Identification of the ferroptosis regulator HELLS with prognostic value for adrenocortical carcinoma based on integrated analysis and experimental validation

Background: For adrenocortical carcinoma (ACC), a rare endocrine malignancy with a high rate of mortality and recurrence, it is difficult for clinicians to predict overall survival and select the most effective treatment. Targeting ferroptosis, a form of cell death, has been reported to be a promising therapeutic strategy for ACC; however, the core ferroptosis regulator and its prognostic value in ACC remain unknown.Methods: RNA sequencing data and clinical information were downloaded from public databases. Differentially expressed gene and survival analyses were performed to identify candidate ferroptosis regulators. A multivariate Cox regression model was used to construct a gene signature, and a nomogram was constructed to predict the overall survival of patients with ACC. Gene set variation analysis (GSVA) was used to identify underlying aberrant pathways and the relative immune cell infiltration levels of each ACC sample. Immunohistochemistry staining was performed in formalin-fixed paraffin-embedded tumor tissue sections.Results: Ultimately, 23 differentially expressed ferroptosis regulators were identified between normal adrenal gland and ACC tissues, and 50 ferroptosis regulators were related to prognosis, with 13 ferroptosis regulators being simultaneously found to satisfy the differential expression and prognostic value. According to the multivariate Cox regression model, a ferroptosis regulator signature was constructed from 3 genes in The Cancer Genome Atlas (TCGA; hazard ratio =9.01; P=1.39x10-10), and the area under the curve (AUC) values of 3-, 5-, 8-year overall survival were 0.924, 0.906, and 0.866, respectively. The survival analysis and the receiver operating characteristic (ROC) analysis validated the prognostic value of the ferroptosis regulator signature in 3 validation datasets. Moreover, metabolism-, E2F-, MYC-, and G2/M checkpoint- related pathways and aberrant immune cell infiltration levels were identified as being responsible for the different prognosis of risk groups in ACC. HELLS was found to be a significantly differentially expressed ferroptosis-suppressor gene with a prognostic value in ACC and to be highly associated with immune cell infiltration levels and multiple biological functions.Conclusions: A ferroptosis regulator signature showed promising power for predicting the prognosis of ACC, and HELLS was identified as a hub ferroptosis regulator in the initiation and progression of ACC.