Suppression of Nuclear Factor-κB by Glucocorticoid Receptor Blocks Estrogen-Induced Apoptosis in Estrogen-Deprived Breast Cancer Cells.

Our clinically relevant finding is that glucocorticoids block estrogen (E2)-induced apoptosis in long-term E2-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E2, but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E2, leading to the blockade of E2-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E2-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 E2-induced apoptosis in patients with aromatase inhibitor-resistant breast cancer.

Modulation of nuclear factor-kappa B activation by the endoplasmic reticulum stress sensor PERK to mediate estrogen-induced apoptosis in breast cancer cells.

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/EBPß), 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.

Conditional cell reprogramming involves non-canonical ß-catenin activation and mTOR-mediated inactivation of Akt.

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 ß-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that ß-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that ß-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, ß-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active ß-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of ß-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.

Integration of Downstream Signals of Insulin-like Growth Factor-1 Receptor by Endoplasmic Reticulum Stress for Estrogen-Induced Growth or Apoptosis in Breast Cancer Cells.

UNLABELLED: Estrogen (E2) exerts a dual function on E2-deprived breast cancer cells, with both initial proliferation and subsequent induction of stress responses to cause apoptosis. However, the mechanism by which E2 integrally regulates cell growth or apoptosis-associated pathways remains to be elucidated. Here, E2 deprivation results in many alterations in stress-responsive pathways. For instance, E2-deprived breast cancer cells had higher basal levels of stress-activated protein kinase, c-Jun N-terminal kinase (JNK), compared with wild-type MCF-7 cells. E2 treatment further constitutively activated JNK after 24 hours. However, inhibition of JNK (SP600125) was unable to abolish E2- induced apoptosis, whereas SP600125 alone arrested cells at the G2 phase of the cell cycle and increased apoptosis. Further examination showed that inhibition of JNK increased gene expression of TNFα and did not effectively attenuate expression of apoptosis-related genes induced by E2. A notable finding was that E2 regulated both JNK and Akt as the downstream signals of insulin-like growth factor-1 receptor (IGFIR)/PI3K, but with distinctive modulation patterns: JNK was constitutively activated, whereas Akt and Akt-associated proteins, such as PTEN and mTOR, were selectively degraded. Endoplasmic reticulum-associated degradation (ERAD) was involved in the selective protein degradation. These findings highlight a novel IGFIR/PI3K/JNK axis that plays a proliferative role during the prelude to E2-induced apoptosis and that the endoplasmic reticulum is a key regulatory site to decide cell fate after E2 treatment. IMPLICATIONS: This study provides a new rationale for further exploration of E2-induced apoptosis to improve clinical benefit.

Identification of gene regulation patterns underlying both oestrogen- and tamoxifen-stimulated cell growth through global gene expression profiling in breast cancer cells.

PURPOSE: A c-Src inhibitor blocks oestrogen (E2)-induced stress and converts E2 responses from inducing apoptosis to growth stimulation in E2-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 E2-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 E2 stimulated cell growth in a concentration-dependent manner. Expression profiling showed a remarkable overlap in genes regulated in the same direction by E2 and 4-OHT. Pathway enrichment analysis of the 280 genes commonly deregulated in MCF-7:PF cells by 4-OHT and E2 revealed functions mainly related to membrane, cytoplasm and metabolic processes. Further analysis of 98 genes up-regulated by both 4-OHT and E2 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 E2 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.

A molecular model for the mechanism of acquired tamoxifen resistance in breast cancer.

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-1Rß). 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-1Rß but increased phosphorylation of IGF-1Rß. 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-1Rß. 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.

Inhibition of c-Src blocks oestrogen-induced apoptosis and restores oestrogen-stimulated growth in long-term oestrogen-deprived breast cancer cells.

PURPOSE: Our publications demonstrate that physiological concentrations of oestrogen (E2) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E2-deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E2-induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E2, a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E2 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 E2 alone decreased cell growth. In contrast, a combination of PP2 and E2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E2, which could be blocked by the pure antioestrogen ICI182,780. One major change was that PP2 collaborated with E2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1Rß). Blockade of IGF-1Rß completely abolished E2-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 E2-induced apoptosis and enhance E2-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.

c-Src modulates estrogen-induced stress and apoptosis in estrogen-deprived breast cancer cells.

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.