Despite progress in understanding the biology of adrenocortical carcinoma (ACC), treatment options have not dramatically changed in the last three decades, nor have we learned how to avoid some of its long-term side effects. Our goal was to improve the understanding of immune pathways that may include druggable targets to enhance immune responses of patients with ACC, focusing on immune evasion and the activation of immune cells against ACC. Our strategy was aimed at improving insight regarding gene expression without steroid interference. Using approaches based on high and low steroid phenotypes (HSP and LSP, respectively), we characterized immune pathways using The Cancer Genome Atlas (TCGA) ACC cohort data. Although previous studies have suggested that patients with ACC receive minimal benefit from immunotherapy, high expression of immune modulators was noted in patients with LSP, suggesting the activation of these biomarkers may be an important adjuvant therapy target after clearance of excess glucocorticoids. In addition, patients with LSP ACC had higher immune cell infiltration than patients with HSP ACC and other cancer subtypes. Our findings can be summarized as follows (1): we confirmed and improved the definition of two immune response pathways to ACC (HSP and LSP) based on in silico transcriptome analysis (2), we demonstrated the steroid profile should be considered, otherwise analyses of ACC immune characteristics can generate confounding results (3), among the overexpressed immunotherapy targets, we demonstrated that LSP was rich in PDCD1LG2 (PD-L2) and both HSP and LSP overexpressed CD276 (B7-H3), which was associated with resistance to anti-PD1 therapy and may have accounted for the modest results of previous clinical trials, and (4) identification of patients with LSP or HSP ACC can be used to help determine whether immunotherapy should be used. In conclusion, we highlighted the differences between LSP and HSP, drawing attention to potential therapeutic targets (CD276, PDCD1, and PDCD1LG2). Treatments to reduce immune evasion, as well as the use of other natural and pharmacological immune activators, should include prior pharmacological inhibition of steroidogenesis. Attempts to combine these with tumor cell proliferation inhibitors, if they do not affect cells of the immune system, may produce interesting results.

We reconstructed a transcriptional regulatory network for adrenocortical carcinoma (ACC) using transcriptomic and clinical data from The Cancer Genome Atlas (TCGA)-ACC cohort. We investigated the association of transcriptional regulatory units (regulons) with overall survival, molecular phenotypes, and immune signatures. We annotated the ACC regulons with cancer hallmarks and assessed single sample regulon activities in the European Network for the Study of Adrenal Tumors (ENSAT) cohort. We found 369 regulons associated with overall survival and subdivided them into four clusters: RC1 and RC2, associated with good prognosis, and RC3 and RC4, associated with worse outcomes. The RC1 and RC3 regulons were highly correlated with the ‘Steroid Phenotype,’ while the RC2 and RC4 regulons were highly correlated with a molecular proliferation signature. We selected two regulons, NR5A1 (steroidogenic factor 1, SF-1) and CENPA (Centromeric Protein A), that were consistently associated with overall survival for further downstream analyses. The CENPA regulon was the primary regulator of MKI-67 (a marker of proliferation KI-67), while the NR5A1 regulon is a well-described transcription factor (TF) in ACC tumorigenesis. We also found that the ZBTB4 (Zinc finger and BTB domain-containing protein 4) regulon, which is negatively associated with CENPA in our transcriptional regulatory network, is also a druggable anti-tumorigenic TF. We anticipate that the ACC regulons may be used as a reference for further investigations concerning the complex molecular interactions in ACC tumors.