Acquisition Of Tamoxifen Resistance Research Articles

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity.

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy.

Riluzole Suppresses Growth and Enhances Response to Endocrine Therapy in ER+ Breast Cancer.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.

HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer.

Tamoxifen is a commonly used drug to treat estrogen receptor-positive patients with breast cancer. Despite the outstanding efficacy of tamoxifen, approximately one-third of patients develop resistance toward it, thereby presenting a therapeutic challenge. HOX genes may be involved in the acquisition of tamoxifen resistance. In this study, we identified HOXA5, a member of the HOX gene family, as a marker of tamoxifen resistance. Using ChIP assay, we found that HOXA5 expression was significantly overexpressed in tamoxifen-resistant MCF7 (TAMR) breast cancer cells because of reduced H3K27me3 binding. HOXA5 upregulation resulted in activation of the PI3K/AKT signaling cascade, which in turn, led to p53 and p21 reduction, ultimately making the TAMR cells less apoptotic. Furthermore, elevated HOXA5 expression resulted in breast cancer cells acquiring more mesenchymal-like and stem cell traits associated with aggressive breast cancer phenotypes. In conclusion, our results delineate a mechanism by which HOXA5 promotes tumorigenesis, cancer progression, and tamoxifen resistance in breast cancer cells.

Long Non-coding RNA BLACAT1 Induces Tamoxifen Resistance in Human Breast Cancer by Regulating miR-503/Bcl-2 Axis.

IntroductionAt present, drug resistance remains a major obstacle for breast cancer (BCa) patients who receive tamoxifen (TAM) chemotherapy. In this study, we aimed to investigate the functional role of long non-coding RNA BLACAT1 in the acquisition of TAM resistance in BCa.MethodsTAM-resistant BCa cells were derived by exposure to 1 μM of TAM for 6 months. The expression levels of BLACAT1 and miR-503 were detected by RT-qPCR analysis. Chemosensitivity of BCa cells to TAM was measured by MTT assay. Apoptosis of BCa cells was detected by flow cytometric analysis, and the expression levels of apoptosis-related proteins were detected by Western blot analysis. The direct binding relation between BLACAT1 and miR-503 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assay.ResultsOur findings showed that BLACAT1 was significantly upregulated in TAM-resistant BCa cells (MCF-7/TR and T47D/TR), and BLACAT1 knockdown markedly reduced the TAM resistance in these cells. Importantly, we observed that BLACAT1 might function as a competing endogenous RNA of miR-503 in MCF-7/TR and T47D/TR cells, thereby increasing the expression of oncogenic Bcl-2 protein. Rescue experiments showed that miR-503 inhibition partly blocked the inhibitory effect of BLACAT1 knockdown on TAM resistance of MCF-7/TR and T47D/TR cells.ConclusionTo conclude, this study revealed that overexpressed BLACAT1 induces TAM resistance in human BCa partly by regulating miR-503/Bcl-2 axis, potentially benefiting BCa treatment in the future.

Abstract 2121: Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer

Abstract Estrogen receptor (ER)-positive breast cancer accounts for almost 75% of all breast cancers. Tamoxifen has been used for over 40 years to treat early, locally advanced and metastatic ER-positive breast cancer; however, patients develop resistance over time. Furthermore, metastatic recurrence, the major cause of death, is common in ER-positive breast cancer patients whose cancer progressed on tamoxifen and other hormone therapies. In this line, we and others reported epithelial-mesenchymal (EMT)-like changes upon acquisition of tamoxifen resistance hinting towards the co-occurrence of resistance and metastatic abilities of the cells. However, little is known about common molecular mediators of drug resistance and metastatic recurrence. Therefore, we addresses the question of how we concurrently overcome tamoxifen resistance and prevent lethal metastatic recurrence whereby eliminating the mortality associated with metastatic breast cancer. Combining whole-transcriptome sequencing and downstream pathway analysis, we identified cyclic AMP (cAMP) signaling to be the most significantly altered pathway in acquired tamoxifen resistant breast cancer cells. We found that PDE4D (Phosphodiesterase 4D), which hydrolyzes cAMP, was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. We demonstrated that PDE4D inhibition overcomes tamoxifen resistance in cell lines and tumor xenografts via cAMP induced endoplasmic reticulum stress and cell death. Importantly, our TamR cells have partial EMT and enrichment of TGF-beta signaling, and PDE4D inhibition in combination with tamoxifen inhibited cell migration in vitro and blocked TGF-beta induced EMT. RNA-Seq experiment also identified one of the highly oncogenic long non-coding RNAs (lncRNA) to be upregulated in TamR cells, and its targeting inhibits PDE4D and leads to tamoxifen sensitization. We showed that higher expressions of both PDE4D and the candidate lncRNA predict worse survival in tamoxifen-treated breast cancer patients. Currently, we are testing the tamoxifen sensitizer and metastasis blocker functions of PDE4D and our candidate lncRNA using patient-derived xenograft (PDX) models of metastatic ER-positive breast cancer. Overall, our results suggest that targeting PDE4D, or its upstream regulatory lncRNA, can simultaneously overcome tamoxifen resistance and prevent metastatic recurrence in ER-positive breast cancer. This is then expected to dramatically reduce mortality rates among ER-positive breast cancer patients in future. Citation Format: Pelin Gulizar Ersan, Ozge Saatci, Oguzhan Tarman, Rasmi Mishra, Nevin Belder, Unal Metin Tokat, Yasser Riazalhosseini, Ozgur Sahin. Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2121.

Anticancer effect of metformin on estrogen receptor-positive and tamoxifen-resistant breast cancer cell lines.

Acquisition of tamoxifen resistance (TR) during anti-estrogenic therapy using tamoxifen is a major obstacle in the treatment of estrogen receptor (ER)-positive breast cancer. As a biguanide derivative, metformin is commonly used to treat type II diabetes. It has recently emerged as a potential anticancer agent. The objective of the present study was to investigate the anticancer activity of metformin in relation to ERα expression and its signaling pathway in ERα-positive MCF-7 and MDA-MB-361 breast cancer cells as well as TR MCF-7 breast cancer cells. Metformin inhibited both protein and mRNA levels of ERα in the presence or absence of estrogen (E2) in the MCF-7, TR MCF-7 and MDA-MB-361 cells. Metformin repressed E2-inducible estrogen response element (ERE) luciferase activity, protein levels and mRNA levels of E2/ERα-regulated genes [including c-Myc, cyclin D1, progesterone receptor (PR) and pS2] to a greater degree than tamoxifen, resulting in inhibition of cell proliferation of MCF-7, TR MCF-7 and MDA-MB-361 cells. Collectively, our results suggest that one of the anticancer mechanisms of metformin could be attributable to the repression of expression and transcriptional activity of ERα. Metformin may be a good therapeutic agent for treating ERα-positive breast cancer by inhibiting the expression and function of ERα. In addition, metformin may be useful to treat tamoxifen-resistant breast cancer.

Abstract 710: CRP induction in tamoxifen-resistant breast cancer cells

Abstract Breast cancer is one of the most common cancer types among post-menopausal women. Tamoxifen has been known as the most effective therapeutic option for estrogen receptor-positive breast cancer. One of the main issues of breast cancer is acquisition of tamoxifen resistance, and if it does, the way to treat tamoxifen-resistant breast cancer has not been discovered. It has been recently suggested that c-reactive protein (CRP), inflammatory marker found in many diseases may be related to breast cancer progression. Here, we found that tamoxifen-resistant breast cancer (TAMR-MCF-7) cells expresses much higher protein and mRNA level of CRP compared to parent breast cancer (MCF-7) cells. CRP knock-down by lentiviral shRNA did not affect cell proliferation rate of TAMR-MCF-7 cells. One of the main characteristics of tamoxifen-resistant breast cancer is higher migratory ability. Through trans-well migration assay, we have discovered that CRP alone increases migratory function of MCF-7 cells. Moreover, CRP knock-down significantly reduced cell migration of TAMR-MCF-7 cells. It has been recently reported that sphingosine-1-phosphate (S1P) induces CRP in mammary epithelial cells and ultimately causes cancer cell transformation. S1P treatment increased CRP expression in MCF-7 cells. Protein expression of sphingosine kinase (Sphk) 1 and 2, which catalyzes conversion of sphingosine to S1P, was also increased in TAMR-MCF-7 cells. Among five S1P receptors, we also discovered that S1P receptor 3 (S1Pr3) is related with CRP induction in TAMR-MCF-7 cells. Taken together, tamoxifen-resistant breast cancer expresses high level of CRP and S1P-S1Pr3 plays a key role in CRP induction in TAMR-MCF-7 cells. Higher levels of CRP increases migratory function of tamoxifen-resistant breast cancer cells. CRP may be an important therapeutic target for the treatment of tamoxifen-resistant breast cancer. Citation Format: Min Chang Choi, Aree Moon, Keon Wook Kang. CRP induction in tamoxifen-resistant breast cancer cells. [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 710. doi:10.1158/1538-7445.AM2015-710

Abstract P1-12-12: The insulin like growth factor axis and development of tamoxifen resistance in breast cancer

Abstract Background Insulin-like growth factors (IGF). IGFs are potent mitogens for breast epithelial cells that modulate the action of AKT, which has been reported to be associated with tamoxifen resistance. To date, however, anti-IGF strategies have proved disappointing in clinical trials We have investigated whether the IGFBP family of proteins, which modulate the activity of IGF, may play a role in tamoxifen resistance, opening up the route for alternative anti-IGF based therapies. Methodology We investigated the expression of IGF axis genes in parental and tamoxifen-resistant (TamR) MCF-7 cells using qRT-PCR. Gene and protein expression was confirmed using ELISA, Western, and ligand blotting. shRNA transfection was used to silence candidate IGF axis genes in both cell lines. Cell sensitivity 4-hydroxytamoxifen (4-HT) was investigated by WST-1 cell proliferation assay. Results IGF-IR, IGF-2R, IGFBP-2, -4 and -5 genes had the highest expression levels. IGFBP-5 was down-regulated 7-fold while IGFBP-2 was up-regulated by 2-fold in TamR v wt cells. Changes in IGFBP-5 and IGFBP-2 gene expression were mirrored in protein levels measured in conditioned media by ELISA, Western and Ligand blot. Importantly knock down of IGFBP-2 in TamR cells restored sensitivity to 4-HT suggesting a causal role for IGFBP-2 in the acquisition of tamoxifen resistance. Conclusion IGFBP-5 and IGFPB-2 are reciprocally regulated on the acquisition of tamoxifen resistance by MCF-7 cells. Preliminary studies suggest that IGFBP-2 may play a role in the development of tamoxifen resistance in vitro. Citation Format: Yousef M Hawsawi, James Beattie, Reem El-Gendy, Valerie Speirs, Christopher Twelves. The insulin like growth factor axis and development of tamoxifen resistance in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-12.

Effect of angiotensin receptor blockade on prevention and reversion of tamoxifen-resistant phenotype in MCF-7 cells.

Tamoxifen (TAM) is a standard adjuvant endocrine therapy in postmenopausal breast cancer patients, but innate or acquired TAM resistance has remained to be a therapeutic challenge for clinicians. The aim of this study was to explore the possible participation of renin-angiotensin system (RAS) in the acquisition of TAM resistance and try to prevent and regress the resistance using an angiotensin II receptor type-1 (AGTR1) blocker, losartan. Establishment of TAM-resistant (TAM-R) cells was accomplished by continuous exposure of MCF-7 cells to 1μmol/L TAM. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to determine cell growth. Moreover, messenger RNA (mRNA) expression levels of AGTR1 and angiotensin II receptor type-2 (AGTR2) were measured by quantitative real-time polymerase chain reaction. A significant increase of AGTR1 and AGTR2 transcripts was observed in TAM-R cells compared to MCF-7 cells. Interestingly, losartan-TAM combination effectively resensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest an important role of RAS in acquired TAM resistance and targeting of RAS by losartan may overcome TAM resistance phenomenon and provide a novel avenue for treatment of resistant breast cancers.

Abstract 2108: Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer

Abstract Introduction: Although the presence of nuclear estrogen receptor (ERα) is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signalling. We have already shown that arginine methylation of ERα is essential for E2-non genomic pathway triggered by ERα interaction with PI3K and c-Src thereby propagating the signal to downstream transduction cascades (Le Romancer et al, Mol Cell, 2008). Recently, we have found that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy (Poulard et al, Embo Mol Med, 2012). In addition, the cross talk between ERα and growth factor signalling has emerged as a critical factor in endocrine resistance. The aim of this study was to investigate the receptor cytoplasmic signalling in human ERα-positive breast cancer with acquired resistance to tamoxifen. Experimental procedures: The activation of cytoplasmic ER signalling pathway was investigated in tamoxifen resistant MCF-7 cells and in patient-derived xenografts (PDX) of ERα-positive breast cancer with acquired resistance in vivo to tamoxifen. In addition, tamoxifen-resistant PDX models were treated with tamoxifen, fulvestrant and everolimus, during 90 days. ERα interactions with Src and PI3K were investigated by Proximity Ligation Assay (PLA) in formalin-fixed tumors, a technology which enables to investigate protein/protein interactions in tissues. Results: Interestingly, we detected an increase of ERα/Src/PI3K expression in tamoxifen resistant versus parental MCF-7 cells. This result was confirmed in one PDX model where we found a significant increase of ERα/Src/PI3K expression in the tamoxifen-resistant tumor compared to the corresponding parental xenograft. Finally, the analysis of treated tumors revealed a significant decrease of ERα/Src/PI3K expression in the fulvestrant-treated tumors as well as in tumors treated by the everolimus and fulvestrant combination. In conclusion these results show that ERα cytoplasmic signalling is associated to acquisition of tamoxifen resistance both in cell line and patients-derived xenografts. In addition, ERα cytoplasmic signalling appears to be affected in tumors responding to alternative treatments such as fulvestrant or everolimus. Targeted therapies against ERα, Src and PI3K are in progress and could constitute a new approach to circumvent resistance to endocrine therapies. Citation Format: Coralie Poulard, Juliette Rambaud, Pascale Cohen, Antimo Migliaccio, Laura Corbo, Elisabetta Marangoni, Muriel Le Romancer. Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer. [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 2108. doi:10.1158/1538-7445.AM2014-2108

The role of captopril and losartan in prevention and regression of tamoxifen-induced resistance of breast cancer cell line MCF-7: An in vitro study

Innate and acquired tamoxifen (TAM) resistance in estrogen receptor positive (ER+) breast cancer is an important problem in adjuvant endocrine therapy. The underlying mechanisms of TAM resistance is yet unknown. In the present study, we evaluated the role of renin–angiotensin system (RAS) in the acquisition of TAM resistance in human breast cancer cell line MCF-7, and the potential role of captopril and captopril+losartan combination in the prevention and reversion of the TAM resistant phenotype. MCF-7 cells were continuously exposed to 1μmol/L TAM to develop TAM resistant cells (TAM-R). MTT cell viability assay was used to determine the growth response of MCF-7 and TAM-R cells, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess angiotensin I converting enzyme (ACE), angiotensin II receptor type-1 and type-2 (AGTR1 and AGTR2) mRNA expressions. Preventive and therapeutic effects of RAS blockers – captopril and losartan – were examined on MCF-7 and TAM-R cells. Based on qRT-PCR, TAM-R cells compared to MCF-7 cells, had a mean±SD fold increase of 319.1±204.1 (P=0.002) in production of ACE mRNA level, 2211.8±777.9 (P=0.002) in AGTR1 mRNA level, and 265.9±143.9 (P=0.037) in production of AGTR2 mRNA level. The combination of either captopril or captopril+losartan with TAM led to the prevention and even reversion of TAM resistant phenotype.

Estrogen receptor α is the major driving factor for growth in tamoxifen-resistant breast cancer and supported by HER/ERK signaling

Resistance to tamoxifen is a major clinical challenge in the treatment of breast cancer; however, it is still unclear which signaling pathways are the major drivers of tamoxifen-resistant growth. To characterize resistance mechanisms, we have generated different tamoxifen-resistant breast cancer cell lines from MCF-7. In this model, we investigated whether signaling from human epidermal growth factor receptors (HERs), their downstream kinases, or from the estrogen receptor α (ERα) was driving tamoxifen-resistant cell growth. Increased expression of EGFR and increased phosphorylation of HER3 were observed upon acquisition of tamoxifen resistance, and the extracellular activated kinase (ERK) signaling pathway was highly activated in the resistant cells. The EGFR inhibitor gefitinib and the ERK pathway inhibitor U0126 resulted in partial and preferential growth inhibition of tamoxifen-resistant cells. All the tamoxifen-resistant cell lines retained ERα expression but at a lower level compared to that in MCF-7. Importantly, we showed via ERα knockdown that the tamoxifen-resistant cells were dependent on functional ERα for growth and we observed a clear growth stimulation of resistant cell lines with clinically relevant concentrations of tamoxifen and 4-OH-tamoxifen, indicating that tamoxifen-resistant cells utilize agonistic ERα stimulation by tamoxifen for growth. The tamoxifen-resistant cells displayed high phosphorylation of ERα at Ser118 in the presence of tamoxifen; however, treatment with U0126 neither affected the level of Ser118 phosphorylation nor expression of the ERα target Bcl-2, suggesting that ERK contributes to cell growth independently of ERα in our cell model. In support of this, combined treatment against ERα and ERK signaling in resistant cells was superior to single-agent treatment and as effective as fulvestrant treatment of MCF-7 cells. Together, these findings demonstrate that ERα is a major driver of growth in tamoxifen-resistant cells supported by HER/ERK growth signaling, implying that combined targeting of these pathways may have a clinical potential for overcoming tamoxifen resistance.

Abstract 3570: Phospho-dependent regulation of ERRγ expression, transcriptional activity, and Tamoxifen resistance in ER+ breast cancer.

Abstract Selective estrogen receptor modulators (SERMs) such as Tamoxifen (TAM) can significantly improve breast cancer-specific survival for women with ER-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor with broad, structural similarities to classical ER that is widely implicated in the transcriptional regulation of energy homeostasis. We previously reported that ERRγ is upregulated during the acquisition of TAM resistance in ER+ breast cancer cell lines, exogenous expression of ERRγ is sufficient to induce TAM resistance, and ERRγ mRNA is significantly increased in tumor samples from women with ER+ breast cancer who relapse following TAM treatment. Because ERRγ has no known ligand, our recent studies have focused on understanding how the expression and activity of this orphan nuclear receptor is regulated, and how this contributes to the TAM resistant phenotype. We have found that TAM-resistant breast cancer cells in which endogenous ERRγ is upregulated show a concomitant hyper activation of p44/p42 ERK. Furthermore, ERK activity (but not that of JNK or p38 MAPK) directly enhances ERRγ protein stability via Serines 57, 81, and/or 219 of the receptor. Phospho-deficient ERRγ is impaired in its ability to induce TAM resistance, and this is associated with a significant reduction in transcriptional activity at the estrogen-related response element (ERRE) half-site, in particular. This led us to hypothesize that ERRγ action at ERREs is most relevant to the development of TAM resistance. We examined a meta-list of validated, ERRE-containing ERR target genes in association with distant metastasis-free survival (DMFS) within 5 years of primary diagnosis in each of 3 publicly available clinical datasets comprised of ER+ breast cancer patients treated with TAM, and obtained a list of 37 differentially expressed targets. The proximal promoter regions of these 37 genes are enriched for binding sites for ELK1, a well-known ERK substrate. Sub-network analysis reveals enrichment of genes whose protein products regulate the mitochondrial unfolded protein response (UPRmito). We also performed differential dependency network (DDN) analysis in an independent dataset using the full meta-list of ERRγ target genes and also identified genes associated with UPRmito. These data suggest a potentially novel role for ERK-mediated regulation of ERRγ in mitochondrial protein folding in TAM-resistant breast cancer. Citation Format: Rebecca B. Riggins, Mary M. Heckler, Hemang Thakor, Cara C. Schafer, Salendra Singh, Ye Tian, Yuriy Gusev, Subha Madhavan, Yue Wang. Phospho-dependent regulation of ERRγ expression, transcriptional activity, and Tamoxifen resistance in ER+ breast cancer. [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 3570. doi:10.1158/1538-7445.AM2013-3570

Abstract A1: Functional significance of miR-10b in tamoxifen resistance of breast cancer cells

Abstract Tamoxifen is a conventional treatment option for breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors. However, a significant number of these patients eventually develop resistance to tamoxifen during therapy, and thus the management of such tamoxifen-resistant patients remains a clinical challenge. In an effort to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed increased expression of microRNA 10b (miR-10b) in ER-positive MCF-7 cells-derived tamoxifen-resistant cells (MCF7TR). The miR-10b has been implicated in invasion and metastasis but its role, if any, in drug resistance of breast cancer cells has never been investigated. To ascertain the mechanistic role of miR-10b in tamoxifen resistance, we downregulated its levels in MCF7TR cells and in addition we also overexpressed miR-10b by transfecting MCF-7 cells with pre-miR-10b. We found that the downregulation of miR-10b in MCF7TR cells led to increased sensitivity to tamoxifen whereas overexpression of miR-10b in MCF-7 cells led to increased resistance to tamoxifen. During our mechanistic investigation, we found that increased expression of miR-10b downregulated the expression of HDAC4 in MCF7TR cells. Inactivation of HDAC4 using HDAC4-specific siRNA transfection in MCF-7 cells led to the acquisition of tamoxifen resistance. Moreover, further reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen resistance. In summary, we have identified miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance, and thus targeting this nexus could become a novel targeted therapeutic approach for the treatment of tamoxifen-resistant breast cancer.

Src kinase promotes adhesion-independent activation of FAK and enhances cellular migration in tamoxifen-resistant breast cancer cells

Src kinase is intimately involved in the control of matrix adhesion and cell migration through its ability to modulate the activity of focal adhesion kinase (FAK). In light of our previous observations that acquisition of tamoxifen resistance in breast cancer cells is accompanied by elevated Src kinase activity, we wish to investigate whether FAK function is also altered in these cells and if this leads to an enhanced migratory phenotype. In in vitro adhesion assays, tamoxifen-resistant (TamR) MCF7 cells had a greater affinity for the matrix proteins fibronectin, laminin, vitronectin and collagen and subsequently demonstrated a much greater migratory capacity across these substrates compared to their weakly-migratory, endocrine-sensitive counterparts. Additionally, elevated levels of activated Src in TamR cells promoted an increase in FAK phosphorylation at Y861 and Y925 and uncoupled FAK activation from an adhesion-dependent process. Inhibition of Src activity using the Src/Abl inhibitor AZD0530 reduced FAK activity, suppressed cell spreading on matrix-coated surfaces and significantly inhibited cell migration. Our data thus suggest that Src kinase plays a central role in the enhanced migratory phenotype that accompanies endocrine resistance through its modulation of FAK signalling and demonstrates the potential use of Src inhibitors as potent suppressors of tumour cell migration.

Elevated Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells

Src kinase plays a central role in growth factor signalling, regulating a diverse array of cellular functions including proliferation, migration and invasion. Recent studies have demonstrated that Src activity is frequently elevated in human tumours and correlates with disease stage. We have previously demonstrated that, upon acquisition of tamoxifen resistance, MCF7 cells display increased epidermal growth factor receptor (EGFR) activation and a more aggressive phenotype in vitro. Since tumours exhibiting elevated EGFR signalling may possess elevated levels of Src activity, we wished to investigate the role of Src in our MCF7 model of endocrine resistance. Src kinase activity was significantly elevated in tamoxifen-resistant (TamR) cells in comparison to wild type MCF7 cells. This increase was not due to elevated Src protein or gene expression. Treatment of TamR cells with the novel Src inhibitor, AZD0530, significantly reduced the amount of activated Src detectable in both cell types whilst having no effect on total Src levels. AZD0530 significantly suppressed the motile and invasive nature of TamR cells in vitro, reduced basal levels of activated focal adhesion kinase (FAK) and paxillin and promoted elongation of focal adhesions. Furthermore, the use of this compound in conjunction with the EGFR inhibitor, gefitinib, was markedly additive towards inhibition of TamR cell motility and invasion. These observations suggest that Src plays a pivotal role in mediating the motile and invasive phenotype observed in endocrine-resistant breast cancer cells. The use of Src inhibitors in conjunction with EGFR inhibitors such as gefitinib may provide an effective method with which to prevent cancer progression and metastasis.

Tamoxifen resistance in breast cancer cells is accompanied by an enhanced motile and invasive phenotype: inhibition by gefitinib ('Iressa', ZD1839).

Despite an initial response to antihormonal therapies, the development of resistance will occur in a significant number of breast cancer patients. The mechanisms that underlie acquired resistance are not yet clear. Using a previously established in vitro cell model of tamoxifen resistance in MCF7 cells, shown to display autocrine epidermal growth factor receptor (EGFR) signalling, we assessed how resistance might modulate their metastatic phenotype in vitro, as metastatic disease is the single most important factor affecting the mortality of cancer patients. Furthermore, we investigated the effect of the EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib ('Iressa', ZD1839; AstraZeneca), on this behaviour. The acquisition of tamoxifen resistance in MCF7 cells was accompanied by a dramatic and significant increase in their invasive and motile nature. The affinity of these cells for matrix components was also enhanced. Inhibition of EGFR signalling with gefitinib reduced both basal and TGF-alpha-stimulated invasion and motility and reduced cell-matrix adhesion. In conclusion, we demonstrate here that resistance to tamoxifen in breast cancer cells is accompanied by a significant increase in their basal motile and invasive activity, properties associated with increased metastatic potential. Inhibition of EGFR signalling by gefitinib significantly inhibited cell motility and invasion thus suggesting a role for the EGF receptor in the aggressive phenotype of tamoxifen-resistant breast cancer cells.

Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells.

Oestrogen receptor (ER) levels are usually maintained on acquisition of tamoxifen resistance in the clinic, however, tumour re-growth is associated with increased expression of epidermal growth factor receptor (EGFR) and activation of the mitogen activated protein kinase (MAPK) pathway. In the present study we have used the ER down-regulator fulvestrant ('Faslodex') to investigate the influence of the ER on growth of a tamoxifen-resistant (TAM-R) human breast cancer cell line. Expression levels of ER mRNA and protein were equivalent in parental wild-type MCF-7 (WT) and TAM-R cells. Fulvestrant eliminated ER protein expression and inhibited proliferation in both cell lines. The growth inhibitory effects of fulvestrant were associated with a decrease in basal EGFR, c-erbB2 and ERK1/2 activity in TAM-R but not WT cells. ER functionality as determined by oestrogen response element (ERE)-luciferase reporter activity and expression of PgR, pS2 and transforming growth factor alpha (TGFalpha) was significantly reduced in TAM-R compared to WT cells and was further decreased by fulvestrant treatment in both cell lines. Epidermal growth factor (EGF) and TGFalpha significantly increased EGFR/MAPK pathway activity in both cell lines. Ligand-induced EGFR/MAPK activation promoted TAM-R cell growth in both the absence and presence of fulvestrant, whereas no proliferative activity was observed under the same conditions in WT cells. These results suggest that the ER modulates EGFR/MAPK signalling efficiency in TAM-R cells possibly through the regulation of TGFalpha availability. This effect may be overcome by the action of exogenous EGFR ligands, which strengthen EGFR/MAPK signalling activity to generate endocrine-insensitive cell growth.

Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone

Tamoxifen resistance is a serious clinical problem commonly encountered in the management of patients with breast cancer. The mechanisms leading to its development are unclear. Tamoxifen acts via multiple pathways and has diverse effects. Hence transformation from a tamoxifen-sensitive to a resistant phenotype could involve multiple genetic events. Knowledge of the genetic pathways leading to resistance may facilitate the development of novel therapeutic strategies. In this study, a variation of conventional comparative genomic hybridization (CGH) has been employed to detect genetic alterations associated with tamoxifen resistance. MCF-7, a tamoxifen-sensitive human breast cancer cells line, and its tamoxifen-resistant clone, CL-9 were used. Both cell lines showed extensive areas of concordance but consistent differences were seen with the acquisition of tamoxifen resistance. These differences included the amplification of 2p16.3∼p23.2, 2q21∼q34, 3p12.3∼p14.1, 3p22∼p26, 3q, 12q13.2∼q22, 13q12∼q14, 17q21.3∼q23, 20q11.2∼q13.1 and 21q11.2∼q21 as well as the deletion of 6p21.1, 6p23∼p25, 7q11.1∼q31, 7q35∼q36, 11p15, 11q24, 13q33, 17p, 18q12∼q21.1, 19p, 19q13.3, 22q13.1∼q13.2. These findings were supported by conventional cytogenetics and chromosome painting. The regions identified by CGH potentially harbor genes that could be important in the development of tamoxifen resistance.

Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes.

Several lines of evidence indicate that the nuclear receptor corepressor (N-CoR) complex imposes ligand dependence on transcriptional activation by the retinoic acid receptor and mediates the inhibitory effects of estrogen receptor antagonists, such as tamoxifen, suppressing a constitutive N-terminal, Creb-binding protein/coactivator complex-dependent activation domain. Functional interactions between specific receptors and N-CoR or SMRT corepressor complexes are regulated, positively or negatively, by diverse signal transduction pathways. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in a mouse model system for human breast cancer. Our data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways.