BCAR1 Research Articles

ELF5 Suppresses Estrogen Sensitivity and Underpins the Acquisition of Antiestrogen Resistance in Luminal Breast Cancer

Author SummaryThe molecular subtypes of breast cancer are distinguished by their intrinsic patterns of gene expression and can be used to group patients with different prognoses and treatment options. Although molecular subtyping tests are currently under evaluation, some of them are already in use to better tailor therapy for patients; however, the molecular events that are responsible for these different patterns of gene expression in breast cancer are largely undefined. The elucidation of their mechanistic basis would improve our understanding of the disease process and enhance the chances of developing better predictive and prognostic markers, new therapies, and interventions to overcome resistance to existing therapies. Here, we show that the transcription factor ELF5 is responsible for much of the patterning of gene expression that distinguishes the breast cancer subtypes. Additionally, our data suggest that ELF5 may also be involved in the development of resistance to therapies designed to stop estrogen stimulation of breast cancer. These effects of ELF5 appear to represent a partial carryover into breast cancer of its normal role in the mammary gland, where it is responsible for the development of milk-producing structures during pregnancy.

Breast-cancer anti-estrogen resistance 4 (BCAR4) encodes a novel maternal-effect protein in bovine and is expressed in the oocyte of humans and other non-rodent mammals

Does BCAR4 have a role in mammalian embryo development? Expression, localization and functional data support that BCAR4 is a maternal-effect protein in non-rodent mammals. BCAR4 was previously identified as an oocyte-specific gene in cattle, and as a marker of certain breast tumors in humans. Human oocytes were obtained from patients undergoing IVF, but had failed to mature after ovarian stimulation. Dog oocytes were obtained from ovariectomized bitches. Pig, horse and bovine ovaries were obtained from commercial slaughterhouses for extraction of immature oocyte-cumulus complexes. In vivo matured bovine matured oocytes were obtained after ovulation induction and ovulation inducing treatment of Montbeliard heifers. Expression at the RNA level was analyzed by reverse transcription coupled to polymerase chain reaction. Western blot and immunolabeling coupled to confocal or electronic microscopy were used to analyze bovine protein expression and intracellular localization. For the functional approach, short-interfering RNA were microinjected into mature bovine oocytes, followed by IVF; cleavage and embryo development were recorded. The BCAR4 gene is conserved in mammalian species from various orders and has been lost in rodents after divergence with lagomorphs. The transcript is expressed in the oocytes of humans and domestic species. We bring the first experimental evidence of the BCAR4 protein in mammals. In cattle, the protein is not detected in immature oocytes but starts to be synthesized during maturation, increases in the zygote and persists until the morula stage. The protein is detected throughout the cytoplasm in mature oocytes, concentrates in and around the pronuclei in the zygote, and appears to shuttle in and out of the nuclei starting in the 2-cell embryo; BCAR4 is also present at the junctions between blastomeres from 2-cell to morula. In our functional approach, targeting the BCAR4 transcript by small-interfering RNA significantly compromised development to the morula or/and blastocyst stages (P < 0.05, logistic regression). As indicated above, protein expression and function were investigated in cattle and mostly in vitro matured oocytes were used. This study provides a novel candidate gene whose mutation or deregulation may underlie certain cases of unexplained female infertility.

Expression of breast cancer anti-estrogen resistance 1 in relation to vascular endothelial growth factor, p53, and prognosis in esophageal squamous cell cancer

The purpose of this study was to clarify the role of breast cancer anti-estrogen resistance 1 (BCAR1) expression in relation to vascular endothelial growth factor (VEGF), p53, and proliferation in esophageal squamous cell cancer (ESCC). Expression of BCAR1, VEGF, p53, and the ki-67 proliferative index were examined by tissue microarray and immunohistochemistry in 106 specimens with ESCC and matched adjacent normal tissues. Among them, 40 cases were simultaneously examined by Western blot. Both Western blot and immunohistochemistry showed that BCAR1 expression was substantially higher in ESCC than in adjacent normal tissues (P < 0.001). BCAR1 expression was significantly connected with degree of tumor differentiation, with poorly differentiated tumors showing higher BCAR1 expression (P < 0.001). BCAR1 expression was significantly and positively correlated with VEGF and p53 expression levels (r= 0.541, P < 0.001; r= 0.374; P < 0.001) but not proliferative index (r= 0.44; P= 0.066). Additionally, a significant relationship was also observed between VEGF and p53 (r= 0.321; P= 0.001). Kaplan-Meier survival analysis revealed that patients with high BCAR1 expression had significantly shorter survival times than those with low BCAR1 expression levels (median survival 40 months vs. 27 months, P= 0.09). Multivariate analysis also revealed that levels of BCAR1 expression (hazard ratio 2.250, P= 0.015) was a significant and independent prognostic indicator. High expression of BCAR1 is associated with elevated VEGF and p53 expression levels, as well as poor prognosis in ESCC. Therefore, BCAR1 may be a potential candidate for predicting prognosis and a new therapy target for ESCC.

BCAR1 (breast cancer anti-estrogen resistance 1)

Review on BCAR1 (breast cancer anti-estrogen resistance 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract 1584: MHC-restricted phosphopeptides as broad-based immunotherapeutic targets for cancer

Abstract Identification of the antigens (Ags) recognized by CD8+ T cells specific for melanoma and other types of cancer cells has been motivated by the expectation that they will serve as the basis for a therapeutic vaccine or will be useful for the stimulation of more effective CD8+ CTL for adoptive immunotherapy. Few of the currently identified Ags are derived from source proteins directly associated with transformation or metastasis, and many are self-Ags which may be subject to tolerance mechanisms. We have defined a new array of phosphorylated peptides presented by HLA-A1, -A2, -A3, -B7, -B44 and HLA-B27 that are presented on melanomas from multiple patients at &amp;gt;1 copy per cell and derived from source proteins associated with tumorigenesis and metastasis. We evaluated the immunogenicity of several of the HLA-A2-restricted phosphopeptide Ags in an HLA-transgenic murine model system and in vitro human T cell cultures, evaluated the presence of the phosphorylated source protein or display of the phosphorylated epitope on cancers of different histological types, and investigated the ability of phosphopeptide-specific CD8+ T cells to prevent outgrowth of cancer cells in a xenogenic model. Evaluation of metastatic melanoma tissue microarrays and cancer cell lysates demonstrated that the phosphorylated source proteins for β-catenin (Ser33-phosphorylated), Breast cancer anti-estrogen resistance −3 (BCAR3; Thr130-phosphorylated), and Insulin receptor substrate-2 (IRS2; Ser1100-phosphorylated) were broadly expressed in melanomas and other cancers but not normal tissues. In addition, these epitopes can be utilized to elicit phosphopeptide-specific T cells in both murine HLA-A2 transgenic mice in vivo and human in vitro culture. The phospho-β-catenin, phospho-IRS2 and phospho-BCAR3-specific-T cells that were generated differentiate between the phosphorylated and non-modified forms of their respective peptides and recognize the endogenously processed and presented phosphopeptide on the surface of cancer cells. Finally, adoptive transfer of these phosphopeptide-specific T cells slowed outgrowth of melanoma tumors implanted in immunodeficient NOD.SCID.IL2Rα-/- mice. Thus, we have defined a new set of post-translationally modified epitopes derived from source proteins associated with tumorigenesis and metastasis that are immunogenic and capable of slowing tumor outgrowth in a xenograft model. Based on these results, we are currently planning a clinical trial utilizing these HLA-A2-restricted phosphopeptides in melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1584. doi:1538-7445.AM2012-1584

Protein pathway activation mapping reveals molecular networks associated with antiestrogen resistance in breast cancer cell lines

Previously, we have identified a panel of breast cancer antiestrogen resistance (BCAR) genes. Several of these genes have clinical relevance because mRNA or protein levels associate with tamoxifen resistance or tumor aggressiveness. We postulated that changes in activation status of protein signaling networks induced by BCAR genes may provide better insight into the mechanisms underlying antiestrogen resistance. Key signal transduction pathways were analyzed for changes in activation or expression using reverse-phase protein microarrays probed with 78 antibodies against signaling proteins with known roles in tumorigenesis. We used ZR-75-1-derived cell lines transduced with AKT1, AKT2, BCAR1, BCAR3, BCAR4, EGFR, GRB7, HRAS, HRAS(v12) or HEF1 and MCF7-derived cell lines transduced with BCAR3, BCAR4 or EGFR. In the antiestrogen-resistant cell lines, we observed increased phosphorylation of several pathways involved in cell proliferation and survival. All tamoxifen-resistant cell lines contained high levels of phosphorylated AKT and its biochemically linked substrates Forkhead box O1/3. The activation of ERBB2, ERBB3 and the downstream modulators focal adhesion kinase and SHC were activated in cells with overexpression of BCAR4. Remarkable differences were observed for the levels of activated AMPK alpha1, cyclins, STAT5, STAT6, ERK1/2 and BCL2. The comparison of the cell signaling networks in estrogen-dependent and -independent cell lines revealed biochemically linked kinase-substrate markers that comprised systemically activated signaling pathways involved in tamoxifen resistance. Our results show that this model provides insights into the molecular and cellular mechanisms of breast cancer progression and antiestrogen resistance. This knowledge may help the development of novel targeted treatments.

BCAR1 expression improves prediction of biochemical reccurence after radical prostatectomy

Because prostate cancer exhibits a great variability in clinical outcome, biomarkers that can be used in daily practice are needed to better stratify patients into prognostic groups. Since steroid hormones play a central role in the development and progression of prostate cancer, we aimed to analyze in a matched nested case-control study the value of molecules involved in steroid signaling, to predict recurrence after radical prostatectomy, independently from standard prognostic tools. Among 1,200 patients treated by radical prostatectomy with negative margins with at least 4 years follow-up, 121 prostate cancers with biochemical relapse were matched after pathological reassessment with 121 cancers with identical clinicopathological features but without relapse. Immunohistochemistry was performed on tissue microarrays, using antibodies directed against molecules involved in androgen and estrogen signaling, including hormone receptors, enzymes (such as the five alpha reductases 1,2 and 3, aromatase, alpha-keto reductase 1C3 and squalene epoxidase), the breast cancer antiestrogen resistance 1 (BCAR1), and the proliferation marker Ki67. The median follow-up for patients without recurrence was 7 years. Both cell proliferation and BCAR1 expression were significantly associated with biochemical relapse, in univariate and multivariate analysis. In subgroup analysis, the sole predictive marker in patients with well-differentiated prostate cancer was BCAR1 (P = 0.004), whereas only proliferation (P = 0.001) was significantly associated with relapse in less-differentiated prostate cancer patients. BCAR1 is an independent predictor of recurrence after radical prostatectomy for "low risk" prostate cancer. The use of this biomarker may enable more individualized treatment approaches.

P4-01-12: The Unfolded Protein Response (UPR) and Pro-Survival Autophagy Contributes to Antiestrogen Resistance in Breast Cancer.

Abstract In 2011, most of the 192,000 newly diagnosed cases of invasive breast cancer will be estrogen receptor alpha positive (ER+). Endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (FAS; Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole or Exemestane is the least toxic and most effective means to manage the hormone-dependent breast cancer in such patients. However, resistance to endocrine therapy remains a significant clinical problem. Previously, we have shown that antiestrogen resistant breast cancer cells over-express X-Box Binding Protein 1 (XBP1), a transcription factor that belongs to the basic region/leucine zipper (bZIP) family. XBP1(U) and XBP1(S) variants result from an unconventional splicing of the XBP1 mRNA by IRE1alpha. In the unfolded protein response (UPR), endoplasmic reticulum proteins (IRE1alpha, ATF6 and PERK) sense cellular stress to regulate the accumulation of unfolded proteins. Initially a compensatory mechanism allowing cells to recover normal endoplasmic reticulum function, prolonged UPR may induce cell death; this is often dependent upon which arm of the UPR predominates. XBP1 is an obligate component in both the IRE1alpha and ATF6 arms of the UPR. In this study, we show that in MCF7/LCC9 [FAS resistant; TAM cross-resistant] cells, there is an increase in UPR signaling as detected by increased expression of BiP/GRP78. Transient expression of XBP1(S) in MCF7/LCC1 [antiestrogen sensitive] cells show decreased sensitivity to FAS that correlated with increased levels of both basal and FAS-induced autophagy as measured by cleaved LC3BII protein fragment, GFP-LC3 activity, and reduced expression of p62/SQSTM1. Furthermore, we show that MCF7/LCC9 cells survive by activating pro-survival autophagy through UPR that is regulated by the transcription factor c-MYC. An inhibitor of c-MYC, 10058-F4, synergized with FAS to inhibit proliferation in MCF7/LCC9 cells by preventing pro-survival autophagy and increasing apoptosis. Thus, our study shows that antiestrogen resistant breast cancer cells evade cell death by activating XBP1-mediated UPR that results in c-MYC-mediated prosurvival autophagy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-12.

Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells

Here, we show that tamoxifen resistance is induced by cancer-associated fibroblasts (CAFs). Coculture of estrogen receptor positive (ER+) MCF7 cells with fibroblasts induces tamoxifen and fulvestrant resistance with 4.4 and 2.5-fold reductions, respectively, in apoptosis compared with homotypic MCF7 cell cultures. Treatment of MCF7 cells cultured alone with high-energy mitochondrial “fuels” (L-lactate or ketone bodies) is sufficient to confer tamoxifen resistance, mimicking the effects of coculture with fibroblasts. To further demonstrate that epithelial cancer cell mitochondrial activity is the origin of tamoxifen resistance, we employed complementary pharmacological and genetic approaches. First, we studied the effects of two mitochondrial “poisons,” namely metformin and arsenic trioxide (ATO), on fibroblast-induced tamoxifen resistance. We show here that treatment with metformin or ATO overcomes fibroblast-induced tamoxifen resistance in MCF7 cells. Treatment with the combination of tamoxifen plus metformin or ATO leads to increases in glucose uptake in MCF7 cells, reflecting metabolic uncoupling between epithelial cancer cells and fibroblasts. In coculture, tamoxifen induces the upregulation of TIGAR (TP53-induced glycolysis and apoptosis regulator), a p53 regulated gene that simultaneously inhibits glycolysis, autophagy and apoptosis and reduces ROS generation, thereby promoting oxidative mitochondrial metabolism. To genetically mimic the effects of coculture, we next recombinantly overexpressed TIGAR in MCF7 cells. Remarkably, TIGAR overexpression protects epithelial cancer cells from tamoxifen-induced apoptosis, providing genetic evidence that increased mitochondrial function confers tamoxifen resistance. Finally, CAFs also protect MCF7 cells against apoptosis induced by other anticancer agents, such as the topoisomerase inhibitor doxorubicin (adriamycin) and the PARP-1 inhibitor ABT-888. These results suggest that the tumor microenvironment may be a general mechanism for conferring drug resistance. In summary, we have discovered that mitochondrial activity in epithelial cancer cells drives tamoxifen resistance in breast cancer and that mitochondrial “poisons” are able to re-sensitize these cancer cells to tamoxifen. In this context, TIGAR may be a key “druggable” target for preventing drug resistance in cancer cells, as it protects cancer cells against the onset of stress-induced mitochondrial dys-function and aerobic glycolysis.

Low Energy Focused Ultrasound (LOFU) Sensitize Breast Cancer Cells to Tamoxifen

Splicing of X-box binding protein1 (XBP1) is implicated in up-regulation of estrogen receptors leading to antiestrogen resistance in breast cancer. Unfolded protein response (UPR) is an adaptive survival mechanism in cancer cells, where XBP-1 splicing is induced. UPR can either increase the folding capacity of cells or initiate apoptosis when the misfolded protein burden is overwhelming. We hypothesized that therapeutic LOFU would induce protein misfolding, increase endoplasmic reticulum stress and sensitize breast tumor cells to tamoxifen.

Abstract B58: Differential mechanisms control steady state expression of breast cancer antiestrogen resistance 3 (BCAR3) in non-invasive and invasive breast cancer cell lines

Abstract The overexpression of breast cancer antiestrogen resistance 3 (BCAR3) has been shown to reduce the antagonistic effects of tamoxifen and fulvestrant in estrogen receptor positive (ER+) breast cancer cell lines. BCAR3 also has a role in the regulation of the migratory and invasive potential of both ER+ and ER- breast cancer cells. A tight correlation between BCAR3 expression and breast cancer cell phenotype exists such that non-invasive, early stage cells express very low levels whereas more invasive, mesenchymal-like cells express very high levels of BCAR3. Because of this correlation, it is important to understand the temporal and spatial regulation of BCAR3. In this study, we set out to define potential mechanisms that regulate BCAR3 expression to better understand, predict, and reverse the consequences of elevated expression. In MCF7 and T47D cells (non-invasive, early stage ER+), high plating density coincident with abundant cell-cell junctions resulted in low levels of both BCAR3 mRNA and protein. Conversely, BCAR3 expression was found to be elevated under conditions in which cell-cell junctions were reduced (low density plating). In the more invasive BT549 and MDA-MB-231 cell lines, BCAR3 mRNA and protein expression was found to be dependent upon the stable expression of its binding partner p130Cas (Cas). Interestingly, we previously showed that BCAR3 expression positively correlates with the tyrosine phosphorylation state of Cas, activation of the tyrosine kinase c-Src, and the physical interaction between Cas and c-Src. We are currently investigating whether cell-cell junction formation and differential expression alters the subcellular localization of BCAR3. Taken together, we propose a role for BCAR3 in driving the transition from localized disease to a more invasive and aggressive breast tumor phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B58.

Characterization of BCAR4, a novel oncogene causing endocrine resistance in human breast cancer cells

Resistance to the antiestrogen tamoxifen remains a major problem in the management of estrogen receptor-positive breast cancer. Knowledge on the resistance mechanisms is needed to develop more effective therapies. Breast cancer antiestrogen resistance 4 (BCAR4) was identified in a functional screen for genes involved in tamoxifen resistance. BCAR4 is expressed in 27% of primary breast tumors. In patients treated with tamoxifen for metastized disease high BCAR4 mRNA levels are associated with reduced clinical benefit and progression-free survival. Regarding tumor aggressiveness high BCAR4 mRNA levels are associated with a shorter metastasis free survival and overall survival. In the present study, we investigated the role of BCAR4 in endocrine resistance. Forced expression of BCAR4 in human ZR-75-1 and MCF7 breast cancer cells resulted in cell proliferation in the absence of estrogen and in the presence of various antiestrogens. Inhibition of estrogen receptor 1 (ESR1) expression with small interfering RNA (siRNA), implied that the BCAR4-induced mechanism of resistance is independent of ESR1. Highly conserved BCAR4 homologues of rhesus monkey, green monkey, and the less conserved common marmoset gene induced tamoxifen-resistant cell proliferation, in contrast to the distant BCAR4 homologues of bovine and rabbit. Injection of BCAR4-expressing ZR-75-1 cells into nude mice resulted in rapidly growing tumors. In silico analysis showed that BCAR4 mRNA is highly expressed in human placenta and oocyte, and absent in other normal tissues. In conclusion, BCAR4 is a strong transforming gene causing estrogen-independent growth and antiestrogen resistance, and induces tumor formation in vivo. Due to its restricted expression, BCAR4 may be a good target for treating antiestrogen-resistant breast cancer.

Abstract 358: Identification of signaling pathways activated by BCAR4 expression in human breast cancer

Abstract Background: The development of resistance to the antiestrogen tamoxifen remains a major challenge in the management of estrogen receptor alpha (ER)-positive breast cancer. Understanding the mechanisms leading to resistance is essential to develop more efficient therapies. In a functional screening for genes causing tamoxifen resistance, we have identified the breast cancer antiestrogen resistance 4 (BCAR4) gene. BCAR4 mRNA is expressed in 27% of primary breast tumors. High BCAR4 mRNA levels predict resistance to tamoxifen treatment and poor outcome, and are associated with a shorter metastasis-free survival and overall survival. Forced expression of BCAR4 in the estrogen-dependent breast cancer cell line ZR-75-1 induced antiestrogen resistance and phosphorylation of ERBB2, ERBB3, and their down-stream mediators ERK1/2 and AKT. The knockdown of ERBB2 or ERBB3 with specific siRNA inhibited cell proliferation, confirming the role of these receptors in BCAR4-induced tamoxifen resistance in vitro. These findings suggest that tumors expressing BCAR4 may rely on ERBB2/ERBB3 signaling and patients with such tumors may be eligible to receive ERBB-targeted therapy. Although ER is functional in ZR/BCAR4 cells, the knockdown of the receptor had no effect on the proliferation rate of BCAR4-expressing cells, showing that this mechanism of resistance is independent of ER signaling. Objective: To identify signaling pathways activated by BCAR4 expression and investigate the consequences of perturbations of the pathways. Experimental design: Reverse-phase protein microarray (RPPA) using phospho-specific antibodies was done to examine the activation status of several key signaling molecules in a cellular model for endocrine resistance of breast cancer. A panel of ZR-75-1-derived cell lines, each containing a different transgene conferring anti-estrogen resistance, was analyzed by RPPA. Results: By measuring the activity of a large number of phospho-protein isoforms in cell lysates from vector-containing control and BCAR4-expressing cells (ZR/BCAR4), it was confirmed that ERBB2 and ERBB3 were strongly phosphorylated in ZR/BCAR4 cells. This was observed in estradiol-containing medium and in medium supplemented with tamoxifen. Furthermore, many established down-stream signaling molecules of ERBB2/3 were found to be activated. Conclusions: ERBB2/ERBB3signaling is strongly activated in cells with forced expression of BCAR4 and completely independent of estrogen signaling. Both ERBB2 and ERBB3 are required for BCAR4-mediated tamoxifen resistance. Our results suggest that patients with ER-positive breast tumors expressing BCAR4 may benefit from ERBB-targeted therapies combined with tamoxifen treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2011-358

Abstract 2070: XBP-1 promotes cell survival by activating the unfolded protein response (UPR) in antiestrogen resistance in breast cancer

Abstract Most of the 192,000 newly diagnosed cases of invasive breast cancer in 2011 will be estrogen receptor positive alpha positive (ER+). Endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), e.g., Letrozole (LET), is the least toxic and most effective means to manage the hormone-dependent breast cancer in such patients. However, resistance to endocrine therapy remains a significant clinical problem. Our group has shown that antiestrogen resistant breast cancer cells over-express X-Box Binding Protein 1 (XBP1), a transcription factor that belongs to the basic region/leucine zipper (bZIP) family. XBP1(U) and XBP1(S) variants result from an unconventional splicing of the XBP1 mRNA by IRE1alpha. In the unfolded protein response (UPR), endoplasmic reticulum proteins (IRE1alpha, ATF6 and PERK) sense cellular stress to regulate the accumulation of unfolded proteins. Initially a compensatory mechanism allowing cells to recover normal endoplasmic reticulum function, prolonged UPR may induce cell death; this is often dependent upon which arm of the UPR predominates. XBP1 is an obligate component in both the IRE1alpha and ATF6 arms of the UPR. Our study shows that antiestrogen resistant breast cancer cells survive by activating pro-survival autophagy through XBP1-mediated UPR. MCF7/LCC9 [Faslodex resistant; TAM cross-resistant; high level of XBP1(S)] cells showed an increase in UPR signaling as detected by increased expression of BiP/GRP78. Antiestrogen-sensitive cells that over-express XBP1(S) show decreased sensitivity to Faslodex and show increased levels of basal and Faslodex-induced autophagy as measured by cleaved LC3BII protein fragment, GFP-LC3 activity, and reduced expression of p62/SQSTM1. Thus, we conclude that antiestrogen resistant breast cancer cells evade cell death by activating the XBP1-regulated arms of the UPR that results in prosurvival autophagy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2070. doi:10.1158/1538-7445.AM2011-2070

Abstract 3138: FOXC1 represses estrogen receptor-α expression via increasing NF-κB activity in breast cancer: A novel mechanism to explain antiestrogen resistance

Abstract The Forkhead-box transcription factor FOXC1 has recently been identified as a critical marker for human basal-like breast cancer, which lacks or under-expresses estrogen receptor-α (ERα). Here we show a consistent inverse correlation between FOXC1 expression and ERα expression in multiple cDNA microarray data sets of human breast cancer. Overexpression of FOXC1 in ERα-positive breast cancer cells induces cell growth, migration, and invasion, but downregulates ERα mRNA and protein levels and consequently reduces cellular responses to estradiol and tamoxifen. This could explain why tamoxifen-refractory breast cancer cells exhibit higher FOXC1 levels as compared with tamoxifen-sensitive parental cells. We also found that FOXC1 induces NF-κB activation, which is known to be associated with ER-negative breast cancer, by increasing Pin1-mediated p65 protein stability and thus p65 protein levels. NF-κB mediates, at least in part, the regulation of ERα by FOXC1, because inhibition of NF-κB by an IκBα super-repressor and small-molecule inhibitors attenuated the suppression of ERα expression by FOXC1. Furthermore, the importance of the NF-κB pathway in FOXC1-stimulated cell growth was demonstrated by increased cell sensitivity to pharmacologic inhibition of NF-κB. Taken together, these results reveal a novel ERα-regulating mechanism that may explain the loss or low expression of ERα in basal-like breast cancer and also may provide new insight into mechanisms for antiestrogen resistance in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3138. doi:10.1158/1538-7445.AM2011-3138

Abstract 1738: Histone deacetylase inhibitor mediated reversal of acquired endocrine therapy resistance in breast cancer cell lines

Abstract Although nearly 70% of breast cancers are estrogen receptor (ER) positive, endocrine therapy using the selective ER modulator tamoxifen or the selective ER down regulator fulvestrant have proved to be beneficial in less than 50% of these patients. Moreover, most ER positive patients that initially respond to tamoxifen eventually acquire resistance. Numerous studies have focused on determining the mechanism of anti-estrogen resistance which involve cellular changes such as variation in uptake/metabolism of tamoxifen, expression of altered ER proteins, ligand-independent ER activation, loss of ER expression and/or variation in recruitment of cofactors and altered cross-talk with other growth factor signaling pathways. Recently, an alternative strategy to modulate ER signaling has been the use of histone deacetylase (HDAC) inhibitors. Preclinical studies have shown that with the addition of HDAC inhibitors, the anti-tumor activity of tamoxifen is potentiated. In these cells, apoptotic cell death is induced, which cannot be rescued by estrogen, indicating the potential to reverse hormone therapy resistance, a strategy currently being evaluated in several clinical trials. Our laboratory is interested in exploring the potential of HDAC inhibition for the reversal of anti-estrogen resistance in ER-positive breast cancer. To this end, we have generated several MCF-7 cell lines resistant to various levels of tamoxifen; up to 10 micromolar. Our preliminary data demonstrate the addition of an HDAC inhibitor to tamoxifen reverses resistance. Significant effects on proliferation and viability are observed in these cells with clinically relevant concentrations of pan-HDAC inhibitors. Furthermore, with the addition of an HDAC inhibitor to tamoxifen, colony formation is attenuated. Initial characterization of these resistant cells indicated a several fold higher growth rate than the parental cells but a similar growth rate in presence and absence of the ligand estrogen, suggesting the possibility of loss of estrogen-dependence. Both the parental and resistant cells, in presence of estrogen, show a stabilization of the estrogen receptor protein by tamoxifen, which is not altered by the HDACi, suggesting an effect that goes beyond modulation of ER protein level. Tamoxifen or the combination in the resistant cells do not decrease Cyclin D1 protein level as is seen in parental cells, suggesting the involvement of pathways not involving ER. Our previous data using siRNA against HDAC2 indicate sensitization of ER positive breast cancer cells to tamoxifen-mediated apoptosis suggesting the importance of HDAC2. We plan to study the importance of ER, in resistant cells with varied ER status, in mediating the hormone therapy response and to identify the potential involvement of other signaling pathways, using both HDAC inhibitors and selectively depleting HDAC2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2011-1738

Breast Cancer Anti-Estrogen Resistance Protein 1 (BCAR1/p130cas) in Pulmonary Disease Tissue and Serum

The purpose of the study was to evaluate clinical presentation of breast cancer anti-estrogen resistance protein 1 (BCAR1, also known as p130cas) expression in pulmonary diseases, and to assess its potential as a molecular marker for diagnosis and prognosis. Between March 2008 and August 2010, we enrolled a total of 80 patients (group A) with non-small-cell lung cancer (NSCLC), 48 patients (group B) with pulmonary tuberculosis (including 27 cases of tuberculoma and 21 cases of cavitary pulmonary tuberculosis), and 32 patients (group C) with other benign pulmonary mass (hamartoma in 15 cases, inflammatory pseudotumor in 10 cases, fibroid tumor in 7 cases). Additionally, 160 healthy age- and sex-matched volunteers were recruited as healthy controls. Tissue BCAR1 expression was investigated by using tissue microarray and immunohistochemistry. BCAR1 and tumor markers (carcinoma embryonic antigen [CEA] and the cancer antigens CA19-9 and CA125) in serum were assayed by using ELISA and immunoradiometrics, respectively. BCAR1 expression was detected (either in the nucleus, the cytoplasm, or both) in tumor cells in 79 of the 80 NSCLC cases in group A, and in fibroblasts in 41 of the 48 pulmonary tuberculosis cases in group B. However, it was not detected in the normal adjacent tissue in 70 of the 80 cases in group A and in 47 of the 48 cases in group B. In group C, BCAR1 expression was negative in all 32 cases. Additionally, we investigated adjacent tissue with acute or chronic inflammation in 20 cases from group C, and found no expression of BCAR1. Serum BCAR1 levels were significantly higher in patients with NSCLC than in the control group, increased gradually with the progression of tumor staging, and decreased after removal of the tumors. The levels were significantly lower in bronchioloalveolar carcinoma than in other subtypes of carcinoma (Mann-Whitney U test, Z = -5.089; p < 0.001). Serum BCAR1 levels were significantly higher in patients with pulmonary tuberculosis than in the control group, were positively and significantly correlated with the diameter of the tuberculosis lesion (Spearman's rho, correlation coefficient 0.753; p < 0.001), and decreased after removal of the tuberculosis lesions. The levels were significantly higher in patients with cavitary pulmonary tuberculosis than in those with tuberculoma (517.6 ± 326.5 vs 282.2 ± 137.6; Student's t-test, t = -3.387; p = 0.001). In group C, there was no appreciable difference in serum BCAR1 levels compared with the matched controls (222.8 ± 111.0 vs 201.6 ± 35.7; Dunnett's T3 test, p = 0.993). The discrimination power of combining BCAR1 and tumor markers in NSCLC versus benign lung diseases was higher than that of sole use of BCAR1 as a marker (maximal sum of sensitivity and specificity: 1.538 vs 1.237). We conclude that a combined assay of serum BCAR1 and traditional tumor markers is potentially applicable for distinguishing NSCLC from benign lung diseases. However, the clinical utility of serum BCAR1 as a molecular marker for prognosis in NSCLC or pulmonary tuberculosis requires further clarification and verification.

Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue

Syk, a non-receptor tyrosine kinase, is an essential signaling molecule in B cells and other hematopoietic cells. Recently, its unexpected diverse functions were recognized in the regulation of cellular adhesion, innate immune recognition, vascular development, and carcinogenesis. Despite its pleiotropic role, only a few substrate proteins have been identified. To find new substrate proteins for Syk, we performed a systemic in vitro kinase assay using GST fusion peptides to determine the substrate specificity surrounding the tyrosine residue to be phosphorylated. Substitution of amino acid residues surrounding tyrosine 178 of BLNK, a principal Syk substrate in B cell receptor-mediated signaling, revealed that acidic residues at sites −5 to −1 were necessary for phosphorylation by Syk. Valine at site +1 was also influential in phosphorylation and a substitution of Pro on site +3 to a basic amino acid residue, Lys, resulted in attenuated phosphorylation. On the basis of these results, a general consensus phosphorylation motif for Syk was determined and several new candidate target proteins were identified in protein database searches. Of the candidate proteins, BCAR-1 (breast cancer anti-estrogen resistance 1) was confirmed to be phosphorylated by Syk in an in vitro kinase assay using a full-length protein of BCAR-1. Furthermore, BCAR-1 was tyrosine phosphorylated upon the overexpression of Syk in HEK-293T cells. These results suggest that more Syk substrates can be found using an in vitro kinase approach and show for the first time that BCAR-1 is a physiological substrate of Syk.

Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin

The coexpression of erbB3 and erbB2 is frequently observed in breast cancer; and erbB3 has a critical role in erbB2 promotion of breast cancer progression and anti-estrogen resistance. In this study, we determine the role of erbB3 in erbB2-mediated paclitaxel resistance in breast cancer cells. The overexpression of exogenous erbB3 via either stable or transient transfection in erbB2-overexpressing, but not epidermal growth factor receptor (EGFR)-expressing, breast cancer cells significantly decreases paclitaxel-induced growth inhibition and apoptosis. Consistently, knockdown of erbB3 expression with a specific short hairpin RNA (shRNA) in breast cancer cells with coexpression of both erbB2 and erbB3 enhances paclitaxel-induced apoptosis evidenced by increased DNA fragmentation, poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and -8. Furthermore, while forced overexpression of erbB3 increases, specific knockdown of erbB3 decreases the expression levels of Survivin only in the erbB2-overexpressing breast cancer cells. Targeting Survivin with specific shRNA overcomes paclitaxel resistance without effect on the expression levels of either erbB2 or erbB3. Mechanistic studies indicate that the specific phosphoinositide 3-kinase (PI-3K), Akt and mammalian target of rapamycin (mTOR) inhibitors, but not the mitogen-activated protein kinase kinase (MEK) inhibitor, not only abrogate erbB3-mediated upregulation of Survivin, but also reinforce the erbB2/erbB3-coexpressing breast cancer cells to paclitaxel-induced growth inhibition. These data demonstrate that heterodimerization of erbB2/erbB3 is a prerequisite for erbB2 tyrosine kinase activation; and elevated expression of erbB3 is required for erbB2-mediated paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway-dependent upregulation of Survivin. Our studies suggest that new strategies targeting erbB3 or Survivin may enhance the efficacy of chemotherapeutic agents against erbB2-overexpressing breast cancer.

Abstract 3668: Relevance of BCAR4 in tamoxifen resistance and tumor aggressiveness of breast cancer

Abstract Background: Tamoxifen is effective in the treatment of estrogen receptor alpha (ER)-positive breast cancer. However, almost all responding patients develop resistance. Understanding the mechanisms leading to resistance is crucial for the development of more efficient therapies. In a functional screen for genes causing tamoxifen resistance, we have identified the breast cancer antiestrogen resistance 4 (BCAR4) gene. Forced expression of BCAR4 in estrogen-dependent breast cancer cell lines induced antiestrogen resistance. Objective: We explored whether BCAR4 mRNA levels in primary tumors predict tamoxifen resistance and/or tumor aggressiveness, and searched for signaling pathways involved in BCAR4-induced resistance to tamoxifen. Experimental design: BCAR4 mRNA levels were measured by quantitative RT-PCR. In 280 ER-positive primary tumors from patients receiving tamoxifen as first-line monotherapy for advanced disease, the associations between BCAR4 mRNA levels and progression-free survival (PFS) and clinical benefit were evaluated. BCAR4 mRNA levels in primary tumors from 506 patients with lymph node-negative, ER-positive cancer were evaluated for their association with distant metastasis-free survival (MFS). None of them received systemic adjuvant therapy, thereby preventing confounding effects of systemic treatment. In order to study BCAR4 function, we forced the expression of BCAR4 in estrogen-dependent ZR-75-1 breast cancer cells, and analyzed changes in phospho-protein expression. Results: In the study for the association of BCAR4 with tamoxifen resistance, high BCAR4 mRNA levels were significantly associated with a shorter PFS and poor clinical benefit (measurable tumor response or no change &amp;gt; 6 months). Multivariate analysis including the traditional predictive factors indicated that a high BCAR4 mRNA level is an independent predictive factor for PFS. In the assessment of tumor aggressiveness, high BCAR4 mRNA levels were associated with a shorter MFS and overall survival (OS). Multivariate analyses showed that a high BCAR4 mRNA level was an independent prognostic factor for MFS and OS. Forced expression of BCAR4 induced phosphorylation of ERBB2, ERBB3, and their down-stream mediators ERK1/2 and AKT in ZR-75-1 cells. Proliferation of these cells was inhibited by the knockdown of ERBB2 or ERBB3 with specific siRNAs, confirming the role of these receptors in BCAR4-induced tamoxifen resistance. The knockdown of ER had no effect on the proliferation of BCAR4-expressing cells, indicating that this mechanism of resistance is independent of ER signaling. Conclusions: Our data indicate that high BCAR4 mRNA levels predict resistance to tamoxifen treatment and poor outcome. The results of our experimental cell model also suggest that tumors expressing BCAR4 may rely on ERBB2/ERBB3 signaling. Therefore, patients with tumors expressing BCAR4 may be eligible to receive ERBB-targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3668.