Adrenal cortical carcinoma (ACC) is a sporadic adrenocortical endocrine tumor with an annual incidence of 0.5 to 2 cases per million. The number of cases in the female population generally outnumbers that of the male population (1.5:1) (1, 2). The clinical manifestations of ACC can be in three different forms. Symptoms related to hormone excess occur in 60% of the patients with ACC. Another 20% of patients with ACC have abdominal pain or fullness due to tumor growth, and the remaining 20% ​​ have unrelated symptoms detected by abdominal imaging (3–5). The prognosis of ACC is also extremely poor since the clinical presentation of ACC is often difficult to determine, and most patients are diagnosed in the late metastatic stage of the disease. Although ACC patients with the locoregional disease are treated with surgery, approximately 75% of patients experience recurrence after treatment (6). In addition, for advanced metastatic disease, the median overall survival was 12–15 months, and the 5-year overall survival was <15% (7, 8). Patients with ACC did not benefit from improvements in overall tumor treatment when compared to those with other tumor types. Evidence suggests that chemotherapy and radiation therapy are ineffective in most ACC cases. Mitotane, the only drug currently approved by the U.S. Food and Drug Administration for ACC, is usually only temporarily effective, and its use is often accompanied by significant side effects (9, 10). Complete tumor resection remains the only curative treatment (11). Therefore, in the current situation of uncertain therapeutic effects, limited toxic drugs, and high risk of disease recurrence, it is important to systematically analyze targeted prediction and prognostic markers in patients with ACC.

Bromodomain and extracellular terminal (BET) family consists of four members, including BRD2, BRD3, BRD4, and BRDT, which play key roles in various cell processes, including cell cycle, apoptosis, migration, and invasion (12). Therefore, the BET family can enhance its carcinogenic function by increasing its expression or promoting the transcriptional activity of carcinogenic factors. BET family is over-expressed in a variety of human cancers, which are closely related to human carcinogenesis (13). They are considered to be attractive therapeutic targets for selectively inhibiting cancer patients (14). However, BRDT is primarily expressed in germ cells, and BRD2, BRD3, and BRD4 are ubiquitously expressed. Among them, BRD4 is the most extensively studied in tumor research. BRD2, BRD3, and BRD4 can specifically recognize acetylated lysine residues and regulate the replication and transcription of related genes, thereby affecting key processes in cell carcinogenesis (15). They have two tandem bromodomains (BD1 and BD2) that regulate gene expression by binding to promoters and super-enhancer regions and promoting transcriptional elongation in cancer. The inhibition of BRD2, BRD3, and BRD4 shortens signaling between super-enhancer regions and oncogene target promoters, resulting in cell-specific inhibition of oncogene expression and ultimately cancer cell death. They contain multiple acetylated residues and activate transcription by binding to acetylated lysine residues on target proteins and mediating chromatin decompression (16). Studies have reported that BRD4 inhibitors treat cancer by interfering with the interaction between BRD4 and acetyl lysine on target proteins (17). Currently, more than 30 targeted inhibitors have shown significant inhibitory effects against various tumors in preclinical and clinical trials (18). Therefore, BRD2, BRD3, and BRD4 may be biomarkers for some cancers, and the development of their inhibitors may be a potential strategy for cancer treatment.

The role of BRD2, BRD3, and BRD4 in ACC is not well understood. Therefore, this study systematically analyzed the expression, gene regulatory network, prognostic value, and target prediction of BRD2, BRD3, and BRD4 in ACC patients, elucidated the association between BET family and ACC, and identified potential new targets for ACC therapy.

The expressions of BRD2 and BRD3 in patients with ACC still remain unclear. BRD4 expression is reported to be significantly upregulated in ACC (19). However, the expression of BRD4 has not been reported for the individual cancer stages of patients with ACC. We first compared the expression levels of BRD2, BRD3, and BRD4 in ACC patients with different cancer stages and found that BRD3 and BRD4 transcript levels were significantly upregulated in patients with ACC. Furthermore, we found a significant positive correlation between the expression of BRD4 and the pathological stage of patients with ACC. In cancer patients with high BRD4 expression, increased BRD4 activity is associated with higher expression of oncogenes such as MYC, NOTCH3, and NRG1 (20). BRD4-driven oncogenes promote tumor cell proliferation, metastasis, and increased chemoresistance (20). Our results also revealed that the expression of BRD2, BRD3, and BRD4 was altered by 5%, 5%, and 12%, respectively, in patients with ACC, with the type of genetic alteration mainly including high and low RNA levels. Increased expression of BRD3 and BRD4 caused by genetic changes may also be an important factor. However, this requires further investigation. Finally, we assessed the prognostic value of BRD2, BRD3, and BRD4 expression in ACC patients. Our results showed that ACC patients with low BRD2, BRD3, and BRD4 expression had longer survival than those with high BRD2, BRD3, and BRD4 expression. BRD2, BRD3, and BRD4 may serve as potential prognostic markers in patients with ACC.

Our results showed a gene alteration frequency of ≥25.00%, ≥25.00%, and ≥44.44% for the 50 most frequently altered neighboring genes of BRD2, BRD3, and BRD4, respectively, in patients with ACC. We further evaluated the potential interactions between BRD2, BRD3, BRD4, and their neighboring genes. We found that BRD2, BRD3, BRD4, and their neighboring genes were linked to a complex interaction network through co-expression, physical interactions, and shared protein domains. Next, we evaluated the functions of BRD2, BRD3, and BRD4 and their neighboring genes. Our results showed that the biological processes related to BRD2 and its neighboring genes mainly include adenylate cyclase-activating G protein-coupled receptor signaling pathway, generation of precursor metabolites and energy, and lymphocyte chemotaxis. These biological processes affecting tumor proliferation, invasion, and metastasis have been reported (21–23). Furthermore, the biological processes related to BRD3 and its neighboring genes mainly include angiogenesis and microtubule cytoskeleton organization involved in mitosis. Abnormal angiogenesis and mitosis are the key factors of tumor occurrence and development (24, 25). Moreover, our results showed that the biological processes related to BRD4 and its neighboring genes mainly include positive regulation of histone modification and phagocytosis. Histone modification is a reversible process mediated by epigenetic enzymes (26). Histone methylation and acetylation are two important chemical modifications. They play important roles in transcriptional activation/inactivation, chromosomal packaging, and DNA damage/repair (27). The molecular functions of BRD2 and its neighboring genes include endopeptidase activity and channel activity. Various endopeptidase activities affect tumorigenesis and prognosis. Prolyl endopeptidase is a serine peptidase involved in the differentiation, development, and proliferation of various tissues. Recent studies have shown that the expression and activity of this cytoplasmic enzyme increases in colorectal cancer and affects the prognosis of colon cancer patients (28). The molecular functions of BRD3 and its neighboring genes include cell adhesion molecule binding, calcium ion binding, and voltage-gated monoatomic ion channel activity. Cell adhesion is a key mediator of cancer progression and promotes cancer hallmarks including immune escape and metastatic spread (29). Calcium (Ca) ion is a key secondary messenger in excitable and unexcitable cells. Ca signaling has received limited attention as a potential target for anticancer therapy (30). However, the molecular functions of BRD4 and its neighboring genes include aromatase activity, G protein-coupled serotonin receptor activity, and integrin binding. Recently, it has been proved that ACC cell overexpress aromatase and estrogen receptor, and estrogen synthesized by aromatase can enhance the proliferation of ACC cells (31). ACC can cause Cushing’s syndrome. However, some studies have shown that several receptors, including G protein-coupled serotonin receptors, are frequently co-expressed in patients with Cushing’s syndrome (32). Integrins are cell-adhesion receptors. However, integrin expression patterns are frequently altered in cancer. The expression of certain integrins is associated with increased metastasis and decreased cancer (33). Chemokine signaling pathway, thiamine metabolism, and olfactory transduction were enriched as per the KEGG pathway analysis for BRD2, BRD3, BRD4, and their neighboring genes. The tumor microenvironment consists of stromal cells and tumor cells, which interact with each other through complex crosstalk mediated by a variety of growth factors, cytokines, and chemokines. Chemokines play a key role in promoting tumor cell proliferation (34). Thiamine supplementation may contribute to tumor cell survival, proliferation, and chemotherapy resistance. However, some studies suggest that thiamine may exhibit some antitumor effects. The role of thiamine in cancer is controversial (35). Olfactory receptors specifically expressed in certain cancer cells, such as prostate-specific G protein-coupled receptor 1 (PSGR1), are significantly elevated in prostate cancer. NDRG1 affects oncogenic signaling pathways and tumor progression (36). There is no relevant research on whether olfactory receptors are specifically expressed in ACC patients. Taken together, the functions and signaling pathways involving BRD2, BRD3, BRD4, and their neighboring genes may be involved in the occurrence and progression of ACC. The regulation of these genes and signaling pathways may be a potential treatment strategy for ACC.

Next, we explored the transcription factor and miRNA targets of BRD2, BRD3, and BRD4 in patients with ACC. We found that SP1, NPM1, STAT3, and TP53 are the key transcription factors of BRD2, BRD4, and their neighboring genes in patients with ACC. SP1 is a well-known member of the transcription factor family, which plays an important role in cell growth, differentiation, apoptosis, and carcinogenesis (37). However, its role in the ACC has not been reported. Studies have shown that STAT3 can promote angiogenesis in patients with ACC, thereby making STAT3 a selective target for molecular-targeted therapy of ACC (38). ACC is a rare tumor type associated with TP53 mutations. Studies have shown that genetic susceptibility caused by mutations in TP53 is associated with approximately 50% of childhood ACC cases but only 3–6% of adult cases (39). The relationship between NPM1 and ACC has not yet been reported, but may be a new potential therapeutic target for ACC. Furthermore, our results showed that miR-142-3P, miR-224, miR-323, miR-484, miR-18A, miR-18B, miR-7, miR-519C, miR-519B, miR-519A, miR-17-5P, miR-20A, miR-106A, miR-106B, miR-20B, miR-519D, and miR-493 are targets of BRD2, BRD3, and BRD4 in patients with ACC. They are associated with tumor cell proliferation, migration, invasion, and drug resistance, and may be promising targets for cancer therapy (40–42). However, their relationship with ACC has not yet been reported. Our results suggest that these transcription factor and miRNA targets of BRD2, BRD3, BRD4, and their neighboring genes may be potentially therapeutic in treating ACC.

We explored the correlation between the differentially expressed genes and BET family expression in patients with ACC. We found that the expression of 19,339 genes was correlated with BRD2, BRD3, and BRD4 expression. Among them, ZSCAN12, DHX16, PRPF4B, EHMT1, CDK5RAP2, POMT1, WIZ, ZNF543, and AKAP8 were the top nine genes whose expressions were positively correlated with the expression of BRD2, BRD3, and BRD4. Therefore, targeting these cells may provide additional therapy for ACC. Tumor immune infiltration is closely related to the clinical prognosis (43). As expected, the expression levels of BRD2, BRD3, and BRD4 in patients with ACC positively correlated with B cell and dendritic cell infiltration levels. Targeting BRD2, BRD3, and BRD4 or their related regulatory targets may be a feasible strategy for reducing immune cell infiltration levels in patients with ACC. We further evaluated the inhibitory effect of BRD4-targeting drug PFI-1 and (BRD2, BRD3, and BRD4)-targeted drug I-BET-151 on ACC lines. We found that PFI-1 and I-BET-151 inhibited 914 and 899 cancer cell lines, respectively. Among them, I-BET-151 had a better inhibitory effect on the SW13 cell line than PFI-1. In short, PFI-1 and I-BET-151 exhibited broad-spectrum inhibitory effects on cancer cells. Therefore, finding inhibitors of BRD2, BRD3, and BRD4 or their regulatory targets may be an important strategy for the treatment of ACC.

In conclusion, this study systematically analyzed the expression, gene regulatory network, prognostic value, and target prediction of BRD2, BRD3, and BRD4 in patients with ACC, elucidated the association between BRD2, BRD3, BRD4 and ACC, and provided insights into the mechanism and treatment of ACC.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

LD and YS performed data analysis work and aided in writing the manuscript. YS designed the study and assisted in writing the manuscript. QL, ZZ, JC, ZS, QX, XL, YC, LL, JZ edited the manuscript. All authors contributed to the article and approved the submitted version.