Age-related Changes in the Adrenal Cortex: Insights and Implications.

Aging is characterized by a gradual decline in physiological function. This process affects all organs including the adrenal cortex, which normally functions to produce essential steroid hormones including mineralocorticoids, glucocorticoids, and androgens. With increasing age, features such as reduced adrenal cortex size, altered zonation, and increased myeloid immune cell infiltration substantially alter the structure and function of the adrenal cortex. Many of these hallmark features of adrenal cortex aging occur both in males and females, yet are more enhanced in males. Hormonally, a substantial reduction in adrenal androgens is a key feature of aging, which is accompanied by modest changes in aldosterone and cortisol. These hormonal changes are associated with various pathological consequences including impaired immune responses, decreased bone health, and accelerated age-related diseases. One of the most notable changes with adrenal aging is the increased incidence of adrenal tumors, which is sex dimorphic with a higher prevalence in females. Increased adrenal tumorigenesis with age is likely driven by both an increase in genetic mutations as well as remodeling of the tissue microenvironment. Novel antiaging strategies offer a promising avenue to mitigate adrenal aging and alleviate age-associated pathologies, including adrenal tumors.

Senescence-induced immune remodeling facilitates metastatic adrenal cancer in a sex-dimorphic manner.

Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

Sublethal Hyperthermia Transiently Disrupts Cortisol Steroidogenesis in Adrenocortical Cells.

Primary aldosteronism is the most common cause of secondary hypertension. The first-line treatment adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone-producing adenomas, while preserving adjacent normal adrenal cortex. To determine the extent of damage to adrenal cells upon exposure to hyperthermia, the steroidogenic adrenocortical cell lines H295R and HAC15 were treated with hyperthermia at temperatures between 37 and 50°C with the effects of hyperthermia on steroidogenesis evaluated following stimulation with forskolin and ANGII. Cell death, protein/mRNA expression of steroidogenic enzymes and damage markers (HSP70/90), and steroid secretion were analyzed immediately and 7 days after treatment. Following treatment with hyperthermia, 42°C and 45°C did not induce cell death and were deemed sublethal doses while ≥50°C caused excess cell death in adrenal cells. Sublethal hyperthermia (45°C) caused a significant reduction in cortisol secretion immediately following treatment while differentially affecting the expression of various steroidogenic enzymes, although recovery of steroidogenesis was evident 7 days after treatment. As such, sublethal hyperthermia, which occurs in the transitional zone during thermal ablation induces a short-lived, unsustained inhibition of cortisol steroidogenesis in adrenocortical cells in vitro.

Mitotane Targets Lipid Droplets to Induce Lipolysis in Adrenocortical Carcinoma.

INTRODUCTION: Adrenocortical carcinoma (ACC) is a rare aggressive cancer with low overall survival. Adjuvant mitotane improves survival but is limited by poor response rates and resistance. Mitotane's efficacy is attributed to the accumulation of toxic free cholesterol, predominantly through cholesterol storage inhibition. However, targeting this pathway has proven unsuccessful. We hypothesize that mitotane-induced free-cholesterol accumulation is also mediated through enhanced breakdown of lipid droplets. METHODOLOGY: ATCC-H295R (mitotane-sensitive) and MUC-1 (mitotane-resistant) ACC cells were evaluated for lipid content using specific BODIPY dyes. Protein expression was evaluated by immunoblotting and flow cytometry. Cell viability was measured by quantifying propidium iodide-positive cells following mitotane treatment and pharmacological inhibitors of lipolysis. RESULTS: H295R and MUC-1 cells demonstrated similar neutral lipid droplet numbers at baseline. However, evaluation of lipid machinery demonstrated distinct profiles in each model. Analysis of intracellular lipid droplet content showed H295R cells preferentially store cholesteryl esters, whereas MUC-1 cells store triacylglycerol. Decreased lipid droplets were associated with increased lipolysis in H295R and in MUC-1 at toxic mitotane concentrations. Pharmacological inhibition of lipolysis attenuated mitotane-induced toxicity in both models. CONCLUSION: We highlight that lipid droplet breakdown and activation of lipolysis represent a putative additional mechanism for mitotane-induced cytotoxicity in ACC. Further understanding of cholesterol and lipids in ACC offers potential novel therapeutic exploitation, especially in mitotane-resistant disease.

Liver X receptor inhibition potentiates mitotane-induced adrenotoxicity in ACC.

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first-line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40 and 50 µM) induced dose-dependent cell death; however, in MUC-1, this only occurred at a supratherapeutic concentration (200 µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1; however, combination treatment with mitotane attenuated this effect. Intracellular free-cholesterol levels were associated with increased cytotoxicity in H295R (r2 = 0.5210) and MUC-1 (r2 = 0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich, whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.