Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer

Abstract

Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis.Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis.Cluster analysis of the whole patient cohort identified a good prognosis group (mean survival=146 months 95% CI 127-165 months) that had a significantly better survival (δ2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group (mean survival=107 months, 95% CI 98-123 months). For the mismatch repair proficient cohort, the good prognosis group had a significantly better survival (δ2=8.985, p=0.003, HR=1.845, 95% CI 1.227-2.774) than the poor prognosis group. Multi-variate analysis showed that cluster group was independently prognostically significant in both the whole patient cohort (p=0.02, HR=1.554, 95% CI 1.072-2.252) and the mismatch repair proficient group (p=0.04, HR=1.530, 95% CI 1.014-2.310).Individual oxysterol metabolising enzymes are overexpressed in colorectal cancer and an oxysterol metabolising enzyme expression profile associated with prognosis has been identified in the whole patient cohort and in mismatch repair proficient colorectal cancers.

The expression of brown fat‐associated proteins in colorectal cancer and the relationship of uncoupling protein 1 with prognosis

“…To overexpress each protein, HEK293 cells had been transiently transfected using the full length reading frame cDNA plasmid of the corresponding gene (Novus Biologicals, Oxfordshire, UK and OriGene Technologies Rockville, USA). The immunoblotting was carried out as previously described …”

“…Similarly, a tissue microarray was constructed consisting of 549 primary tumour samples from the validation cohort. For each tissue sample, two representative 1 mm cores were obtained from the corresponding formalin fixed, paraffin embedded block to construct the tissue microarrays …”

“…The immunoblotting was carried out as previously described. 22 The initial immunohistochemical evaluation of each of the antibodies and their tissue specificity was performed using a multi-tissue microarray which was designed to include a wide range of normal tissue and tumour types (Supporting Information Table S3).…”

“…For each tissue sample, two representative 1 mm cores were obtained from the corresponding formalin fixed, paraffin embedded block to construct the tissue microarrays. 21,22,24 Immunohistochemistry Monoclonal antibody characterisation using multi-tissue microarray. Immunohistochemistry was performed using a Dako autostainer (Dako universal staining system, Dako/Agilent Technologies LDA UK Limited, Cheadle, UK).…”

The Role of Oxysterols in Human Cancer

“…The expression level of oxysterol metabolizing cytochrome P450 proteins was evaluated in colorectal carcinoma samples and the overexpression of some enzymes and association with cancer prognosis was found, e.g., the levels of the 27-HC metabolizing enzymes CYP27A1 and CYP7B1 showed similar association to patient prognosis as in breast cancer patients – higher expression of CYP27A1 was associated with poor outcome of patients and increased level of CYP7B1 was associated with good prognosis of patients [53], suggesting a potential role of 27-HC in patients with colorectal carcinoma.…”