*3.4.1 MMP1 and MMP13: collagenases*

*Advances in the Molecular Understanding of Colorectal Cancer*

HSP60 levels in CRC compared to controls (P = 0.0001) [83].

HSP60 is a chaperone protein with functions including transport and mitochondrial protein folding. This protein has been associated with a wide range of cancers including prostate, breast, cervical bladder, hepatic and CRC [71]. HSP60 is also elevated in non-cancer conditions such as chronic hepatitis and liver cirrhosis [80]. 2DE coupled to LC-MS–MS/MS using 28 histological adenocarcinoma samples and a 789 patient IHC validation set revealed that HSP60 was overexpressed along with S100A9 and translationally controlled tumor protein (p < 0.001 for each) [81]. This study also identified the beta subunit of 14-3-3 as a prognostic marker [69]. Additionally, IHC and immunoblot were used to demonstrate, in histological samples of 44 patients, that HSP60 was elevated in tumor tissues and there was a significant association between HSP60 levels, tumor differentiation, and tumor stage [82]. Comparison of colonic tumor samples and matched normal tissue confirmed overexpression of HSP60 (3.25-fold change ratio) with 2D-DIGE and immunoblotting) [83]. The authors of this study also developed an immunoassay for serum HSP60 detection, confirming a statistically significant elevation in serum

HSP70 has 13 subgroup family members. It is associated with cytosolic calcium level homeostasis and, inhibition of HSP70 expression, has been shown to stimulate release of intra-cellular calcium in cell culture. Calcium induces cell death by the caspase dependent mechanism in CRC cell lines, and functions in the stabilization of lysosomes and inhibition of apoptosis [84]. Importantly, in other types of cancer such as pancreatic and prostate cancer, HSP70 has been shown to upregulate cell survival [84]. In a study of 33 CRC patient plasma samples, using ELISA assays, serum levels of HSP70 were significantly elevated (≥2.25 ng/ml) in cancer patients compared to healthy controls, The sensitivity and specificity of elevated serum HSP70 in the CRC group was reported as 96.77% and 96.96% respectively [85]. It has been further demonstrated using ELISA testing that high serum concentration of HSP70 is associated with increased mortality (p = 0.005) [86]. Additionally the use of immunostaining has shown that mitochondrial HSP70 overexpression correlates to poor survival (p = 0.04) [87]. Independent IHC analyses of 81 primary CRC tissues revealed that HSP70, as well as HSP110, overexpression is associated with highly advanced clinical stages and positive lymph node involvement [88].

HSP90 activates Hypoxia-inducible factor-1 and Nuclear Factor-κB which in turn regulate epithelial to mesenchymal transition, invasion and motility of CRC [89]. HSP90 has been shown in various studies to be overexpressed in CRC and may serve as a potential biomarker for CRC. In a small study of histological adenocarcinoma samples with an iTRAQ labeling method and QStar LC-MS/MS approach, a total of 82 altered proteins were found in CRC patients, which included overexpression of HSP90α and significant downregulation of Gelsolin. The results also suggested that HSP70 had decreased expression in the same samples [90]. Further validation using immunoprecipitation, MALDI-TOF-MS and immunoblotting confirmed that HSP90α is overexpressed in tumor cells and is correlated with poor prognosis and metastatic disease [91]. Plasma HSP90α serum levels were also significantly elevated in an analysis of 77 CRC patients compared to controls [92],

thus highlighting the potential biomarker utility of this protein.

*3.3.3 HSP60*

*3.3.4 HSP70*

*3.3.5 HSP90*

**146**

MMP1 functions to degrade type I, II and III collagen. MMP13 is structurally similar to MMP1, and likewise it also cleaves collagens, as well as degrading extracellular matrix proteins including fibrillar collagen, fibronectin, tenascin C and aggrecan core protein 1 [94]. Demonstration by immunostaining of 133 CRC samples showed that MMP1 expression was significantly correlated with hematogenous colorectal metastasis [95]. Increased expression is also associated with poor prognostic factors such as invasion level, lymph node and hepatic metastasis [96]. Similarly, MMP13 overexpression in CRC has also been shown to be associated with poor prognosis [93].
