TR4 nuclear receptor enhances prostate cancer initiation via altering the stem cell population and EMT signals in the PPARG-deleted prostate cells

Abstract

A recent report indicated that the TR4 nuclear receptor might suppress the prostate cancer (PCa) initiation via modulating the DNA damage/repair system. Knocking-out peroxisome proliferator-activated receptor gamma (PPARG), a nuclear receptor that shares similar ligands/activators with TR4, promoted PCa initiation. Here we found 9% of PCa patients have one allele of PPARG deletion. Results from in vitro cell lines and in vivo mouse model indicated that during PCa initiation TR4 roles might switch from suppressor to enhancer in prostate cells when PPARG was deleted or suppressed (by antagonist GW9662). Mechanism dissection found targeting TR4 in the absence of PPARG might alter the stem cell population and epithelial-mesenchymal transition (EMT) signals. Together, these results suggest that whether TR4 can enhance or suppress PCa initiation may depend on the availability of PPARG and future potential therapy via targeting PPARG to battle PPARG-related diseases may need to consider the potential side effects of TR4 switched roles during the PCa initiation.

Keywords: PPARG; Prostate cancer; TR4.

Figure 1. 9% of PCa patients have PPARG deletion

69 PCa samples were tested by FISH. 6 out of 69 cancer specimens showed one allele PPARG deletion while none of the normal compartments of those 69 samples showed the PPARG deletion. The statistic results were calculated using Fisher's exact test. * P<0.05.

Figure 2. TR4 increases PPARG null normal prostate epithelial cell growth and transformation

(A-B) Cell growth was assayed by MTT in the PPARG null normal prostate epithelial cell line mPrE^−/−. TR4 knockdown (siTR4) compared to scramble control (Scr) shown in (A) and TR4 over-expressed (TR4) compared to vector control (Vec) shown in (B). The inset graphs represent the TR4 mRNA level by QPCR. (C-D) Anchorage independent cell growth was assayed by agarose colony formation. TR4 knockdown compared to scramble control shown in (C) and TR4 over-expressed compared to vector control shown in (D). Right panels show the quantification of the colony numbers. **P<0.01, ***P< 0.001

Figure 3. TR4 increases PPARG null normal prostate epithelial cell tumor formation

10⁶ of mPrE^−/− cells were inoculated subcutaneously in nude mice (N=4 in each group). (A) TR4 knocked-down mPrE^−/− cells (siTR4) were inoculated into the left side compared to the vector control inoculated into the right side (Scr). The mice were sacrificed at 14 weeks. The inset pictures represent the tumors removed from the mice. The quantification data of tumor weights were shown in the bottom panel. P-values < 0.05 (*) were calculated by Student's t test. (B) TR4 over-expressed mPrE^−/− cells (TR4) were inoculated into the right side compared to the vector control inoculated into the left side (Vec). The mice were sacrificed at 7 weeks. The black circles represent the xenograft tumors. The quantification data of tumor weights were shown in the bottom panel. P-values < 0.01 (**) were calculated by Student's t test.

Figure 4. TR4 increases prostate trans-differentiation in PPARG null normal prostate epithelial cell

The tumors from the xenografted nude mice in Figure 3 were collected and processed. H&E staining were performed in the scramble control (Scr), TR4 knockdown (siTR4), vector control (Vec), and TR4 over-expressed (TR4) tumor samples. The black arrow indicates the luminal structure and the red arrow indicates the muscular structure.

Figure 5. TR4 increases stem cell population in PPARG null normal prostate epithelial cell

Scramble control (Scr), TR4 knockdown (siTR4), vector control (Vec), and TR4 over-expressed (TR4) mPrE^−/− cell lines were stained by CD44 and Sca1 antibodies. The right panels next to CD44 or Sca1 show the images merged with DAPI staining.

Figure 6. TR4 increases EMT in PPARG null normal prostate epithelial cell

mPrE^−/− cell lines with TR4 knockdown (siTR4) or over-expressed TR4 (TR4) were assayed for TR4 and the EMT related proteins E-Cadherin, Vimentin, and N-Cadherin expression by Western Blot.