Proteomic Profiling of Cold Thyroid Nodules

Abstract

Cold thyroid nodules (CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs (10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs.

Evidence for a role of the amyloid precursor protein in thyroid carcinogenesis

“…The molecular pathogenesis of benign CTNs is unknown. We recently performed a proteomic study of benign CTNs, where we found evidence for increased expression of APP, APP-binding protein and APP-p3-component in CTNs (Krause et al 2007b). Since CTNs exhibit certain signs of dedifferentiation (e.g.…”

“…In a recent proteomic study, we have identified an increased expression of APP, the amyloid precursor-binding protein 1 (APPBP1) and the APP-p-component in benign cold thyroid nodules (CTNs) compared with normal thyroid tissue (Krause et al 2007b). The molecular aetiology of CTNs is unknown and at present it is unclear, if the CTN represents a truly benign thyroid neoplasm or a lesion with the potential for further dedifferentiation.…”

Dissecting Molecular Events in Thyroid Neoplasia Provides Evidence for Distinct Evolution of Follicular Thyroid Adenoma and Carcinoma

“…18 Fifty micrograms of protein, adapted to an equal thyroglobulin amount, was subjected to 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-TCTP antibody (Abcam, Cambridge, UK). Rabbit IgG conjugated with peroxidase was used as secondary antibody (1.0000; Cell Signaling, Charlottesville, VA) and was visualized by enhanced chemiluminescence (Pierce, Milwaukee, WI).…”

“…18 The following primary antibodies were used: antiphospho-Chk2 (Thr68), anti-phospho-H2AX (Ser139) (both from Cell Signaling Technology), anti-I2 protein phosphatase 2A (PP2A), and anti-DJ-1 (both from Abcam). For controls, tissues from colon carcinomas (for ␥-H2AX and pChk2), Graves' disease (for DJ-1), and breast carcinomas (for I2PP2A) were used.…”

“…Up-regulation of DJ-1 has previously been shown in benign CTNs compared with corresponding surrounding tissues. 18 DJ-1 (PARK7) is a protein that plays a dual role in cellular biology because a loss or gain of its function drives abnormal cellular responses, leading either to cell death (in neurodegenerative disease) or cell survival (in cancer). Subsequent immunohistochemistry confirmed the observed up-regulation of DJ-1 in FTC compared with NT ( Figure 5A).…”