In this study, for the first time, we performed a 2-DE-based proteomic study to compare the protein profiling of ACC and normal adrenocortical tissues. A panel of protein markers were identified to be differently expressed. For only a few samples could be included in traditional proteomic studies, we adopted a “sample pool” strategy to increase the sample size. This strategy could also decrease the sample heterogeneity in some extent. To validate the results of proteomic analysis, we further validate three biomarkers calreticulin, prohibitin and HSP60 in a larger size of samples by immunohistochemestry. These proteins were selected for the following reasons: first, these biomarkers have a relatively high expression level in ACC, compared with normal adrenocortical tissues; secondly, previous studies have indicated that these genes are involved in the malignant progression of multiple cancers, but have not been evaluated in ACC; third, commercial antibodies for immunohistochemistry are available.
Consistent with our proteomic findings, we confirmed calreticulin, prohibitin and HSP60 overexpressed in ACC tumors than normal adrenocortical tissues. It has been suggested that the protein profiling of benign tumors partly resemble their malignant counterparts . A candidate marker elevated in both ACC and ACA would lower their specificity in ACC diagnosis. Therefore, we further compared the expression of calreticulin, prohibitin and HSP60 in ACA and ACC. We found that HSP60 was overexpressed in both ACC and ACA, compared with their normal controls, which would lower its further utility as a candidate biomarker for ACC. Different from HSP60, ACC tissues had significantly higher expression levels of calreticulin and prohibitin than ACA, supporting their utility as specific biomarkers for ACC tumors.
Calreticulin was first identified as a Ca2+ binding protein, and has been implicated in many cellular functions and pathophysiological process such as cell adhesion, autoimmunity and heat shock [12, 13]. Elevated expression of calreticulin has been reported in multiple cancers, and it is proposed that the upregulation of calreticulin seems to be induced by cellular stress from cancers . Our results indicated that calreticulin correlated to tumor stage of ACC in clinical samples. However, the exact mechanisms for its increases in ACC are as yet undetermined.
Prohibitin has been shown to localize to mitochondria, and has been identified to be up-regulated in many cancers in previous studies . However, experimental data about its role in tumorigenesis is conflicting. Several studies have suggested that prohibitin effects as a tumor suppressor , while other data indicated that prohibitin is required for the activation of several central signaling pathways related to carcinogenesis such as RAS-induced RAF-MEK-ERK activation . Our findings supported that prohibitin upregualted in ACC tumors and its roles in ACC carcinogenesis deserves further investigation.
Except above three markers, we also identified other 17 up-regulated proteins in ACC, most of which (such as transitional endoplasmic reticulum ATPase and 14-3-3 protein epsilon) have been demonstrated to be involved in cancer carcinogenesis in other cancers in previous studies [18–20], but have not been reported to be associated with ACC. Therefore, these proteins might also be novel potential candidate markers for ACC, and deserves further investigation in the future.