Abstract
Biomarkers come in many forms including DNA (BRAF T1899A), RNA (RET/PTC or Pax8-PPARγ rearrangements, mRNA levels of multiple transcripts), and proteins. They all have the potential to serve as markers but, of these, proteins offer some unique benefits. First, proteins are amenable to high-throughput, cost-effective, standardized methods of measurement such as immunohistochemistry, immunocytochemistry, and enzyme-linked immunosorbent assay. Second, there is important functional information that is only available at the protein level. For example, while a human cell contains only about 20,000 specific genes, downstream co- and post-translational events mean that many more proteins are expressed and these incorporate functionally important modifications. Analysis of proteins therefore offers insights in cellular function (and dysfunction) that are inaccessible at the genomic level.
Proteomics is the name given to a set of analytical strategies that can simultaneously identify and quantify thousands of protein components in a biological sample. There is, however, no single approach that optimally meets this demanding objective; instead, several complementary technologies have emerged, each offering distinctive strengths and weaknesses. These approaches are now facilitating new biomarker discoveries in many areas of medicine. Studies of the thyrocyte and thyroid cancer cell proteome are in their infancy compared with studies of the genome and transcriptome.
In this issue of Thyroid, Sofiadis and colleagues (1) use one of these strategies, surface-enhanced laser desorption ionization–time of flight mass spectrometry (SELDI-TOF), to explore the proteome of normal thyroid tissue, papillary thyroid carcinoma, and other tumor types. SELDI utilizes protein chips with different chromatographic surfaces (hydrophobic, ionic, hydrophilic) to capture a specific set of proteins for analysis by mass spectrometry. While the approach is fast and easy to perform and has unquestionably significant strengths compared with the alternatives, SELDI offers limited coverage of the proteome. It is limited by protein charge and size, and the peaks are characterized only by their masses. Nevertheless, in their study the authors were able to perform detailed analysis of cytosolic and nuclear extracts from papillary and follicular thyroid tumors and matched normal thyroid tissue. They found 13 protein peaks between 10.1 and 13.9 kDa that differed in abundance between papillary thyroid carcinoma (PTC) and follicular tumors. They hypothesized that the peak cluster at m/z 10,200 corresponded to the Ca+2-binding protein S100A6. This was subsequently confirmed by immunoprecipitation and liquid chromatography–tandem mass spectrometry. Western blotting also confirmed a significantly higher expression of S100A6 in PTC in comparison with the other tumor groups or normal tissue. Immunohistochemical analysis on 98 tumors showed that PTC cases had significantly stronger cytosolic staining and a larger proportion of stained nuclei than follicular tumors. Finally, they showed that the BRAF mutation was not significantly associated with S100A6 protein levels but that, as had been shown previously in lung tumors and cell lines (2,3), there existed different forms of S100A6 in thyroid tumors.
These findings are important because they build on and add to our slowly evolving understanding of the biochemistry of thyroid cancer. Notably, our group adopted a different proteomic strategy to examine papillary and follicular thyroid cancer (4,5). We separated intact, labeled proteins by differences in gel electrophoresis two-dimensional gel separation and then identified differentially abundant proteins by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass fingerprints. In these studies, we identified S100A6 as a potential marker of PTC along with a list of other candidates (4). The work of Sofiadis et al. (1) provides independent verification of the previous identification of S100A6 as a potential marker of PTC by our group (4) and others (6) and importantly, provides independent validation based on their immunohistochemical studies.
There have been other studies in the thyroid field that have exploited proteomic discovery approaches. In one of the first studies, Srisomsap and colleagues (7) compared different thyroid tissue and tumor samples (normal, Graves, multinodular goiter, follicular adenoma, follicular carcinoma, and papillary carcinoma) using a parallel two dimensional gel electrophoresis (2DGE) approach followed by electrospray mass spectrometry. They identified 32 abundant proteins in the different tissues. Most notably, they identified a group of distinct spots that were all variant forms of cathepsin B. These proteins were more abundant in PTC and differed in post-translational modifications (glycosylation), which would not have been detected by genomic approaches. Proteomic analyses have also been carried out in human thyroid carcinoma cell lines (8 –10), FRTL-5 cells (11), human tumor tissues (3,9,12,13), and fine-needle aspiration biopsies (14,15).
What are we to make of all these findings? The first and most obvious lesson is that no single biomarker study leads to the discovery and validation of clinically useful biomarkers for thyroid cancer, or for that matter, any other disease. The process of validation in particular is slow, but it is optimal when it draws on the cumulative findings of many investigators, sometimes working on seemingly disparate problems. In the present instance, for example, the work of Sofiadis and colleagues (1) provides independent verification for what we and others have found and this should catalyze larger scale validation studies targeted at S100A6. Furthermore, other evidence is accumulating that S100A6 may be involved in cancer. For example, it has recently been shown that S100A6 binds to p53 and affects its activity (16).
More work is clearly required. Slowly we are building a list of candidate proteins, bringing us a step closer to identifying useful molecular markers for thyroid neoplasms and deepening our understanding of thyroid cancer biology.