Tumor stage-dependent expression of autophagy proteins in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with poor yet heterogeneous prognosis, mostly due to its complex and incompletely understood molecular background. Autophagy is a multi-step catabolic process with a dual role in tumorigenesis, acting either as a tumor suppressor or promoter in a context-dependent manner. Therefore, our aim was to investigate the expression of key proteins involved in different autophagy steps in both adrenocortical adenomas (ACA) and ACC, to further understand the role of autophagy in this type of tumors, particularly its involvement in the pathophysiology of ACC.

Autophagy status was evaluated in ACA (n=20) and ACC (n=29) by assessing the expression of autophagy-related protein 5 (ATG5), microtubule associated protein 1 light chain 3 beta (LC3B), and sequestosome 1 (p62/SQSTM1), using immunohistochemistry. Additionally, in vitro experiments, including migration and invasion assays, were conducted in JIL-2266 and H295R cell lines to investigate the impact of autophagic flux inhibition in ACC cell behavior.

LC3B punctate staining was present in 89% of ACC and 25% of ACA, with significantly higher LC3B expression in ACC compared to ACA (14.81 ± 2.26% vs 2.05 ± 1.00%, p < 0.0001). ACC with ENSAT stage of 1-2 exhibited significantly higher LC3B expression compared to ACC with ENSAT 3-4 (19.17 ± 1.05% vs 12.62 ± 4.00%, p = 0.02). Similarly, non-metastatic ACC showed a significantly higher percentage of LC3B positive cells than metastasized ACC (18.77 ± 3.52% vs 5.83 ± 1.70%, p = 0.004). In vitro, inhibition of autophagy significantly reduced cell migration and invasion in ACC cells. Higher cytoplasmic p62/SQSTM1 levels were found in ACC with advanced disease (ENSAT 3-4 vs ENSAT 1-2: 72.07 ± 3.61% vs 51.33 ± 9.24%, p = 0.02). No significant differences were observed for ATG5.

Our findings indicate a tumor stage-dependent role of autophagy in ACC and show that autophagy may play a role in ACC molecular pathophysiology. A punctate LC3B expression accumulation, associated with less aggressive malignant features, namely absence of metastasis, appears to result from a blockade at the late stages of autophagy. Whereas active autophagy may be associated with a more aggressive cellular phenotype in ACC, particularly by promoting migration and invasion.