It has been suggested that the environmental factor BPA may play an important role in the initiation (Ho et al. ) and progression of PCa and in hormonal therapy bypass (Wetherill et al. ). At the level of PCa cells, BPA was able to induce androgen-independent tumor cell proliferation and reduced therapeutic efficacy in xenograft models (Wetherill et al. ). While these data point toward the potential for BPA to assist tumor cells in escaping therapy, the molecular mechanisms of this process were not well-known.
This report demonstrates for the first time that the environmentally relevant concentrations of BPA (1–10 nM) induce cell migration by modulating the cell calcium signalling. These data highlight the previously unrecognized action of BPA in the progression of human PCa, thereby providing strong support for the growing recognition of the adverse effects of BPA on human health.
Invasion and metastasis are major events underlying cancer morbidity and mortality (Molloy and Van ’t [Veer 2008]). Because of the widespread metastasis in advanced cancer patients, where a resistance is observed to conventional therapies, the mortality rate remains extremely high and warrants new strategies to intervene in the metastatic cascade. Thus, an enhanced understanding of the molecular events in the pathogenesis of PCa will offer improved diagnosis, prognosis, therapy and prevention measures of the disease, that will ultimately help us to eliminate PCa metastasis. A common regulatory point in several signal transduction pathways is intracellular calcium homeostasis. One approach could be to focus on the intracellular signalling pathways underlying the metastatic process. Several data have clearly shown the involvement of calcium entry in cancer and non-cancer cell migration (Bisaillon et al. ; Li et al. ; Schaff et al. ; Yang et al. ).
In the present work, we present conclusive evidence for the first time that the pre-treatment of human PCa cells with environmentally relevant concentrations of BPA (1–10 nM) induces their migration (Figure 1). By calcium imaging technique, we show that BPA pre-treatment induces an amplification of Store-Operated Calcium Entry (SOCE) in LNCaP cells (Figure 2). RT-PCR and Western blot experiments allowed us to identify those ion channel proteins up-regulated by BPA pre-treatments. These channels include Orai1, a protein known to constitute an important actor in SOCE in various cell systems including human PCa cells (Figure 4). Meanwhile, in our studies, we failed to observe any direct effect of BPA on the rate of basal calcium (Figure 2) whereas in other cell systems, BPA or its derivatives induced a calcium increase. In TM4 Sertoli cells, a direct application of a derivative compound of BPA, Tetrabromobisphenol A (TBBPA), a commonly used brominated flame retardant (BFR), induced an increase in the basal free calcium rate originating from internal stores (Ogunbayo et al. ). In pituitary tumor cells (GH3/B6/F10 rat Somatomammotropes), Kochukov et al. () showed that BPA at a 1 nM concentration induced a great increase in Ca2+ oscillation frequency, the activation of MAPK pathways (ERK1/2) and subsequently a PRL release (Kochukov et al. ). Similar results were reported by Bulayeva et al. () and Wozniak et al. () where the authors demonstrated that the BPA-induced Ca2+ influx was strictly dependent on membrane Estrogen Receptor (mER-α) and mediated by L-type voltage-gated Ca2+ channels in pancreatic β cells (Bulayeva et al. ; Wozniak et al. ). The LNCaP cells used in our present work do not express L-type voltage-gated Ca2+channels (non-excitable cells). This is probably the reason why a direct application of BPA on LNCaP and LNCaP C4.2 cells failed to induce a direct calcium response.
In the present work, when the human PCa cells were pre-treated with BPA (1–10 nM), an increase in SOCE and a remodelling of ion channel expression was observed. The alterations of the ion channel expression could be mediated by a stimulation of a signal transduction pathways leading to the activation of nuclear transcription factors. Published data suggest several transduction pathways activated by BPA. In PCa cells, BPA has been shown to be an agonist for mutant androgen receptor (AR-T877A) expressed in recurrent PCa (Wetherill et al. [2002,, 2005,, 2006]), and in the LNCaP cell line used in our studies. According to the authors, BPA induces cell proliferation in cells expressing the mutated AR. The clinical ramifications of BPA activating tumor-derived mutant ARs and inducing androgen-independent tumor cell proliferation may be substantial, as BPA can reduce therapeutic efficacy in xenograft models (Wetherill et al. ). In our experiments, the DHT induced the expression of Orai1 (Figure 4C) and BPA appears to mimic these effects on the canonical AR ligand (DHT). However, all the effects of BPA could not be mediated by the activation of AR. In a recent work, Hess-Wilson et al. () showed clearly that BPA and DHT elicited distinct transcriptional signatures in PCa cells expressing the BPA-responsive mutant AR-T877A, even if some common genes were activated by both DHT and BPA in LNCaP cells (Hess-Wilson et al. ). These observations could explain the cell migration and Orai1 expression induced by BPA in androgen-independent human PCa cells PC-3 (Figure 8), where the AR is absent. BPA could thus activate other signal transduction pathways than the AR activation, to induce the effects observed in our studies on androgen-insensitive PCa cells. The study of the involvement of other transduction pathways than those involving the AR receptor (growth factor signalling pathways, …) in the effects of BPA in androgen-dependent and androgen-independent cells needs further extensive investigations in the future. In this context, BPA was reported to induce the phosphorylation of extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear translocation of the nuclear factor (NF)-κ B, in mouse hippocampal HT-22 cells (Lee et al. ). Interestingly, functional NF-κ B-binding sites in promoter regions of STIM1 and Orai1 have been identified and the expression of the Orai1 calcium channel was reported to be positively modulated by NF-κB (Eylenstein et al. ). Subsequently, the store-operated Ca2+ entry was similarly increased by overexpression of p65/p50 or p65/p52, and decreased by treatment with the NF-κ B inhibitor, Wogonin. BPA could thus interfere with the growth factors' signal transduction to activate the PI3K/AkT pathway and thereby induce the activation of the NF-k B transcription factor, leading in turn to an up-regulation of the expression of ion channels including Orai1. In this context, authors have shown that the activation of mER-α induces the activation of the PI3K/Akt signalling cascade (Simoncini et al. ; Stirone et al. ) and BPA is shown to activate mER-α (Bouskine et al. ; Quesada et al. ). The activation of the mER-α in androgen-dependent (LNCaP) and androgen-independent (PC-3) PCa cells may thus induce the PI3K/Akt signalling cascade which leads to the activation of the NF-kB transcription factor and Orai1 gene expression. Several works have also demonstrated the stimulation of the PKA/CREB pathways by nanomolar concentrations of BPA through the activation of mER-α (Bouskine et al. ; Quesada et al. ). However, the involvement of this pathway in the expression of ion channels needs further investigation. As shown in Figure 5, BPA pre-treatment induced an increase in BKCa and IKCa1 Ca2+-activated potassium channel expression in LNCaP cells. We previously showed that the IKCa1 Ca2+-activated potassium channels are involved in SOCE in LNCaP and PC-3 human PCa cells (Lallet-Daher et al. ). These potassium channels could constitute a functional complex with Orai1 protein to promote calcium entry and cell migration. A study is in progress in our lab to show the functional up-regulation and involvement of these Ca2+-activated potassium channels (IKCa1 and BKCa) in cell migration in the BPA-treated LNCaP and PC-3 cancer cells.
Receptor-mediated activation of phospholipases C by the factors present in the serum leads to IP3-mediated depletion of Ca2+ from the ER, which in turn stimulates Ca2+ influx through the plasma membrane involving Orai1/STIM1 complex formation (Varnai et al. ). Recent works have elegantly demonstrated that STIM1, Orai1, and SOCE play critical roles in the migration of a number of cell types in cancer and non-cancer cells (Bisaillon et al. ; Li et al. ). These observations support our data where BPA, by up-regulating the ion channels expression increases the SOCE developed by PCa cells in response to factors present in serum.
Our data show clearly that the BPA-induced cell migration is dependent on the calcium entry and the use of pharmacological tools suggests the involvement of SOCE channels in the effects of BPA on cell migration (Figure 7). Increase in cytoplasmic calcium induced by BPA may have several types of impacts which trigger cell migration, including the induction of the up-regulation of their gene and protein expression, secretion and the activation of the enzymes such as metalloproteinases (MMP2, MMP9), which are involved in cell migration. The MMP proteins are clearly shown to be dependent on calcium for their expression (calcium/Calcineurin/NFAT, …), their processing and their activity (Collier et al. ; Mukhopadhyay et al. ; Stetler-Stevenson et al. ). These observations suggest that the increase in calcium entry induced by BPA pre-treatment could promote all these processes leading to cell migration.
For the first time, we also demonstrate the expression of Orai1 proteins in human PCa tissues (Figure 4F). As shown, a strong immuno-staining of Orai1 protein was found in epithelial cells of the acini and also in the stromal cells. Thus, stromal cells could also be influenced by BPA impregnation. Given the importance of the epithelium-stroma (reactive stroma) in the progression of cancer, the potential effects of BPA on calcium signalling and on the secretion of growth factors by these cells need further investigation.