Sinclair R. Dundas

Mortalin is over-expressed by colorectal adenocarcinomas and correlates with poor survival

Using comparative proteomic analysis we have identified over‐expression of mortalin in colorectal adenocarcinomas. Mortalin, also known as mitochondrial heat‐shock protein 70 (mhsp 70), is involved in cell cycle regulation with important roles in cellular senescence and immortalization pathways. It is known to bind to and inactivate wild‐type tumour suppressor protein p53 and influences the Ras‐Raf‐MAPK pathway. By immunostaining a colorectal cancer tissue microarray linked to a patient database, we further found that mortalin over‐expression correlates with poor patient survival and, in multivariate analysis, is independent of standard prognostic variables (p = 0.04). Our findings demonstrate that mortalin over‐expression may predict outcome in colorectal cancer and suggest that this protein is involved in colorectal neoplasia. Our experimental approach emphasises the analytical power of combining proteomics with tissue microarray analysis in the context of a well‐defined tumour database. Copyright

Matrix Metalloproteinase/Tissue Inhibitors of Matrix Metalloproteinase Phenotype Identifies Poor Prognosis Colorectal Cancers

Purpose: The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes involved in tumor invasion; several individual members of which have been implicated in tumor prognosis. These enzymes and their physiologic inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), act in a coordinated manner to form an integrated system. Therefore, to understand their role in tumor invasion, it is necessary to evaluate them collectively. Experimental Design: In this study all of the major members of the matrix metalloproteinase (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, MT1-MMP and MT2-MMP)/tissue inhibitor of matrix metalloproteinase (TIMP-1, TIMP-2, and TIMP-3) system have been investigated by immunohistochemistry in a series (n = 90) of stage III (Dukes’ C) colorectal cancers. An immunohistochemical score based on the intensity of immunoreactivity and proportion of immunoreactive cells was established for each MMP and TIMP. Results: The MMP/TIMP profile defined by hierarchical cluster analysis of the immunohistochemical score identifies a distinct group of colorectal cancers with poor prognosis (log-rank test, 12.22, P = 0.0005). The median survival time of patients in this survival group was 18 months compared with a median survival of 49 months in the “good” survival group. Multivariate analysis showed that this profile was independently the most significant prognostic factor (P = 0.001). Conclusions: This study has identified that the MMP/TIMP profile is an independent indicator of poor prognosis in colorectal cancer.

Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer

Heterogeneous ribonucleoprotein K (hnRNP K) is a member of the hnRNP family which has several different cellular roles including transcription, mRNA shuttling, RNA editing and translation. Several reports implicate hnRNP K having a role in tumorigenesis, for instance hnRNP K increases transcription of the oncogene c-myc and hnRNP K expression is regulated by the p53/MDM 2 pathway. In this study comparing normal colon to colorectal cancer by proteomics, hnRNP K was identified as being overexpressed in this type of cancer. Immunohistochemistry with a monoclonal antibody to hnRNP K (which we developed) on colorectal cancer tissue microarray, confirmed that hnRNP K was overexpressed in colorectal cancer (P<0.001) and also showed that hnRNP K had an aberrant subcellular localisation in cancer cells. In normal colon hnRNP K was exclusively nuclear whereas in colorectal cancer the protein localised both in the cytoplasm and the nucleus. There were significant increases in both nuclear (P=0.007) and cytoplasmic (P=0.001) expression of hnRNP K in Dukes C tumours compared with early stage tumours. In Dukes C patients good survival was associated with increased hnRNP K nuclear expression (P=0.0093). To elaborate on the recent observation that hnRNP K is regulated by p53, the expression profiles of these two proteins were also analysed. There was no correlation between hnRNP K and p53 expression, however, patients who presented tumours that were positive for hnRNP K and p53 had a poorer survival outcome (P=0.045).

Cytochrome P450 Profile of Colorectal Cancer: Identification of Markers of Prognosis

Purpose: The cytochromes P450 (P450) are a multigene family of enzymes with a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, carcinogens, and endogenous compounds. The purpose of this study was to define the P450 profile of colorectal cancer and establish the prognostic significance of expression of individual P450s in colorectal cancer. Experimental Design: Immunohistochemistry for a panel of 23 P450s was done on a colorectal cancer tissue microarray consisting of 264 primary colorectal cancers, 91 lymph node metastasis, and 10 normal colorectal samples. The intensity of immunoreactivity in each sample was established by light microscopy. Results: The most frequently expressed form of P450 in normal colon was CYP3A4. In primary colorectal cancer, several P450s (CYP1B1, CYP2S1, CYP2U1, CYP3A5, and CYP51) were present at a significantly higher level of intensity compared with normal colon. P450 expression was also detected in lymph node metastasis and the presence of several P450s (CYP1B1, CYP2A/2B, CYP2F1, CYP4V2, and CYP39) in the lymph node metastasis strongly correlated with their presence in corresponding primary tumors. The presence of strong CYP51 (log-rank = 12.11, P = 0.0005) or strong CYP2S1 (log-rank = 6.72, P = 0.0095) immunoreactivity were associated with poor prognosis. CYP51 was also an independent marker of prognosis (P = 0.009). Conclusions: The expression of individual P450s has been established in colorectal cancer. Several P450s show increased expression in colorectal cancer. High expression of CYP51 or CYP2S1 were associated with poor prognosis and CYP51 is an independent marker of prognosis.

Profiling markers of prognosis in colorectal cancer.

PURPOSE: Colorectal cancer is one of the most common forms of cancer in developed nations and the incidence of this disease is increasing. There is a need to further stratify prognostically distinct groups of colorectal cancer, and the purpose of this study was to identify prognostically significant immunohistochemical marker profiles in colorectal cancer. EXPERIMENTAL DESIGN: In this study, a range (n = 23) of markers [pRb, p16, p21, p27, p53, proliferating cell nuclear antigen, cyclin D1, bcl-2, epidermal growth factor receptor, C-erb-B2, topoisomerase-I, liver fatty acid-binding protein, matrix metalloproteinases (MMP) 1-3, 7, 9, and 13, MT1-MMP, MT2-MMP, and tissue inhibitors of MMP 1-3] of putative prognostic significance have been investigated by immunohistochemistry on formalin-fixed, wax-embedded sections in a series (n = 90) of stage III (Dukes C) colorectal cancers. An immunohistochemical score based on the intensity of immunoreactivity and, where relevant, the proportion of immunoreactive cells was established for each marker. RESULTS: Unsupervised two-dimensional hierarchical cluster analysis identified three distinct cluster groups (designated groups 1-3) with different marker profiles. There were significant survival differences between groups 1 and 2 (log rank = 11.48; P = 0.0007) and between groups 1 and 3 (log rank = 8.32; P = 0.0039). Multivariate analysis showed that the complete marker profile was independently the most significant prognostic factor (hazard ratio, 2.27; 95% confidence interval, 1.15-4.48; P = 0.004). CONCLUSIONS: This study has identified an immunohistochemical marker profile of colorectal cancer and showed that it is an independent indicator of prognosis in this type of cancer.

Liver fatty acid binding protein expression in colorectal neoplasia

Liver fatty acid binding protein is a member of the fatty acid binding group of proteins that are involved in the intracellular transport of bioactive fatty acids and participate in intracellular signalling pathways, cell growth and differentiation. In this study we have used proteomics and immunohistochemistry to determine the changes in liver fatty acid binding protein in colorectal neoplasia. Comparative proteome analysis of paired samples colorectal cancer and normal colon identified consistent loss of liver fatty acid binding protein (L-FABP) in colorectal cancer compared with normal colon. To identify the changes in liver fatty acid binding protein expression during colorectal cancer development and progression the cell-specific expression of L-FABP was determined by immunohistochemistry in a series of colorectal cancers and colorectal adenomas. Decreased L-FABP immunoreactivity was significantly associated with poorly differentiated cancers (P<0.001). In colorectal adenomas there was a significant trend towards decreased staining of L-FABP in the larger adenomas (P<0.001). There was consistent L-FABP immunostaining of normal surface colonocytes. This study demonstrates that loss of L-FABP occurs at the adenoma stage of colorectal tumour development and also indicates that L-FABP is a marker of colorectal cancer differentiation.

Laser Capture Microdissection: Applications in Urological Cancer Research

There has been a rapid advancement in the number of sophisticated techniques used to study the molecular processes underlying carcinogenesis and tumour progression. Nevertheless, the heterogeneous nature of normal tissues and tumours complicates meaningful molecular analysis. For example, study of tumour gene expression at the mRNA level may be complicated by contaminant RNA derived from connective tissue, blood vessels and inflammatory cells. This problem has been diminished with the development of laser capture microdissection (LCM), which can directly isolate pure populations of cells visualised on tissue sections. The purpose of this review is to outline the principles of LCM and to indicate its applications in urological cancer research.

Mortalin Expression in Normal and Neoplastic Tissues

The purpose of this chapter is to discuss the distribution of mortalin in different normal tissues and tumour types highlighting its role in neoplasia. Correlating mortalin expression with tumour grade and stage and its influence on cancer metastasis will also be reviewed along with the emerging role mortalin has as a clinically relevant therapeutic target and prognostic marker.