The evolution from benign adenoma to colorectal carcinoma (CRC) involves specific nuclear events that change the cellular DNA structure and function toward a malignant phenotype. These changes include chromosomal instability (CIN) and microsatellite instability (MIN)1 and may involve the multiple nuclear “housekeeping” or chromatin binding proteins (CBP) that interact with DNA, maintaining structure, replication and repair processes. Drawing from their earlier research,2 Knol et al.3 used subcellular fractionation to examine changes in CBP profiles in CRC tissues compared with those in pre-cancerous adenomas.
The researchers used stored tissues surgically resected from patients undergoing treatment for colorectal disease (adenoma = 3, CRC = 5). Tissues were snap-frozen on removal and a histopathologist confirmed cancer morphology. The researchers determined the CIN and MIN subtype status of each CRC specimen and ensured that cancer cell content was above 70%.
Using their previously validated technique, the research team homogenized and fractionated the tissues to obtain the CBP nuclear extracts. They confirmed their precision using polyacrylamide gel electrophoresis for separation and identification of proteins involved, along with their nuclear involvement.
Following in-gel digestion, the peptides underwent nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) in an LTQ FT hybrid mass spectrometer (Thermo Scientific), with data analyzed against the human IPI database using the Sequest program (Thermo Scientific). Knol and co-workers used a label-free approach to quantify the peptides identified.
The researchers identified a total of 309 proteins differentially expressed by adenoma and CRC tissues. By constructing a nuclear annotation database, the researchers further reduced their list to 169 nuclear-associated proteins. Of these, 79 were significantly more abundant in CRC samples and 90 were increased in adenoma specimens. Where mRNA results were available for individual proteins (n=44), Knol and co-workers found that 59% of these showed concordant molecular and proteomics results.
Results were also submitted for BiNGO4 and DAVID (Database for Annotation, Visualization and Integrated Discovery) gene ontology analyses, using the STRING tool (Search Tool for the Retrieval of Interacting Genes/Proteins) to determine protein–protein interactions. By focusing on the nuclear-localized proteins, the researchers found their results clustered in various functional groups for each sample set.
Proteins increased in the CRC samples grouped into three main clusters: ribosome and RNA-related actions (ribsome synthesis, RNA processing, splicing events, translation activity); cytoskeleton factors; and glycolytic enzymes, possibly reflecting change in cancer cell metabolism. Other notable functions represented by proteins included control of apoptosis, DNA replication regulation and ubiquitin-associated proteasomal processing.
In the pre-malignant adenoma samples, the researchers found that the 90 proteins showing increased abundance were involved in chromosome/chromatin organization and included various histones. They also noted proteins involved in DNA repair and RNA processing.
In summarizing their results, the researchers comment that their research model identifies nuclear proteins that could play a role in the change from a pre-malignant status to malignancy in colon cancer. They describe their work as showing “a specific shift in the chromatin landscape going from adenomas to carcinomas,” suggesting that further exploration here could yield new therapeutic options, as well as valuable biomarker discoveries.
1. Diep, C.B., et al. (2006) “The order of genetic events associated with colorectal cancer progression inferred from meta-analysis of copy number changes,” Genes, Chromosomes and Cancer, 45 (pp. 31–41).
2. Albrethsen, J., et al. (2010) “Subnuclear proteomics in colorectal cancer: Identification of proteins enriched in the nuclear matrix fraction and regulation in adenoma to carcinoma progression,” Molecular and Cellular Proteomics, 9(552) (pp. 988–1005).
3. Knol, J.C., et al. (2013, December) “Proteomics of differential extraction fractions enriched for chromatin-binding proteins from colon adenoma and carcinoma tissues,” Biochimica Biophysica Acta, available at http://dx.doi.org/10.1016/j.bbapap.2013.12.006.
4. Maere, S., Heymans, K., & Kuiper, M. (2005) “BiNGO: a Cytoscape plugin to assess overrepresentation of Gene Ontology categories in biological networks,” Bioinformatics, 21 (pp. 3448–9).