Cutaneous fatty acid-binding protein (C-FABP) or FABP5, is a FABP family member that can bind to long chain fatty acids with high affinity. C-FABP was identified by our research group as a gene involved in malignant progression of prostate cancer and able to promote the growth of primary tumours and induce metastasis when transfected into rat benign Rama 37 model cells. It was demonstrated that C-FABP was a prognostic marker for patient outcome and a target of tumour-suppression for prostate cancer. As an initial step to understanding the complex molecular mechanisms involved in the cancer promoting activity of C-FABP, this study investigated the possible relationship between tumorigenicity-promoting activity of C-FABP and its fatty acid-binding capability. After single and double mutations were generated in the fatty acid binding motif of the C-FABP cDNA, wild type and mutant C-FABP cDNAs were excised from the mammalian vector pIRES2-EGFP and inserted into pBluescript cloning vector to generate a complimentary restriction sites at both ends of the cDNAs. The C-FABP cDNAs were excised from the pBluescript vector with KpnI and PstI and inserted into pQEs expression vector to form three constructs that express the wild type and two mutant C-FABPs (C-FABP-WT, C-FABP-R109A and C-FABP-R109/129A), respectively. SDS-PAGE and sequence analysis confirmed the correct insertion of C-FABPs into expression vector pQE. The pure recombinant proteins were subsequently produced and purified. The importance of fatty acid-binding activity to the cancer promoting function was assessed by comparing the cancer promoting abilities exerted by C-FABPs with different fatty acid-binding capabilities. To test whether the recombinant proteins produced were biological active, the fatty acid binding ability of wild type and mutant C-FABPs were tested. When fatty acid binding ability of the wild type C-FABP is set at 1, the average fatty acid binding ability the C-FABP-R109A and C-FABP-R109/129A were significantly reduced to 0.32% and 0.09%, respectively (Student t-test, P<0.005). These results suggested that fatty acid binding ability of C-FABP depends on the structured integrity of the binding motif. Thus, changing one amino acid in the motif significantly reduced the fatty acid binding ability by 68%, and changing two amino acids almost completely abolished the fatty acid binding ability of C-FABP. To access the importance of the structural integrity of the fatty acid binding motif to the tumorigenicity-promoting activity of C-FABP in prostate cancer, the effect of wild type and mutant C-FABPs on cell proliferation, invasion and colony formation as indication of tumourigencity were analysed. The average growth rate of cells stimulated with C-FABP-WT was significantly increased by 17% (Student t-test, P<0.05) when compared to control cells. Whereas, the average growth rate of cells stimulated with C-FABP-R109A and C-FABP-R109/129A were significantly reduced by 33% and 47%, respectively (Student t-test, P<0.005) when compared to control cells. The invasiveness of cells stimulated with C-FABP-WT, C-FABP-R109, C-FABP-R109/129A and the control cells were 256±40, 163±32, 80±26 and 96.6±15.2, respectively. The number of invaded cells stimulated with C-FABP-WT was the highest in all cell lines and more than 2.6-fold higher than the number of invaded cells from control (Student t-test, P<0.05). The average number of colonies produced in the soft agar by selected cells stimulated with C-FABP-WT, C-FABP-R109A, C-FABP-R109/129A and control were 1050±132.29, 283±76.38, 157±38.1 and 155±68.74, respectively. In comparison with the control, the average number of colonies produced by C-FABP-R109A stimulated cells was increased by 45% (Student t-test, P<0.01) whereas the average number of colonies produced by C-FABP-R109/129A stimulated cells was at same level as the control cells (Student t-test, P>0.5). The most significant change occurred in C-FABP-WT stimulated cells that produced more than a 6.7-fold (85%) increase in the number of colonies formed in soft agar when compared to controls (Student t-test, P<0.001). These results showed that the increased wild type C-FABP stimulation in prostate cell line significantly increased cell proliferation, invasiveness, and tumorigenicity. Whereas, the increased expression of both mutant forms of C-FABP did not significantly affect these characteristics. Overall, this study has confirmed that the biological potential of C-FABP to promote tumourigencity of prostate cancer cells depends on its ability of binding to fatty acids. Thus, C-FABP may facilitate cancer development through a mechanism involving transportation of intracellular fatty acids into cells. These results were supported by our recent data obtained from in-vivo studies performed in a nude mouse model.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:559410 |
| Date | January 2011 |
| Creators | Malki, Mohammed Imad |
| Contributors | Ke, Youqiang. ; Foster, Christopher S. |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/5213/ |
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