Robert X.-D. Song

Estrogen rapid action via protein complex formation involving ERα and Src

This review provides insight into biomolecular knowledge regarding the non-genomic actions of estrogen in hormone-dependent breast cancer, particularly its role in the rapid stimulation of pathways that transmit signals to increase cell division or decrease programmed cell death. Until recently, attention to estrogenic effects focused primarily on events in the nucleus, where most estrogen receptors (ERα and β) reside. However, a fraction of ERα associated with the cell membrane also participates in rapid estrogen-induced cell membrane-mediated events via formation of a protein complex with many signaling molecules, leading to activation of the mitogen-activated protein kinase and Akt signaling pathways. Understanding the mechanisms underlying these relationships, with the aim of abrogating specific steps, should lead to more targeted strategies to treat hormone-dependent breast cancer.

Membrane Initiated Estrogen Signaling in Breast Cancer

Abstract Recent research has focused on effects of the estrogen receptor acting at the level of the cell membrane in breast cancer. In this review we describe 17beta-estradiol (E2)-initiated membrane signaling pathways involving the activation of several kinases that contribute to the regulation of cell proliferation and prevention of apoptosis. Although classical concepts had assigned priority to the nuclear actions of estrogen receptor, recent studies document the additional importance of estrogen receptor residing in or near the plasma membrane. A small fraction of estrogen receptor is associated with the cell membrane and mediates the rapid effects of E2. Unlike classical growth factor receptors, such as insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), estrogen receptor has no transmembrane and kinase domains and is known to initiate E2 rapid signals by forming a protein complex with many signaling molecules. The formation of the protein complex is a critical step, leading to the activation of the MAPK1/3 (also known as MAP kinase) and AKT1 (also known as Akt) pathways. A full understanding of the mechanisms underlying these relationships, with the ultimate aim of abrogating specific steps, should lead to more-targeted strategies for treatment of hormone dependent-breast cancer.

Membrane association of estrogen receptor α mediates estrogen effect on MAPK activation

Estrogen rapidly activates MAPK in many cell types but the mechanisms have not been fully understood. We previously demonstrated that 17-β-estradiol (estradiol) rapidly induced membrane translocation of estrogen receptor α (ERα) and activated MAPK in MCF-7 breast cancer cells. This study further determines the cause and effect relationship between the presence of membrane ERα and MAPK activation. ERα with a membrane localization signal (HE241G-mem) was expressed and compared with the ones in nucleus (HEGO) or cytosol (HE241G) localization. Confocal microscopy showed that HE241G-mem was expressed in the cell membrane as well as in the cytosol in COS-1 cells. HE241G localized in the cytosol and HEGO in the nucleus. Functional studies showed that only membrane ERα, not cytosol and nuclear ones, responded to estradiol by inducing MAPK phosphorylation. HE241G-mem neither increased basal nor estradiol-induced ERE promoter activation, indicating no transcriptional action involved. Our data support the view that membrane-associated ERα is critical in estrogen-initiated MAPK activation.

Adaptive hypersensitivity to estrogen: mechanism for sequential responses to hormonal therapy in breast cancer.

Clinical observations demonstrate that women with breast cancer often respond to subsequent endocrine manipulation after resistance to initial hormonal therapy develops. As a mechanistic explanation for these findings, we hypothesized that human breast tumors can adapt in response to the pressure exerted by endocrine therapy with development of hypersensitivity to estradiol. To understand the signaling pathways responsible, we examined estrogenic stimulation of cell proliferation in a model system and provided in vitro and in vivo evidence that long-term deprivation of estradiol (LTED) causes adaptive hypersensitivity. Even though the estrogen receptor α (ERα) is markedly up-regulated in LTED cells, the enhanced responses to estradiol do not appear to involve mechanisms acting at the level of transcription of estrogen-regulated genes. We found that ERα co-opts a classical growth factor pathway and induces rapid nongenomic effects that are enhanced in LTED cells. Estradiol binds to cell membrane-associated ERs, physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb2 and Sos, which result in the rapid activation of mitogen-activated protein kinase. These nongenomic effects of estradiol produced biological effects, as evidenced by Elk-1 activation and by morphological changes in cell membranes. The mechanistic pathways involved in adaptive hypersensitivity suggest that inhibitors of the mitogen-activated protein kinase and phosphatidylinositol-3-OH kinase pathways might prevent the development of adaptive hypersensitivity and allow more prolonged efficacy of endocrine therapies.

Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells

Activation of IGF-1R can activate metalloproteinases which release heparin-binding EGF (Hb-EGF) and lead to EGFR-dependent MAPK activation in certain tissues. We postulated that this pathway is operative in E(2)-induced MAPK activation in breast cancer tissues. As evidence, we showed that E(2) rapidly induced the phosphorylation of both IGF-1R and EGFR and that siRNA knockdown or selective inhibitors against either growth factor receptor inhibited E(2)-induced MAPK activation. The selective inhibitors or knockdown of either IGF-1R or EGFR significantly inhibited cell growth and reversed cell death protection induced by E(2) in MCF-7 cells. Our data support the conclusion that the IGF-1R acts upstream of EGFR in a linear pathway which mediates E(2) action on MAPK activation, cell growth stimulation and anti-apoptosis in breast cancer cells. During the process of development of tamoxifen resistance this pathway is up-regulated with increased sensitivity to activate EGFR for cell growth and protection against apoptosis. Surprisingly, translocation of ERalpha out of the nucleus into the cytoplasm, mediated by c-Src, occurs during development of resistance. This effect can be abrogated by administration of the c-Src inhibitor, PP2 which also restores sensitivity to tamoxifen.

Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells.

As breast cancer cells develop secondary resistance to estrogen deprivation therapy, they increase their utilization of non-genomic signaling pathways. Our prior work demonstrated that estradiol causes an association of ERalpha with Shc, Src and the IGF-1-R. In cells developing resistance to estrogen deprivation (surrogate for aromatase inhibition) and to the anti-estrogens tamoxifen, 4-OH-tamoxifen, and fulvestrant, an increased association of ERalpha with c-Src and the EGF-R occurs. At the same time, there is a translocation of ERalpha out of the nucleus and into the cytoplasm and cell membrane. Blockade of c-Src with the Src kinase inhibitor, PP-2 causes relocation of ERalpha into the nucleus. While these changes are not identical in response to each anti-estrogen, ERalpha binding to the EGF-R is increased in response to 4-OH-tamoxifen when compared with tamoxifen. The changes in EGF-R interactions with ERalpha impart an enhanced sensitivity of tamoxifen-resistant cells to the inhibitory properties of the specific EGF-R tyrosine kinase inhibitor, AG 1478. However, with long term exposure of tamoxifen-resistant cells to AG 1478, the cells begin to re-grow but can now be inhibited by the IGF-R tyrosine kinase inhibitor, AG 1024. These data suggest that the IGF-R system becomes the predominant signaling mechanism as an adaptive response to the EGF-R inhibitor. Taken together, this information suggests that both the EGF-R and IGF-R pathways can mediate ERalpha signaling. To further examine the effects of fulvestrant on ERalpha function, we examined the acute effects of fulvestrant, on non-genomic functionality. Fulvestrant enhanced ERalpha association with the membrane IGF-1-receptor (IGF-1-R). Using siRNA or expression vectors to knock-down or knock-in selective proteins, we further demonstrated that the ERalpha/IGF-1-R association is Src-dependent. Fulvestrant rapidly induced IGF-1-R and MAPK phosphorylation. The Src inhibitor PP2 and IGF-1-R inhibitor AG1024 greatly blocked fulvestrant-induced ERalpha/IGF-1-R interaction leading to a further depletion of total cellular ERalpha induced by fulvestrant and further enhanced fulvestrant-induced cell growth arrest. More dramatic was the translocation of ERalpha to the plasma membrane in combination with the IGF-1-R as shown by confocal microscopy. Taken in aggregate, these studies suggest that secondary resistance to hormonal therapy results in usage of both IGF-R and EGF-R for non-genomic signaling.

The role of adapter protein Shc in estrogen non-genomic action.

Breast cancer is one of the most common malignancies in the United States. Seventy percent of breast cancers are hormone-responsive due to the presence of estrogen receptors ERalpha and ERbeta, which are important diagnostic and therapeutic targets in cancer treatment. Estrogen acts through its receptors, which reside on the cell membrane as demonstrated recently and in the nucleus, leading to cancer cell proliferation and protection from cell death. The membrane ERalpha has been reported in MCF-7 human breast cancer cells and is believed to mediate estrogen effects to activate mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3-kinase). Activation of many growth factor receptors require adapter proteins to delivery the upstream signals to downstream kinases, such as MAP kinase. Both Shc and the p85alpha subunit of PI3-kinase are adapter proteins. In addition to their roles in transducing signals from membrane growth factor receptors, they have been demonstrated to interact with ERalpha in an estrogen dependent manner. In this review, the role of Shc in mediating estrogen effects on MAP Kinase regulation, cell growth and anti-apoptosis will be discussed. The possible role of PI3-kinase in estrogen rapid action is also reviewed in brief.

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER(+) breast cancer cells.

Postmenopausal women with estrogen receptor positive (ER+) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17β -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER+ postmenopausal breast cancer cells.

Adaptive mechanisms induced by long-term estrogen deprivation in breast cancer cells.

Clinical observations suggest that human breast tumors can adapt in response to endocrine therapy by developing hypersensitivity to estradiol. To understand the mechanisms responsible, we examined estrogenic stimulation of cell proliferation in a model system and provided evidence that long-term deprivation of estradiol causes adaptive hypersensitivity. The enhanced responses to estradiol do not involve mechanisms acting at the level of transcription of estrogen regulated genes. We found no evidence of hypersensitivity when examining the effects of estradiol on regulation of c-myc, pS2, progesterone receptor, several ER reporter genes or c-myb in hypersensitive cells. On the other hand, deprivation of breast cells long term was found to up-regulate a separate pathway whereby the estrogen receptor co-opts a classical growth factor pathway and induces rapid non-genomic effects. Through this pathway, estradiol caused rapid activation of mitogen-activated protein (MAP) kinase. In exploring the mechanisms mediating this event, we found that estradiol binds to cell membrane associated estrogen receptors and causes phosphorylation of Shc, an adaptor protein usually involved in growth factor signaling pathways. ERalpha was found to complex with Shc under these conditions. In turn, Shc bound Grb-2 and Sos which resulted in the activation of MAP kinase. The pure antiestrogen, ICI 182,780, blocked several steps in the rapidly responding ER alpha, Shc, MAP kinase pathway. These non-genomic effects of estradiol produced biologic effects by activating Elk and by inducing morphologic changes in cell membranes. Using confocal microscopy, we demonstrated that estradiol caused a rapid alteration in membrane ruffling, the formation of pseudopodia and translocation of ER alpha to regions contiguous with the cell membrane. These morphologic effects could be blocked with a pure anti-estrogen. We conclude that long-term estradiol deprived cells utilize both genomic (transcriptional) and rapid, non-genomic estradiol induced pathways. We postulate that synergy between these two pathways acting at the level of the cell cycle is responsible for adaptive hypersensitivity.

The Role of Adapter Proteins in Eraα Membrane Association and Function

Breast cancer is one of the most common malignancies in the United States. Forty percent of breast cancers are hormone-responsive due to the presence of estrogen receptors ERα and ERβ. Estrogen acts through its receptors residing on the cell membrane as demonstrated recently, leading to cancer cell proliferation and cell death protection. The membrane ERα has been reported in MCF-7 human breast cancer cells. A large body of evidence has linked ERα, an important diagnostic and therapeutic target in breast cancer, to the activation of the mitogen-activated protein (MAP) kinase and Phosphoinositide 3-kinase (PI3-kinase) pathways. Growth factor-induced MAP kinase activation involves signaling via the adapter proteins. Both She and the p85α subunit of Phosphoinositide 3-kinase (PI3-kinase) are adapter proteins and have been demonstrated to interact with ERα in an estrogen dependent manner. In this chapter, the role of She and p85α in mediating estrogen effects on the regulation of MAP Kinase, PI3-kinase, cell growth and apoptosis will be discussed.