BCAR1 Research Articles

Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor β/Smad signaling and associates with favorable breast cancer disease outcomes.

IntroductionThis study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor β (TGFβ) pathway, which induces potent migration and invasion responses in breast cancer cells.MethodsWe surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFβ/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFβ-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging–based matrix degradation assay, respectively.ResultsRelatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFβ through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFβ/Smad signaling pathway.ConclusionBCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFβ/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3’s molecular mechanism of action and highlight BCAR3 as a novel TGFβ/Smad antagonist in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0476-9) contains supplementary material, which is available to authorized users.

Breast cancer anti-estrogen resistance 4 (BCAR4)

Breast cancer anti-estrogen resistance 4 (BCAR4)

Altered protein expression profile associated with phenotypic changes in lung fibroblasts co-cultured with gold nanoparticle-treated small airway epithelial cells

Despite the availability of toxicity studies on cellular exposure to gold nanoparticles (AuNPs), there is scarcity of information with regard to the bystander effects induced by AuNPs on neighboring cells not exposed to the NPs. In this study, we showed that exposure of small airway epithelial cells (SAECs) to AuNPs induced changes in protein expression associated with functional effects in neighboring MRC5 lung fibroblasts in a co-culture system. Uptake of 20 nm size AuNPs by SAECs was first verified by focused ion beam scanning electron microscopy. Subsequently, pretreated SAECs were co-cultured with unexposed MRC5 lung fibroblasts, which then underwent proteome profiling using a quantitative proteomic approach. Stable-isotope labeling by amino acids in cell culture (SILAC)–based mass spectrometry identified 109 proteins (which included 47 up-regulated and 62 down-regulated proteins) that were differentially expressed in the lung fibroblasts co-cultured with AuNP pretreated SAECs. There was altered expression of proteins such as Paxillin, breast cancer anti-estrogen resistance 1 and Caveolin-1, which are known to be involved in the cell adhesion process. Morphological studies revealed that there was a concomitant increase in cell adhesion and altered F-actin stress fiber arrangement involving vinculin in the lung fibroblasts. It is likely that phenotypic changes observed in the underlying lung fibroblasts were mediated by AuNP-induced downstream signals in the pretreated SAECs and cell–cell cross talk.

Abstract 4451: Novel orphan G-protein-coupled receptor is oncogenic and mediates anti-estrogen resistance in human mammary carcinoma

Abstract Alterations in cellular pathways related to both endocrine and angiogenic factors may contribute to breast cancer progression. Inhibition of the elevated levels of these pathways is associated with clinical benefits. However, underlying mechanisms by which endocrine-related pathways and angiogenic factor signaling cooperatively promote breast cancer progression remain unclear. In this study, we report that novel orphan G-protein-coupled receptor (GPCR), GPR YJ, is oncogenic and mediates anti-estrogen resistance in breast cancer. GPR YJ is overexpressed in breast cancer cells and tissues, where elevated expression correlates with high tumor grade. Also, GPR YJ induces the expression of several angiogenic factors (VEGF, bFGF and IL-8), as well as ErbB (amphiregulin, epiregulin and neuregulins), which activate their respective pathways, leading to increased cell proliferation and survival, enhanced anchorage-independent growth, and promoted migration and invasion. In vivo, GPR YJ promotes the formation of large, well-vascularized tumors that metastasize to distant organs such as lymph nodes, lung and liver. Thus, deregulated expression of GPR YJ contributes to breast cancer progression and metastasis by inducing several growth factors that alter tumor-specific cell fates and the tumor stromal microenvironment. Citation Format: Yu Jin Lee, Kyeong Jin Shin, Soo-Ah Park, Sung Ho Ryu, Pann-Ghill Suh. Novel orphan G-protein-coupled receptor is oncogenic and mediates anti-estrogen resistance in human mammary carcinoma. [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 4451. doi:10.1158/1538-7445.AM2014-4451

Association of the Breast Cancer Antiestrogen Resistance Protein 1 (BCAR1) and BCAR3 Scaffolding Proteins in Cell Signaling and Antiestrogen Resistance

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.

Transcriptional regulation prediction of antiestrogen resistance in breast cancer based on RNA polymerase II binding data

BackgroundAlthough endocrine therapy impedes estrogen-ER signaling pathway and thus reduces breast cancer mortality, patients remain at continued risk of relapse after tamoxifen or other endocrine therapies. Understanding the mechanisms of endocrine resistance, particularly the role of transcriptional regulation is very important and necessary.MethodsWe propose a two-step workflow based on linear model to investigate the significant differences between MCF7 and OHT cells stimulated by 17β-estradiol (E2) respect to regulatory transcription factors (TFs) and their interactions. We additionally compared predicted regulatory TFs based on RNA polymerase II (PolII) binding quantity data and gene expression data, which were taken from MCF7/MCF7+E2 and OHT/OHT+E2 cell lines following the same analysis workflow. Enrichment analysis concerning diseases and cell functions and regulatory pattern analysis of different motifs of the same TF also were performed.ResultsThe results showed PolII data could provide more information and predict more recognizably important regulatory TFs. Large differences in TF regulatory mode were found between two cell lines. Through verified through GO annotation, enrichment analysis and related literature regarding these TFs, we found some regulatory TFs such as AP-1, C/EBP, FoxA1, GATA1, Oct-1 and NF-κB, maintained OHT cells through molecular interactions or signaling pathways that were different from the surviving MCF7 cells. From TF regulatory interaction network, we identified E2F, E2F-1 and AP-2 as hub-TFs in MCF7 cells; whereas, in addition to E2F and E2F-1, we identified C/EBP and Oct-1 as hub-TFs in OHT cells. Notably, we found the regulatory patterns of different motifs of the same TF were very different from one another sometimes.ConclusionsWe inferred some regulatory TFs, such as AP-1 and NF-κB, cooperated with ER through both genomic action and non-genomic action. The TFs that were involved in both protein-protein interactions and signaling pathways could be one of the key resistant mechanisms of endocrine therapy and thus also could be new treatment targets for endocrine resistance. Our flexible workflow could be integrated into an existing analytical framework and guide biologists to further determine underlying mechanisms in human diseases.

Abstract P5-09-11: BP1, a homeoprotein, regulates estrogen receptor alpha and induces estrogen independence

Abstract Introduction: BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors. Moreover, 100% of inflammatory breast tumors are BP1 positive. These data led us to define the mechanism of BP1-related tumorigenesis and aggressiveness in breast cancer. Materials and Methods: MCF-7/O1, O2 and O4 cells overexpressing BP1 and control V1 and V2 cells were tested for growth in estrogen free media, malignant potential and invasiveness using cell viability assays, soft agar assays and matrigel assays, respectively. To determine the influence of BP1 overexpression on tumor characteristics, empty vector cells (V1) and overexpressor cells (O2 and O4) were injected into the fat pads of athymic nude mice. Mice were supplemented with estrogen pellets or were unsupplemented. Chromatin immunoprecipitation assays (ChIP) were used to validate the binding of pBP1 to ERa and EP300. The effects of BP1 expression on ERα and EP300 were investigated using immunoblotting and qRT-PCR. Effects of BP1 overexpression on tamoxifen sensitivity were measured using the MTT assay. Results: Cells overexpressing BP1 showed higher viability (p<0.05) when grown in the absence of serum, greater invasive potential (p<0.05) and formed larger and more rapidly growing colonies (p<0.0001) compared with cells containing the empty vector. Tumors were larger in mice receiving O1, O2 or O4 cells than in mice receiving V1 or V2 cells (p<0.0001). There was also a positive correlation between BP1 mRNA levels and tumor size in patients (p = 0.01). 20% of mice injected with O2 or O4 developed tumors in the absence of estrogen, in contrast to control mice which did not develop tumors. Several mechanisms of estrogen independence related to BP1 were established: (1) an indirect mechanism, in which BP1 stabilizes ERα protein by transcriptional activation of p300, and (2) a direct mechanism, in which BP1 binds to the first intron of ERα and upregulates ERα RNA and protein expression. In addition, breast cancer anti-estrogen resistance 1 (BCAR1) RNA expression was higher in O2 as compared to V1 cells. Consistent with these findings, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition. Conclusion: High BP1 levels can lead to estrogen independence in ER positive breast cancer cells and tumors in mice by at least two mechanisms, indirect and direct, and are associated with resistance to tamoxifen. These results suggest that BP1 may be an important therapeutic target in ER positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-11.

Functional roles of BCAR3 in the signaling pathways of insulin leading to DNA synthesis, membrane ruffling and GLUT4 translocation

Breast cancer anti-estrogen resistance 3 (BCAR3) is an SH2-containing signal transducer and is implicated in tumorigenesis of breast cancer cells. In this study, we found that BCAR3 mediates the induction of ERK activation and DNA synthesis by insulin, but not by IGF-1. Specifically, the SH2 domain of BCAR3 is involved in insulin-stimulated DNA synthesis. Differential tyrosine-phosphorylated patterns of the BCAR3 immune complex were detected in insulin and IGF-1 signaling, suggesting that BCAR3 is a distinct target molecule of insulin and IGF-1 signaling. Moreover, microinjection of BCAR3 inhibitory materials inhibited membrane ruffling induced by insulin, while this did not affect insulin-mediated GLUT4 translocation. Taken together, these results demonstrated that BCAR3 plays an important role in the signaling pathways of insulin leading to cell cycle progression and cytoskeleton reorganization, but not GLUT4 translocation.

ELF5, normal mammary development and the heterogeneous phenotypes of breast cancer

SUMMARY The ETS transcription factor ELF5 specifies the formation of the secretory cell lineage of the mammary gland during pregnancy, by directing cell fate decisions of the mammary progenitor cells. The decision-making activity continues in breast cancer, where in luminal breast cancer cells forced ELF5 expression suppresses estrogen sensitivity and shifts gene expression toward the basal molecular subtype. The development of anti-estrogen resistance in luminal breast cancer is accompanied by increased expression of ELF5 and acquired dependence on ELF5 for continued proliferation, providing a potential new therapeutic target or prognostic marker to improve the treatment of this stage of the disease. Forced ELF5 expression suppresses the mesenchymal phenotype, making cells more epithelial and producing lower rates of invasion and motility. Conversely, loss of ELF5 promotes metastasis, with a clear corollary in the claudin-low subtype of breast cancer, which does not express ELF5 and is highly metastatic, or during the final stages of tumor progression, where loss of ELF5 expression may be involved in the acquisition of the lethal phenotype. In circumstances where ELF5 expression increases in parallel with metastatic potential, such as anti-estrogen resistant luminal breast cancers and basal breast cancer, there is much more to be understood about ELF5 and metastasis.

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.

Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression

Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.

Breast Cancer Antiestrogen Resistance 3 (BCAR3) Promotes Cell Motility by Regulating Actin Cytoskeletal and Adhesion Remodeling in Invasive Breast Cancer Cells

Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.

Targeting GRP78 and Antiestrogen Resistance in Breast Cancer

Breast cancer is the most prevalent cancer in women, with over 200,000 new cases diagnosed each year. Over 70% of breast cancers express the estrogen receptor-α, and drugs targeting these receptors such as tamoxifen or Faslodex(®) often fail to cure these patients. Many estrogen receptor-positive tumors lose drug sensitivity, making endocrine resistance a major clinical problem. Recently, investigation into the molecular mechanisms of endocrine resistance has highlighted a causative role of the unfolded protein response in antiestrogen resistance. In particular, the master regulator of the unfolded protein response, GRP78, was observed to be elevated in endocrine-resistant breast cancer and directly affected antiestrogen therapy responsiveness. GRP78 was found to impact many different cellular processes that may affect breast cancer survival. Recently, various compounds have been reported to affect GRP78 activity and it may be advantageous to combine these drugs with antiestrogens to overcome endocrine therapy resistance.

Abstract 3778: Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells.

Abstract Metastatic breast cancer is currently incurable and associated with a 5-year survival rate of only 23%. Thus it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility in order to improve patient survival. The adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) functions in cellular processes that contribute to cell motility. Previous work from our group has shown that elevated BCAR3 protein levels enhance breast cancer cell motility, while depletion of BCAR3 decreases cell motility and invasion in vitro. Here, we show BCAR3 controls membrane protrusion, Rac1 activity, and adhesion disassembly in invasive breast cancer cells in response to adhesion signals. Conversely, RhoA signaling pathways appear to predominate when BCAR3 is depleted from these cells, as evidenced by an increase in ROCK-mediated myosin light chain phosphorylation and the presence of stress fibers and large ROCK/mDia1-dependent focal adhesions. Thus, through its ability to tip the balance in favor of Rac1 signaling, BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells, thereby promoting a more invasive, pro-migratory phenotype. Interestingly, we demonstrate that BCAR3 also controls actin cytoskeletal and adhesion remodeling in invasive breast cancer cells in response to epidermal growth factor. Considering that BCAR3 protein levels are elevated in advanced breast cancer cells lines, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process. BCAR3 function is intimately linked to two other proteins, the adaptor molecule p130Cas (Cas) and the non-receptor tyrosine kinase c-Src. While BCAR3 protein levels have yet to be thoroughly assessed in human breast tumors, high expression of Cas and/or c-Src is associated with more aggressive breast cancer behaviors. Future work will determine whether this BCAR3/Cas/c-Src signaling network serves as a useful biomarker for invasive disease, and whether BCAR3 promotes breast tumor progression and metastasis in mouse models of human breast cancer. Citation Format: Ashley L. Wilson, Randy S. Schrecengost, Michael S. Guerrero, Keena S. Thomas, Amy H. Bouton. Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive 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 3778. doi:10.1158/1538-7445.AM2013-3778

Abstract 2638: The BCAR3/Cas/c-Src signaling node in breast cancer progression.

Abstract Despite advances in early detection and improved adjuvant therapies, breast cancer remains the second leading cause of cancer death among women mainly due to metastatic spread of the disease. The ability of cancer cells to migrate and invade is important in the process of metastasis. Studies have shown that the adaptor protein Breast Cancer Antiestrogen Resistance 3 (BCAR3) is highly expressed in invasive breast cancer cell lines and functions as a positive regulator of migration and invasion. To date, BCAR3 has only one clearly established binding partner in breast cancer cells, the adaptor protein, p130Cas (Cas). Studies of human breast tumors have shown a positive correlation between high levels of Cas protein expression and poor clinical outcome in patients (Dorssers LC et al, 2004). Additionally, the protein tyrosine kinase c-Src is a C-terminal binding partner of Cas. c-Src kinase activity promotes enhanced Cas signaling and is associated with breast cancer progression and poor clinical outcome (Blscardi JS et al, 1999). These data suggest a critical role for the BCAR3/Cas/c-Src signaling node in breast tumor progression. We hypothesize that enhanced signaling through the BCAR3/Cas/c-Src network in breast epithelial cells is critical for the transition to an invasive cancer cell phenotype. In this work, we compared the relationship between these proteins in non-tumorigenic mammary epithelial cells (MCF10A) and invasive breast cancer cells (BT549 and MDA-MB-231). As is the case for invasive breast cancer cells, we found that the majority of BCAR3 in MCF10A breast epithelial cells is in complex with Cas. However, less Cas is found in association with c-Src in MCF10A cells compared to more invasive breast cancer cells. These finding suggest that despite some similarities in BCAR3, Cas, and c-Src signaling between non-tumorigenic and invasive breast cancer cells, both the physical interplay between these proteins and the signaling pathways activated downstream become dysregulated as non-tumorigenic cells progress to a fully oncogenic state. A better understanding of BCAR3/Cas/c-Src signaling in non-tumorigenic versus invasive breast tumor cells is necessary to determine whether this signaling network contributes to breast tumor progression and metastasis. Citation Format: Allison M. Batties, Michael S. Guerrero, Ashley L. Wilson, Keena S. Thomas, Amy H. Bouton. The BCAR3/Cas/c-Src signaling node in breast cancer progression. [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 2638. doi:10.1158/1538-7445.AM2013-2638

Abstract 5397: MYC-driven glutamine metabolism promotes antiestrogen resistance in breast cancer.

Abstract About 70% of newly diagnosed cases of invasive breast cancer in the U.S. will be estrogen receptor alpha positive (ER+). Endocrine therapy is the least toxic and most effective means to manage the hormone-dependent breast cancer, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (FAS; Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole (LET). However, advanced ER+ breast cancer that has become resistant to endocrine therapy remains a significant clinical problem. Here we show that in breast cancer cells and rat tumors with acquired antiestrogen resistance, MYC, a oncogenic transcription factor, is overexpressed. Inhibition of MYC with small molecule inhibitor or siRNA resensitizes resistant cells to antiestorgens by inducing apoptosis. MYC inhibition in resistant cells also inhibited glutamine uptake and decreased levels of ASCT2/SLC1A5, a glutamine transporter. Resistant cells showed significant increase in cell proliferation in response to glutamine than sensitive cells. Moreover, resistant cells showed increased sensitivity to an inhibitor of glutaminase, GLS, an enzyme that hydrolysis of glutamine to glutamate. Thus, MYC promotes increased dependence on glutamine in antiestrogen resistant cells, and targeting glutamine metabolism could help circumvent antiestrogen resistance. Overreaching goal of this study is to and to identify effective therapies to treat endocrine resistant breast cancer. Citation Format: Ayesha N. Shajahan, Katherine L. Cook, Jessica L. Schwartz-Roberts, Ahreej E. Eltayeb, Diane M. Demas, Anni Warri, Leena Hilakivi-Clarke, Steven Metallo, Robert Clarke. MYC-driven glutamine metabolism promotes antiestrogen resistance in 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 5397. doi:10.1158/1538-7445.AM2013-5397

Abstract 4349: Genome-wide kinase and phosphatase siRNA screen identifies clinically relevant targets in IGF1R signaling-mediated antiestrogen resistance in breast cancer.

Abstract Antiestrogen resistance of breast cancer has been linked to increased growth factor receptor signaling such as invoked by the insulin-like growth factor 1 (IGF-1) receptor (IGF1R). We have previously shown that increased IGF1R signaling by ectopic IGF1R overexpression in MCF7 cells (MCF7-IGF1R) directly drives an estrogen receptor (ER)-independent cell proliferation in MCF7 cells that leads to antiestrogen resistance. To identify components of the downstream IGF1R signaling pathways that mediate antiestrogen resistance, we have performed a human genome-wide kinase and phosphatase siRNA screen under tamoxifen resistance condition (1 μM 4-OH-tamoxifen, 1 nM estradiol, and 100 ng/ml IGF-1) in MCF7-IGF1R cells. We identified 174 kinases and phosphatases that increased or decreased tamoxifen resistance. Of these, 14 significantly correlated with the time to disease progression of metastatic breast cancer patients treated with first-line tamoxifen monotherapy, including ADRA1B, BMPR1B, CDK10, CDK5, EIF2AK1 and MAP2K5. Low expression of these six genes in patients correlated with short time to disease progression and silencing of these genes in MCF7-IGF1R cells further increased proliferation under tamoxifen resistance condition. Low expression of the other eight genes in patients correlated with longer time the time to disease progression and silencing of these genes in MCF7-IGF1R cells decreased proliferation under tamoxifen resistance condition Therefore, these latter genes may represent novel drug targets that may be useful to restore tamoxifen sensitivity in breast cancer. Knockdown of two of these genes suppressed IGF1R-mediated effects and restored the anti-proliferative effect of tamoxifen in a dose dependent manner, suggesting synthetic lethality between the knockdowns and tamoxifen. Further study of one of these genes revealed that stable knockdown inhibits proliferation and three-dimensional growth under tamoxifen resistance condition, accompanied with an increase in G1/G0 arrest and mild induction of apoptosis. These effects are independent of PI3K/AKT and MAPK/ERK signaling pathways, and suggest that an as yet unknown pathway plays a decisive role in tamoxifen resistance induced by IGF1R signaling. Citation Format: John H. Meerman, Yinghui Zhang, Maurice P. Jansen, Ramakrishnaiah Siddapa, Sreenivasa Ramaiahgari, Leo Price, Erik Danen, Els M. Berns, John Foekens, Bob van de Water. Genome-wide kinase and phosphatase siRNA screen identifies clinically relevant targets in IGF1R signaling-mediated antiestrogen resistance in 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 4349. doi:10.1158/1538-7445.AM2013-4349

Abstract 4046: Estrogen receptor-α coordinates the unfolded protein response,autophagy, and reactive oxygen species generation to regulate breast cancer survival.

Abstract Of the almost 240,000 new breast cancers diagnosed in 2011 within the USA, approximately 70% of these cases express the estrogen receptor-α (ER). ER targeted therapies such as tamoxifen (TAM) or faslodex (ICI) are often used to treat this breast cancer subtype. However, the curative potential of these interventions is frequently limited in patients due to resistance. Understanding how antiestrogen resistance occurs could lead to improved approaches to eradicate ER+ breast cancers. Increased unfolded protein response (UPR, an endoplasmic reticulum stress pathway) or autophagy (a “self” eating process used to clear dysfunctional protein or organelles) was previously shown to promote antiestrogen resistance in ER+ breast cancer. Using the antiestrogen sensitive MCF7-LCC1 (LCC1) and resistant MCF7-LCC9 (LCC9) cell lines, we determined the effect of antiestrogens and ERα on autophagy and UPR signaling. Suprisingly, ablation of ERα through RNAi potentiated LCC1 to antiestrogen-mediated cell death and resensitized LCC9 cells to endocrine therapy. While ICI stimulated both autophagy and UPR signaling, RNAi knockdown of ERα in LCC1 and LCC9 cells inhibited ICI-mediated UPR signaling and concurrently stimulated autophagy. Furthermore, we showed that ERα knockdown stimulated ROS production and potentiated the cell death response to antiestrogen therapy. ERα knockdown, but not ICI treatment, reduced nuclear Nrf2 (a UPR-induced antioxidant signaling protein) and increased cytosolic KEAP1 (an inhibitor of Nrf2), which may lead to the observed increase in ROS production. Autophagy inhibition through ATG7 silencing increases cell death and apoptosis in LCC1 ERα knockdown cells, showing that autophagy induction by antiestrogens is prosurvival in this context. These data suggests that UPR and autophagy stimulated by antiestrogen therapy results from two different mechanisms of ER regulation. We show that antiestrogens promote the accumulation of ERα in the cytosol that is associated with increased UPR signaling, while the inhibition of ERα promotes prosurvival autophagy. The inherent stimulation of resistance mechanism signaling through antiestrogen treatment suggests that combining autophagy or UPR inhibitors with anti-estrogens may greatly reduce the development of acquired antiestrogen resistance in breast cancer. Citation Format: Pamela A. Clarke, Katherine Cook, Rong Hu, Jessica L. Schwartz, Mones Abu-Asab, Anni Warri, Robert Clarke. Estrogen receptor-α coordinates the unfolded protein response,autophagy, and reactive oxygen species generation to regulate breast cancer survival. [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 4046. doi:10.1158/1538-7445.AM2013-4046

Abstract 3638: Hypermethylation of BRCA1, and hypomethylation and overexpression of unfolded protein response (UPR) regulators EIF2AK3, IKBKB and NFkB in breast cancer may originate from elevated in utero estrogenic environment.

Abstract High in utero estrogenic environment increases later breast cancer risk in women and preclinical models. This exposure also causes permanent epigenetic alterations in the mammary glands in animals that are inherited not only by the daughters, but the granddaughters and great granddaughters as well. Further, we have found that both de novo tamoxifen resistance and recurrence of breast cancers are significantly increased in rats exposed to excessive levels of estradiol (E2) in utero. Treatment with histone deacetylase and DNA methyltransferase inhibitors prevents the resistance to antiestrogen therapy in the E2 exposed rats. In this study, we investigated whether DNA methylation patterns are altered in daughters who are naturally exposed to high in utero E2 levels. Mothers’ E2 levels were determined on gestation weeks 8-12 and 26-28, and buccal swab DNA was obtained from 22 of their one-year-old daughters. Analysis using Illumina Infinium Methylation 450 arrays was performed and the results were explored by comparing methylation patterns among daughters whose mothers had the highest versus lowest quintile (3-fold lower) of E2 levels during pregnancy. A total of 223 genes exhibited significantly higher, and 666 genes significantly lower, DNA methylation patterns in the top E2 quintile daughters, compared with the bottom E2 quintile daughters. Of the hypermethylated genes in the buccal swab DNA, 23% were also more methylated in tumors compared with normal mammary tissues in data from women in the Cancer Genome Atlas (TCGA). Of the hypomethylated genes in buccal swab DNA, 25% were hypomethylated in the tumors in TCGA. Further, 21% of the methylated genes were confirmed to be down-regulated, and 31% of the hypomethylated genes were over-expressed in TCGA breast tumors. The most strongly hypermethylated gene in the high E2 exposed daughters was BRCA1 (3.7-fold, p<0.006), and this gene was also methylated in TCGA tumors. However, BRCA1 mRNA expression was not reduced in breast tumors in TCGA, and was only marginally reduced in the mammary glands of rats or mice exposed to excess E2 in utero. Among the hypomethylated genes were several members of the unfolded protein response (UPR) pathway, including EIF2AK3 (p<0.007), IKBKB (p<0.01) and NFkB (p<0.05); these UPR genes were also significantly up-regulated in the breast tumors in TCGA. Our findings thus suggest that epigenetically-induced alterations in the expression of UPR genes originate from in utero estrogenic exposures. Since abnormalities in UPR are linked to breast cancer risk and antiestrogen resistance, hypomethylation of UPR genes may partially explain high breast cancer risk and increased de novo and acquired tamoxifen resistance in in utero estrogen exposed females. Citation Format: Leena A. Hilakivi-Clarke, Xiao Zhang, Riitta Luoto, Nguyen Nguyen, Xiyuan Zhang, Jason Xuan, Jin Lu, Alan Zwart, Robert Clarke. Hypermethylation of BRCA1, and hypomethylation and overexpression of unfolded protein response (UPR) regulators EIF2AK3, IKBKB and NFkB in breast cancer may originate from elevated in utero estrogenic environment. [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 3638. doi:10.1158/1538-7445.AM2013-3638

Abstract 1190: Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy .

Abstract To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer we performed microarray profiling and identified 366 genes with altered expression in 4 tamoxifen-resistant (TamRs) vs. the parental tamoxifen-sensitive MCF-7/S0.5 cell lines. Most of these genes were functionally linked to cell growth, proliferation, death and control of gene expression and included FYN, PRKCα, ITPR1, DPYD, DACH1, GBP1 and PRLR. Increased expression of FYN was observed in TamR cells and treatment with FYN-specific interfering RNA or a specific Src family kinase (SFK) inhibitor restored the anti-proliferative effect of tamoxifen in these cells. TamR cells also expressed increased levels of phosphorylated breast cancer antiestrogen resistance protein 1 (pBCAR1), an important downstream SFK substrate in the MAPK pathway. Evaluation of the association of subcellular localization of FYN in cancer tissue and clinical endpoints was made in 2 cohorts of ER+ breast cancer patients, one with advanced disease (N=47) and the other with early breast cancer (N=76). The result showed that plasma membrane-associated FYN expression in the cancer cells strongly correlated with longer progression-free survival compared to FYN expression not associated with the plasma membrane (p<0.0002) in the advanced disease cohort. Similarly, in the early breast cancer patients, plasma membrane associated FYN expression was associated with longer metastasis-free survival (p<0.003) and overall survival (p<0.0005) independent of tumor size, grade and node status. Our results indicate that FYN plays an important role in tamoxifen resistance, and its subcellular location in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer. Citation Format: Daniel Elias Roro, Henriette Vever, Henrik Ditzel. Gene expression profiling identified FYN as an important gene in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy . [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 1190. doi:10.1158/1538-7445.AM2013-1190