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(A) The JNK inhibitor effectively blocked phosphorylation of JNK. MCF-7:2A cells were treated with vehicle (0.1% DMSO) or SP600125 (10-5 mol/L) for 24 hours. p-JNK was examined by Western blot. Total JNK was measured as loading control. (B) The JNK inhibitor could not block E2-induced apoptosis in MCF-7:2A cells. MCF-7:2A cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), SP600125 (10-5 mol/L), and E2 (10-9 mol/L) plus SP600125 (10-5 mol/L) for 6 days. Annexin V binding assay was used to detect apoptosis. p

Basal expression levels of stress-related genes in MCF-7:5C and MCF-7:2A cells.

Legends of supplementary Figures

PDF file - 53K, 7A, Inhibitors of Akt could not block growth inhibition induced by E2 in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), LY294002 (10-6mol/L), and E2 (10-9 mol/L) plus LY294002 (10-6mol/L) respectively. Cells were harvested after 7 days treatment and total DNA was determined using a DNA fluorescence quantitation kit. 7B, Inhibitor of MAPK could not block growth inhibition induced by E2 in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), U0126 (5x10-6 mol/L), and E2 (10-9 mol/L) plus U0126 (5x10-6 mol/L) respectively. Cells were harvested after 7 days treatment and total DNA was determined using a DNA fluorescence quantitation kit. All data shown were representative of at least three separate experiments with similar results.

PDF file - 82K, 6A, The c-Src inhibitor blocked tumor necrosis factor (TNF) super family genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol. LTA (TNF super family member 1) gene was detected by real-time PCR. P

PDF file - 77K, 1A, The c-Src inhibitor blocked E2 activated non-genomic pathway. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 10 minutes and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 1B, E2 rapidly activated MAPK and c-Src. MCF-7:5C cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 1C, E2 stimulated c-Src after 24 hours treatment. MCF-7:5C cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated c-Src was examined by immunoblotting with primary antibody. Immunoblotting for total c-Src was used for loading control. 1D, Quantification of Annexin V binding assay. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours and the cells were harvested for Annexin V binding assay through flow cytometry. The percentage of Annexin V binding was compared with control. P

PDF file - 47K, 5A, The oxidative stress indicator HMOX1 expressed in wild-type MCF-7 cells after E2 treatment. Wild-type MCF-7 cells were cultured in E2 free medium for three days. Then, cells were plated in six-well plates. After one day, cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cells were harvested in TRIzol for real-time PCR. P

PDF file - 76K, The c-Src inhibitor blocked apoptosis-related genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT(10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol for real-time PCR. 4A, PPP1R15A (GADD34) gene. 4B, BCL2L11 (Bim) gene. 4C, NUAK2 gene. 4D, PMAIP1 (Noxa) gene. P

PDF file - 104K, 2A, Activation of pS2 by different concentrations of estrogen dendrimer conjugate (EDC). MCF-7:5C cells were treated with vehicle (0.1% MeOH), different concentrations of EDC, E2, and empty dendrimer as indicated for 8 hours in triplicate. Cells were harvested in TRIzol for real-time PCR. 2B, Cell growth after EDC treatment. MCF-7:5C cells were treated with vehicle (0.1% MeOH), different concentrations of EDC, E2, and empty dendrimer as indicated for 7 days in triplicate. Cells were harvested and total DNA was determined using a DNA fluorescence quantitation kit. 2C, The c-Src inhibitor blocked EDC activated non-genomic pathway. MCF-7:5C cells were treated with vehicle (0.1% MeOH), EDC (10-8 mol/L), PP2 (5x10-6 mol/L), EDC (10-8 mol/L) plus PP2 (5x10-6 mol/L) respectively for 15 minutes and the cell lysates were harvested. Phosphorylated MAPK was examined by immunoblotting with primary antibody. Immunoblotting for total MAPK was used for loading control. 2D, EDC rapidly activated MAPK and c-Src in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% MeOH) and EDC (10-8 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 2E, EDC activated signaling pathways after 24 hours. MCF-7:5C cells were treated with vehicle (0.1% MeOH), EDC (10-8 mol/L), PP2 (5x10-6 mol/L), EDC (10-8 mol/L) plus PP2 (5x10-6 mol/L) respectively for 24 hours and 48 hours. Cell lysates were harvested. Phosphorylated MAPK, Akt, and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK, Akt, and c-Src were used for loading controls.

PDF file - 59K, Sequences of Primers.

Our clinically relevant finding is that glucocorticoids block estrogen (E(2))-induced apoptosis in long-term E(2)-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E(2) widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), interleukin-6 (IL-6), and tumor necrosis factor α (TNFα) in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone (Dex) upregulated FADS1 and IL-6 but downregulated TNFα expression. Furthermore, Dex was synergistic or additive with E(2) in upregulating FADS1 and IL-6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E(2) in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, Dex effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E(2), but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E(2). Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that Dex could not reverse IRE1α-mediated degradation of phosphoinositide 3-kinase (PI3K)/Akt-associated signal pathways activated by E(2). Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E(2), leading to the blockade of E(2)-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E(2)-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E(2)-induced apoptosis in aromatase inhibitor-resistant breast cancer patients.

Stress responses are critical for estrogen (E2)-induced apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is an important therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unknown. Here, we demonstrated that E2 differentially modulates NF-κB activity according to treatment time. E2 initially has significant potential to suppress NF-κB activation; it completely blocks tumor necrosis factor alpha (TNFα)-induced activation of NF-κB. We found that E2 preferentially and constantly enhances the expression of the adipogenic transcription factor CCAAT/enhancer binding protein beta (C/EBPβ), which is responsible for the suppression of NF-κB activation by E2 in MCF-7:5C cells. Interestingly, NF-κB p65 DNA-binding activity is increased when E2 is administered for 48 h, leading to the induction of TNFα and associated apoptosis. Blocking the nuclear translocation of NF-κB can completely prevent the induction of TNFα and apoptosis induced by E2. Further examination revealed that protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response (UPR), plays an essential role in the late activation of NF-κB by E2. This modulation between PERK and NF-κB is mainly mediated by a stress responsive transcription factor, transducer and activator of transcription 3 (STAT3), independently of the classic canonical IκBα signaling pathway. Thus, inhibition of PERK kinase activity completely blocks the DNA binding of both STAT3 and NF-κB, thereby preventing induction of NF-κB-dependent genes and E2-induced apoptosis. All of these findings suggest that PERK is a key regulator to convey stress signals from the endoplasmic reticulum to the nucleus and illustrate a crucial role for the novel PERK/STAT3/NF-κB/TNFα axis in E2-induced apoptosis in E2-deprived breast cancer cells.

Purpose Oestrogen (E2)-stimulated growth re-emerges after a c-Src inhibitor blocking E2-induced apoptosis. A resulting cell line, MCF-7:PF, is selected with features of functional oestrogen receptor (ER) and over-expression of insulin-like growth factor-1 receptor beta (IGF-1Rβ). We addressed the question of whether the selective ER modulator (SERM), 4-hydroxytamoxifen (4-OHT) or other SERMs could target ER to prevent E2-stimulated growth in MCF-7:PF cells. Methods Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was measured through real-time RT-PCR. Recruitment of ER or nuclear receptor coactivator 3 (SRC3) to the promoter of ER-target gene was detected by chromatin-immunoprecipitation (ChIP). Results 4-OHT and other SERMs stimulated cell growth in an ER-dependent manner. However, unlike E2, 4-OHT suppressed classical ER-target genes as does the pure antioestrogen ICI 182,780 (ICI). ChIP assay indicated that 4-OHT did not recruit ER or SRC3 to the promoter of ER-target gene, pS2. Paradoxically, 4-OHT reduced total IGF-1Rβ but increased phosphorylation of IGF-1Rβ. Mechanistic studies revealed that 4-OHT functioned as an agonist to enhance the non-genomic activity of ER and activate focal adhesion molecules to further increase phosphorylation of IGF-1Rβ. Disruption of membrane-associated signalling, IGF-1R and focal adhesion kinase (FAK), completely abolished 4-OHT-stimulated cell growth. Conclusions This study is the first to recapitulate a cellular model in vitro of acquired tamoxifen resistance developed in athymic mice in vivo. Importantly, it provides a rationale that membrane-associated pathways may be valuable therapeutic targets for tamoxifen resistant patients in clinic.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing in vitro into an undifferentiated adult stem cell-like state that is characterized by long-term proliferation. This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions. Due to the ability to modulate the proliferation and differentiation of the cells, this method is referred to as conditional reprogramming and it is increasingly being used in studies of tumor heterogeneity, personalized medicine and regenerative medicine. However, little is known about the biology of these conditionally reprogrammed (CR) cells. Previously we showed that β-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that β-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that β-catenin is activated via a non-canonical pathway that is independent of Wnt and Akt/GSK-3. Active Akt actually decreases due to increased mTOR signaling, with a consequent increase in dephosphorylated, active GSK-3. Despite the increase in active GSK-3, β-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active β-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of β-catenin. Moreover, we demonstrate similar results using human prostate and breast cells, indicating that these changes are not restricted to ectocervical epithelial cells and may represent a more fundamental property of conditional reprogramming.

Stress responses are critical for estrogen (E2) to induce apoptosis in E2-deprived breast cancer cells. Nuclear factor-kappa B (NF-κB) is well known as a therapeutic target to prevent stress responses in chronic inflammatory diseases including cancer. However, whether E2 activates NF-κB to participate in stress-associated apoptosis in E2-deprived breast cancer cells is unclear. We demonstrated that E2 differentially modulates NF-κB activity in E2-deprived breast cancer cells according to the treatment time. Because E2 initially has significant potential to down modulate the NF-κB activation, it completely suppresses the tumor necrosis factor alpha (TNFα)-induced NF-κB activation. We found that E2 preferentially and constantly enhances the expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) which is responsible for suppression of NF-κB activation by E2 in MCF-7:5C cells. The mTOR signaling pathway promotes repression of NF-κB by C/EBPβ which is confirmed by the evidence that inhibition of mTOR is synergistic with E2 to upregulate NF-κB-dependent genes, such as TNFα. Interestingly, NF-κB p65 activity is upregulated when E2-treatment is administered for 48 hours, leading to induction of TNFα. Blocking the nuclear translocation of NF-κB completely prevents E2 from induction of TNFα and apoptosis. Importantly, protein kinase RNA-like endoplasmic reticulum kinase (PERK), a stress sensor of unfolded protein response, is activated by E2 and plays an essential role in increasing NF-κB p65 DNA binding through the activation of STAT3, independently of canonical IκBα signal pathway. Thus, inhibition of PERK kinase activity completely blocks nuclear activation of NF-κB and NF-κB-dependent induction of TNFα, thereby preventing E2-induced apoptosis. All of these findings illustrate a crucial role for the novel PERK/NF-κB/TNFα axis in E2-induced apoptosis which is integrally modulated by the stress responsive transcription factor C/EBPβ and endoplasmic reticulum stress. This study provides an important rationale for exercising caution in clinical trials when considering targeting PERK or NF-κB following the development of acquired resistance to aromatase inhibitors whereas mTOR may be a target to enhance the therapeutic effects of E2 in antihormone resistant breast cancer. Citation Format: Ping Fan, Amit K. Tyagi, Fadeke A. Agboke, Niranjana Pokharel, V. Craig Jordan. Integral modulation of nuclear factor-kappa B activation by C/EBPβ and the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in estrogen-deprived breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2332. doi:10.1158/1538-7445.AM2017-2332

The discovery of the first nonsteroidal antiestrogen ethamoxytriphetol (MER25) in 1958, opened the door to a wide range of clinical applications. However, the finding that ethamoxytriphetol was a “morning after” pill in laboratory animals, energized the pharmaceutical industry to discover more potent derivatives. In the wake of the enormous impact of the introduction of the oral contraceptive worldwide, contraceptive research was a central focus in the early 1960’s. Numerous compounds were discovered e.g., clomiphene, nafoxidine, and tamoxifen, but the fact that clinical studies showed no contraceptive actions, but, in fact, induced ovulation, dampened enthusiasm for clinical development. Only clomiphene moved forward to pioneer an application to induce ovulation in subfertile women. The fact that all the compounds were antiestrogenic made an application in patients to treat estrogen responsive breast cancer, an obvious choice. However, toxicities and poor projected commercial returns severely retarded clinical development for two decades. In the 1970’s a paradigm shift in the laboratory to advocate long term adjuvant tamoxifen treatment for early (non-metastatic) breast cancer changed medical care and dramatically increased survivorship. Tamoxifen pioneered that paradigm shift but it became the medicine of choice in a second paradigm shift for preventing breast cancer during the 1980’s and 1990’s. This was not surprising as it was the only medicine available and there was laboratory and clinical evidence for the eventual success of this application. Tamoxifen is the first medicine to be approved by the Food and Drug Administration (FDA) to reduce the risk of breast cancer in women at high risk. But it was the re-evaluation of the toxicology of tamoxifen in the 1980’s and the finding that there was both carcinogenic potential and a significant, but small, risk of endometrial cancer in postmenopausal women that led to a third paradigm shift to identify applications for selective estrogen receptor (ER) modulation. This idea was to establish a new group of medicines now called selective ER modulators (SERMs). Today there are 5 SERMs FDA approved (one other in Europe) for applications ranging from the reduction of breast cancer risk and osteoporosis to the reduction of menopausal hot flashes and improvements in dyspareunia and vaginal lubrication. This article charts the origins of the current path for progress in women’s health with SERMs.

Purpose A c-Src inhibitor blocks oestrogen (E 2 )-induced stress and converts E 2 responses from inducing apoptosis to growth stimulation in E 2 -deprived breast cancer cells. A reprogrammed cell line, MCF-7:PF, results in a functional oestrogen receptor (ER). We addressed the question of whether the selective ER modulator 4-hydroxytamoxifen (4-OHT) could target ER to prevent E 2 -stimulated growth in MCF-7:PF cells. Methods Expression of mRNA was measured through real-time RT-PCR. Global gene expression profile was analysed through microarray. Transcriptome profiles were screened by RNA-sequencing. Results Unexpectedly, both 4-OHT and E 2 stimulated cell growth in a concentration-dependent manner. Expression profiling showed a remarkable overlap in genes regulated in the same direction by E 2 and 4-OHT. Pathway enrichment analysis of the 280 genes commonly deregulated in MCF-7:PF cells by 4-OHT and E 2 revealed functions mainly related to membrane, cytoplasm and metabolic processes. Further analysis of 98 genes up-regulated by both 4-OHT and E 2 uncovered a significant enrichment in genes associated with membrane remodelling, cytoskeleton reorganisation, cytoplasmic adapter proteins, cytoplasm organelle proteins and related processes. 4-OHT was more potent than E 2 in up-regulating some membrane remodelling molecules, such as EHD2 , FHL2 , HOMER3 and RHOF . In contrast, 4-OHT acted as an antagonist to inhibit expression of the majority of enriched membrane-associated genes in wild-type MCF-7 cells. Conclusions Long-term selection pressure has changed the cell population responses to 4-OHT. Membrane-associated signalling is critical for 4-OHT-stimulated cell growth in MCF-7:PF cells. This study provides a rationale for the further investigation of target therapy for tamoxifen resistant patients.

Purpose Our publications demonstrate that physiological concentrations of oestrogen (E 2 ) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E 2 -deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E 2 -induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E 2 , a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E 2 on MCF-7:5C cells. Methods Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was detected through real-time polymerase chain reaction (PCR). Cell cycles were analysed by flow cytometry. Results Long-term treatment with PP2 alone or E 2 alone decreased cell growth. In contrast, a combination of PP2 and E 2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E 2 , which could be blocked by the pure antioestrogen ICI182,780. One major change was that PP2 collaborated with E 2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1Rβ). Blockade of IGF-1Rβ completely abolished E 2 -stimulated growth in MCF-7:PF cells. Furthermore, combination treatment up-regulated transcription factors, Twist1 and Snail, and repressed E-cadherin expression which made MCF-7:PF cells display a characteristic phenotype of epithelial–mesenchymal transition (EMT). Conclusions These data illustrate the role of the c-Src inhibitor to block E 2 -induced apoptosis and enhance E 2 -stimulated growth. Caution must be exercised when considering c-Src inhibitors in clinical trials following the development of acquired resistance to aromatase inhibitors, especially in the presence of the patient’s own oestrogen.

The emergence of anti-estrogen resistance in breast cancer is an important clinical phenomenon affecting long-term survival in this disease. Identifying factors that convey cell survival in this setting may guide improvements in treatment. Estrogen (E2) can induce apoptosis in breast cancer cells that have been selected for survival after E2 deprivation for long periods (MCF-7:5C cells), but the mechanisms underlying E2-induced stress in this setting have not been elucidated. Here, we report that the c-Src kinase functions as a key adapter protein for the estrogen receptor (ER, ESR1) in its activation of stress responses induced by E2 in MCF-7:5C cells. E2 elevated phosphorylation of c-Src, which was blocked by 4-hydroxytamoxifen (4-OHT), suggesting that E2 activated c-Src through the ER. We found that E2 activated the sensors of the unfolded protein response (UPR), IRE1α (ERN1) and PERK kinase (EIF2AK3), the latter of which phosphorylates eukaryotic translation initiation factor-2α (eIF2α). E2 also dramatically increased reactive oxygen species production and upregulated expression of heme oxygenase HO-1 (HMOX1), an indicator of oxidative stress, along with the central energy sensor kinase AMPK (PRKAA2). Pharmacologic or RNA interference–mediated inhibition of c-Src abolished the phosphorylation of eIF2α and AMPK, blocked E2-induced ROS production, and inhibited E2-induced apoptosis. Together, our results establish that c-Src kinase mediates stresses generated by E2 in long-term E2-deprived cells that trigger apoptosis. This work offers a mechanistic rationale for a new approach in the treatment of endocrine-resistant breast cancer. Cancer Res; 73(14); 4510–20. ©2013 AACR.

Inflammation is critical for estrogen (E2) to undertake apoptosis in E2-deprived breast cancer cells. However, it remains unclear how E2 induces inflammation. Here, we demonstrate that E2 increases lipid metabolism and accumulates unfolded proteins in the endoplasmic reticulum in E2-deprived breast cancer cells. These two major alterations result in different inflammatory responses. E2 rapidly increases lipid metabolism related genes such as fatty acid desaturase 1 (FADS1) and CCAAT/enhancer binding protein beta (CEBPB) to modulate adipose inflammation related factors, e.g. IL6/IL6R after two hours treatment, which regulate the cellular proliferation or differentiation. Simultaneously, accumulation of the unfolded protein activates unfolded protein response (UPR) sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring protein 1 alpha (IRE1α) within few hours after exposure to E2. Importantly, inhibition of PERK, rather than IRE1α, is able to completely prevent E2 from induction of late event inflammatory genes associated with apoptosis, such as tumor necrosis factor (TNF) superfamily members, BCL2L11 (Bim), and HMOX1,which are activated by E2 after 48 hours. Consistently, nuclear factor-kappa B (NF-κB) is activated when TNFα is maximal after E2 treatment. Further examination revealed that an anti-inflammatory agent, dexamethasone (Dex), activates glucocorticoid receptor (GR) transcriptional activity and blocks E2-induced apoptosis. These effects can be reversed by the knockdown of GR. A notable mechanistic change is that Dex effectively blocks the phosphorylation of PERK and produces equivalent inhibitory effects on the apoptosis-associated inflammatory genes as the PERK inhibitor. However, Dex has an additional potential to activate lipid metabolism and are additive with E2 to elevate FADS1 and adipokine IL6/IL6R. All of these data, for the first time, describe the mechanisms underlying E2-induced inflammation in E2-deprived breast cancer. It provides important information to restrict the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in estrogen deprived breast cancer patients. Citation Format: Ping Fan, Fadeke A. Agboke, Amit Kumar Tyagi, V. Craig Jordan. Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2863.

Recent clinical trials have demonstrated that estrogen (E2) alone reduces breast cancer incidence in postmenopausal women and has therapeutic effects on aromatase inhibitor resistant patients which both are related with the effect of E2-induced apoptosis. We have shown that E2 induces apoptosis in long-term E2 deprived breast cancer cells (MCF-7:5C) through stress responses, but the molecular mechanism underlying E2-induced stress remains to be elucidated. Here, we report that E2 activated the sensors of unfolded protein response (UPR) inositol-requiring protein 1 alpha (IRE1α) and PRK-like endoplasmic reticulum kinase (PERK) within 24 hours. Knockdown of PERK and IRE-1α through small interferon RNAs (siRNA) partially prevented E2-induced apoptosis, which suggested that endoplasmic reticulum stress was involved in the E2-induced apoptosis. Further examination showed E2 activated both classical estrogen responsive element (ERE) pathway and nonclassical activating protein-1 (AP-1) pathway in MCF-7:5C cells. Classical ERE regulated genes were not directly involved in E2-induced apoptosis. However, the transcription factor c-Fos acted as a critical trigger involved in stress responses induced by E2 in MCF-7:5C cells. E2 immediately elevated c-Fos expression in MCF-7:5C cells but not in wild-type breast cancer cells and 4-hydroxytamoxifen blocked this stimulation which suggested that E2 activated c-Fos through estrogen receptor (ER). E2 increased phosphorylation of c-Jun after 24 hours treatment but did not significantly enhance the abundance of c-Jun as c-Fos. c-Fos protein forms stable heterodimers with c-Jun which preferentially bind to phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE). Interestingly, low doses of TPA which activated TRE activity could induce apoptosis and activated apoptosis-related genes similar to E2 in MCF-7:5C cells. Knockdown of c-Fos and c-Jun with specific siRNAs blocked E2-induced apoptosis, which confirmed the activation of AP-1 played a critical role in the process of apoptosis induced by E2. Although c-Fos closely associates with c-Jun to form a stable heterodimer, they had differential functions in modulation of UPR and other nuclear transcription factors. Knockdown of c-Fos but not c-Jun could inhibit the activation of UPR by E2 in MCF-7:5C cells. Furthermore, downregulation of c-Fos abolished the expression of multiple nuclear transcription factors such as SRC-3, NF-κB, and p300 etc in MCF-7:5C cells. Overall, these data illustrate that c-Jun might provide a dimerizing site for c-Fos, whereas c-Fos was identified as a pivotal regulatory target of E2 in MCF-7:5C cells to trigger the apoptotic cascades. This study provides an important rationale for further exploration of E2-induced apoptosis in endocrine resistant breast cancer to improve clinical benefit. Citation Format: Ping Fan, Fadeke Agboke, Obi L. Griffith, Russell E. McDaniel, Xiaojun Zou, Karen Creswell, Joe W. Gray, Virgil Craig Jordan. Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 830. doi:10.1158/1538-7445.AM2013-830