Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with an incidence of 0.7–2 cases per million annually (1, 2). Despite complete surgical resection of early-stage tumors, ACC patients remain at high risk for relapse and metastasis (1, 3). The 5-year survival rate for ACC patients who undergo complete surgical resection is approximately 30% (4, 5). However, recent data, including our findings, indicate that survival rates in adult ACC may range from 40% to 60%, depending on individual prognostic factors (6). For patients with metastatic ACC, unfortunately, the 5-year survival rate remains under 15% (7–9). Approximately one-third of ACC patients present with locally advanced cancer or synchronous metastasis at diagnosis (10), and more than half will develop distant metastases even after complete resection of the primary tumor (5, 11). Most deaths due to ACC are directly associated with metastatic disease (12).

Current treatment options for metastatic ACC remain limited and demonstrate suboptimal efficacy. The only FDA-approved drug, mitotane, primarily used as adjuvant therapy, has shown inconsistent survival benefits and significant toxicity, including gastrointestinal and neurological side effects (13). Combination chemotherapy regimens, such as EDP-M (etoposide, doxorubicin, cisplatin, and mitotane), provide moderate response rates but are associated with substantial toxicity, limiting their long-term use (14). Furthermore, targeted therapies, including IGF1R inhibitors and mTOR inhibitors, as well as immune checkpoint inhibitors, have demonstrated limited clinical efficacy, with response rates typically below 10% (15, 16). These challenges underscore the urgent need for novel molecular targets and therapeutic strategies to improve outcomes for metastatic ACC patients.

To enhance understanding of ACC and improve patient survival, substantial research has focused on its molecular mechanisms. Key factors in ACC pathogenesis include overexpression of IGF2, constitutive activation of WNT/β-catenin signaling (1, 17), and inactivation of the TP53/RB pathway (18). Gara et al. found that metastatic ACC exhibits a higher mutation rate and greater tumor heterogeneity than primary ACC, highlighting the insufficiency of studying primary tumors alone to fully understand metastasis in ACC patients (12).

In recent decades, genome-wide association studies have transformed our understanding of cancer biology and treatment discovery. Weighted gene co-expression network analysis (WGCNA) is a powerful bioinformatics tool that constructs gene co-expression modules, which can be summarized by module eigengenes and intramodular hub genes (19). WGCNA enables exploration of network module structure, relationships between genes and modules, inter-module associations, and gene or module ranking with respect to a sample trait, ultimately generating testable hypotheses for independent data validation (19). WGCNA has been successfully applied to cancers such as lymphoma, prostate cancer, and colon cancer (20–23); however, its application to ACC metastasis remains unexplored.

In this study, we applied WGCNA to transcriptional profiling data from The Cancer Genome Atlas (TCGA) for ACC to identify hub genes associated with metastasis. Additionally, we used the Drug Gene Interaction Database (DGIdb, http://dgidb.org/) to identify potential therapeutic agents that may prevent or treat metastatic ACC, aiming to extend patient survival.

In this study, we applied WGCNA to analyze ACC transcriptional data from TCGA, successfully identifying seven hub genes—ZWINT, CDK1, TPX2, TYMS, CCNA2, CCNB1, and BIRC5—closely associated with metastasis in adult ACC. These genes were significantly upregulated in the metastatic group, and survival analysis confirmed their association with patient prognosis. Further, using the DGIdb, we identified potential drugs targeting these hub genes, offering promising insights for therapeutic interventions.

ACC treatment options, especially for metastatic disease, remain limited. Epidemiological data show minimal improvement in survival outcomes for ACC patients from 1993–1998 to 2005–2010 (8). The sole FDA-approved drug, mitotane, has demonstrated uncertain benefits regarding overall survival (13). Other second- and third-line therapies for metastatic ACC also provide limited benefits (25). Despite the efficacy of PD-L1 antibodies in many cancers, a phase Ib trial in 50 metastatic ACC patients yielded only a 6% response rate (3 partial responses) (15). Similar limitations are observed with IGF1R-mTOR inhibitors (3.9% response rate) (3), tyrosine kinase inhibitors (TKIs, 1.4%) (3), and gemcitabine-based chemotherapy (4.9%) (16). Thus, discovering new molecular targets and more effective drugs for metastatic ACC is essential. Our study, by identifying key genes and possible drug targets for metastatic ACC, offers insights that may enhance patient outcomes.

The seven hub genes identified here contribute to several processes crucial for malignancy, including cell cycle progression, DNA repair, and replication fidelity. Based on GSEA results, these genes are implicated in malignant transformation pathways, such as cell cycle regulation, mismatch repair, DNA replication, and nucleotide excision repair. Using Gene Expression Profiling Interactive Analysis (GEPIA) and the UniProt knowledgebase, we investigated each hub gene’s function. We found that CDK1 and CCNA2 are essential regulators of the G1/S and G2/M cell cycle transitions, while CCNB1 controls the G2/M checkpoint. ZWINT encodes a protein crucial for kinetochore function, and TPX2 is involved in spindle microtubule organization during mitosis, being exclusively expressed in proliferating cells. These five genes are closely tied to cell cycle regulation and mitosis, aligning with known observations that Ki-67, a cell proliferation marker, is highly expressed in metastatic ACC (26, 27). TYMS encodes an enzyme that has been widely studied as a target for cancer chemotherapy, particularly as the primary site of action for drugs like 5-fluorouracil (5-FU) and folate analogs. Overexpression of TYMS is associated with resistance to 5-FU and poor prognosis in colorectal, breast, and other cancers (28). Given that mitotane is currently the only approved drug for metastatic ACC and its mechanism remains unclear, the overexpression of TYMS in metastatic ACC may potentially impact the efficacy of mitotane and other chemotherapies, a hypothesis warranting further investigation.

BIRC5 has been shown to regulate key pathways involved in EMT, including Wnt/β-catenin and TGF-β signaling, which contribute to metastatic progression in ovarian and lung cancers. Its role in promoting apoptosis resistance and cell survival has also been implicated in pancreatic and nasopharyngeal cancers (29–31). By stabilizing β-catenin and enhancing TGF-β signaling, BIRC5 facilitates the loss of epithelial markers such as E-cadherin and the gain of mesenchymal markers such as vimentin, thereby promoting cellular plasticity and migration. These mechanisms, extensively studied in ovarian and lung cancers, suggest a similar role for BIRC5 in ACC metastasis. Our study confirms that high expression of BIRC5 is significantly associated with poor prognosis in ACC patients, consistent with its role in promoting metastasis and apoptosis resistance in other cancer types. Future studies should focus on validating these pathways in ACC using functional assays, such as gene knockdown or overexpression, and exploring their therapeutic implications through pathway-specific inhibitors. ZWINT, a kinetochore-associated protein, plays a pivotal role in chromosome segregation and mitotic progression. Its overexpression has been linked to tumor aggressiveness in prostate and hepatocellular carcinomas (32, 33). Our findings suggest that ZWINT may contribute to the metastatic potential of ACC by regulating mitotic progression, although further validation is needed to confirm its precise role in ACC metastasis. CDK1, a master regulator of the cell cycle, is frequently overexpressed in metastatic cervical, breast, and neuroblastoma cancers, where it drives G2/M transition and tumor proliferation (34–36). CDK1 interacts with cyclins such as CCNB1 and CCNA2 to form complexes that regulate cell cycle progression and genomic stability. CDK1 was identified as an independent prognostic factor for poor survival in ACC patients, highlighting its critical role in driving metastatic progression. Targeting CDK1 with specific inhibitors may offer a promising therapeutic strategy for metastatic ACC. CCNA2 is overexpressed in metastatic breast, lung, and bladder cancers (37, 38), while CCNB1 contributes to metastasis in cervical, breast, and ovarian cancers (39, 40). These interactions reinforce the interconnected roles of these hub genes in cancer metastasis, including ACC. TYMS, involved in nucleotide biosynthesis and DNA repair, is overexpressed in metastatic breast, lung adenocarcinoma, and colorectal cancers (41–43). Its elevated expression has been linked to drug resistance, particularly in fluoropyrimidine-based chemotherapy, highlighting its dual role in metastasis and therapy resistance. TPX2, a microtubule-associated protein, is overexpressed in metastatic hepatocellular carcinoma and clear cell renal cell carcinoma (44, 45), underscoring its potential as both a biomarker and therapeutic target in aggressive tumors. These findings reinforce our hypothesis that these hub genes contribute critically to the metastatic progression of ACC.

A recent study by Behan et al. applied CRISPR-Cas9 screening across 324 cancer cell lines, identifying prioritized therapeutic targets (46). Among our seven hub genes, TYMS was categorized as a Group 1 priority target (targets with approved drugs), TPX2 and CCNA2 as Group 2 targets (potentially tractable but lacking approved drugs), and ZWINT as a Group 3 target (promising but with limited tractability data). These findings suggest that several of our identified hub genes are strong candidates for therapeutic targeting in metastatic ACC. Future directions should aim to validate these findings through experimental studies, particularly by assessing the mRNA and protein expression levels of these hub genes in tumor tissues and cell lines from diverse populations, including Chinese cohorts. Such validation is critical for determining their relevance across ethnic groups and for confirming their potential as diagnostic biomarkers and therapeutic targets. Moreover, functional studies (e.g., knockdown and overexpression experiments) will provide deeper insights into the mechanisms by which these genes contribute to ACC metastasis and drug resistance.

Our use of the DGIdb further revealed several drug candidates for these hub genes, including antibiotics, hormones, NSAIDs, and TKIs. For example, TYMS can be targeted by trimethoprim and dexamethasone, while BIRC5 is targeted by plicamycin and sulindac. With manageable side effects, these drugs might offer long-term treatment options for metastatic ACC if proven effective, especially those targeting TYMS as a Group 1 candidate. Plicamycin, in particular, has shown antitumor effects in ACC cell lines NCI-H295R, BD140A, and SW-13 (47). Additionally, TKIs such as lapatinib, which have shown efficacy in other cancers, could potentially benefit metastatic ACC patients (48). Further studies to assess these drugs’ effectiveness in ACC models are warranted.

To our knowledge, this is the first study using WGCNA to investigate metastatic ACC. By identifying and verifying seven hub genes, this study provides insights into potential diagnostic biomarkers and therapeutic targets for ACC metastasis. We also identified several potential drugs with manageable side effects that may represent viable long-term treatment options. However, our study has limitations: due to the rarity of ACC, the availability of external validation cohorts and tumor specimens is restricted, which limits the immediate feasibility of performing in vitro experiments or animal model testing of the identified drugs. These validations are necessary to confirm the generalizability and reliability of our findings. We are actively working on obtaining additional samples and expanding our research cohort for future validation efforts. These validations are a key component of our ongoing and future research, which we plan to complete as part of our next steps. Future research should aim to validate these findings through large-scale external cohorts and functional assays in ACC cell lines or animal models. These studies will help assess the efficacy of the potential therapeutic agents identified in this study and further explore their role in improving survival outcomes for metastatic ACC. Moreover, the investigation of biological roles of these hub genes in ACC metastasis, especially through gene knockdown or overexpression studies, will be crucial in understanding their contributions to tumor progression and drug resistance. Large-scale validation studies across diverse populations are also necessary to assess the reliability of these hub genes as diagnostic biomarkers and therapeutic targets. The identified hub genes, including TYMS and BIRC5, may serve as potential candidates for clinical trials aimed at evaluating targeted therapies to improve metastatic ACC treatment outcomes.

We applied WGCNA to RNA-seq data of 73 ACC cases from TCGA database and selected the most significant module concerned with metastasis. With a series of selections, we identified seven hub genes: ZWINT, CDK1, BIRC5, CCNA2, CCNB1, TYMS and TPX2. These seven hub genes may play a key role in ACC metastasis and may serve as promising biomarkers for metastatic ACC. Targeting drugs of them may serve as potential prevention or treatment options for metastasis ACC with tolerable side-effects. Our results may contribute to the exploration of the further and more in-depth research of metastasis ACC.