The androgen receptor (AR) is the classical target for prostate cancer treatment [1
], and estrogens and their receptors have recently been implicated in prostate cancer development and tumor progression [2
]. Initially, prostate cancer depends on androgen to evolve, but it can gradually progress to an androgen-independent form of the disease, also known as castration-resistant prostate cancer (CRPC) [2
]. The molecular mechanisms involved in this stage of the disease are not fully understood and the current therapies are insufficient to improve the survival of patients.
Previous studies from our laboratory have already shown that, in androgen-independent prostate cancer cells PC-3 [6
] and DU-145 [7
], estrogen receptors (ER) ERα (ESR1) and ERβ (ESR2) are mostly located outside the nucleus of these cells, indicating the activation of rapid signaling pathways. In fact, the activation of these receptors increases the phosphorylation of ERK1/2 (extracellular signal-regulated kinase1 and 2) in both cell lines [6
] and the phosphorylation of AKT (serine/threonine kinases) in PC-3 cells [8
]. Furthermore, in PC-3 cells, the activation of ERβ decreases N-cadherin [9
] and increases non-phosphorylated β-catenin levels [8
]. The activation of ERβ promotes the increase of migration, invasion, and anchorage-independent growth of PC-3 cells through β-catenin pathway. The activation of ERα also plays a role on invasion and anchorage-independent growth of PC-3 cells [10
]. However, the molecular mechanisms of the crosstalk between ER and β-catenin pathways in this cell remains to be elucidated.
It is important to mention that 17β-estradiol impacts normal and malignant tissue development via ERα and ERβ, either through ligand-activated transcriptional regulation (genomic pathway) or by triggering cytoplasmic-signaling cascades (nongenomic pathway). The possible convergence of genomic and nongenomic pathways on target genes is an attractive mechanism by which ER can finely regulate gene expression in different cells [11
]. Indeed, evidence indicates that a pool of ER located in the cytoplasm and/or at the plasma membrane forms multiprotein complexes leading to the activation of downstream signaling molecules. ER may interact with SRC (non-receptor tyrosine kinase) out of the nucleus to activate extranuclear signaling pathways, such as ERK1/2 and AKT, in breast cancer cells [11
]. In addition, the extranuclear complex of ERα:SRC:PLCγ (phospholipase Cγ) plays a role in activation of the tumor-protective anticipatory UPR (unfolded protein response) (UPR), thereby increasing the resilience of breast cancer cells [13
]. SRC, which is induced by various cellular signal molecules and has a great effect in regulating numerous processes, including cell growth, differentiation, adhesion and the migration signaling pathway [14
], is highly expressed in several prostate cancer cell lines [17
]; as well as in most tissues obtained from prostate cancer [17
]. Studies have also demonstrated that the phosphorylation of tyrosine 654 from β-catenin by SRC reduces the association of β-catenin with E-cadherin and α-catenin [21
]. When SRC phosphorylates E-cadherin at residue Y860 and β-catenin at residue Y654, no interaction between E-cadherin/β-catenin occurs. β-catenin is then degraded or remain stabilized in the cytoplasm, and E-cadherin will be directed via Haikai for degradation [23
]. Whether ER activates SRC and plays a role in regulating the expression and/or activity of β-catenin remains to be investigated.
SRC can combine with another non-receptor protein tyrosine kinase, FAK (focal adhesion kinase) to form a dual-kinase complex, which coordinate cell behavior through regulating downstream pathways and molecules, including AKT, p38 and ERK [24
]. PI3K (phosphatidylinositol 3-kinase) and AKT are also involved in the development of prostate cancer and CRPC, but their functions are not yet fully elucidated [25
]. The most common change in PI3K signaling in patients with advanced prostate cancer is the bi-allelic loss of tumor suppressor PTEN (phosphatase and tensin homolog) that occurs in 50% of patients. PTEN is the negative regulator of PI3K and its inactivation, by mutation or loss, results in the accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of AKT [26
]. The phosphorylation of AKT activates mTOR (mammalian target of rapamycin), which leads to cell division [27
]. In vitro studies with prostate cancer cells have shown that PI3K/AKT/mTOR signaling is not only involved with proliferation [28
] and apoptosis [29
], but also with migration and invasion [30
Therefore, this study aimed to examine the role of estrogen receptor in the activation of SRC, and the involvement of SRC and PI3K/AKT on invasion and colony formation of the androgen-independent prostate cancer cells PC-3.
Recent studies of rapid actions mediated by estrogen in the prostate and its relationship with the development of prostate cancer or with CRPC are emerging. Our laboratory showed that in androgen-independent prostate cancer PC-3 and DU-145 cells, the estrogen receptors ERα and ERβ are mostly located outside the cell nucleus [6
]. The activation of ERα and ERβ can activate rapid cell signaling pathways in these cells, including an increase in the phosphorylation of ERK1/2 in PC-3 and DU-145 cells [6
] and AKT in PC-3 cells [7
], but not in DU-145 cells [7
]. We now report that ER induces activation of SRC and PI3K/AKT, increases the expression of the non-phosphorylated β-catenin and enhances the invasion and colony formation of the PC-3 cells.
SRC and the non-receptor protein tyrosine kinases are downstream targets for cell surface receptors, and function as a link between the membrane receptors and the cytoplasmic signaling machinery, thereby regulating many fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals [15
]. All these processes, if deregulated, lead to tumor progression [35
]. E2-ERα complex can enhances kinase activity by inducing binding of phosphotyrosine 537 of ERα to SH2 domain of SRC, changing the inactive conformation of SRC to active conformation [38
]. In fact, in the present study, using the PC-3 cells, the activation of ER by E2, ERα- (PPT) or ERβ-selective agonists (DPN) leads to the phosphorylation of SRC (Tyr419). Furthermore, the selective inhibitor for SRC-family kinases (PP2) blocked the invasion of the PC-3 cells stimulated by DPN or PPT and also the invasion of the DU-145 cells stimulated by E2, indicating the involvement of ERβ-SRC and ERα-SRC on the invasion of both cells. In addition, activation of both ER by E2 increases the size and number of the colony formed by PC-3 cells [10
] and present study. The pretreatment with the selective inhibitor for SRC-family kinases PP2 blunted these effects induced by E2, indicating the involvement of ER/SRC on tumor formation in vitro. The possible convergence of nongenomic and genomic pathways on target genes involved with invasion and colony formed by androgen-independent prostate cancer cells may be occurring.
It is important to mention that the activation of ERβ increases the expression of the non-phosphorylated β-catenin [8
], and present data and ERβ-β-catenin-TCF/LEF complex is involved in proliferation of the PC-3 cells [8
], migration, invasion, size and number of the colony formed by PC-3 cells [10
]. In the present study, the pretreatment with the selective inhibitor for SRC-family kinases PP2 blocked the effect induced by DPN on expression of the non-phosphorylated β-catenin, indicating the involvement of SRC in the regulation of this protein. It is important to mention that the antibody used in the detection of the non-phosphorylated β-catenin is against the Ser33/37/Thr41 region, this region may be phosphorylated by GSK3β [39
] and not by SRC.
SRC can phosphorylate β-catenin in Tyr654 [23
]. Phosphorylation of β-catenin by members of the SRC family reduces the association of β-catenin with E-cadherin and α-catenin [21
] increasing the levels of cytoplasmic β-catenin and translocation to the nucleus, where it interacts with the TCF/LEF transcription factor, resulting on the activation of target genes [40
]. Whether ERβ-SRC complex also plays a role on the activation of β-catenin in Tyr654 in PC-3 cells remains to be determined. In the present study, we show that ERβ induces activation of SRC (30 min) (rapid action, nongenomic) and increases the levels of the expression of the non-phosphorylated β-catenin in the cytoplasm of PC-3 cells (2 h, genomic action).
Consistent with our results, study has shown that selective inhibitor for SRC-family kinases PP2 suppressed migration, invasion, and angiogenesis of PC3 and LNCAP cells via FAK [41
]. Whether 17β-estradiol-ER-SRC plays a direct role or together with β-catenin and/or FAK on migration, invasion, and angiogenesis of PC3 cells remains to be explored.
Recently, new emerging roles for SRC have been described in the nuclear compartment. In the nucleus of normal and cancer cells, SRC is involved in several activities involving both its enzymatic activity as tyrosine kinase and its capability to interact with other protein thereby forming protein complexes. SRC participates in the regulation of chromatin reorganization and transcriptional activity of transcription factors, and it is surely involved in the oncogenic transformation of tumoral cells, by repressing some oncosuppressors [42
]. The roles of SRC in the nuclear compartment of the prostate cancer cells remains to be explored.
Although several partners of extranuclear ER have been described in different cell types, the most conserved partners are SRC and PI3K [43
]. The pathway, characterized by the formation of ER/SRC/PI3K and the subsequent activation of AKT, is present in normal breast tissue and is hyperactivated in aggressive breast tumors [44
]. In PC-3 cells, our laboratory showed that E2 increases the phosphorylation of AKT [8
]. In the present study was shown that the inhibitors of PI3K or AKT blocked the increase in cell invasion stimulated by DPN (100%) or PPT (80%), suggesting the involvement of ERβ-PI3K/AKT and ERα-PI3K/AKT on the invasion potential of PC-3 cells. In addition, the activation of ERβ by DPN or ERα by PPT increases the size and number of the colony formed by PC-3 cells [10 and present study). These effects were also blocked by inhibitors of PI3K or AKT.
It is also important to mention that the phosphorylation of the β-catenin in the Ser552 by AKT may increase β-catenin/TCF-LEF activation possibly by association with histone acetylase [23
]. In addition, coactivators recruited by β-catenin can determine which target genes are activated, and this differential recruitment can be regulated by phosphorylation [45
]. Whether the complex ER/PI3K and the subsequent activation of AKT plays a role in expression and/or activation of β-catenin remains to be explored.
In conclusion, this study provides novel insights into molecular mechanisms of ER in androgen-independent prostate cancer cells. In PC-3 cells, ER activates rapid responses molecules, including SRC and PI3K/AKT. SRC is involved on the expression of the non-phosphorylated β-catenin. These events enhance the tumorigenic potential of prostate cancer cells PC-3, increasing cell proliferation, migration, invasion, and tumor formation. The complete mechanism by which ER are involved in CRPC is not fully understood, but it represents a promising new therapeutic avenue for advanced prostate cancer.