with the PPREs in the promoter region of VEGF gene
The results in this study combined with the results in some of our previous studies suggested that large amount of fatty acids transported by the elevated level of C-FABP may stimulate and activate their nuclear receptor PPARγ which triggers a chain of molecular events that lead to the up-regulation of some down-stream cancer-promoting genes, and a major such gene is VEGF167, 168, 189, 190, 231. Although it was demonstrated that androgen can mediate the up-regulation of VEGF expression in androgen-dependent cells through the Sp1/Sp3 binding site in VEGF core promoter 236, it was not previously known how PPARγ exactly up- regulated VEGF in prostate cancer cells. Some studies showed that regulatory effect of PPARγ ligands on VEGF gene expression in human endometrial cells was modulated through
PPREs in the promoter region of VEGF gene 248. Could PPARγ up-regulate VEGF expression and promote angiogenesis in prostate cancer cells through acting with the PPREs in the promoter region of VEGF gene, as it does in endometrial cells?
To answer this question, a luciferase reporter gene system was employed to test the relationship between the presence or absence of PPRE and the level of VEGF expression when prostate cancer cells were treated with PPARγ agonists or antagonists. Although the promoter region of VEGF gene is relatively long and contains many sequences of PPREs, the efficiency in promoting the reporter gene expression generated by the full length (2274bp) and the efficiency produced by a truncated (790bp) segment of VEGF promoter region were very similar 248. Thus in this study, a truncated DNA segment containing 2 PPREs, rather
than the full-length of the promoter region, was used to assess whether PPARγ up-regulates
VEGF gene through PPREs in promoter region. The pGL3-promoter-luciferase plasmid was used to make 3 reporter-gene constructs by ligating (to the up-stream of the 5’- end) 3 different DNA fragments from the VEGF promoter region: Construct A (Wild type) containing a 805bp segment of VEGF promoter region which includes 2 PPREs; Construct B (Mutant1) containing a 805bp segment of DNA from the same region with both PPREs
mutated; and construct C (Mutant2) containing a 393bp sequence of DNA from VEGF
promoter region in which both PPREs were deleted. Following cell lines were chosen to perform transient transfection experiments: 22RV1 cells, a moderately malignant cell line which expresses relatively low levels of C-FABP and PPARγ 110; PC3-M cells, which expresses very high levels of C-FABP and PPARγ 5, 115; PC3-M-3 cells, a PC3-M-derived cell line established by knocking down C-FABP gene 168; and PC3-M-PPARγ-si-H, a PC3- M- derived cell line established by suppressing PPARγ expression (section 3.8.1).
When Wild type and the control plasmids were co-transfected into the 22RV1 cells, which expressed low levels of PPARγ and C-FABP, and treated with PPARγ agonist (Rosiglitazone), the relative level of luciferase activity was significantly increased from 2.62 to 4.25; whereas in cells transfected with other constructs which did not contain PPREs, the luciferase activity was not remarkably changed (Fig. 3.28, A). Similarly, when the co- transfected highly malignant PC3-M cells, which expressed high levels of both PPARγ and C-FABP, were treated with a PPARγ antagonist (GW9662), the luciferase level was remarkably reduced to the level similar to that in Mutant1 transfectants or that in PPARγ- suppressed cells (PC3-M-PPARγ-si-H).These results suggested that increased VEGF expression in prostate cancer cells was due to the interaction between PPARγ with the PPREs in VEGF promoter region to activate and up-regulate VEGF expression.
When the co-transfected 22RV1 cells (with control vector and Wild type, Mutant1 and Mutant2 plasmids, respectively) were treated with wild type recombinant C-FABP, the level of luciferase activity of the Wild type transfectants was increased by 62%(Fig. 3.29, A). No increment was observed in either Mutant1 or Mutant2 transfectants (Fig. 3. 29, A). This result suggested that C-FABP can promote VEGF expression only at the presence of PPREs. When the same transfectants were treated with the mutant C-FABP which is incapable of binding fatty acids, no increment was observed in any of the 3 transfectants. This result indicated that it was the fatty acids transported by C-FABP that activated PPARγ and up- regulated VEGF through PPREs. This was further confirmed by the result that when C-FABP was knocking down (PC3-M-3 cells), the luciferase activity was reduced by 35% compared to the co-transfected PC3-M cells, as the suppressed C-FABP expression (Fig. 3.29, B). After treating 22RV1 co-transfectants (transfected with Wild type) with PPARγ agonist (Rosiglitazone) and recombinant C-FABP, levels of luciferase activity of VEGF promoter were increased by 60% and 55%, respectively (Fig. 3.30, A); while subjecting them to a combination of both treatments could only raise the level of luciferase activity to 65% (Fig. 3.30, A). There was no significant difference between the level of luciferase activity obtained by combined PPARγ agonist and recombinant C-FABP treatment and that obtained by each of individual treatment. This result suggested that in 22RV1 cells expressing low levels of PPARγ and C-FABP, both PPARγ agonist and recombinant C-FABP can effectively increase VEGF expression. Alternatively, levels of luciferase activities in PC3-M co-transfectants (transfected with Wild type) treated with PPARγ antagonist (GW9662), the untreated PC3- M-PPARγ-si-H co-transfectants (transfected with Wild type) and PC3-M-3 co-transfectants (transfected with Wild type) were reduced by 32.5%, 35% and 34%, respectively (Fig. 3.28, B) (Fig. 3.29, B). This result suggested that in PC3-M transfectant cells, which expressed high levels of both PPARγ and C-FABP, suppressing the biological activity of PPARγ by
either its antagonist or knocking down its mRNA by RNAi (as seen in PC3-M-PPARγ-si-H cells) can reduce the level of VEGF expression. Furthermore, suppression of C-FABP
expression in PC3-M cells can also reduce the level of VEGF (as seen in PC3-M-3). The difference between the level of luciferase activity in the untreated C-FABP-knockdown PC3- M-3 cells and that in the cells treated with PPARγ antagonist was not significantly different. This result indicated that when VEGF was already reduced by suppressing C-FABP, little further reduction can be achieved by further treatment with PPARγ antagonist. Combined these results together, it is clear that both C-FABP and PPARγ may be involved in an identical signalling pathway which regulated VEGF promoter activity in prostate cancer cells. Based on these results, the detailed route of the proposed fatty acid-C-FABP-PPARγ-VEGF axis that leading to a facilitated malignant progression of the prostate cancer cells can be shown by following illustration (Fig. 4.1):
Figure 4.1: Schematic illustration of “C-FABP (fatty acids)-PPARγ-VEGF” axis.
The excessive amount of intracellular fatty acids was transported by over-expressed C-FABP into the cancer cells to activate their nuclear receptors PPARγ which then up-regulated VEGF
gene through acting with PPREs in the promoter region of VEGF. Secreted VEGF promoted angiogenesis by facilitating vasculature network formation (paracrine pathway). It also directly promoted tumorigenicity by stimulating VEGFR-2 (autocrine pathway).
Long chain fatty acids
C-FABP
PPARγ
PPRE
VEGF gene
Secreted VEGF Endothelial cells tube formation (angiogenesis) VEGF
4.6 Androgen regulated VEGF activity in androgen-dependant prostate cancer cells