Impaired Expression of Humanin during Adrenocortical Carcinoma

Abstract

The discovery of mitochondria-derived peptides (MDPs) has provided a new perspective on mitochondrial function. MDPs encoded by mitochondrial DNA (mtDNA) can act as hormone-like peptides, influencing cell survival and proliferation. Among these peptides, humanin has been identified as a crucial factor for maintaining cell survival and preventing cell death under various conditions. Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy that results from adrenal hormone dysfunction. This study aimed to investigate humanin expression in the adrenal tissue and serum of patients with ACC. For the first time, our study revealed significant reduction in the mRNA expression of humanin in patients with ACC compared to healthy controls. However, no significant changes were observed in the serum humanin levels. Interestingly, we identified a positive correlation between patient age and serum humanin levels and a negative correlation between tumor size and LDL levels. While the impaired expression of humanin in patients with ACC may be attributed to mitochondrial dysfunction, an alternative explanation could be related to diminished mitochondrial copy number. Further investigations are warranted to elucidate the intricate relationship among humanin, mitochondrial function, and ACC pathology.

Keywords: ACC; humanin; mitochondria.

Figure 1

Adrenal expression of humanin mRNA in patients with adrenocortical carcinoma (n = 14) compared to normal adrenals (n = 6). The bar plot displays the median with interquartile range. Each dot represents humanin expression in individual patients. Statistical differences were determined by using the Mann−Whitney U nonparametric test (** p < 0.01).

Figure 2

The unchanged level of humanin protein (ng/mL) in serum of ACC patients (n = 26) compared to healthy controls (n = 10). Humanin levels were measured by using the ELISA test and analyzed based on the presence of the disease (A) and disease stage (B). Statistical differences were determined by using the Mann−Whitney U test (A) or the Kruskal–Wallis test (B).

Figure 3

Correlations of humanin protein expression level in serum of ACC patients (n = 26) with (A) glucose level (R = 0.46, p = 0.017), (B) tumor size (R = −0.36, p = 0.066), (C) LDL serum level (R = −0.44, p = 0.065), and (D) patients age (R = 0.6, p = 0.00093). Each dot represents an individual ACC patient. The orange line represents the regression line.

Figure 4

The correlations of humanin protein expression level in serum with clinical characteristics of ACC patients (n = 26). Each dot represents an individual ACC patient. The orange line represents the regression line.

Figure 5

The tissue microarray slide (TMA) was used to analyze the expression of humanin protein in the human adrenal gland disease spectrum, focusing on various types of adrenal cancer. The localization of humanin protein was visualized through immunohistochemical staining (DAB) (A). The relevant types of adrenal cancer progression are indicated on the TMA map using distinct colors (B).

Figure 6

Densitometric analysis of humanin protein expression in the tissue microarray slide of the adrenal gland disease spectrum compared to adrenal tissue (healthy control). The expression of humanin protein was analyzed in tissues from normal adrenal (n = 8), adjacent normal (n = 6), adrenal cortical hyperplasia (n = 4), adrenal cortical adenoma (n = 34), adrenal cortical adenocarcinoma (n = 10), pheochromocytoma (n = 30), neuroblastoma (n = 3), and ganglioneuroma (n = 1), using duplicate cores per case. The expression of humanin protein is decreased according to ACC progression (A,B). Correlations of tissue humanin expression level with age of adrenal gland disease spectrum patients (C). No significant alterations were observed based on the biological sex of patients with ACC (D). The boxplot displays each group with its median and interquartile range (IQR) (A,B,D). Individual patient densitometric data were added to the corresponding boxplots and represented as dots. The Kruskal–Wallis (KW) test was used to compare groups, followed by the Dunn post hoc test. Differences between groups were denoted with letter annotation, where different letters mark statistically significant differences between compared groups.

Figure 7

Immunohistochemical analysis of humanin protein of the progression of ACC (stage II (A–F), stage III (G–I), stage IV (J–L)). Brown staining (B,C,E,F,H,I,K,L) represents humanin protein (red arrows), located typically cellularly with hematoxylin counterstain (nucleus). The negative control of adrenal gland tissue (A,D,G,J). The images were captured using 20× (A,B,D,E,G,H,J,K) and 40× (C,F,I,L) objective.

Figure 8

Receiver operating characteristic (ROC) curves for normal vs. cancer (A) and pheochromocytoma vs. cancer (B) illustrating the potential of HN immunoreactivity as a biomarker in adrenal cancer. The area under curve (AUC) values are shown in graphs.