles of the other three HARGs in EWS are still unclear. Further research is warranted to investigate their functions.