Research Projects

Colorectal cancer research – Prof Jonathan Blackburn

Colorectal cancer research – Prof Jonathan Blackburn

Prof Jonathan Blackburn

Professor Jonathan Michael Blackburn

Project Title

Dysregulation of CT antigen expression in HNPCC and microbially-associated colorectal cancers

Project Description

The aim of this project is to investigate whether, in certain colorectal cancers, there is a causal relationship with infection of the colonic mucosa by specific bacterial pathogens, including Enteropathogenic Escherichia coli, Enterotoxigenic Bacteroides fragilis, Streptococcus gallolyticus and Enterococcus faecalis.  We also aim to investigate whether such bacterial-driven colorectal cancers show similar mechanisms of tumourigenesis and pathogenesis to that of a familial form of colorectal cancer, hereditary nonpolyposis colorectal cancer (HNPCC).  In particular, we are interested in studying whether bacterial infection of the colonic mucosa can cause alterations in components of the human DNA mismatch repair system, leading to microsatellite instability, and hence to cancer progression in those individuals.

In this project, we aim to use state-of-the-art DNA copy number profiling, transcriptomic & epigenomic techniques to study in parallel the differential expression of all genes present in colorectal tumour and normal biopsy samples, our aim being to uncover new insights regarding the underlying mechanisms and origins of CRC.

Summary of original objectives of the project

The causal link between specific pathogens and numerous cancers has now been firmly established. Irrefutable evidence exists that a significant percentage of cervical cancers are directly caused by infection with human papillomavirus (HPV) [1]. Similarly, Helicobacter pylori is a known risk factor for the development of gastric cancer and is considered a class I carcinogen by the WHO [2,3]. Recent studies, both epidemiological and in vitro, suggest for the first time that a subset of sporadic colorectal cancer may be caused by Enteropathogenic Escherichia coli (EPEC) infection in susceptible individuals [4,5,6]. In fact, a recent in vitro study has shown that EPEC cause cellular defects in DNA mismatch repair that in some ways mimic those found in hereditary nonpolyposis colorectal cancer (HNPCC) [5].

The aim of this project is to validate these preliminary findings in vitro, as well as to investigate possible mechanisms whereby EPEC infection may cause cancer initiation and/or progression using high-throughput genomic analysis of tissue samples gathered from colonic resection of colorectal cancer patients. This project offers a unique opportunity to shift the traditional therapeutic focus in cancer biology to antimicrobial and/or vaccine-based strategies. If our hypothesis holds true, the implications for early diagnosis and prevention of at least a subset of colorectal cancers are profound.

Our objectives are to harness recent technological advances in the field of high throughput genomics and to apply a bioinformatics approach to interpreting and integrating genomic, proteomic and glycomic datasets in order to uncover new insights with regards to the etiology of CRC, specifically related to bacterial contribution to disease, which have previously remained obscure. This approach, in addition to resolving false positives in each of the datasets, will increase our power of inference with regards to the etiology of this disease since we are now considering this problem from a systems biological perspective.

References:

[1] M Schi, PE Castle, J Jeronimo, AC Rodriguez & S Wacholder (2007) Lancet 370, 890-907.

[2] A Manuel, D Machado, C Figueiredo, R Seruca & L Juel (2010) Biochimica et Biophysica Acta.
doi:10.1016/j.bbcan.2010.01.007.

[3] R Vogelmann, & MR Amieva (2007) Current Opinion in Microbiology 10, 76-81.

[4] JF Reid, et al. (2009) Genes, Chromosomes & Cancer 48, 953-962.

[5] ODK Maddocks, AJ Short, MS Donnenberg, S Bader & DJ Harrison (2009) PloS One 4, e5517.

[6] A Swidsinski, et al. (1998) Gastroenterology 115, 281-286.

Non-scientific report

Colorectal cancer is the third leading cause of cancer death in both men and women in the US, and an estimated 49,960 deaths were expected from the disease in 2008. In South Africa, colorectal cancer ranks in the top five most common cancers with 1 in 97 men and 1 in 162 women at risk of developing colorectal cancer. The 5-year survival rate for CRC patients is greater than 90% when tumours are detected at a localised early stage – however, that rate drops to 40-65% after the cancer has spread regionally and to 10% after distant metastases of the original tumour. Unfortunately, only 39% of CRCs are in fact diagnosed at an early stage, mainly due to low rates of screening.  These figures emphasise the need for a better understanding of the underlying causes of different classes of CRC as a means to enable early detection and prevention of CRC.

The aim of this project is therefore to investigate whether, in certain colorectal cancers, there is a causal relationship with infection of the gut mucosa by specific bacterial pathogens, including Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Bacteroides fragilis (ETBF), Streptococcus gallolyticus and Enterococcus faecalis.  We also aim to investigate whether such bacterial-driven colorectal cancers show similar mechanisms of tumourigenesis and pathogenesis to that of a familial form of colorectal cancer, hereditary nonpolyposis colorectal cancer (HNPCC).  In particular, we are interested in studying whether bacterial infection of the gut mucosa can cause alterations in components of the human DNA mismatch repair system, leading microsatellite instability and hence to cancer progression in those individuals.

We have performed quantitative bacterial detection of ETBF and EPEC in 30-paired patient samples. The results show ETBF- and EPEC-infection in at least 40% and 10% of our cohort respectively.  Interestingly, there seems to be a positive correlation between EPEC infection and microsatellite instability.  These finding warrant further investigation through a larger cohort.

To date, we have analysed differences in the expression level of every human gene that exist between tumour and normal biopsy samples obtained from a historical cohort of 18 colorectal cancer patients undergoing surgery.  To facilitate this process we have had to develop a method for assessing the validity of gene expression data generated from patient samples of variable RNA quality.  Our new method will be useful for any study based on ex vivo samples. We have also carried out genotyping analyses to identify differences amongst 2.7 million human genetic markers that exist between the tumour and normal biopsy samples from the same patients.

Preliminary analyses using our own, as well as external transcriptomic datasets demonstrate marked immunological response in MSI-positive compared to MSI-negative samples with components specifically related to bacterial recognition. We are currently analysing epigenomic data, which we recently obtained using state-of-the art Illumina HumanMethylation450k BeadChips. By integrating transcriptomic and epigenomic data, we intend to gauge the potential of CRC-associated pathogens to induce aberrant epigenetic changes in host cells that may promote tumourigenesis.

In due course, our study should lead to an improved understanding of the underlying molecular origins of colorectal cancer in individual patients and as such could lead to new and improved diagnosis, treatment and prevention strategies in the future.  Specifically, if our hypothesis – that bacterial infection can play a causative role in initiating colorectal cancers – holds true, our research could offer the opportunity to shift the traditional therapeutic focus in cancer treatment away from targeting human effector proteins downstream of the initial stimuli – an approach which often fails due to the robust nature of cancer systems – and towards an antimicrobial strategy for certain patients.

Peer-reviewed Publications

Quality assessment and data handling methods for Affymetrix Gene 1.0 ST arrays with variable RNA integrity. Viljoen KS &  Blackburn JM. BMC Genomics 2013, 14:14 doi:10.1186/1471-2164-14-14

Manuscripts

Prospects of ‘omics-based molecular approaches in colorectal cancer diagnosis and treatment in the developing world: a case study in Cape Town South Africa. Adeola H, Goosen RW, Goldberg P & Blackburn JM. In “Colorectal Cancer – Surgery, Diagnostics and Treatment” (ISBN 980-953-307-958-0). In the press.

Copy number profiling of primary colorectal cancer in a South African cohort by Affymetrix® Cytogenetics Whole-genome 2.7M microarrays. Goosen RW, Robertson B, Goldberg P, Ramesar R & Blackburn JM. Manuscript in preparation


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