Luminal Breast Tumors Research Articles

MicroRNA-100 targets IGF1R and regulates migration in a breast cancer model of epithelial-mesenchymal transition

Abstract. Luminal breast tumors typically have a lower risk of metastasis due to the epithelial nature of the cells. Strong adhesion between epithelial cancer cells lowers their motility, migration rate, and invasive potential. Despite the nature of epithelial breast tumors, they may still undergo metastasis, which is thought to be initiated by epithelial-mesenchymal transition (EMT). EMT is a process through which epithelial cells acquire the phenotype of mesenchymal cells, which have enhanced invasive potential in cancer. MicroRNAs, small regulatory RNAs that inhibit gene expression, play a role in the regulation of EMT. This study demonstrates that microRNA-100 (miR-100) is upregulated in mesenchymal breast cancer cells (MCF-7M) versus epithelial breast cancer cells (MCF-7) via a mammosphere-induced EMT. MiR-100 may play a role in EMT by targeting the insulin like growth factor 1 receptor (IGF1R). Inhibition of miR-100 in MCF-7M cells significantly increased endogenous IGF1R expression. IGF1R has been ...

PIPPing on AKT1: How Many Phosphatases Does It Take to Turn off PI3K?

In this issue of Cancer Cell, Ooms and colleagues show that the lipid phosphatase PIPP/INPP5J, frequently inactivated in triple-negative breast cancers, functions as a tumor suppressor by specifically modulating the activity of AKT1 in the context of oncogenic PI3K signaling, leading to inhibition of metastatic dissemination.

Targeting IL13Ralpha2 activates STAT6-TP63 pathway to suppress breast cancer lung metastasis.

IntroductionBasal-like breast cancer (BLBC) is an aggressive subtype often characterized by distant metastasis, poor patient prognosis, and limited treatment options. Therefore, the discovery of alternative targets to restrain its metastatic potential is urgently needed. In this study, we aimed to identify novel genes that drive metastasis of BLBC and to elucidate the underlying mechanisms of action.MethodsAn unbiased approach using gene expression profiling of a BLBC progression model and in silico leveraging of pre-existing tumor transcriptomes were used to uncover metastasis-promoting genes. Lentiviral-mediated knockdown of interleukin-13 receptor alpha 2 (IL13Ralpha2) coupled with whole-body in vivo bioluminescence imaging was performed to assess its role in regulating breast cancer tumor growth and lung metastasis. Gene expression microarray analysis was followed by in vitro validation and cell migration assays to elucidate the downstream molecular pathways involved in this process.ResultsWe found that overexpression of the decoy receptor IL13Ralpha2 is significantly enriched in basal compared with luminal primary breast tumors as well as in a subset of metastatic basal-B breast cancer cells. Importantly, breast cancer patients with high-grade tumors and increased IL13Ralpha2 levels had significantly worse prognosis for metastasis-free survival compared with patients with low expression. Depletion of IL13Ralpha2 in metastatic breast cancer cells modestly delayed primary tumor growth but dramatically suppressed lung metastasis in vivo. Furthermore, IL13Ralpha2 silencing was associated with enhanced IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and impaired migratory ability of metastatic breast cancer cells. Interestingly, genome-wide transcriptional analysis revealed that IL13Ralpha2 knockdown and IL-13 treatment cooperatively upregulated the metastasis suppressor tumor protein 63 (TP63) in a STAT6-dependent manner. These observations are consistent with increased metastasis-free survival of breast cancer patients with high levels of TP63 and STAT6 expression and suggest that the STAT6-TP63 pathway could be involved in impairing metastatic dissemination of breast cancer cells to the lungs.ConclusionOur findings indicate that IL13Ralpha2 could be used as a promising biomarker to predict patient outcome and provide a rationale for assessing the efficacy of anti-IL13Ralpha2 therapies in a subset of highly aggressive basal-like breast tumors as a strategy to prevent metastatic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0607-y) contains supplementary material, which is available to authorized users.

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

Size does not matter: commensal microorganisms forge tumor-promoting inflammation and anti-tumor immunity.

Recent studies have demonstrated that the commensal microbiota are indispensable for the maintenance of immune homeostasis, orchestration of immune responses against pathogens and most recently during cancer immunotherapy and malignant progression of extraintestinal tumors. Here we discuss the recent findings that a common genetic variation in TLR5 influences the progression and outcome of ovarian, sarcoma, and luminal breast tumors and the implications of these findings in light of recent publications describing the role of the commensal microbiota in control of the systemic immune system.

Glycoproteomic Comparison of Clinical Triple-Negative and Luminal Breast Tumors

Triple-negative (TN) breast cancer accounts for ∼ 15% of breast cancers and is characterized by a high likelihood of relapse and a lack of targeted therapies. In contrast, luminal-type tumors that express the estrogen and progesterone receptors (ER+/PR+) and lack expression of human epidermal growth factor receptor 2 (Her2-) are treated with targeted hormonal therapy and carry a better prognosis. To identify potential targets for the development of future therapeutics aimed specifically at TN breast cancers, we have used a hydrazide-based glycoproteomic workflow to compare protein expression in clinical tumors from nine TN (Her2-/ER-/PR-) and nine luminal (Her2-/ER+/PR+) patients. Using a label-free LC-MS based approach, we identified and quantified 2264 proteins. Of these, 90 proteins were more highly expressed and 86 proteins were underexpressed in the TN tumors relative to the luminal tumors. The expression level of four of these potential targets was verified in the original set of tumors by Western blot and correlated well with our mass-spectrometry-based quantification. Furthermore, 30% of the proteins differentially expressed between luminal and TN tumors were validated in a larger cohort of 406 TN and 469 luminal tumors through corresponding differences in their mRNA expression in publically available microarray data. A group of 29 of these differentially expressed proteins was shown to correctly classify 88% of TN and luminal tumors using microarray data of their associated mRNA levels. Interestingly, even within a group of TN breast cancer patients, the expression levels of these same mRNAs were able to significantly predict patient survival, suggesting that these proteins play a role in the aggressiveness seen in TN tumors. This study provides a comprehensive list of potential targets for the development of diagnostic and therapeutic agents specifically aimed at treating TN breast cancer and demonstrates the utility of using publicly available microarray data to further prioritize potential targets.

Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells.

IntroductionLuminal, estrogen receptor-positive (ER+) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models.MethodsImmunocompromised, ovariectomized (ovx’d) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER+PR+ cytokeratin 5-negative (CK5−) E3 and MCF-7 cells, basal ER−PR−CK5+ estrogen withdrawn-line 8 (EWD8) cells, and basal ER−PR−CK5− MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx’d mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers.ResultsER−PR− cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx’d mice injected with ER+PR+ cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER+PR+CK5− cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER−PR−CK5+ cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx’d control mice injected with ER+PR+ cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER+ but PR−. Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration.ConclusionsWe conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER−PR− subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx’d mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0489-4) contains supplementary material, which is available to authorized users.

Gene expression abnormalities in histologically normal breast epithelium from patients with luminal type of breast cancer.

The gene expression profile of breast cancer has been described as a great breakthrough on the way to comprehend differences in cancer origin, behavior and therapy. However, gene expression profile in histologically normal epithelium (HNEpi) which could harbor genetic abnormalities predisposing breast tissue to develop malignancy was minor scope for scientists in the past. Thus, we aimed to analyze gene expressions in HNEpi and breast cancer tissue (BCTis) in order to establish its value as potential diagnostic marker for cancer development. We evaluated a panel of disease-specific genes in luminal type (A/B) of breast cancer and tumor surrounding HNEpi by qRT-PCR Array in 32 microdissected samples. There was 20.2 and 2.4% deregulation rate in genes with at least 2-fold or 5-fold over-expression between luminal (A/B) type breast carcinomas and tumor surrounding HNEpi, respectively. The high-grade luminal carcinomas showed higher number of deregulated genes compared to low-grade cases (50.6 vs. 23.8% with at least 2-fold deregulation rate). The main overexpressed genes in HNEpi were KLK5, SCGB1D2, GSN, EGFR and NGFR. The significant differences in gene expression between BCTis and HNEpi samples were revealed for BAG1, C3, CCNA2, CD44, FGF1, FOSL1, ID2, IL6R, NGFB, NGFR, PAPPA, PLAU, SERPINB5, THBS1 and TP53 gene (p < 0.05) and BCL2L2, CTSB, ITGB4, JUN, KIT, KLF5, SCGB1D2, SCGB2A1, SERPINE1 (p < 0.01), and EGFR, GABRP, GSN, MAP2K7 and THBS2 (p < 0.001), and GSN, KLK5 (p < 0.0001). The ontological gene analyses revealed high deregulations in gene group directly associated with breast cancer prognosis and origin.

Abstract 3559A: Molecular subtype-specific methylation of the miR-29c promoter in breast cancer correlates with basal-like pattern of gene expression

Abstract MicroRNA-29c (miR-29c) has been shown to have altered expression in several different cancer types, including gastric, leukemia, thyroid, and gliomas. Our lab has previously identified miR-29c as a significantly down-regulated miRNA in basal-like breast tumors and demonstrated an association with cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in breast cancer. Expression of miR-29c was determined using qRT-PCR in breast cancer cell lines of the basal, luminal, and claudin-low subtypes. miR-29c promoter was located using CAGE-tag analysis and ChIP assays to determine transcription start sites and RNA polymerase II binding sites, respectively. CpG islands were predicted using methylation software, and primers were designed to amplify those regions for sequencing after bisulfite treatment. The percent of methylated CpGs was compared to expression levels in breast cancer cell lines and results were validated using primary tumors in the TCGA dataset. miR-29c has lower expression in basal-like cell lines compared to claudin-low and luminal cell lines. To determine if the changes in expression are driven by changes in promoter activity, we first identified the promoter of miR-29c at the region upstream of the gene on Chromosome 1q32. We then identified potential CpG islands in the promoter region. Bisulfite sequencing of DNA from breast cancer cell lines revealed a significant difference in percent of methylation between the different subtypes. The methylation of the promoter in basal, claudin-low, and luminal subtypes was 77.1%, 60.0%, and 16.6%, respectively (p=.002). The percent of methylation was inversely correlated with expression of miR-29c in the luminal and basal subtypes (R2=.68). An analysis of TCGA data for methylation at several CpG islands in the miR-29c promoter region in breast tumors confirmed a statistically significant mean difference of methylation of basal-like tumors versus Her2, luminal A and luminal B tumors. We have identified the location of the miR-29c promoter and determined that miR-29c is expressed lower in basal-like breast cancer cell lines. We have also determined that the miR-29c promoter in basal-like breast cancer cell lines has a higher percentage of methylation at CpG islands compared to luminal, which is correlated with expression differences between these two subtypes. In addition, basal-like and luminal breast tumors from TCGA have patterns of methylation in the promoter region that correlate with our cell line data. More work needs to be done to understand how methylation of mir-29c in basal-like tumors contributes to the aggressiveness of the disease and whether it is a potential predictive biomarker for resistance to chemotherapy. Citation Format: Elizabeth Carla Poli, Rachel Tolbert, Jing Zhang, Yoo-Jeong Han, Olufunmilayo I. Olopade. Molecular subtype-specific methylation of the miR-29c promoter in breast cancer correlates with basal-like pattern of gene expression. [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 3559A. doi:10.1158/1538-7445.AM2014-3559A

JARID1B Is a Luminal Lineage-Driving Oncogene in Breast Cancer

Recurrent mutations in histone-modifying enzymes imply key roles in tumorigenesis, yet their functional relevance is largely unknown. Here, we show that JARID1B, encoding a histone H3 lysine 4 (H3K4) demethylase, is frequently amplified and overexpressed in luminal breast tumors and a somatic mutation in a basal-like breast cancer results in the gain of unique chromatin binding and luminal expression and splicing patterns. Downregulation of JARID1B in luminal cells induces basal genes expression and growth arrest, which is rescued by TGFβ pathway inhibitors. Integrated JARID1B chromatin binding, H3K4 methylation, and expression profiles suggest a key function for JARID1B in luminal cell-specific expression programs. High luminal JARID1B activity is associated with poor outcome in patients with hormone receptor-positive breast tumors.

Identification and validation of oncologic miRNA biomarkers for luminal A-like breast cancer.

IntroductionBreast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.MethodsBlood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.ResultsMicroarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.ConclusionThis study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.

Abstract S3-05: Patient-derived xenograft study reveals endocrine therapy resistance of ER+ breast cancer caused by distinct ESR1 gene aberrations

Abstract Endocrine therapy resistance occurs in 50% of estrogen receptor positive (ER+) luminal breast cancers but the underlying mechanisms are poorly understood. To gain insight, we have taken advantage of whole-genome and RNA sequencing data of five late-stage hormone-resistant luminal breast tumors all of which have been successfully established as patient-derived mouse xenograft (PDX) models. Here we describe genetic alterations in ESR1-the gene coding for ERa-in three of these five tumors. They include an ESR1 point mutation (Y537S), a gene translocation causing an in-frame fusion between N-terminal ER and C-terminal Yes-associated protein 1 (YAP1), and ESR1 gene amplification. Functional characterization of the ESR1(Y537S) mutant and ESR1-YAP1 fusion in ER+ cell lines indicated that they both possess constitutive transcriptional activity and drive hormone-independent cell proliferation, mirroring the endocrine resistance of the originating tumors and the estradiol-independent growth of the PDX tumors. ESR1 (Y537S), a known gain-of-function experimental mutation in the ligand-binding domain of ER is not seen in primary breast cancer (TCGA data), suggesting it is a mutation associated with acquired resistance. Regarding the ESR1-YAP1 fusion gene, the truncated N-terminal ER fragment lacks the hormone-dependent transactivation domain (AF2) and the ligand binding domain but retains the hormone-independent transactivation domain (AF1) and therefore drives resistance to all endocrine approaches. As for ESR1 gene amplification, the tumor of origin, though resistant to aromatase inhibition, paradoxically responded to estradiol treatment, and this was recapitulated in the PDX model. Interestingly, acquired ESR1 gene amplification also occurred in long-term estrogen-deprived breast cancer MCF-7 cells which similarly regress upon estradiol exposure. Thus, ESR1 amplification may be a biomarker for paradoxical therapy with estradiol. Together, our focused study of advanced endocrine resistant luminal breast tumors revealed three distinct mutational mechanisms affecting the ESR1 gene that drive endocrine therapy resistance. Prevalence studies using RNAseq are underway to determine the frequency of somatic changes in the ESR1 gene in advanced breast cancer samples and additional ER+ PDX models. These additional data will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-05.

Prognostic relevance at 5 years of the early monitoring of neoadjuvant chemotherapy using 18F-FDG PET in luminal HER2-negative breast cancer

The objective of this study was to evaluate, in the luminal human epidermal growth factor receptor 2 (HER2)-negative breast cancer subtype, the prognostic value of tumour glucose metabolism at baseline and of its early changes during neoadjuvant chemotherapy (NAC). This prospective study included 61 women with hormone-sensitive HER2-negative breast cancer treated with NAC. (18)F-Fluorodeoxyglucose (FDG) positron emission tomography (PET) was performed at baseline. Hepatic activity was used as a reference to distinguish between low metabolic and hypermetabolic tumours. In hypermetabolic tumours, a PET exam was repeated after the first course of NAC. The relative change in the maximum standardized uptake value of the tumour (∆SUV) was calculated. Nineteen women had low metabolic luminal breast cancers at baseline, correlated with low proliferation indexes. Forty-two women had hypermetabolic tumours, corresponding to more proliferative breast cancers with higher Ki-67 expression (p = 0.017) and higher grade (p = 0.04). The median follow-up period was 64.2 months (range 11.5-93.2). Thirteen women developed recurrent disease, nine of whom died. Worse overall survival was associated with larger tumour size [>5 cm, hazard ratio (HR) = 6.52, p = 0.009] and with hypermetabolic tumours achieving a low metabolic response after one cycle of NAC (ΔSUV < 16%, HR = 10.63, p = 0.004). Five-year overall survival in these poor responder patients was 49.2%. Overall survival in women with low metabolic tumours or hypermetabolic/good response tumours was 100 and 96.15%, respectively. In luminal HER2-negative breast tumours, tumour metabolism at baseline and changes after the first course of NAC are early surrogate markers of patients' survival. A subgroup of women with hypermetabolic/poorly responding tumours, correlated with poor prognosis at 5 years, can be identified early. These results may guide future studies by tailoring the NAC regimen to the metabolic response.

Abstract A023: Investigating differential expression of microRNAs in the luminal breast cancer subtype

Abstract Breast Cancer is a heterogeneous disease, and tumors vary in pathological and clinical characteristics making it important that tumors be accurately characterized at diagnosis. Current methods reveal a number of breast cancer subtypes correlating with clinical factors; however, patients with the luminal subtype have a range of outcomes. Our hypothesis is that microRNA (miRNA) expression profiling may provide a method of discriminating tumors in this subtype into good and poor prognosis groups, as well as reveal potential biological factors affecting prognosis. Expression profiling was performed on miRNAs from 39 primary ER+, HER2- luminal breast tumors from a prospective cohort of women with node negative breast cancer with a median follow-up time of 116 months. Of the 39 tumors, 19 were from patients who eventually experienced a recurrence and 20 were from matched patients who remained disease-free. Total RNA was extracted from the specimens and the miRNAs quantified using TaqMan Array MicroRNA Cards. Results analyzed using significance analysis of microarrays (SAM) revealed nine miRNAs with a standard t-test p-value less than 0.05 and ranked within the top 20, designated as significantly differentially expressed between the two groups. Four of these, miR-135a, miR-140-5p, miR-200a, and miR-218, were selected for validation on the same samples using singleplex quantitative real-time PCR conducted in triplicate, and confirmed to be significantly less expressed in tumors in patients who had experienced recurrence compared to tumors from patients without recurrence. Functional analysis of miR-200a and miR-218 is being performed in MCF-7 breast cancer cells to determine the role they play in luminal breast cancer recurrence. Validated targets of these miRNAs suggest involvement in epithelial to mesenchymal transition and cellular migration, invasion, and proliferation. The activity of these miRNAs is being studied in vitro and altered expression of miRNA targets is being confirmed. Future studies to examine whether these modifications result in changes in tumorgenicity phenotypes will be addressed by assaying cell proliferation, migration, and invasion. This project has the potential to aid in improving breast cancer prognostication in the clinical setting, improve our knowledge of the role if miRNAs in luminal breast cancer, and identify miRNAs suitable as possible diagnostic biomarkers or novel therapeutic targets. Citation Format: Dylan A. Ehman, Dushanthi Pinnaduwage, Shelley B. Bull, Irene L. Andrulis. Investigating differential expression of microRNAs in the luminal breast cancer subtype. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A023.

Cyclin D1-Dependent Induction of Luminal Inflammatory Breast Tumors by Activated Notch3

Accumulating evidence suggests that Notch3 (N3) is involved in breast cancer development, but its precise contributions are not well understood. Here, we report that pregnant mice expressing an activated intracellular form of N3 (N3(IC)) exhibit a cyclin D1-dependent expansion of premalignant CD24(+) CD29(low) luminal progenitors with enhanced differentiation potential in vitro and in vivo. Parous mice developed luminal mammary tumors in a cyclin D1-dependent manner. Notably, mice expressing higher levels of N3(IC) exhibited tumors resembling inflammatory breast cancer that frequently metastasized. N3(IC)-induced tumors contained a large percentage of tumor-initiating cells, but these were reduced significantly in tumors derived from N3(IC) transgenic mice that were heterozygous for cyclin D1. After transplantation in the presence of normal mammary cells, N3(IC)-expressing tumor cells became less malignant, differentiating into CK6(+) CK18(+) CK5(-) alveolar-like structures akin to expanded luminal progenitors from which they were likely derived. Taken together, our results argue that activated N3 signaling primarily affects luminal progenitors among mammary cell subsets, with more pronounced levels of activation influencing tumor type, and provide a novel model of inflammatory breast cancer.

Abstract A053: ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

Abstract HER3/ErbB3 is required in luminal mammary epithelial cells (MECs). Given that breast cancer phenotypes may reflect biological traits of the MECs from which they originate, we tested the hypothesis that HER3/ErbB3 drives luminal breast cancer growth. We found higher ERBB3 expression and frequent ERBB3 gene copy gains in clinical Luminal A/B breast cancers over other molecular subtypes. ErbB3 increased growth of luminal breast cancer cells, while ErbB3 targeting using U3-1287, a fully-human monoclonal anti-ErbB3 antibody, decreased three-dimensional colony growth, increased apoptosis, and decreased tumor growth in vivo. In response to the anti-endocrine fulvestrant, ErbB3 expression increased in clinical breast tumors, as did phosphatidylinositol 3-kinase (PI3K)/mTOR signaling. However, ErbB3 targeting with U3-1287 blocked compensatory PI3K/mTOR signaling in fulvestrant-treated tumor cells, transcriptional responses to fulvestrant, tumor cell survival and tumor growth. U3-1287 produced these effects in tumor cells with and without genetic PI3K pathway aberrations (PIK3CAH1047R, PIK3CAE545K, PTEN loss). Although fulvestrant increased phosphorylation of all ErbB family RTKs and insulin-like growth factor-1 receptor, phospho-RTK upregulation was not seen in tumors treated with U3-1287 and fulvestrant. Therefore, luminal breast cancer cells utilize ErbB3 for growth, survival, and to limit response to fulvestrant, thus defining a novel role for ErbB3 in breast cancer. Citation Format: Meghan M. Morrison, Jamie Stanford, Christian Young, David Vaught, Andrew Williams, michelle williams, Chuck M. Perou, Carlos L. Arteaga, Rebecca S. Cook. ErbB3 downregulation enhances luminal breast tumor response to antiestrogens. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A053.

Prognostic relevance at 5 years of the early monitoring of neoadjuvant chemotherapy using FDG PET in luminal HER2-negative breast cancer.

11007 Background: To evaluate, in the luminal breast cancer subtype, the prognostic value of tumor glucose metabolism at baseline and of its changes after one cycle of neoadjuvant chemotherapy (NAC). Methods: This prospective study included 61 women with immunophenotypically defined luminal HER2-negative breast cancer treated with NAC. 18F-FDG PET was performed at baseline. Hepatic activity was used as a reference to distinguish between low-metabolic and hypermetabolic tumors. In hypermetabolic tumors, a PET exam was repeated after the first course of NAC. The relative change in the maximal Standardized Uptake Value of the tumor (ΔSUV), corresponding to the metabolic response, was calculated. Results: Forty-two women had hypermetabolic tumors at baseline, corresponding to more proliferative breast cancers with higher Ki-67 expression (p=0.017) and higher grade (p=0.04). Nineteen women had low-metabolic tumors with lower proliferation indexes. Worse overall survival was associated with larger tumor size (&gt;5cm, HR=6.52, P=0.009) and with hypermetabolic tumors achieving a low metabolic response after one cycle of NAC (ΔSUV&lt;16%, HR=10.63, P=0.004). Five-year overall survival in these poor-response patients was 49.22% (95% CI=[14.76%-76.90%]). In contrast, overall survival in women with low-metabolic tumors or hypermetabolic/good-response tumors (ΔSUV≥16%) was good, 100% and 96.15%, respectively (95% CI=[75.69%-99.45%]). Conclusions: In luminal HER2-negative breast tumors, tumor metabolism at baseline and changes after the first course of NAC are surrogate markers of patients’ survival. A subgroup of women with hypermetabolic/bad-responding tumors correlated with poor prognosis can be identified. These results may create the ability to tailor the NAC regimen to the metabolic response at an early stage.

Abstract 2401: Evaluation of circulating serum Amphiregulin levels in patients with breast cancer.

Abstract Amphiregulin (AREG), a ligand for Epidermal Growth Factor Receptor (EGFR), has been implicated as an effector of ERα signaling in mammary gland development and as a candidate ERα effector in human breast cancers. Analysis of 13 luminal subtype breast cancer cell lines and human breast tumors showed a strong correlation between ERα status and high AREG levels. A pilot study using healthy and breast cancer women's serum showed that circulating AREG levels were higher in the ERα positive patients as compared to healthy individuals. Therefore we hypothesized that Amphiregulin is a potential biomarker and/or therapeutic target for ERα positive breast cancers. To test our hypothesis, we are currently evaluating AREG as a potential serum biomarker of breast cancer progression and as an indicator of response to treatment by analyzing a cohort of breast cancer patients (n=225) stratified by stage of disease and subtypes (hormone receptor status). In this study, we want to determine if tumor-derived AREG is released into the systemic circulation and whether it may be a useful serum biomarker for the presence of breast cancer and for response to therapy. In the first phase, serum Amphiregulin was measured by an ELISA assay to establish the reference range in 125 healthy females. Fifty-five percent of the healthy women had no detectable circulating AREG (n=69), and only ten women had levels exceeding 500 pg/mL. Serum AREG levels did not vary significantly during the menstrual cycle. We determined that the 95th and 90th percentile was &amp;gt;1575 pg/ml and &amp;gt;471 pg/ml, respectively, to use as a comparison against the AREG levels of the breast cancer patients. We are analyzing serum AREG levels in breast cancer patient serum samples of three different cohorts: women with no evidence of disease after more than 1 year post-surgery (Cohort 1), women with locally advanced or stage IV with disease progression (Cohort 2) and women with operable disease that will have surgery and subsequent chemotherapy and/or endocrine therapy (Cohort 3). Each cohort was further stratified by hormone receptor status. In addition to determining the baseline AREG levels, for Cohort 3, AREG levels will be measured post-surgery and at different timepoints during treatment. Interim analysis of the different cohorts showed a significance difference (p&amp;lt;0.05) between healthy individuals and patients with ERα+ breast tumors when comparing detectable versus undetectable AREG levels (threshold of detection: 20 pg/ml). Interestingly, longitudinal sampling of one patient with very high levels (18,300 pg/ml at diagnosis) showed a 37% decline following tumor resection and a 60% decline after 9 months of endocrine therapy. In summary, determining the potential role of AREG as predictive biomarker for therapy and/or as therapeutic target by the completion of this clinical study, will contribute to the quest for better predictive biomarkers in breast cancer and better management of the disease. Citation Format: Esther A. Peterson, Paraic A. Kenny. Evaluation of circulating serum Amphiregulin levels in patients with 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 2401. doi:10.1158/1538-7445.AM2013-2401

Abstract 3569: Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.

Abstract Progesterone receptors (PR) are emerging as important breast cancer drivers. Elevated mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2) signaling is common in breast cancer and these kinases drive direct PR-B, but not PR-A, Ser294 phosphorylation. Post-translational modifications, including phosphorylation and SUMOylation, dramatically impact PR transcriptional activity. Phospho-Ser294 PRs are less SUMOylated on Lys388 (i.e. a repressive modification). Thus, active mitogenic kinase signaling provides a mechanism for PR derepression (i.e. transcriptional activation) during breast cancer progression. A broad range of PR protein expression is observed clinically; PR-negative tumors are generally more aggressive. However, clinical (IHC) assays do not measure phospho-PRs, which turnover rapidly and may appear undetectable, confounding the true level of activated PR within tumors. A PR gene signature would provide a valuable marker of PR contribution to breast cancer progression. Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild type or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Target genes sensitive to changes in PR SUMOylation included those required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors were preferentially recruited to enhancer regions of derepressed genes. SUMO-deficient (i.e. phospho-Ser294) PR gene signatures were significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Furthermore, treatment with antiprogestin or MEK inhibitors abrogated expression of SUMO-sensitive PR target-genes and inhibited progestin-driven proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells. Similarly, treatment of T47D cells expressing deSUMOylated PR with ERBB2 inhibitors blocked progestin-dependent growth. Potential mechanisms of differential promoter selection were investigated. Notably, genes preferentially up-regulated by SUMO-deficient PR contain RUNX binding motifs located near their transcriptional start sites. Herein, we demonstrated that RUNX2 directly interacts with PR. Further, RUNX2 knockdown in breast cancer cells reduced SUMO-deficient PR dependent gene expression. Our studies suggest that deSUMOylated PR cooperates with RUNX2 to mediate promoter selective gene expression. We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters PR target gene selection in breast cancer cells, in part via regulation of PR interactions with gene-specific co-factors, such as RUNX2. Patients whose ER+/PR+ tumors are driven by hyperactive phospho-PRs may benefit from endocrine (antiestrogen) therapies that also contain an antiprogestin. Citation Format: Todd P. Knutson, Andrea R. Daniel, Danhua Fan, Kevin A. Silverstein, Kyle R. Covington, Suzanne A. Fuqua, Carol A. Lange. Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during 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 3569. doi:10.1158/1538-7445.AM2013-3569

Progression of Luminal Breast Tumors Is Promoted by Ménage à Trois between the Inflammatory Cytokine TNFαand the Hormonal and Growth-Supporting Arms of the Tumor Microenvironment

Breast cancer progression is strongly linked to inflammatory processes, aggravating disease course. The impacts of the inflammatory cytokine TNFα on breast malignancy are not fully substantiated, and they may be affected by cooperativity between TNFα and other protumoral mediators. Here, we show that together with representatives of other important arms of the tumor microenvironment, estrogen (hormonal) and EGF (growth-supporting), TNFα potently induced metastasis-related properties and functions in luminal breast tumor cells, representing the most common type of breast cancer. Jointly, TNFα + Estrogen + EGF had a stronger effect on breast cancer cells than each element alone, leading to the following: (1) extensive cell spreading and formation of FAK/paxillin-enriched cellular protrusions; (2) elevated proportion of tumor cells coexpressing high levels of CD44 and β1 and VLA6; (3) EMT and cell migration; (4) resistance to chemotherapy; (5) release of protumoral factors (CXCL8, CCL2, MMPs). Importantly, the tumor cells used in this study are known to be nonmetastatic under all conditions; nevertheless, they have acquired high metastasizing abilities in vivo in mice, following a brief stimulation by TNFα + Estrogen + EGF. These dramatic findings indicate that TNFα can turn into a strong prometastatic factor, suggesting a paradigm shift in which clinically approved inhibitors of TNFα would be applied in breast cancer therapy.