*3.6.2 Complement component C9*

*Advances in the Molecular Understanding of Colorectal Cancer*

Tissue inhibitors of metalloproteases (TIMPs) have been implicated in tumourigenesis. TIMP1 overexpression is associated with advanced stages of CRC [109]. IHC studies have demonstrated a significant correlation between TIMP2 expression in inflammatory cells, increasing tumor size, lymph node involvement and presence of metastasis [110]. TIMP3 has been described as independent prognostic marker for CRC, where strong cytoplasmic staining has been associated with longer

Annexins are phospholipid-binding membrane-binding calcium regulated proteins from a multigene family. They function in membrane processes such as structural control as well as cell transport and as linkers between membranes, or between membranes and cytoskeleton as well as calcium regulated exocytosis. In humans the annexin family consists of subfamilies; A1–A11 and A13 [112]. The sensitivities of annexins A3, A4, and A11 peptides for detecting early-stage CRC have been reported to exceed those of CEA, and as such these peptides are promis-

A shotgun proteomics analysis (LC-MS/MS) of extracellular vesicle proteins with selected reaction monitoring performed on CRC cell culture lines has demonstrated annexin A3, annexin A4, and annexin A11 overexpression, particularly in early stage CRC patients. Reported sensitivities of annexin A3, A4, A11 for stage one disease are in the range of 82.1–85.7%, and for stage two disease between 89.3– 96.4% [26], therefore highlighting a potential role for these annexins as an early stage disease biomarker. Notably, the same study reported the sensitivity of CEA for early stage disease to be as low as 38.8% [26]. Importantly, progressive increases in annexin A3 abundance have been shown to strongly correlate with disease progres-

Confirmation of Annexin A2 overexpression in a small cohort of histological samples has been described using a 2D-LC-MS/MS approach with iTRAQ labeling; with results being validated with immunoblot and IHC [90]. Conversely a study examining serum levels of Annexin A2 found that the protein was significantly lower in CRC patients compared to healthy controls; Annexin A2 levels were also inversely related to tumor size and stage [113]. In addition to Annexin A2, altered Annexin A4 expression has been demonstrated in CRC via the application of a label free LC-MS/MS approach, with validation in CRC serum samples confirming its overexpression and thus potential as a biomarker of the disease [114]. Annexin A10 is not frequently overexpressed in CRC with an estimated elevation being recorded in only 5.8% of patients. However, it too is associated with poor overall survival and poorer progression-free survival particularly in late stage cancers. As such, Annexin

A10 may be considered as a prognostic marker when present [115], similarly annexin A13 expression is associated with lymph node metastasis, however it is not

Complement component C3 (C3), and its fragment C3 anaphylatoxin (C3a), overexpression has been demonstrated in fecal, serum and histological samples from CRC patients. C3 is also a component of the innate immune system, with

associated with tumor stage or differentiation [116].

**3.6 Complement component proteins**

*3.6.1 Complement component C3*

sion from normal tissue, to adenoma and finally to carcinoma [26].

*3.4.5 Tissue inhibitors of metalloproteases*

survival in rectal cancer patients [111].

ing biomarkers for early detection of CRC [26].

**3.5 Annexins**

**148**

Complement component C9 (C9) is a constituent of the complement system that has important functions in the innate immune system. It is a terminal constituent of the membrane attack complex (MAC) and thereby aids in immune system response to cell death [121]. Changes in C9 expression have been described in both fecal and plasma samples, in a series of 315 stool samples using a combination of LC separation, LTQ-FT hybrid MS and QE-Label free-MS; C9 and C3 in addition with S100A8, S100A9 were found to be overexpressed [118]. A UHPLC–LC-MS approach and plasma-based immunoassay using 187 proteins previously described in the literature, demonstrated significant elevation of C9 in CRC plasma samples [23]. Similarly, an analysis of 31 CRC plasma samples revealed overexpression of C9 compared to healthy controls as well as reduced expression of Apolipoprotein AI [122].

## **3.7 S100 proteins**

S100 are a family calcium-binding proteins, which consists of 24 members subdivided into three groups; broadly those with intracellular regulatory functions only, extracellular functions only and those with both intracellular and extracellular functions [123]. The proteins, S100A8 and S100A9, form a heterocomplex that is postulated to function in myeloid differentiation, cell transport, nuclear factor interaction and calcium related phagocytosis [123]. Mouse models have demonstrated accumulation of S100A8/A9 positive cells in areas of dysplasia and adenoma as well as promotion of MAPK and NF-κB activation signaling pathways [124]. IHC staining has demonstrated overexpression of S100A8, S100A9, Adenosylhomocysteinase (AHCY) and Nm23-H1 in CRC tumor cell cytoplasm, in the same study, S100A8 and S100A9 were also significantly increased in the plasma of CRC patients [36]. S100A8 has also been shown to have increased expression at progressive CRC stages (Duke's A-D) compared with controls [125]. Minichromosome maintenance complex component 4 (MCM4) and S100A9 overexpression have also been shown in proximal colonic fluid mouse proteome, using label free MS [126]. The same study identified Chitinase 3 like 1 (CHI3L1) protein overexpression in adenomas and advanced adenomas and CRC, the overexpression was further confirmed to be present in the serum of all three patient subtypes compared to controls [126]. A 2DGE LC-MS/MS based analysis of Dukes stage B CRC also identified S100A9, HSP60 and TCTP as overexpressed proteins. In addition to histological and plasma samples, S100A8 and S100A9 have been shown to be overexpressed in fecal samples also using a LC-MS/MS approach [118, 119]. Additionally, S100A11 has been identified among a cohort of 23 upregulated proteins in CRC samples using a combined targeted LC-MS/MS and SRM approach [22].
