Antiestrogen Resistance Research Articles

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.

Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis

Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.

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.

Effect of nonmalignant accessory cells from the metastatic microenvironment on breast cancer cell resistance to antiestrogens.

11039 Background: To address the role of the local metastatic microenvironment in the antiestrogen (AE) resistance exhibited by disseminated breast cancer (BrCa), we assembled heterotypic in vitro three-dimensional (3D) tissue cultures comprised of estrogen receptor-positive (ER+) BrCa cells and non-malignant accessory cells from organs frequently targeted by metastatic disease, to model the composition and architecture of metastatic lesions. Methods: MCF7 cells expressing luciferase, cultured alone or with accessory cells, were exposed to dose-ranges of hydroxytamoxifen (4-OHT), fulvestrant or raloxifene. MCF7 cells were also injected s.c. in nude mice either alone or with HS-5 bone marrow stromal cells (BMSCs). Tumor spheroid viability or xenograft growth were measured by bioluminescence. Results: MCF7 response to AEs in 3D conditions (collagen type I and/or Matrigel) was marked by acinar differentiation of tumor spheroids that resembles normal breast epithelium. Co-culture of MCF7 and immortalized accessory cells from the bone (BMSCs; HOBIT and hFOB osteoblasts), brain (SVGp12 astrocytes), liver (THLE3 hepatocytes), and lung (MRC9 fibroblasts) in 3D (but not 2D) conditions, conferred resistance to 4-OHT, raloxifene, and fulvestrant at clinically relevant doses. Heterotypic xenografts (MCF7 + BMSCs) had reduced response to tamoxifen compared with monotypic xenografts (MCF7 alone). BMSCs induced in MCF7 cells downregulation of TGFβ2; upregulation of a transcriptional signature enriched for genes associated with high-grade tumors, including genes involved in interferon response, signaling through EGFR superfamily members, NFkB and other antiapoptotic pathways; and elevated c-Myc protein levels. Exogenous TGFβ2 or neutralizing antibodies against INFγ partially re-sensitized MCF7 cells to 4-OHT in co-cultures. Conclusions: Our results indicate that accessory cells representing the microenvironment of metastatic sites confer AE resistance to BrCa cells in 3D tissue cultures and xenograft models. The mechanisms of stroma-induced AE resistance may reveal new therapeutic targets for refractory BrCa patients with metastatic disease.

Endocrine Resistance: What Do We Know?

Adjuvant therapy with antiestrogens targeting estrogen receptor α (ER) signaling prevents disease recurrence in many patients with early-stage ER+ breast cancer. However, a significant number of cases exhibit de novo or acquired endocrine resistance. While other clinical subtypes of breast cancer (HER2+, triple-negative) have disproportionately higher rates of mortality, ER+ breast cancer is responsible for at least as many deaths because it is the most common subtype. Therefore, identifying mechanisms that drive endocrine resistance is a high clinical priority. A large body of experimental evidence indicates that oncogenic signaling pathways underlie endocrine resistance, including growth factor receptor tyrosine kinases (HER2, epidermal growth factor receptor [EGFR], fibroblast growth factor receptor 1/2 [FGFR], insulin-like growth factor-1 receptor [IGF-1R]/ insulin receptor [InsR]), PI3K/AKT/ mTOR, MAPK/ERK, Src, CDK4/CDK6, and ER itself. Combined targeting of ER and such pathways may be the most effective means to combat antiestrogen resistance, and clinical trials testing such strategies show promising results. Herein, we discuss pathways associated with endocrine resistance, biomarkers that may be useful to predict response to targeted agents, and avenues for further exploration to identify strategies for the treatment of patients with endocrine-resistant disease.

Reduced expression of miR-200 family members contributes to antiestrogen resistance in LY2 human breast cancer cells.

IntroductionThe role of miRNAs in acquired endocrine-resistant breast cancer is not fully understood. One hallmark of tumor progression is epithelial-to-mesenchymal transition (EMT), characterized by a loss of cell adhesion resulting from reduced E-cadherin and increased cell mobility. miR-200 family members regulate EMT by suppressing expression of transcriptional repressors ZEB1/2. Previously we reported that the expression of miR-200a, miR-200b, and miR-200c was lower in LY2 endocrine-resistant, mesenchymal breast cancer cells compared to parental, endocrine sensitive, epithelial MCF-7 breast cancer cells. Here we investigated the regulation of miR-200 family members and their role in endocrine-sensitivity in breast cancer cells.ResultsmiR-200 family expression was progressively reduced in a breast cancer cell line model of advancing endocrine/tamoxifen (TAM) resistance. Concomitant with miR-200 decrease, there was an increase in ZEB1 mRNA expression. Overexpression of miR-200b or miR-200c in LY2 cells altered cell morphology to a more epithelial appearance and inhibited cell migration. Further, miR-200b and miR-200c overexpression sensitized LY2 cells to growth inhibition by estrogen receptor (ER) antagonists TAM and fulvestrant. Knockdown of ZEB1 in LY2 cells recapitulated the effect of miR-200b and miR-200c overexpression resulting in inhibition of LY2 cell proliferation by TAM and fulvestrant, but not the aromatase inhibitor exemestane. Demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) in combination with histone deacetylase inhibitor trichostatin A (TSA) increased miR-200b and miR-200c in LY2 cells. Concomitant with the increase in miR-200b and miR-200c, ZEB1 expression was decreased and cells appeared more epithelial in morphology and were sensitized to TAM and fulvestrant inhibition. Likewise, knockdown of ZEB1 increased antiestrogen sensitivity of LY2 cells resulting in inhibition of cell proliferation.ConclusionsOur data indicate that reduced miRNA-200b and miR-200c expression contributes to endocrine resistance in breast cancer cells and that the reduced expression of these miR-200 family members in endocrine-resistant cells can be reversed by 5-aza-dC+TSA.

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 LB-254: XBP1-NFkappaB signaling promotes antiestrogen resistance in breast cancer animal model.

Abstract There are nearly 3 million breast cancer survivors in the United States, with more than 230,000 women being newly diagnosed with invasive breast cancer each year. Of total breast cancer patients, around 70 percent are estrogen receptor (ER) positive and often receive endocrine therapy. Endocrine resistance, either de novo or acquired, is a major hurdle for treating ER positive breast cancer patients. Unfolded protein response (UPR) is known to be activated and promote endocrine resistance in breast cancer cells. As an essential component of URP signaling, X-box binding protein 1 (XBP1) was shown up-regulated in endocrine resistant breast cancer. XBP1 was demonstrated as an important mediator for endocrine resistance in vitro cell culture model in our previous findings. In this study, the effect of XBP1 in endocrine resistance in animal model was examined. XBP1 contains two alternative splice variants: XBP1(S) and XBP1(U). In order to differentiate the role of each variant, we generated XBP1 mutant constructs that overexpress specifically only either the XBP1(S) or XBP1(U). The XBP1 over-expressing human breast cancer cells have been injected into the mammary fat pad of female nude mice. The preliminary data shows that the over-expression of both XBP1 splice variants promotes tumor growth in vivo. More importantly, tumors with over-expressed XBP1 show less sensitivity to tamoxifen treatment. Our previous finding revealed that NFkappaB signaling is regulated by XBP1 and required for antiestrogen resistance in cultured cells. In the current study, we have treated XBP1-overexpressed breast tumor bearing animals with NFkappaB inhibitor Parthenolide, and find that it enhances tamoxifen sensitivity. In conclusion, our data demonstrate that XBP1-NFkappaB signaling plays an important role in antiestrogen resistance in vivo. Citation Format: Rong Hu, Alan Zwart, Anni Warri, Robert Clarke. XBP1-NFkappaB signaling promotes antiestrogen resistance in breast cancer animal model. [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 LB-254. doi:10.1158/1538-7445.AM2013-LB-254

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 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 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 3563: Deep kinome sequencing identifies a novel D189Y mutation in the Src family kinase LYN as a possible mediator of antiestrogen resistance in ER+ breast cancer .

Abstract Estrogen receptor-positive (ER+) breast tumors adapt to hormone deprivation and acquire resistance to aromatase inhibitors (AIs). The proliferative rate of tumor cells measured by Ki67 after short-term treatment with an AI has been proposed as a surrogate endpoint of long term patient outcome. To identify kinase mutations associated with resistance to endocrine therapy, we performed deep-kinome sequencing on 4 ER+/HER2– breast tumors that retained high Ki67 scores (14.8 to 24.5%) after two weeks of pre-surgical therapy with letrozole. Genomic DNA from the surgically excised tumors was deep sequenced using a capture approach with oligonucleotides hybridizing to 612 genes, including 517 kinases with ≥300x coverage. All 4 tumors contained an activating mutation in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K. One tumor also contained a novel D189Y (565G>T) somatic mutation in the SH2 domain of LYN, a member of the Src family kinases (SFKs; variant frequency of 8%). Reverse-phase protein array (RPPA) analysis available in 10 tumors in this study revealed a significant correlation (p=0.006) between Y416 P-Src (which detects all SFKs) and a high post-letrozole Ki67 score. A siRNA screen targeting 779 kinases identified LYN as one of the top hits whose knockdown significantly reduced growth of ER+ MCF-7 cells with acquired resistance to estrogen deprivation. LYN mRNA expression was also upregulated in these cells. We next analyzed the Cancer Genome Atlas (TCGA) SNP data to detect copy number changes for 444 tumors where corresponding gene expression data were available. Copy number increases (>0.8 log2 ratio over normal matched DNA) in LYN were present in approximately 10% of breast cancers, with the highest copy number gains observed in luminal B tumors. In contrast, other SFKs showed less frequent copy number increases (YES1 <1%, SRC 2%, FYN 1%). Finally, we investigated the role of D189Y LYN in antiestrogen-resistant breast cancer using ER+/PIK3CA mutant MCF-7 cells transduced with GFP (control), wild-type (WT) LYN or D189Y LYN vectors. Overexpression of WT or mutant LYN resulted in increased phosphorylation of Src (at Y416), FAK, IGF-IR, EGFR, STAT3, AKT and MAPK. LYND189Y increased phosphorylation of FAK, EGFR, and IRS-1 as compared to LYNWT. Although stable transduction of either LYNWT or LYND189Y accelerated MCF-7 cell growth in estrogen-depleted medium, the mutant was more potent than LYNWT at inducing this effect. Further, LYND189Y overexpression rendered treatment with the ER downregulator fulvestrant or the PI3K inhibitor BKM120 less effective, as well as rescued apoptosis induced by BKM120. These results suggest, first, that LYN plays a role in escape from estrogen deprivation in a subset of ER+ breast cancers. Second, ER+ breast cancers harbor multiple molecular alterations capable of mediating hormone-independent growth. Citation Format: Emily M. Fox, Justin M. Balko, Carlos L. Arteaga. Deep kinome sequencing identifies a novel D189Y mutation in the Src family kinase LYN as a possible mediator of antiestrogen 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 3563. doi:10.1158/1538-7445.AM2013-3563

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 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

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

GX15-070 (Obatoclax) Induces Apoptosis and Inhibits Cathepsin D- and L–Mediated Autophagosomal Lysis in Antiestrogen-Resistant Breast Cancer Cells

In estrogen receptor-positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX15-070 has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here, we show that GX15-070 is more effective in reducing the cell density of antiestrogen-resistant breast cancer cells versus sensitive cells and that this increased sensitivity of resistant cells to GX15-070 correlates with an accumulation of autophagic vacuoles. Formation of autophagosomes in GX15-070-treated cells was verified by changes in expression of the lipidation of microtubule-associated protein-1 light chain-3 and both confocal and transmission electron microscopy. While GX15-070 treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulate. Moreover, GX15-070 exposure leads to a reduction in cathepsin D (CTSD) and L (CTSL1) protein expression that would otherwise digest autolysosome cargo. Thus, GX15-070 has dual roles in promoting cell death: (i) directly inhibiting antiapoptotic BCL2 family members, thereby inducing apoptosis; and (ii) inhibiting downstream CTSD and CTSL1 protein expression to limit the ability of cells to use degraded material to fuel cellular metabolism and restore homeostasis. Our data highlight a new mechanism of GX15-070-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer.