Identification of Genetic Changes that Promote the Spread of Cancer in the Body
Prof P Rudland,* Prof R Sibson,* Dr R Barraclough,*
and
Mr R Zakaria *†, Mr M Jenkinson*†
University of Liverpool* and
Walton Centre NHS Foundation Trust†
Project Identification of Genetic Changes that Promote the Spread of Cancer in the Body
Background to Metastasis
The most important property of a cancer is its ability to spread and disseminate or metastasise to other parts of the body. It is this process of dissemination or metastasis that is largely responsible for the deaths of cancer patients. This is because the primary tumour can be removed successfully by surgery, but if the disseminated cancer has spread to other parts of the body, then surgery alone with not eradicate the disease. The patient is then usually treated throughout the entire body by chemicals which largely antagonise and kill growing cells. However, these chemicals are not selective; they will kill normal growing cells as well. Hence a sufficiently high dose cannot be given to kill the cancer cell without seriously harming the patient.
Discovery of Metastasis-Inducing Genes
To understand the problem of metastasis at the genetic level, Prof Rudland and colleagues developed a cell line from a benign rat breast tumour which, when reintroduced into the same colony of rats produced only benign nonmetastatic breast tumours(Ref 1, 2). This cell line was then used as a test-bed to identify metastasis-inducing genes (MIGs) by introducing them by a process called transfection into the genome of these benign cells. These cells were then reintroduced into the rats to identify those genes that induced the benign cells to disseminate and metastasise. (Ref 3-6). Antibodies were then raised to the protein products of these genes, the so called metastasis-inducing proteins or MIPs and used to locate and measure the levels of these proteins in primary tumours of breast cancer patients. When these proteins and their mRNAs occurred in statistically higher levels in the primary breast carcinomas, the patients were found to die prematurely of metastatic disease(Ref 6-10). Similar results are being reported in lung, colon, bladder, prostate and ovary cancers.
Aim of Project
Now we need to know why these metastasis-inducing genes are preferentially turned on in some patients contributing to their early death, but not in others enabling them to survive much longer.
Plan of Project
Previously in pilot studies in collaboration with the Beijing Genomics Institute in China using their high throughput DNA sequencing machines, we have identified a change in the position of one of our metastasis-inducing genes, osteopontin in a breast cancer cell line that has the ability to produce metastases in mice. This change was not observed in a nonmetastatic breast cell line. Now we wish to confirm this finding in another metastatic breast cancer cell line called MDA-MB-231 and in two human brain secondary tumours that have been produced by metastatic spread from two patients with primary breast cancer.
At a technical level, the DNA from each of the 3 samples will be fragmented into thousands of small pieces and libraries will be constructed containing all these fragments. To minimise the likelihood of systematic bias in sampling the library of all the different pieces of DNA, four libraries with synthetic matching ends joined to appreciably-sized fragment of sample DNA (e.g 500bp) will be “constructed”. These libraries containing thousands of small fragments of the entire genome of each of our 3 metastatic samples will have their DNA sequence determined in parallel by reading in from their ends on an Illumina HiSeq 2000 machine. Then each DNA fragment will be aligned on the known DNA sequence of the human genome using computer programmes. These programmes will then identify any changes in single DNA residues called point mutations, up to major rearrangements of entire genes on single chromosomesor even between chromosomes, called major structural variations. Thus we are seeking to confirm our original observation in a metastatic human cell line of a major structural variation which translocates one of our metastasis-inducing genes from an inactive to an active region of the genome in another cell line and in human brain metastases of 2 patients with breast cancer. Undoubtedly we shall also discover other differences in and around our metastasis-inducing genes which lead to increases in the production of their protein products. One such example which we have already observed in primary breast cancer is a single base change in the promoter ahead of one of our metastasis-inducing genes which causes an increased synthesis of its protein product and a change in response to chemotherapy in the patient.
In this way genetic differences in three specimens will be identified between the metastatic breast cancer and the normal genome in those genes which are believed to be responsible for metastasis. The results of this project will then lay the basis for a much larger programme investigating these same changes in many more brain metastases and comparing them with their primary tumours. The importance of the results will be to identify genetic changes in the primary tumour which predisposes it to metastasise and kill the patient. In this way aggressive chemotherapy can be given to such high risk patients and at the same time protecting those patients at much lower risk from such therapy. In the long run it may be possible to intervene in the production of the metastasis-inducing protein using targeted gene therapy to prevent their production.
Costs
Isolation of DNA from 3 samples (£70 each) =£210
Full DNA sequencing and bioinformatics analysis
at Beijing Genomics Institute (£3180 each)=£9790
+ Shipment (£250)
Total=£10,000
Selected References
(1)Bennett DC, Peachey LA, Durbin H and Rudland PS. A possible mammary stem cell line. Cell 15, 283-298 (1978).
(2)Dunnington DJ, Monaghan P, Hughes CM and Rudland PS. Phenotypic instability of rat mammary tumour epithelial cells. J. Natl Cancer Inst, 71, 1227-1240 (1982).
(3)Davies BR, Davies MPA, Gibbs FM, Barraclough R and Rudland PS. Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium binding protein but not with the oncogene, EJ ras-1. Oncogene 8, 999-1009 (1993).
(4)Oates AJ, Barraclough R and Rudland PS. Identification of osteopontin as a metastasis-related gene product in a rodent mammary tumour model. Oncogene 13, 97-104 (1996).
(5)Liu D, Rudland PS, Sibson DR and Barraclough R. Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells. Br J Cancer 87, 423-431 (2004).
(6)Wang G, Platt-Higgins A, Carroll J, de Silva Rudland S, Winstanley J, Barraclough R and Rudland PS. Induction of metastasis by S100P in a rat mammary model and its association with poor survival of breast cancer patients. Cancer Res 66, 1199-1207 (2006).
(7)Rudland PS, Platt-Higgins A, Renshaw W, West CR, Winstanley JHR, Robertson L and Barraclough R. Prognostic significance of the metastasis-inducing protein S100A4 in human breast cancer. Cancer Res 60, 1595-1603 (2000).
(8)Rudland PS, Platt-Higgins A, El-Tanani M, de Silva Rudland S, Barraclough R, Winstanley JHR, Howitt R and West CR. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res 62, 3417-3427 (2002).
(9)Barraclough D, Platt-Higgins A, de Silva Rudland S, Barraclough R, Winstanley J, West CR and Rudland PS. The metastasis-associated anterior gradient 2 protein is correlated with poor survival of breast cancer patients. Am J Pathol. 175, 1848-57 (2009).
(10)Rudland PS, Platt-Higgins A, Davies LM, de Silva Rudland S, Wilson JB, Alawadani A, Winstanley J, Liu Barraclough D, Barraclough R, West CR and Jones NJ. Significance of the Fanconi Anaemia FANCD2 protein in sporadic and metastatic human breast cancer. Am J Pathol. 176, 2935-2947 (2010).
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