Mark T. Hayes

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

BackgroundEnvironmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans.ResultsHere, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass.ConclusionsThis is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

Proteomic analysis of advanced colorectal cancer by laser capture microdissection and two-dimensional difference gel electrophoresis.

The emergence of laser capture microdissection (LCM) and two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to greatly improve the accuracy and sensitivity of global protein expression analysis. However, their combined use in profiling tumour proteome has rarely been reported. In this study, we applied these techniques to profile the protein expression changes of the late stage colorectal cancer (CRC) and its liver metastases. The study revealed that both the primary and secondary tumours showed a distinct protein expression profile compared to normal tissues, but were indistinguishable from each other. Differential analysis between the primary tumour and patient-matched normal colon mucosa identified a total of 71 proteins to be altered in CRC. Over 40% of these proteins have been previously reported as CRC-related proteins, validating the accuracy of the current analysis. We have also identified many previously unknown changes including overexpression of ACY1, HSC70, HnRNP I, HnRNP A3, SET, ANP32A and TUFM in CRC, which have been further verified by western blotting and immunohistochemistry. This study demonstrated that LCM in combination with 2D-DIGE is a powerful tool to analyse the proteome of tumour tissues and may lead to the identification of potential novel protein markers and therapeutic targets for cancer.

Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays.

Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 40–50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

TUFM is a potential new prognostic indicator for colorectal carcinoma

Aims: Mitochondrial Tu translation elongation factor (TUFM) is a nuclear encoded protein that participates in mitochondrial polypeptide translation. TUFM has been reported to be over-expressed in many tumour types including colorectal carcinoma (CRC) by proteomics. The present study aims to examine the prognostic implication of TUFM in CRC. Methods: Immunohistochemical staining was performed in tissue microarrays composed of 123 cases of CRC using a polyclonal anti-TUFM antibody. Immunoreactivity was quantified using Image-Pro plus software, and analysed in association with patients’ clinicopathological parameters and survival time. Results: The immunoreactivity of TUFM was negative in 25%, weak in 50% and strong in 25% of CRC cases. TUFM immunoreactivity had no significant association with the clinicopathological parameters examined including TNM stage and grade. However, strong TUFM expression significantly correlated with a higher 5-year recurrence rate (p = 0.024). Kaplan–Meier analysis revealed that patients with strong TUFM expression had significantly shorter cancer-specific survival than patients with negative TUFM (log-rank test, p = 0.038). In multivariate analysis, strong TUFM expression remained a stage-independent unfavourable prognostic indicator (p = 0.024). Conclusions: Increased expression of TUFM is a promising new prognostic indicator for CRC. Selective inhibition of TUFM in tumour cells may present a new avenue for the targeted therapy of this cancer.