DIMENSION IDENTIDAD DE MARCA IMAGEN DE MARCA (META) (REALIDAD ACTUAL)

When 22RV1 cells were co-transfected with different constructs, Wild type, Mutant1 and Mutant2 produced increases in relative luciferase activities (Fig. 3.27) by 260%, 240%, and 30%, respectively. The increment produced by Wild type was similar to that by Mutant1. Mutant1 or Mutant2 did not contain PPREs and luciferase activities did not change when the Mutant1 or Mutant2 transfectants were stimulated with PPARγ agonist (Rosiglitazone). Thus these increment caused by Mutant1 was not produced by the PPREs in the promoter region. This result suggested that there are some other elements, rather than PPREs, in the promoter region of the VEGF gene which can up-regulate VEGF expression in the 22RV1 prostate cancer cells. When PC3-M cells were transfected with different DNA constructs, both Wild type and Mutant1 produced increase in luciferase activity. While 3.3-fold increment was seen when Wild type was transfected, Mutant1 produced a 2-fold increase when compared with control. Mutant1 also produced some increments in PC3-M-PPARγ-si-H and PC3-M-3 transfectants. This result further confirmed that except PPREs, there are some other elements in the promoter region of VEGF gene that can modulate the VEGF expression in the highly malignant PC3-M cells (Fig. 3.27).

The occurrence and development of prostate cancer has been related to the level of circulating male hormone. Thus androgen receptor played a key role in the carcinogenesis of prostate cells 315-317. Prostate cancer cells are generally sensible to the initial androgen blockade treatment, but in the majority of cases, cancer relapses in about 2 years after the initial androgen blockade therapy with a more aggressive form (named castration resistant prostate cancer) (CRPC). The growth and expansion of castration resistant prostate cancer does not completely depend on androgen supply anymore, but in large number of circumstances, continuation of androgen blockade therapy can still suppress the malignant

progression 318, 319. The molecular pathology involved in how the androgen-dependent cancer cells were transformed to androgen-independent or castration resistant cells is not fully understood. Currently several hypothesized mechanisms were proposed to explain this transition, and the most common theory to explain this transition is the androgen receptor (AR) sensitivity amplification theory 320, 321. This hypothesis proposed that when the cancer cells is deprived of androgen supply in the initial round of therapy, cells try to maximise their survival ability by increasing the sensitivity of AR to make use the small amount of androgen escaped from the blockage and thus some survived cells with an increased ability of using micro-quantity of androgen become dominant and castration resistant 322, 323. Some studies also suggested that mutations of AR gene could increase sensitivity to androgen stimulation 324, 325

. Although each current theory can explain certain aspects of this complicated molecular pathology process, no single theory can satisfactorily explain every aspects of the process of transition in the cancer cells from androgen-dependent to androgen-independent form. For example, if AR amplification theory is true, then re-expression of AR in AR- negative cells should increase the malignancy, but several studies on the highly malignant PC3 cells showed that the forced re-expression of AR in PC3 cells actually reduced the malignancy of cells 326-328.

In previous study, an important molecular mechanism involved in VEGF regulation was the androgen-AR initiated Specificity-protein-1 or Specificity-protein-3 (Sp1/Sp3) pathways in androgen-dependent prostate cancer cells. Sp1/Sp3 binding sites are located in the promoter region of VEGF and their regulatory effect on VEGF activity was shown in breast cancer 235, retinoblastoma cancer 329, bronchiolo-alveolar cancer 330 and pancreatic cancer 331. Oestrogen has been reported to up-regulate the expression of VEGF in breast cancer cells via acting with Sp1/Sp3 transcription sites in the core VEGF promoter 235. Similarly, androgen could

mediate the up-regulation of VEGF expression in androgen-dependent prostate cancer cells through Sp1/Sp3 binding sites in the VEGF core promoter region 236.

To investigate the possible regulatory effect of Sp1 transcription binding site on VEGF activity in androgen-dependent prostate cancer cells in comparison with androgen- independent cells, 22RV1 cells and PC3-M cells were transiently transfected with the three luciferase gene constructs. After treating 22RV1 co-transfectants (transfected with Wild type) with GW9662 and Mithramycin A, levels of luciferase activity were reduced by 21% and 34%, respectively (Fig. 3.31, A). Comparing with GW9662, Mithramycin A produced more than 1.6-times reduction in luciferase activity. Subjecting them to a combination of both treatments could reduce the level of luciferase activity to only 36% (Fig. 3.32). Thus level of luciferase activity in 22RV1 co-transfectants significantly reduced after treating with Sp1 inhibitor and PPARγ antagonist. These results indicated that in androgen-dependent 22RV1 cells, both the C-FABP-PPARγ and the androgen-Sp1 pathways played important roles in up- regulating the expression level of VEGF and the androgen-Sp1 route appeared to be even more important than the C-FABP-PPARγ route. Alternatively, when PC3-M co-transfectants (transfected with Wild type) was treated with the Sp1 inhibitor (Mithramycin A), the level of luciferase activity was hardly reduced by only 3% (Fig. 3.31, B). While level of luciferase activity in 22RV1 co-transfectants (transfected with Mutant1) was decreased by 51% after Mithramycin A treatment (Fig. 3.31, A); level in PC3-M co-transfectants (transfected with Mutant1) were only reduced by 15% after the same treatment (Fig. 3.31, B). While significant reduction in luciferase activity in PC3-M co-transfectants was achieved by GW9662 treatment (Fig. 3.28, B), no significant reduction was detected in the level of luciferase activity in PC3-M co-transfectants after treating with Sp1 inhibitor. These results suggested that in the androgen-independent PC3-M cells, the androgen-Sp1 pathway is not important anymore in up-regulating VEGF expression. The extremely high level of VEGF

expression in these cells was caused mainly through C-FABP-PPARγ route. It seemed that in the early stage of prostate cancer when the cancer cells are still responsive to androgen stimulation, the androgen-Sp1 pathway played a dominant role in promoting VEGF expression. As the cells gradually reduced their dependency on androgen supply and until ultimately loss responsive ability to androgen, the role of androgen-Sp1 pathway is gradually reduced and ultimately disappeared completely in AR-negative, androgen-independent cells (Fig. 4.2).

Figure 4.2: Schematic illustration of inter-relationship between androgen-Sp1/Sp3 and C- FABP (fatty acids)-PPARγ signalling route in up-regulating VEGF in androgen-dependant and androgen-independent prostate cancer cells.

Androgen-independent Prostate cancer cells Androgen-dependent

Prostate cancer cells

Androgen- Sp1/Sp3-VEGF pathway

C-FABP(fatty acids)-PPARγ-VEGF pathway

Increase Degree of Malignancy Increased Androgen Dependency

VEGF is a potent angiogenic factor which promotes vasculature formation of vessel network that is essential for growing and expansion of the cancer cells. VEGF can also directly promote malignancy of the cancer cells through an autocrine mechanism to simulate the receptor highly expressed on the surface of the prostate cancer cells (VEGFR-2)332, 333. Based on the results in this study and relevant results from other studies, I propose the following alternative hypothesis to explain the molecular mechanism involved in the crucial transition of the prostate cancer cells from androgen-dependant to androgen-independent state: When the cancer cells are deprived of androgen supply in the initial round of chemotherapy, the cells are desperate to seek for new sources of energy supply and under the heavy selection pressure, most of the cancer cells died due to starvation. However, some of the cancer cells may have survived under the heavy selection pressure by switching their reliance on androgen to fatty acids (transported by C-FABP) as an alternative energy source. These cells may be the so-called castration resistant cells. Although these cells can still use androgen, and although further androgen blockade will kill some more cells, it can also make some other cells more resistant to androgen deprivation by increasing and eventually, completely relying on fatty acids as their energy source. As the consequence of the increased demand of fatty acids and hence the high level of C-FABP during this process, the C-FABP-PPARγ-VEGF axis gradually increases its functional activity and eventually replace the androgen-Sp1/Sp3 pathway to become the dominant route to promote further malignant progression. Consequentially, cancer cells will ultimately become totally androgen-independent after their androgen supply is repeatedly blocked. This hypothesis is further supported by the fact that a fatty acid synthase (FASN) greatly increased in prostate cancer cells 334-337. Based on this alternative hypothesis, disrupting the C-FABP-PPARγ-VEGF axis and cutting off the alternative energy supply of the cancer cells, rather than only blocking the last drop of

androgen, should be the correct way to kill the AR-negative androgen-independent cancer cells.