Bisphenol A promotes testicular seminoma cell proliferation through GPER/GPR30

Dear Sir, Testicular germ cell tumors (TGCT) are the most frequent cancer affecting young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown. Epidemiological, clinical and molecular data1, 2 have suggested that fetal exposure to environmental endocrine disruptors with estrogenic effects could participate in testicular germ cell carcinogenesis by influencing the fate of germ stem cells that share molecular markers with the malignant germ cells.2 However, no animal model supports this hypothesis, and even estrogen dependency of TGCT has not yet been fully demonstrated. Using the JKT-1 cell line derived from a human testicular seminoma3 and seminoma tumors, the most frequent TGCTs, we have previously reported that seminoma cells expressed functional aromatase,4 which is able to convert testosterone into estradiol as well as estrogen receptor beta (ERβ) but not ERα.4 Estradiol 17β (E2) at physiological concentrations was able to suppress in vitro JKT-1 cell proliferation through ERβ.4 However, when E2 was conjugated to bovine serum albumin (E2-BSA), which does not cross the membrane, the effect observed was completely different. E2-BSA was able to stimulate JKT-1 cell proliferation by activating the PKA pathway with a rapid (15 min) phosphorylation of the transcription factor cAMP response element binding protein (CREB), through a membrane G-protein-coupled receptor (GPCR). Similar results were obtained with bisphenol A (BPA) at low (pM to nM) concentrations.5 BPA, a well-recognized estrogenic endocrine disruptor, is used as the monomer to manufacture polycarbonate plastic, and it is released from resin lining of canned foods or beverages or from dental sealants. It is believed to be involved in developmental, reproductive and malignant diseases by mimicking the natural hormone E2 and interfering with endogenous endocrine regulation at specific periods, such as during fetal growth.6 However considering its weak affinity for the classical ERs, which is 1,000–2,000 times lower than E2, it appeared difficult to explain its endocrine disruption in animals and human at the low picomolar or nanomolar concentrations widespread in the environment.6 Thus, some have contested its real estrogenic effect,6, 7 and it has been suggested that BPA could act through completely different receptors.6 To identify the receptors involved in the promoting action of BPA that we observed on seminoma cells, we have screened specific agonists and antagonists. Here, we report that BPA at very low doses, environmentally relevant and found in human blood and urine (nM), was able to stimulate in vitro seminoma cell proliferation through GPER, the G-protein-coupled estrogen receptor. BPA induced a proliferative effect opposite to that induced by E2 and which was not neutralized by ICI 182780, a pure ER antagonist. This effect was identical to the one produced by G1, a specific agonist of GPER.8 G15, a specific antagonist of GPER,9 was able to inhibit BPA-induced promotive effect and even able to reduce the level of proliferation in the presence of BPA when compared to control (Fig. 1). GPER also known as GPR30 is a membrane GPCR completely different from ERβ or ERα, which has been already identified on several malignant cells such as breast and ovarian cancers.8, 10 Effects of estrogens on human testicular seminoma cell (JKT-1) proliferation in vitro. Cell proliferation is assessed after 24-hr exposure to different compounds: estradiol 17β (E2), estradiol 17β linked to BSA (E2-BSA), bisphenol A (BPA), G1 (a specific agonist of GPER), alone (each at 10−9 M) or after a 90-min pretreatment with ICI-182780 (ICI, a specific antagonist of ERβ) or G15 (a specific antagonist of GPER, each at 10−9 M). Values shown are expressed in percent change in cell number compared to control (steroid-free medium containing DMSO for bisphenol A or medium containing ethanol for estrogens, G1 and G15) given as the mean ± SE of three independent experiments. Cell counting was performed using a Malassez hemocytometer and confirmed using Vi-CELL automate (Beckman Coulter, Fullerton, CA). *p < 0.05; **p < 0.001. Our results illustrate the estrogen dependency of human seminoma cells, which express different estrogens receptors (here, ERβ and GPER) like other estrogen-dependent cancers (breast, ovary and endometrium). These receptors can be activated through two different genomic and nongenomic pathways by estrogens and xenoestrogens depending on their respective affinity and the cell microenvironment (expression level and cofactors). In relation to JKT-1 seminoma cells, E2 that has a low affinity for GPER and a high affinity for ERβ induces a suppressive effect. BPA with a low affinity for ERβ and a high affinity for GPER induces a promoting effect. Fetal exposure to BPA in pregnant rodents has been shown to be associated with premalignant lesions in adult breast or prostate cells.6 Free BPA has been found in human cord blood6 illustrating mother exposure and placenta transfer. However whether fetal exposure could participate to seminoma carcinogenesis remains completely to be demonstrated. However, we show here that BPA, a weak estrogenic endocrine disruptor with a low affinity for classical ERs, is able in vitro to promote at very low concentrations (nM), seminoma cell proliferation through GPER, a membrane GPCR, unrelated to ERs. This result will probably modify the way to assess human bisphenol toxicity. G15, a GPER antagonist, neutralizes this effect and reduces proliferation in the presence of BPA, and therefore, it may be a helpful adjuvant in the treatment of TGCT.