The proteomics of colorectal cancer: identification of a protein signature associated with prognosis

Abstract

Colorectal cancer is one of the commonest types of cancer and there is requirement for the identification of prognostic biomarkers. In this study protein expression profiles have been established for colorectal cancer and normal colonic mucosa by proteomics using a combination of two dimensional gel electrophoresis with fresh frozen sections of paired Dukes B colorectal cancer and normal colorectal mucosa (n = 28), gel image analysis and high performance liquid chromatography-tandem mass spectrometry. Hierarchical cluster analysis and principal components analysis showed that the protein expression profiles of colorectal cancer and normal colonic mucosa clustered into distinct patterns of protein expression. Forty-five proteins were identified as showing at least 1.5 times increased expression in colorectal cancer and the identity of these proteins was confirmed by liquid chromatography-tandem mass spectrometry. Fifteen proteins that showed increased expression were validated by immunohistochemistry using a well characterised colorectal cancer tissue microarray containing 515 primary colorectal cancer, 224 lymph node metastasis and 50 normal colonic mucosal samples. The proteins that showed the greatest degree of overexpression in primary colorectal cancer compared with normal colonic mucosa were heat shock protein 60 (p<0.001), S100A9 (p<0.001) and translationally controlled tumour protein (p<0.001). Analysis of proteins individually identified 14-3-3β as a prognostic biomarker (χ² = 6.218, p = 0.013, HR = 0.639, 95%CI 0.448-0.913). Hierarchical cluster analysis identified distinct phenotypes associated with survival and a two-protein signature consisting of 14-3-3β and aldehyde dehydrogenase 1 was identified as showing prognostic significance (χ² = 7.306, p = 0.007, HR = 0.504, 95%CI 0.303-0.838) and that remained independently prognostic (p = 0.01, HR = 0.416, 95%CI 0.208-0.829) in a multivariate model.

Figure 1. Tumour and normal 2D gels.

Representative reference 2D gels of normal colon (A) and colon tumour (C). These are the annotated reference gels created by the Progenesis Same Spots gel image analysis software for the analysis of individual gels. The number of each spot is assigned by the image analysis software. For easier visualisation of individual spots representative non-annotated 2D gels of normal colon and colon tumour are shown in panels B and D respectively. The proteins which were validated by immunohistochemistry have been identified in D.

Figure 2. Hierarchical cluster and principal components analyses of 2D gels.

Representative hierarchical cluster analysis (A) and principal components analysis (B) of normal and tumour gels. Both statistical methods show that the protein expression profiles determined by 2D gel electrophoresis and gel image analysis are distinct in tumour samples compared with normal samples. The lower panel in each figure shows the standardised expression profiles. The figures presented in figure 2 are “screen captures” of the output of analysis by the Progenesis SameSpots software. Both figure 2A and figure 2B represents the results of the same experiment of one case (i.e. one pair of normal gels N1 and N2 and one pair of tumour gels T1 and T2). The lower panel in each part of the figure shows the standardised expression profile and represents proteins of distinct expression plotted vertically with lines ”connecting” the corresponding proteins in each gel. The coloured spots represent interactive spots placed by the software for the user to access each data set and are positioned arbitrarily by the software on the screen.

Figure 3. Immunohistochemistry photomicrographs.

Photomicrographs of the immunohistochemical localisation of individual proteins in normal colon, primary colorectal cancer and lymph node metastasis.

Figure 4. Frequency expression of individual proteins in normal colon, colorectal cancer and metastatic colorectal cancer.

Frequency of expression as evaluated immunohistochemically of individual proteins in A. normal colon, B, primary colorectal cancer and C. lymph node metastasis.

Figure 5. Frequency expression of individual proteins in specific Dukes stages of colorectal cancer.

Frequency of expression of individual proteins as evaluated immunohistochemically in A. Dukes A colorectal cancer, B. Dukes B colorectal cancer and C. Dukes C colorectal cancer.

Figure 6. Survival curves of marker proteins.

The relationship of individual proteins evaluated by immunohistochemistry with survival with different cut-off points. A. 14-3-3β (positive v negative immunoreactivity), B. PHB (positive v negative immunoreactivity), C. IDH1 (negative/weak immunoreactivity v moderate/strong immunoreactivity), D. LDHB (negative/weak immunoreactivity v moderate/strong immunoreactivity), E. TCTP (negative/weak immunoreactivity v moderate/strong immunoreactivity), F. IDH1 (negative/weak/moderate immunoreactivity v strong immunoreactivity), G. MVP (negative/weak/moderate immunoreactivity v strong immunoreactivity), H. survival in each of 10 clusters identified by hierarchical cluster analysis (each cluster is numerically identified and corresponds to the clusters that are identified in the cluster analysis panel of Figure 7), I. survival in 2 clusters- cluster 1 and clusters 2–10 combined and J. two protein signature of 14-3-3β and ALDH1 showing that double negative tumours have a significantly better outcome.

Figure 7. Hierarchical cluster analysis of immunohistochemical marker proteins.

Graphical representation of the immunohistochemistry marker data is shown in the middle panel. The right hand panel shows the results of the hierarchical cluster analysis presented as a dendrogram with 10 individual clusters identified. The left hand panel shows an expanded segment of the graphical representation. Proteins are represented in columns and cases in rows.