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. 2018 Apr 5;39(4):546-555.
doi: 10.1093/carcin/bgy020.

Ethnic differences in TGFβ-signaling pathway may contribute to prostate cancer health disparity

Bethtrice Elliott  1 , DeAdra L Zackery  1 , Vanessa A Eaton  1 , Re'Josef T Jones  1 , Fisseha Abebe  1   2 , Camille C Ragin  3 , Shafiq A Khan  1
Affiliations

Ethnic differences in TGFβ-signaling pathway may contribute to prostate cancer health disparity

Bethtrice Elliott et al. Carcinogenesis. .

Abstract

Epidemiological studies show that the incidence and mortality rates of prostate cancer (PCa) are significantly higher in African-American (AA) men when compared with Caucasian (CA) men in the United States. Transforming growth factor β (TGFβ) signaling pathway is linked to health disparities in AAs. Recent studies suggest a role of TGFβ3 in cancer metastases and its effect on the migratory and invasive behavior; however, its role in PCa in AA men has not been studied. We determined the circulating levels of TGFβ3 in AA and CA men diagnosed with PCa using ELISA. We analyzed serum samples from both AA and CA men diagnosed with and without PCa. We show that AA PCa patients had higher levels of TGFβ3 protein compared with AA controls and CA patients. In fact, TGFβ3 protein levels in serum were higher in AA men without PCa compared with the CA population, which may correlate with more aggressive disease seen in AA men. Studies on AA-derived PCa cell lines revealed that TGFβ3 protein levels were also higher in these cells compared with CA-derived PCa cell lines. Our studies also reveal that TGFβ does not inhibit cell proliferation in AA-derived PCa cell lines, but it does induce migration and invasion through activation of PI3K pathway. We suggest that increased TGFβ3 levels are responsible for development of aggressive PCa in AA patients as a consequence of development of resistance to inhibitory effects of TGFβ on cell proliferation and induction of invasive metastatic behavior.

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Figures

Figure 1.
Figure 1.
Circulating levels of TGFβ3 in men diagnosed with and without PCa (control group) in AA and CA men. Data represents the concentrations of TGFβ3 (pg/ml) determined in each serum sample by ELISA analysis. In the box plots, the horizontal black line marks the median. The total number (N) of participants per group are shown at the bottom within the graph. The Mean ± S.E., median, minimum, maximum and IQR are presented at the bottom outside the graph. ‘a–c’ denote significant (P < 0.05) differences of TGFβ3 levels between the indicated groups. IQR, interquartile range; SE, standard error.
Figure 2.
Figure 2.
Expression of AR and TGFβ signaling components in PCa cell lines derived from CA and AA patients. (A) Expression of TGFβ signaling components’ mRNA determined by RT-PCR in CA- (LNCaP and DU145) and AA- (E006AA, E006AA-HT, MDA-2A and MDA-2B) derived PCa cells. (B) Real-time qPCR analysis of TGFβ1 and TGFβ3 mRNA expression and (C) Expressions of AR and TGFβ signaling component proteins determined by western blot analysis. (D) Densitometry analysis of protein levels normalized to α-Tubulin. LNCaP cells were used as AR (+) control, and DU145 cells were used for AR (−) control. L19, GAPDH and α-Tubulin were used as the loading control for mRNA and protein analysis, respectively. Data represent at least three independent experiments. ‘a–c’ denote significant differences (P < 0.05) among different treatment groups.
Figure 3.
Figure 3.
Effects of TGFβ1 and -β3 on cell proliferation, migration and invasion in PCa cell lines. (A) DU145, E006AA, E006AA-HT, MDA-2A and MDA-2B cells were treated with TGFβ1 and -β3 (5 ng/ml) for 5 days, and the cell number was determined using a Cellometer. Each bar represents the mean ± SEM from at least three independent experiments. ‘a–f’ denote significant differences (P ≤ 0.002) among various cell lines and treatments. (B, C) TGFβ stimulation promotes (B) migration and (C) invasion in E006AA and E006AA-HT cells. Cells were seeded at appropriate densities and allowed to migrate or invade for 24 or 48 h, respectively. TGFβ1 and -β3 (5 ng/ml) were used as chemoattractants, and EGF (10 ng/ml) was used as a positive control. DMEM with 0.1% BSA + 5% FBS was used for the control cells. Data represent at least three independent experiments. ‘a–d’ denote significant differences (P ≤ 0.05) among different treatments and cell lines. (D) Representative images of migrated E006AA cells depicted in (B).
Figure 4.
Figure 4.
The effects of TGFβ on the phosphorylation of AKTser473 and the migration and invasion of AA-derived PCa cells. (A, D) TGFβ1 and -β3 induces the phosphorylation of AKTser473 in E006AA and E006AA-HT cells. PI3K inhibitor blocks TGFβ-induced pAKTser473. Cells were treated with TGFβ1 and -β3 (5 ng/ml) in the presence or absence of specific inhibitors of βRII (LY2157299; 10 µM), Smad3 (SIS3; 3 µM) and PI3K (LY294002 10 µM). Cells treated with EGF were used as positive controls. Total (pan) AKT was used as a loading control. (B, C, E, F) TGFβ promotes cell migration and invasion of E006AA and E006AA-HT cells via the PI3K-signaling pathway. Cells were treated with TGFβ1 and -β3 in the presence or absence of the PI3K inhibitor (LY294002) and allowed to migrate or invade for 24 and 48 h, respectively. Data represent at least three independent experiments. ‘a–d’ denote significant differences (P < 0.001) among different treatment groups.
Figure 5.
Figure 5.
The effect of TGFβ on MMP2 and MMP9 expression in AA-derived PCa cells. E006AA and E006AA-HT cells were treated with TGFβ1 and -β3 in the presence or absence of TGFβRII inhibitor (LY2157299) for 24 h. (A) RT-PCR and (B) qPCR analysis of MMP2 and MMP9 mRNA expression and (C) western blot analysis of MMP2 and MMP9 protein levels in AA-derived PCa cells. (D) Densitometry analysis of protein levels normalized to α-Tubulin. Data represent at least three independent experiments. ‘a–d’ denote significant differences (P < 0.001) among different treatment groups.
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