Abundant data are available regarding the dual roles of ATF3 in the protection of both normal and cancer cells from further transformation and in the promotion of tumor progression [17]. We used bioinformatics methods to analyze published sequencing data to understand the function of ATF3 in HCC and its regulatory network in detail, and thus, guide future studies on HCC and identification of novel biomarkers.
Using ONCOMINE and TIMER datasets, we found that ATF3 levels were down-regulated in human HCC and they were correlated with patient clinical characteristics. We found that low levels of ATF3 were associated with lower OS and DFS in patients with HCC, suggesting that the gene acts as a tumor suppressor. These results were consistent with the results of a previous study that demonstrated low expression of ATF3 at the protein and mRNA levels in HCC [18]. Similarly, ATF3 is down-regulated in esophageal squamous cell carcinoma [19]. It has been reported that the expression of ATF3 is lower in human colorectal tumors than in normal adjacent tissues, while overexpression of ATF3 in vivo can reduce the volume of mouse tumor xenografts by 54% [20]. More importantly, higher ATF3 protein levels were detected in non-enveloped HCC patients, suggesting that ATF3 may be a target for migration inhibition [21]. In present study, low expression of ATF3 was correlated with poor prognosis of HCC in stage 1/3/4, T1-T3, and grade 2, with highest HR for low OS and PFS (Table 1). Taken together, these findings strongly suggest that ATF3 could be a biomarker for the prognosis of HCC.
Conflicting results regarding the expression and function of ATF3 in tumors can be explained by differences in ATF3 expression pattern among tumor types and cell lines. First, diverse signaling molecules and pathways may be involved. ATF3 can be induced by a variety of extracellular stress signals, including MAPK, P53, c-Myc, and TGF-β, which are involved in cell proliferation, differentiation, transformation, and death [22]. Our results suggest that the functional network involving ATF3 participates primarily in cytokine receptor signaling pathway activation. We found that ATF3 in HCC is related to the kinase network, including MAPK8, IKBKB, and CDK3. These kinases regulate genome stability and cell proliferation. Abnormal regulation of various transcription factors, such as NF-κB, AP-1, and Ets, is thought to play an important role in tumorigenesis, and each transcription factor can regulate multiple interacting signaling pathways. Through network analysis, it was predicted that part of the ATF3 transcription complex also contains members of the NF-κB family [23]. These transcription factor signaling pathways provide novel candidate targets for the prevention and treatment of HCC. Second, differences in the effects of ATF3 may be explained by differences in dimer partners. ATF3 can form a homodimer that inhibits transcription and a heterodimer complex with c-Jun or JunD that inhibits or activates target gene expression [24]. In addition, we found a marked positive association between ATF3 expression and NR4A3 in patients with HCC. In aggressive lymphoma, NR4A3 has powerful tumor suppressor function similar to NR4A1 [25, 26]. However, the relationship between NR4A3 and HCC warrants further study. Taken together, our results clearly reveal that ATF3 plays a role in inhibiting tumor growth in patients with HCC.
Using GSEA enrichment for analysis of the target gene set, we determined important target kinases, miRNAs, and transcription factor networks. We found that ATF3 in HCC was associated with a network of kinases, including MAPK, IKBKB, and CDK3. These kinases regulate tumor signaling pathways, NF-κB signaling, and the cell cycle [27–29]. Dysregulation of MAPK signaling pathways in HCC has been reported previously [27]. Our study also identified several miRNAs involved in transcriptional regulation and carcinogenesis. MiR-380-3p was shown to target SOX6 to regulate bactericidal effects by affecting β-catenin MITF transcription and translation, providing insights into the mechanisms by which miR-380-3p controls melanogenesis [30]. To further clarify genetic alterations, functions, and carcinogenic mechanisms of ATF3, we evaluated the frequency of ATF3 mutations in HCC and obtained an estimate of 10% based on publicly available data. In addition, the OS and DFS of a case with a query genetic alteration of ATF3 were lower than those without a query genetic alteration, but the differences were not statistically significant. Another important aspect of this study is that in HCC, ATF3 expression is correlated with TAMs and M1/M2 macrophage infiltration levels. Furthermore, gene markers of different macrophages such as CCL2, NOS2, and CD163 are correlated with ATF3 expression. These results suggest that ATF3 plays an important role in the recruitment of macrophages and in the regulation of immune infiltration in HCC.
Targeted gene analysis from public online database revealed multi-level evidence of the importance of ATF3 in liver cancer development and supported its role as a biomarker in HCC. In addition, ATF3 expression may be involved in the regulation of tumor-related and M1/M2 macrophages. Therefore, ATF3 may play an important role in immune cell infiltration and may serve as a biomarker for prognosis in patients with HCC. An obvious limitation of our study is that transcriptome sequencing can only detect static mutations and cannot directly provide information related to protein activity or expression levels; these issues should be addressed in subsequent research using molecular biology techniques.