The pathological significance of ER extranuclear signaling and its role in the progression to metastasis of breast cancer remain unknown. In this study, using estrogen dendrimers, dominant negative reagents, and pharmacological inhibitors of ER-extranuclear signaling, we found that ER-extranuclear actions play an important role in cell motility and metastases. In addition, we established for the first time that endogenous PELP1 play a critical role in coupling ER-extranuclear signaling to cell motility via ER-Src-PELP1-ILK-Rac/CDC42 pathway.
The proto-oncogene c-Src is a multifunctional intracellular tyrosine kinase implicated in the regulation of a variety of processes including proliferation, differentiation, survival, and motility (26
). Src interacts with multiple cellular factors including HER2, EGFR and ER and breast tumors overexpress Src kinase (27
). PELP1 acts as a scaffolding protein coupling the ER with Src kinase leading to activation of the ER-Src-MAPK pathway (11
). Our data suggest that PELP1 and Src kinase play an essential role in the activation of ER-extranuclear signaling leading to cytoskeleton reorganization and migration. Since breast tumors overexpress Src kinase, deregulation of PELP1 seen in breast tumors can contribute to activation of Src, leading to the progression to metastasis. Pharmacological inhibition of Src using dasatinib significantly inhibited E2-mediated extranuclear actions and reduced E2 mediated migratory potential. These results suggest that the ER-Src-PELP1 axis is a novel target for preventing the emergence of metastatic cells and that dasatinib may have therapeutic utility in blocking ER-positive metastases.
ERα has been implicated in breast cancer progression and majority of the human breast cancers start out as hormone-dependent. Some evidence suggests that the extranuclear effects of estrogen can regulate different cellular processes, such as proliferation, survival, apoptosis (28
). Our results using EDC demonstrates that ER extranuclear signaling has potential to promote cytoskeletal changes, leading to increased cell migration. Findings from these studies also showed that E2 extranuclear signaling promotes formation of signaling complexes that contain PELP1, ER Src, and ILK1 and that extranuclear signaling from this axis play important roles in cytoskeletal rearrangements, motility and metastasis.
We identified ILK1 as a novel interacting protein of PELP1 and demonstrated that ILK1 functions as a downstream effector of ER-extranuclear signaling, leading to cytoskeletal reorganization. ILK1 is known to play an important role in cytoskeleton reorganization and in the activation of Rho GTPases (Rac and CDC42). These effects are reversible upon inhibition of ILK protein expression (29
) The ability of PELP1 to modulate the ILK1 pathway and its potential deregulation in metastatic breast cancer suggest that the modulation of ILK1 pathway may represent one potential mechanism by which PELP1 promotes metastasis in breast cancer cells.
PELP1 is a key component of the ER signalsome in the cytoplasm and is shown to play a role in ER extranuclear actions (8
). PELP1 expression appears to be predominantly in the cytoplasm in a subset of breast tumors. Previous studies showed that PELP1 cytoplasmic locolization excessively promote ER-extranuclear signaling and that such deregulation contributed to tamoxifen therapy resistance (12
). A recent study demonstrated that patients whose tumors had high levels of cytoplasmic PELP1 had a tendency to respond poorly to tamoxifen compared with patients whose tumors had low levels of cytoplasmic PELP1 (30
). In this study, using ligands that uniquely activate ER extranuclear signaling (EDC) and PELP1shRNA or dominant mutants that block PELP1 signaling, we found that E2-driven PELP1-mediated ER-extranuclear actions can promote the cell migratory potential.
Endocrine therapy has also been shown to have a positive effect on the treatment of advanced metastatic disease (3
). A few earlier studies suggested a negative effect of ER signaling on motility and invasion of cells (31
), while several recent studies showed a positive effect of ER signaling on motility (32
). Many metastatic tumors retain ER (34
), and if primary tumors are ER positive, >80% of lymph node metastases and 65-70% of distant metastases retain ER (2
). A clinical correlation has also been reported between ER-positive tumors and the development of bone metastasis (36
). Similarly ER signaling has been shown to enhance lung metastasis by promoting host-compartment response (38
). PELP1 expression is deregulated in metastastic tumors (14
) and PELP1 protein expression is an independent prognostic predictor of shorter breast cancer-specific survival and its elevated expression is positively associated with markers of poor outcome (39
). Our data suggest that ER-extranuclear signaling plays a role in metastasis and PELP1 deregulation commonly seen in metastastic tumors may play a role in metastasis by enhancing ER-extranuclear signaling.
In summary, our data provide the first evidence demonstrating the significance of ER-extranuclear signaling to the metastatic potential of breast cancer cells. Our findings also identified ILK1 as a novel component of ER-PELP1 signalsome that connects ER signaling to cytoskeleton. We hypothesize that the ER-Src-PELP1-PI3K-ILK1 pathway represents a novel target to prevent the emergence of ER-positive metastatic cells via blockage of ER-extranuclear signals in combination with endocrine therapy.