The Arachidonic Acid Pathway: a potential application in the diagnosis and prognosis of prostate cancer

Prostate cancer (PCa) is the most common cancer in men, and in 2012 more than 400,000 cases were diagnosed in Europe. Although the introduction of the PSA test improved the management of the disease, its low specificity has led to overdiagnosis and unnecessary treatments for men considered to be at low risk for progression of the disease. Thus, novel and reliable molecular markers are needed to improve the diagnosis of PCa and also to better differentiate indolent cases of PCa from those that will recur after initial treatment. In this thesis, we aimed to identify and validate novel metabolites and proteins markers in tissue or bio-fluids, which could improve the diagnosis and/or the prognosis of PCa. We used analytical approaches to study the role of selected metabolites and proteins, particularly along the arachidonic acid (AA) pathway.
We used targeted metabolomics approaches in serum to evaluate whether concentrations of molecules in two pathways could be used as markers for PCa. For tryptophan (Trp) and one of its metabolites, kynurenine (Kyn), we used an HPLC method with fluorescence detection on serum samples from older men having low PSA values and no PCa, as well as subjects diagnosed with PCa.
We could not identify statistical differences between the groups and thus we concluded that neither the concentrations of these metabolites, nor the ratio Kyn/Trp, could be used as markers for PCa. Next, we used a UHPLC-MS/MS method to evaluate the concentrations of AA and some of its metabolites in serum from PCa patients at different stages of the disease. We developed a method for the simultaneous quantification of six metabolites belonging to the AA pathway, and we used the results to evaluate PCa diagnosis. Interestingly, we found that a selected group of patients had higher concentrations of hydroxyeicosatetraenoic acid (HETE) metabolites, compared to the control group. We then used the same methodology on an extended and well defined group of patients having different stages of PCa, and we found that concentrations of HETE metabolites are associated with the most aggressive status of the disease. Concomitantly, we also found a reduction in the AA concentration in the same group of patients and these results (which may be related to each other) could be used as an indicator of PCa relapse after radical prostatectomy.
In tissue, we used label free quantitative proteomics to identify proteins in PCa that are associated to diagnosis and prognosis and also to evaluate whether the altered concentrations of AA and its metabolites in serum could be associated with a deregulation of particular proteins in the arachidonic acid (AA) pathway.We analysed the protein fraction from RNA isolation of 67 PCa tissue samples, consisting of 33 normal adjacent tissue (NAP) samples and 34 PCa samples, using nano-LC high resolution mass spectrometry. In addition, we validatedthe expression of four proteins by immunohistochemistry and we used tissue microarrays (TMA) to evaluate the prognostic performance of these markers.
Remarkably, we found that FASN, AGR2, and one protein of the AA pathway: TEBP, were highly upregulated in PCa. In addition, TMA indicated that both low percentage of positive AGR2 tumour cells and low percentage of positive LOX5 tumour cells, are predictors of biochemical recurrence after radical prostatectomy. These results highlight the role of proteins in the AA pathway inthe diagnosis and prognosis of PCa. We have shown that proteins and metabolites in the AA pathway play a key role in PCa.
To complement the structural characterisation of AA in mass spectrometry, we studied the fragmentation of AA in the presence of metal ions such as Ca2+ and Mg2+. We showed that the addition of these metal ions improved the fragmentation of AA in the gas-phase, and in particular such fragmentations provide the exact location of the double bonds in the carbon chain. Thus, this strategy could be further used to analyse extended sets of fatty acids and lipids using mass spectrometry, and also their role in diseases like cancer.
We conclude that we have shown that the experimental methodologies used in this thesis help to understand the role of the fatty acid metabolism and arachidonic acid pathway in PCa. Key metabolites such as HETEs and AA, as well as enzymes along this pathway, could improve the diagnosis and prognosis of PCa, and they could also become important therapeutic targets of the disease.