Proteomic Study in CRC of Paired Tumor Versus Nontumor Tissue May Identify New Biomarkers

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“Gene expression data can be useful for identifying potential biomarkers and pathways, while proteomics studies are needed to validate whether or not differences observed in gene expression are truly reflected at the protein level,” explained Susan Fanayan, PhD, senior research fellow at the Department of Medicine and Health Sciences at Macquarie University, Sydney, Australia. She maintains that the two approaches can be complementary. “In fact in recent years, integrated genomic and proteomic approaches have proven quite useful in providing useful insights that may not be deciphered from individual analysis of genome or proteome.”

Publishing their results in the latest issue of theJournal of Proteomics, Fanayan et al profiled the membrane-enriched proteome of tumor and adjacent nontumor tissues from patients with colorectal cancer (CRC). Specifically, they compared the membrane-enriched proteomes of tumor with nontumor tissue, early-stage versus late-stage cancer, epidermal growth factor receptor positive (EGFR+) versus EGFR negative (EGFR-) cancerous tissue, and analyzed the impact of the deregulated proteins on biological functions and pathways.²

Fanayan explained, “In an earlier study,³we used a similar approach by using CRC cells lines, representing different pathological states of CRC, to gain molecular insights into the mechanisms underlying CRC pathogenesis. We observed significant differences in the expression of several cancer-associated proteins such as EGFR, MET and CEACAM5, between these cell lines,” she said. “We also observed that several pathways such as RHOA signaling and cell adhesion were perturbed in these cell lines. So we set out to determine if these molecular deregulations are also observed in CRC patient tissues compared with healthy individuals.”

The latest study focused on the membrane-enriched proteome, for two reasons. Because of their involvement in many biological functions, membrane proteins are suitable targets for therapeutics, and they are often released into the circulation making many of them prime candidates for biomarker studies. The aim of the study was to provide a detailed map of the altered proteome and associated protein pathways in CRC and reveal opportunities for the identification of new CRC protein markers.²

Patients and Tissues

The study analyzed tissue from eight patients whose ages ranged from 56 years to 78 years. Two specimens were used from each patient (seven male, one female), one of the CRC tumor, and another of a piece of the colon that was not cancerous. The distance between the tumor and nontumor tissues ranged from 5 cm to 22 cm. All the primary tumors were adenocarcinoma; two each of stage IIIB, stage IV, and stage I, and one each of stage IIA and stage IIB. Half of the tumors were EGFR+ and half were EGFR-, determined by western blot analysis and immunohistochemical staining. Tissue analyses were performed using label-free nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS)-based quantitative proteomics and advanced pathway analysis.²

Differential Expression of Proteins in Tumor Tissue Versus Nontumor Tissue

Analysis confirmed differential expression of a total of 948 proteins expressed in tumor tissue and nontumor tissue. They found that 184 proteins were differentially expressed (P≤.05, protein fold change of >1.5). Of these, 69 proteins were upregulated and 115 were downregulated in tumor tissue versus nontumor tissue. A hierarchical cluster analysis of the differentially expressed proteins revealed a clear delineation of tumor and nontumor tissues into separate groups, which according to the authors, indicates a strong CRC association of this proteome subset. The identities and roles of the most upregulated and downregulated proteins in tumor tissue correlated with tumor behavior. Fibronectin (FN1), which is implicated in migration and cell adhesion processes, epithelial to mesenchymal transition (EMT), and progressive disease, was the most upregulated protein (176.8 fold,P= .02) versus nontumor tissue. Other examples of proteins upregulated and downregulated in tumor versus nontumor tissue, are given in the Table.

TABLE:Examples of Proteins Upregulated and Downregulated in Tumor Versus Nontumor Tissue

Examples of Upregulated Proteins With Known Cancer Association

Protein name (gene name)

Examples of Most Downregulated Proteins

Protein name (gene name)

Tenascin (TNC)

Programmed cell death 4 and lectin (PDCD4)

Defensin alpha 1 (DEFA1)

Malectin (MLEC)

Serpin peptidase inhibitor clade H [heat shock protein 47] member 1 (SERPINH1)

Cadherin 17 (CAD17)

Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)

Ezrin (EZR)

Integrin beta 2 (ITGB2)

CD36

Mucin 2 (MUC2)

Galacoside-binding soluble 4 (LGALS4)

Describing their results, among the top ten upregulated proteins in tumor samples, the authors highlighted a preponderance of other EMT signature proteins such as tenascin and collagen. They stated that this agrees with previous reports of their upregulation in serum and tumor tissues in patients with CRC versus controls, leading to the possibility that they may be reliable CRC markers.⁴Taken together with the downregulation of adhesion proteins such as cadherin 17, their findings correlate with work that traditionally showed that downregulation of adhesion proteins, and upregulation of fibronectin and collagen was associated with activation of EMT and poor prognosis.⁵Other upregulated proteins such as the integrins and heatshock proteins are also known to promote tumor invasiveness and disease progression. In contrast, downregulated proteins in tumor tissues included the tumor suppressors PDCD4 and LGALS49 (Table).

Biomarker Analysis

A further analysis of these differentially regulated proteins, using the Ingenuity Pathway Analysis, identified that 26 of them were either established or promising CRC biomarkers for diagnosis, (eg, FN1, TNC, CEAMCAM7), prognosis (eg, EZR) and treatment efficacy prediction (eg, ICAM1). Both PDCD4 and LGALS4 have already been put forward as potential markers of CRC (diagnostic and prognostic).^6,7

Analysis of Networks Associated With the Deregulated Proteins

The researchers then turned their attention to the co-regulated protein/gene networks that contained the deregulated proteins. They found that the regulation of the actin cytoskeleton, and the Rho guanine nucleotide dissociation inhibitor (RhoGDI) pathways were significantly altered, and not surprisingly the ECM pathway was also deregulated (contained the upregulated proteins FN1, TEN, and collagen).²In their discussion, the authors emphasize the importance of the strong association between a deregulated actin cytoskeleton pathway and the invasive and metastatic phases of CRC, and cite work by others demonstrating that RhoGDIs exert influence over cell division, morphology, and migration.^2,8

The Proteomic Profile and Tumor Stage

It proved possible to identify differentially expressed proteins by tumor stage. The team analyzed early stage versus late stage. For this purpose, stages I and II were grouped together as early stage, and stages III and IV were grouped as being late stage, (the analysis could not delineate between stages I and II, and III and IV).²This identified 36 upregulated and 15 downregulated (total 51 differentially expressed proteinsP≤0.05, protein fold change >1.5) proteins in early-stage versus late-stage tumors. Many ribosomal proteins were upregulated along with other tumor-associated proteins such as spectrin alpha nonerythrocytic (SPTAN1) and junction plakoglobin (JUP), and were present in increased concentration in early, but decreased in late stage tissues. Members of the annexin family, known to be involved in proliferation and differentiation,⁹and show increased expression in higher tumor stage,^10,11,12and other cancer associated proteins such as CD46 and serpin peptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin) member 1 (SERPINA1) displayed elevated expression in late-stage tumor tissue.

EGFR Status and Proteomic Profile

Four of the tumors were strongly positive for EGFR staining and the remaining four displayed absent or negligible staining and were designated as EGFR negative. Once again, it was possible to identify differentially expressed proteins. There were 129 differentially expressed proteins between EGFR+ and EGFR- tumors (P≤.05, protein fold change of >1.5), 93 of which were upregulated and 36 downregulated in EGFR+ tumors. Again, those upregulated in EGFR+ tumors were identified as cancer-associated proteins such as KRT20, PSME2 and SPTAN1, while cell adhesion molecules (eg, ICAM1, HLA-DPB1, HLA-B) were among those significantly downregulated, suggesting a role in cancer progression.²

Future Opportunities

Concluding their paper the authors acknowledge that while the molecular alterations observed need to be confirmed in a larger study, “this study provides insights into the deregulated proteins and networks in CRC tumors that could act as likely drivers of CRC onset and progression and may serve as potential CRC markers.”²

Looking to future research Fanayan explained, “We have also used an MS-based glycomic approach to map the N-glycosylation patterns of the same samples and identified CRC-specificN-glycosylation signatures. We hope to test these findings in a larger sample set, covering different CRC stages as well as differential expression status of key CRC signature markers such as EGFR, to obtain a comprehensive multi-omic view of the disease.”

 

References

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8.    Dransart E, Olofsson B, Cherfils J. RhoGDIs revisited: novel roles in Rho regulation.Traffic.2005;6:957-966.

9.    Moss SE, Morgan RO. The annexins.Genome Biol. 2004;5:219 (1-8).

10. Duncan R, Carpenter B, Main LC, et al. Characterisation and protein expression profiling of annexins in colorectal cancer.Br J Cancer.2008;98:426-433.

11. Xue G, Hao LQ, Ding FX, et al. Expression of annexin a5 is associated with higher tumor stage and poor prognosis in colorectal adenocarcinomas.J Clin Gastroenterol.2009;43:831-837.

12. He ZY, Wen H, Shi CB, et al. Up-regulation of hnRNP A1, Ezrin, tubulin ß-2C and Annexin A1 in sentinel lymph nodes of colorectal cancer.World J Gastroenterol.2010;16:4670-4676.