Lipoxygenases and Eicosanoid Receptors in Colorectal Tumors

Chronic inflammation, endogenous mediators of inflammation and tissue regeneration play an essential role in carcinogenesis. This has most convicingly been shown in colorectal tumors where tumor development can be suppressed by non-steroidal anti-inflammatory drugs (NSAIDs). The major mechanism mediating the tumor inhibitory effect of NSAIDs was thought to consist in an inhibition of cyclooxygenases, the enzymes that catalyze the oxygenation of arachidonic acid to prostaglandins. However, recently accumulating data indicate that additional targets have to exist. Two of these are the arachidonic acid metabolizing enzyme 15-lipoxygenase (15-LOX) and peroxisome proliferator activate receptor (PPAR ) that can be activated by LOX products. Both proteins belong to larger protein families whose expression, activity and function in carcinogenesis is still largely unknown. Thorough knowledge about these proteins and the cellular mechanisms they support are a necessary prerequisite of any preventive or therapeutic intervention. This project proposes to investigate LOX- and PPAR-dependent gene expression in colorectal tumor tissue and cell lines to characterize cellular mechanisms and identify targets of prophylactic and therapeutic intervention. Expression of LOX enzymes and PPARs will be assessed and LOX product levels will be measured both in tumor tissue and in a panel of cell lines. To study the cellular mechanisms supported by LOX enzymes, their products and the corresponding PPAR receptors we will overexpress the genes in selected cell lines. The overexpressing cell models will be used to analyze LOX- and PPAR-dependent expression of genes related to growth, tumor progression and metastatic potential. The biological effects will be studied with regard to cell cycle control, cell contact proteins, cell migration and proteolytic activity. In a parallel set of experiments LOX enzymes and PPARs will be downregulated by anti-sense approaches and the cell biological consequences analyzed.. From the results of these experiments we plan to develop a diagnostic micro-array to identify targets of therapeutic or prophylactic intervention. It is expected that such a tool will be useful to devise new individualized strategies of chemoprevention and therapy of colorectal tumors.

Lipoxygenases (LOX) are a family of enzymes catalysing the oxidation of unsaturated (essential) fatty acids at highly specific positions to produce lipid mediators, that have diverse functions in growth regulation, cell death, inflammation and cancer. This project focused on one of the LOX enzymes - 12(S)-LOX that inserts the hydroxy- group in position 12 of arachidonic acid so that its main product is 12(S)-hydroxyeicosatetraenoic acid [12(S)- HETE]. Both enzyme and product have been identified as pro-tumourigenic factors in several tumour types. This project investigated its involvement in colorectal cancer. Our results indicate that the role of 12(S)-LOX in the development of colon cancer is more complex than for other malignancies. The gene is up-regulated in only about 20% of the colon tumour specimens analysed, while it is down-regulated in 60%. This indicates that 12(S)-LOX can have both pro-tumourigenic and anti-tumourigenic effects. We have constructed cell line models to investigate 12(S)-LOX effects and found similarly ambiguous effects of 12(S)-LOX and 12(S)-HETE on the cellular level affecting cell growth and the expression of metastasis- related genes. We found that expression of the cell surface molecules E-cadherin, integrin ß1 and DCC and of the matrix protein laminin 1 was down-regulated. This permits the detachment of cells from the primary tumour and stimulates metastasis. In addition the expression of vascular endothelial growth factor is increased stimulating the invasion of tumour vasculature - a prerequisite of tumour growth and metastasis. On the other hand we also observed anti-tumourigenic effects: tissue-degrading enzymes that may further tumour invasion were down- regulated by 12(S)-LOX. Growth was only minimally affected and growth-related genes were down-modulated. Together our results indicate that 12(S)-LOX may be pro- or anti-tumourigenic depending on the cellular background. 12(S)-LOX has frequently been regarded as a target for the therapy and prevention of cancer, but its role and interaction with cellular factors need to be understood before an intervention regimen can be planed. Our results supply information necessary to characterise 12(S)-LOX effects and demonstrate that interacting cellular factors still have to be investigated.