An alkylphenol mix promotes seminoma derived cell proliferation through an ERalpha36-mediated mechanism

Abstract

Long chain alkylphenols are man-made compounds still present in industrial and agricultural processes. Their main use is domestic and they are widespread in household products, cleansers and cosmetics, leading to a global environmental and human contamination. These molecules are known to exert estrogen-like activities through binding to classical estrogen receptors. In vitro, they can also interact with the G-protein coupled estrogen receptor. Testicular germ cell tumor etiology and progression are proposed to be stimulated by lifelong estrogeno-mimetic exposure. We studied the transduction signaling pathways through which an alkyphenol mixture triggers testicular cancer cell proliferation in vitro and in vivo. Proliferation assays were monitored after exposure to a realistic mixture of 4-tert-octylphenol and 4-nonylphenol of either TCam-2 seminoma derived cells, NT2/D1 embryonal carcinoma cells or testis tumor in xenografted nude mice. Specific pharmacological inhibitors and gene-silencing strategies were used in TCam-2 cells in order to demonstrate that the alkylphenol mix triggers CREB-phosphorylation through a rapid, ERα36-PI3kinase non genomic pathway. Microarray analysis of the mixture target genes revealed that this pathway can modulate the expression of the DNA-methyltransferase-3 (Dnmt3) gene family which is involved in DNA methylation control. Our results highlight a key role for ERα36 in alkylphenol non genomic signaling in testicular germ cell tumors. Hence, ERα36-dependent control of the epigenetic status opens the way for the understanding of the link between endocrine disruptor exposure and the burden of hormone sensitive cancers.

Figure 1. Alkylphenols stimulates TCam-2 and NT2/D1 cell proliferation in vitro and in vivo.

(A) Cells were treated by M4 concentrations ranging from 1×10⁻⁶ to 1×10⁻¹⁴ M or vehicle for 48 h and counted under inverted microscope (see text for details). The DMSO dilution retained and indicated as “vehicle” was similar to the one of M4 having the most important effect: 1 nM M4 corresponding to 0.00001% DMSO. Notably, none of the DMSO doses tested (ranging from 0.01% to 10⁻¹²%) displayed proliferation modulating effect compared to non treated cells. Values indicated are the mean of 6 counts ±SEM. *P<0.05 vs vehicle treated. (B) Exposure to M4 alkylphenol mix stimulates tumor growth in nude male mice, injected 5 times a week with a M4 dose corresponding to human exposure or with a ten-fold higher dose. Tumors were weighed after a 6-week long treatment duration. Indicated values are the mean of at least 10 tumor weights ± SEM.

Figure 2. M4 induces CREB phosphorylation in TCam-2 cells through a GPER-independent pathway.

(A) Cells were treated for 20 minutes with 1.0 nM of M4 and the level of phosphorylated CREB (P-CREB) was assessed by western-blot analyses compared to vehicle treated cells. (B) Western blot analyses of CREB phosphorylation upon exposure to 1.0 nM M4, 10 nM G1, 10 nM G15, or 1.0 nM E₂ for 20 minutes in TCam-2 cells transfected by scrambled siRNA (siCtrl), or GPER-targeted siRNA (siGPER). Alpha tubulin stands for a loading control. Quantification means ± SEM and corresponding statistical analyses from at least three similar experiments are indicated. *P<0.05 vs vehicle treated.

Figure 3. ERα36 mediates M4-induced PI3K-dependent proliferation and CREB-phosphorylation.

(A) neo-TCam-2 or sh36-TCam-2 were previously described [-] : briefly, the neo-TCam-2 cell line was transfected with a control vector whereas sh36-TCam-2 stably expresses ERα36 targeted shRNA. Both cell lines were treated by M4 concentrations ranging from 1×10⁻⁶ to 1×10⁻¹⁴ M or vehicle for 48 h and cells were counted by using an inverted microscope. Values indicated are the mean of 6 counts ±SEM. *P<0.05 vs vehicle treated. (B) neo-TCam-2 or sh36-TCam-2 cell lines were treated for 20 minutes with 1.0 nM of M4 alone or after a 30 minute pre-treatment with 0.2 µM wortmanin. The level of phosphorylated CREB was then assessed by western-blot analysis and compared to vehicle treated cells. Quantification means ± SEM and corresponding statistical analyses from at least three similar experiments are indicated. *P<0.05 vs vehicle treated. (C) TCam-2 and NT2/D1 cells were pre-treated with 0.2 µM wortmanin for 1 hour before a 48 hour exposure to 1 nM M4. Cells were then counted by using an inverted microscope. Quantification means ± SEM. *P<0.05 vs M4 treated.

Figure 4. Venn diagram of M4 regulated genes as revealed by microarray analysis in TCam-2 cells.

860 genes and 369 genes were regulated by at least two-fold (p<0.05) after 1 hour or 24 hour 1 nM M4 treatment, respectively. 264 genes were commonly regulated at both exposure times.

Figure 5. M4 represses the expression of Dnmt3A, Dnmt3B and Dnmt3L.

(A–B). TCam-2 cells were treated for 24 hours with 1.0 nM of M4 and Dnmt3A, 3B or 3L expression was assessed by real-time PCR (A) or western-blot analysis (B). (C) M4-dependent Dnmt3B and Dnmt3L down-regulation was also observed in neo-TCam-2 cells but was impaired after a 30 minute pre-treatment with 0.2 µM wortmanin or in sh36-TCam-2 cells. Quantification means ± SEM and corresponding statistical analyses from at least three similar experiments are indicated. *P<0.05 vs vehicle treated.

Figure 6. M4 modulates the expression of Dnmt3A, Dnmt3B and Dnmt3L through PI3K-dependent signaling.

(A–B). NT2/D1 cells were treated with 1.0 nM of M4 and Dnmt3A, 3B or 3L expression was assessed by real-time PCR (A) or western-blot analysis without or after a 30 minute pre-treatment with 0.2 µM wortmanin (B). Quantification means ± SEM and corresponding statistical analyses from at least three similar experiments are indicated. *P<0.05 vs vehicle treated.