Fotis Nikolos

ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR

IntroductionEpithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.MethodsBasal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.ResultsHere we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.ConclusionsTaken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.

ERβ decreases breast cancer cell survival by regulating the IRE1/XBP-1 pathway

Unfolded protein response (UPR) is an adaptive reaction that allows cancer cells to survive endoplasmic reticulum (EnR) stress that is often induced in the tumor microenvironment because of inadequate vascularization. Previous studies report an association between activation of the UPR and reduced sensitivity to antiestrogens and chemotherapeutics in estrogen receptor α (ERα)-positive and triple-negative breast cancers, respectively. ERα has been shown to regulate the expression of a key mediator of the EnR stress response, the X-box-binding protein-1 (XBP-1). Although network prediction models have associated ERβ with the EnR stress response, its role as regulator of the UPR has not been experimentally tested. Here, upregulation of wild-type ERβ (ERβ1) or treatment with ERβ agonists enhanced apoptosis in breast cancer cells in the presence of pharmacological inducers of EnR stress. Targeting the BCL-2 to the EnR of the ERβ1-expressing cells prevented the apoptosis induced by EnR stress but not by non-EnR stress apoptotic stimuli indicating that ERβ1 promotes EnR stress-regulated apoptosis. Downregulation of inositol-requiring kinase 1α (IRE1α) and decreased splicing of XBP-1 were associated with the decreased survival of the EnR-stressed ERβ1-expressing cells. ERβ1 was found to repress the IRE1 pathway of the UPR by inducing degradation of IRE1α. These results suggest that the ability of ERβ1 to target the UPR may offer alternative treatment strategies for breast cancer.

ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 oncogenic function.

Most (80%) of the triple-negative breast cancers (TNBCs) express mutant p53 proteins that acquire oncogenic activities including promoting metastasis. We previously showed that wild-type ERβ (ERβ1) impedes epithelial to mesenchymal transition (EMT) and decreases the invasiveness of TNBC cells. In the present study we searched for signaling pathways that ERβ1 uses to inhibit EMT and invasion in TNBC cells. We show that ERβ1 binds to and opposes the transcriptional activity of mutant p53 at the promoters of genes that regulate metastasis. p63 that transcriptionally cooperates with mutant p53 also binds to ERβ1. Downregulation of p63 represses the epithelial phenotype of ERβ1-expressing cells and alters the expression of mutant p53 target genes. These results describe a novel mechanism through which ERβ1 can disturb oncogenic signals to inhibit aggressiveness in TNBCs.

ERβ Regulates NSCLC Phenotypes by Controlling Oncogenic RAS Signaling

Non–small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. In addition to the aberrant growth factor signaling, dysregulation of other pathways, such as those mediated by estrogens and their receptors, has been linked to NSCLC initiation and progression. Although the expression of wild-type estrogen receptor β (ERβ1) has been associated with prolonged disease-free survival in patients with NSCLC, the molecular mechanism that accounts for this correlation is unknown. Here, upregulation of ERβ1 reduced proliferation and enhanced apoptosis in the context of mutant RAS. ERβ1 was found to induce apoptosis by stimulating the intrinsic apoptotic pathway that involves BIM, a Bcl-2 proapoptotic family member that is regulated by the extracellular signal–regulated kinase (ERK). Downregulation of EGFR and inactivation of RAS and the downstream components ERK1/2 were found to be involved in the ERβ1-induced apoptosis. Manipulation of EGFR and RAS expression and activity in ERβ1-expressing cells revealed the central role of oncogenic RAS inhibition in the ERβ1-mediated proapoptotic phenotype and EGFR regulation. These results demonstrate that ERβ1 decreases the survival of NSCLC cells by regulating oncogenic RAS signaling. Implications: The ability of ERβ1 to regulate the oncogenic functions of RAS suggests its importance in the biology of NSCLC and its clinical management. Mol Cancer Res; 12(6); 843–54. ©2014 AACR.

Somatic loss of estrogen receptor beta and p53 synergize to induce breast tumorigenesis

BackgroundUpregulation of estrogen receptor beta (ERβ) in breast cancer cells is associated with epithelial maintenance, decreased proliferation and invasion, and a reduction in the expression of the receptor has been observed in invasive breast tumors. However, proof of an association between loss of ERβ and breast carcinogenesis is still missing.MethodsTo study the role of ERβ in breast oncogenesis, we generated mouse conditional mutants with specific inactivation of ERβ and p53 in the mammary gland epithelium. For epithelium-specific knockout of ERβ and p53, ERβF/F and p53F/F mice were crossed to transgenic mice that express the Cre recombinase under the control of the human keratin 14 promoter.ResultsSomatic loss of ERβ significantly accelerated formation of p53-deficient mammary tumors. Loss of the receptor also resulted in the development of less differentiated carcinomas with stronger spindle cell morphology and decreased expression of luminal epithelial markers.ConclusionsOur results show that synergism between ERβ and p53 inactivation functions to determine important aspects of breast oncogenesis and cancer progression.

Pleiotropic signaling evoked by tumor necrosis factor in podocytes

TNF has been implicated in glomerular diseases, but its actions on podocytes are not well understood. Endogenous TNF expression is markedly increased in mouse podocytes exposed to sera from patients with recurrent focal segmental glomerulosclerosis, and TNF is able to increase its own expression in these cells. Exposure of podocytes to TNF increased phosphorylation of NF-κB p65-RelA followed by increased tyrosine phosphorylation of STAT3. STAT3 activation was blocked by the NF-κB inhibitor JSH-23 and by the STAT3 inhibitor stattic, whereas TNF-evoked NF-κB activation was not affected by stattic. TNF treatment increased nuclear accumulation of nuclear factor of activated T cells (NFAT)c1 in podocytes, a process that occurred downstream of STAT3 activation. TNF also increased expression of cyclin D1 but had no effect on cyclin-dependent kinase 4, p27(kip), or podocin. Despite its effects on cyclin D1, TNF treatment for up to 72 h did not cause podocytes to reenter the cell cycle. TNF increased total expression of transient receptor potential (TRP)C6 channels through a pathway dependent on NFATc1 and increased the steady-state expression of TRPC6 subunits on the podocyte cell surface. TNF effects on TRPC6 trafficking required ROS. Consistent with this, La(3+)-sensitive cationic currents activated by a diacylglycerol analog were increased in TNF-treated cells. The effects of TNF on NFATc1 and TRPC6 expression were blocked by cyclosporine A but were not blocked by the pan-TRP inhibitor SKF-96365. TNF therefore influences multiple pathways previously implicated in podocyte pathophysiology and is likely to sensitize these cells to other insults.

ERβ alters the chemosensitivity of luminal breast cancer cells by regulating p53 function

Estrogen receptor α (ERα)-positive breast cancers tend to develop resistance to both endocrine therapy and chemotherapy. Despite recent progress in defining molecular pathways that confer endocrine resistance, the mechanisms that regulate chemotherapy response in luminal tumors remain largely elusive. Luminal tumors often express wild-type p53 that is a major determinant of the cellular DNA damage response. Similar to p53, the second ER subtype, ERβ, has been reported to inhibit breast tumorigenesis by acting alone or in collaboration with p53. However, a synergistic mechanism of action has not been described. Here, we suggest that ERβ relies on p53 to elicit its tumor repressive actions in ERα-positive breast cancer cells. Upregulation of ERβ and treatment with ERβ agonists potentiates the tumor suppressor function of p53 resulting in decreased survival. This effect requires molecular interaction between the two proteins that disrupts the inhibitory action of ERα on p53 leading to increased transcriptional activity of p53. In addition, we show that the same interaction alters the chemosensitivity of endocrine-resistant cells including their response to tamoxifen therapy. Our results suggest a collaboration of ERβ and p53 tumor suppressor activity in breast cancer cells that indicates the importance of ligand-regulated ERβ as a tool to target p53 activity and improve the clinical management of resistant disease.

ERβ Sensitizes NSCLC to Chemotherapy by Regulating DNA Damage Response

The expression of wild-type estrogen receptor β (ESR2/ERβ1) correlates with clinical outcome in patients with non–small cell lung cancer (NSCLC). However, the molecular mechanism that accounts for this association is currently poorly understood. ERβ1 was previously linked to chemotherapy response in patients with breast cancer and in breast cancer cells. The effect of the receptor in NSCLC cells after chemotherapy treatment, a common remedy for advanced NSCLC, has not been studied. Here, upregulation of ERβ1 increases the sensitivity of NSCLC cells to treatment with doxorubicin and etoposide. This effect was primarily observed in p53-defecient NSCLC cells. In these cells, ERβ1 either enhanced G2–M cell-cycle arrest by activating the checkpoint kinase 1 (Chk1) and altering downstream signaling or induced apoptosis. The expression of p63 target genes that control G2–M checkpoint activation was altered by ERβ1 suggesting an ERβ1–p63 transcriptional cooperation in lung cancer cells that affects DNA damage response (DDR). These results suggest involvement of ERβ1 in the mechanism that regulates DNA damage response in NSCLC cells and support the potential predictive and therapeutic value of the receptor in clinical management of the disease. Implications: This study demonstrating the impact of ERβ1 on chemosensitivity of NSCLC cells suggests the predictive value of the receptor for successful response of tumors to chemotherapy and the potential benefit of chemotherapy-treated patients from the use of ER ligands. Mol Cancer Res; 16(2); 233–42. ©2017 AACR.

Abstract 5043: ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 gain-of-function

Triple-negative breast cancers (TNBCs) display distinct metastatic potential and a poor prognosis despite a relative chemosensitivity. Most (80%) of these highly aggressive tumors express mutant p53 proteins that are known to elicit oncogenic activities that contribute to metastasis and chemoresistance. Despite the lack of estrogen receptor α (ERα), TNBCs express wild-type ERβ (ERβ1). We previously showed that ERβ1 impedes epithelial to mesenchymal transition (EMT) in TNBC cells by repressing epidermal growth factor receptor (EGFR) signaling. Our recent findings indicate that ERβ1 regulates EGFR expression and decreases the invasiveness of TNBC cells by interacting with mutant p53 and inhibiting its function. Suppression of a mutant p53 transcriptional program and induction of p63 activity are essential for the anti-migratory activity of ERβ1 in TNBC cells. These results describe an ERβ1/mutant p53 interaction that disturbs oncogenic signals to inhibit aggressiveness in TNBC. Citation Format: Igor Bado, Fotis Nikolos, Gayani Rajapaksa, Jan-Ake Gustafsson, Christoforos Thomas. ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 gain-of-function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5043. doi:10.1158/1538-7445.AM2015-5043

Abstract 507: ERβ1 decreases breast cancer cell survival by regulating the unfolded protein response

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Estrogen receptors (ERs) regulate cellular processes that influence the development and response of breast cancer to hormonal therapy. Unfolded protein response (UPR) has been identified as a key mechanism that determines endocrine responsiveness and cellular survival in breast cancer. UPR assists the cells to cope with the stress caused by the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Although ERα is known to affect the cellular response to ER stress, the role of ERβ in regulating the UPR has not been investigated. Here we show that the wild-type ERβ (ERβ1) enhanced apoptosis in breast cancer cells in response to treatment with the ER stress inducers thapsigargin and bortezomib. Targeting the Bcl-2 in the ER of the ERβ1-expressing cells prevented the apoptosis induced by ER stress but not by non-ER stress apoptotic stimuli indicating that ERβ1 promotes ER stress-regulated apoptosis. ERβ1 promoted ER stress-induced apoptosis by suppressing the inositol-requiring enzyme-1 (IRE-1) pathway of the UPR. ERβ1 repressed the transcription of the IRE-1α gene and the subsequent splicing of the X-box binding protein-1 (XBP-1). These results suggest a potential role of ERβ1 in influencing stress and therapy response in breast cancer by regulating the UPR. Citation Format: Christoforos Thomas, Gayani Rajapaksa, Fotis Nikolos, Igor Bado, Jan-Ake Gustafsson. ERβ1 decreases breast cancer cell survival by regulating the unfolded protein response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 507. doi:10.1158/1538-7445.AM2014-507