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issn:1465-542
1.  Dysregulation of microRNAs in breast cancer and their potential role as prognostic and predictive biomarkers in patient management 
MicroRNAs (miRNAs) are an emerging class of gene expression modulators with relevant roles in several biological processes, including cell differentiation, development, apoptosis, and regulation of the cell cycle. Deregulation of those tiny RNA molecules has been described frequently as a major determinant for the initiation and progression of diseases, including cancer. Not only miRNAs but also the enzymes responsible for miRNA processing could be deregulated in cancer. In this review, we address the role of miRNAs in the pathogenesis of breast cancer, since there are oncogenic, tumor-suppressive, and metastatic-influencing miRNAs. Additionally, the different detection platforms and normalization strategies for miRNAs will be discussed. The major part of this review, however, will focus on the capability of miRNAs to act as diagnostic, predictive, or prognostic biomarkers. We will give an overview of their potential to correlate with response to or benefit from a given treatment and we will consider their ability to give information on prognosis in breast cancer. We will focus on miRNAs validated by more than one study or verified in independent cohorts or where results rely on preclinical as well as clinical evidence. As such, we will discuss their potential use in the personalized management of breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0526-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-015-0526-y
PMCID: PMC4332424
2.  Patient-derived xenograft models of breast cancer and their predictive power 
Despite advances in the treatment of patients with early and metastatic breast cancer, mortality remains high due to intrinsic or acquired resistance to therapy. Increased understanding of the genomic landscape through massively parallel sequencing has revealed somatic mutations common to specific subtypes of breast cancer, provided new prognostic and predictive markers, and highlighted potential therapeutic targets. Evaluating new targets using established cell lines is limited by the inexact correlation between responsiveness observed in cell lines versus that elicited in the patient. Patient-derived xenografts (PDXs) generated from fresh tumor specimens recapitulate the diversity of breast cancer and reflect histopathology, tumor behavior, and the metastatic properties of the original tumor. The high degree of genomic preservation evident across primary tumors and their matching PDXs over serial passaging validate them as important preclinical tools. Indeed, there is accumulating evidence that PDXs can recapitulate treatment responses of the parental tumor. The finding that tumor engraftment is an independent and poor prognostic indicator of patient outcome represents the first step towards personalized medicine. Here we review the utility of breast cancer PDX models to study the clonal evolution of tumors and to evaluate novel therapies and drug resistance.
doi:10.1186/s13058-015-0523-1
PMCID: PMC4323263
3.  Lobular breast cancer: molecular basis, mouse and cellular models 
Infiltrating lobular breast cancer (ILC) is the most common special breast cancer subtype. With mutational or epigenetic inactivation of the cell adhesion molecule E-cadherin (CDH1) being confined almost exclusively to ILC, this tumor entity stands out from all other types of breast cancers. The molecular basis of ILC is linked to loss of E-cadherin, as evidenced by human CDH1 germline mutations and conditional knockout mouse models. A better understanding of ILC beyond the level of descriptive studies depends on physiologically relevant and functional tools. This review provides a detailed overview on ILC models, including well-characterized cell lines, xenograft tumors and genetically engineered mouse models. We consider advantages and limitations of these models and evaluate their representativeness for human ILC. The still incompletely defined mechanisms by which loss of E-cadherin drives malignant transformation are discussed based on recent findings in these models. Moreover, candidate genes and signaling pathways potentially involved in ILC development and progression as well as anticancer drug and endocrine resistance are highlighted.
doi:10.1186/s13058-015-0517-z
PMCID: PMC4320436
4.  Invasive lobular carcinoma of the breast: morphology, biomarkers and ’omics 
Invasive lobular carcinoma of the breast is the most common ‘special’ morphological subtype of breast cancer, comprising up to 15% of all cases. Tumours are generally of a good prognostic phenotype, being low histological grade and low mitotic index, hormone receptor positive and HER2, p53 and basal marker negative, and with a generally good response to endocrine therapy. Despite this, clinicians face countless challenges in the diagnosis and long-term management of patients, as they encounter a tumour that can be difficult to detect through screening, elicits a very invasive nature, a propensity for widespread metastatic colonisation and, consequently, in some studies a worse long-term poor outcome compared with invasive carcinoma of no special type. Here we review the morphological and molecular features that underpin the disparate biological and clinical characteristics of this fascinating tumour type.
doi:10.1186/s13058-015-0519-x
PMCID: PMC4310190
5.  Multigene prognostic tests in breast cancer: past, present, future 
There is growing consensus that multigene prognostic tests provide useful complementary information to tumor size and grade in estrogen receptor (ER)-positive breast cancers. The tests primarily rely on quantification of ER and proliferation-related genes and combine these into multivariate prediction models. Since ER-negative cancers tend to have higher proliferation rates, the prognostic value of current multigene tests in these cancers is limited. First-generation prognostic signatures (Oncotype DX, MammaPrint, Genomic Grade Index) are substantially more accurate to predict recurrence within the first 5 years than in later years. This has become a limitation with the availability of effective extended adjuvant endocrine therapies. Newer tests (Prosigna, EndoPredict, Breast Cancer Index) appear to possess better prognostic value for late recurrences while also remaining predictive of early relapse. Some clinical prediction problems are more difficult to solve than others: there are no clinically useful prognostic signatures for ER-negative cancers, and drug-specific treatment response predictors also remain elusive. Emerging areas of research involve the development of immune gene signatures that carry modest but significant prognostic value independent of proliferation and ER status and represent candidate predictive markers for immune-targeted therapies. Overall metrics of tumor heterogeneity and genome integrity (for example, homologue recombination deficiency score) are emerging as potential new predictive markers for platinum agents. The recent expansion of high-throughput technology platforms including low-cost sequencing of circulating and tumor-derived DNA and RNA and rapid reliable quantification of microRNA offers new opportunities to build extended prediction models across multiplatform data.
doi:10.1186/s13058-015-0514-2
PMCID: PMC4307898
6.  Markers for the identification of late breast cancer recurrence 
Postmenopausal women with early breast cancer are at an ongoing risk of relapse, even after successful surgery and treatment of the primary tumor. The treatment of breast cancer has changed in the past few years because of the discovery of prognostic and predictive biomarkers that allow individualized breast cancer treatment. However, it is still not clear how to identify women that are at high risk of a late recurrence. Clinical parameters are good prognostic markers for early recurrence, but only nodal status and, to a lesser extent, tumor size have proven to be strong prognostic markers for late recurrence. Multi-gene signatures have become widely used for the prediction of overall recurrence risk and tailoring administration of adjuvant chemotherapy, but only a few have been shown to be prognostic for late (distant) relapse. There is a need to accurately identify women who may benefit from extended endocrine therapy but also those who may be spared any additional treatment. Recent results from large clinical trials have shown that the research is going in the right direction, and these results might help to optimize extended endocrine therapy for patients with early breast cancer. However, further research is needed to select individual biomarkers or multi-gene signatures that offer identification of late recurrence specifically and thus justify routine use of these tests in the clinical setting.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0516-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-015-0516-0
PMCID: PMC4307995
8.  Molecular mechanism and clinical impact of APOBEC3B-catalyzed mutagenesis in breast cancer 
Cancer genomic DNA sequences enable identification of all mutations and suggest targets for precision medicine. The identities and patterns of the mutations themselves also provide critical information for deducing the originating DNA damaging agents, causal molecular mechanisms, and thus additional therapeutic targets. A classic example is ultraviolet light, which crosslinks adjacent pyrimidines and leads to C-to-T transitions. A new example is the DNA cytosine deaminase APOBEC3B, which was identified recently as a source of DNA damage and mutagenesis in breast, head/neck, cervix, bladder, lung, ovary, and to lesser extents additional cancer types. This enzyme is normally an effector protein in the innate immune response to virus infection but upregulation in these cancer types causes elevated levels of genomic C-to-U deamination events, which manifest as C-to-T transitions and C-to-G transversions within distinct DNA trinucleotide contexts (preferentially 5’-TCA and 5’-TCG). Genomic C-to-U deamination events within the same trinucleotide contexts also lead to cytosine mutation clusters (kataegis), and may precipitate visible chromosomal aberrations such as translocations. Clinical studies indicate that APOBEC3B upregulation correlates with poorer outcomes for estrogen receptor-positive breast cancer patients, including shorter durations of disease-free survival and overall survival after surgery. APOBEC3B may therefore have both diagnostic and prognostic potential. APOBEC3B may also be a candidate for therapeutic targeting because inhibition of this non-essential enzyme is predicted to decrease tumor mutation rates and diminish the likelihood of undesirable mutation-dependent outcomes such as recurrence, metastasis, and the development of therapy resistant tumors.
doi:10.1186/s13058-014-0498-3
PMCID: PMC4303225
9.  Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death 
Introduction
Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance.
Methods
Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression.
Results
We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells.
Conclusion
Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0506-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0506-7
PMCID: PMC4336497  PMID: 25588716
10.  BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers 
Introduction
Intrinsic or acquired chemoresistance is a major problem in oncology. Although highly responsive to chemotherapies such as paclitaxel, most triple negative breast cancer (TNBC) patients develop chemoresistance. Here we investigate the role of BRCA1-IRIS as a novel treatment target for TNBCs and their paclitaxel-resistant recurrences.
Methods
We analyzed the response of BRCA1-IRIS overexpressing normal mammary cells or established TNBC cells silenced from BRCA1-IRIS to paclitaxel in vitro and in vivo. We analyzed BRCA1-IRIS downstream signaling pathways in relation to paclitaxel treatment. We also analyzed a large cohort of breast tumor samples for BRCA1-IRIS, Forkhead box class O3a (FOXO3a) and survivin expression. Finally, we analyzed the effect of BRCA1-IRIS silencing or inactivation on TNBCs formation, maintenance and response to paclitaxel in an orthotopic model.
Results
We show that low concentrations of paclitaxel triggers BRCA1-IRIS expression in vitro and in vivo, and that BRCA1-IRIS activates two autocrine signaling loops (epidermal growth factor (EGF)/EGF receptor 1 (EGFR)-EGF receptor 2 (ErbB2) and neurogulin 1 (NRG1)/ErbB2-EGF receptor 3 (ErbB3), which enhances protein kinase B (AKT) and thus survivin expression/activation through promoting FOXO3a degradation. This signaling pathway is intact in TNBCs endogenously overexpressing BRCA1-IRIS. These events trigger the intrinsic and acquired paclitaxel resistance phenotype known for BRCA1-IRIS-overexpressing TNBCs. Inactivating BRCA1-IRIS signaling using a novel inhibitory mimetic peptide inactivates these autocrine loops, AKT and survivin activity/expression, in part by restoring FOXO3a expression, and sensitizes TNBC cells to low paclitaxel concentrations in vitro and in vivo. Finally, we show BRCA1-IRIS and survivin overexpression is correlated with lack of FOXO3a expression in a large cohort of primary tumor samples, and that BRCA1-IRIS overexpression-induced signature is associated with decreased disease free survival in heavily treated estrogen receptor alpha-negative patients.
Conclusions
In addition to driving TNBC tumor formation, BRCA1-IRIS overexpression drives their intrinsic and acquired paclitaxel resistance, partly by activating autocrine signaling loops EGF/EGFR-ErbB2 and NRG1/ErbB2-ErbB3. These loops activate AKT, causing FOXO3a degradation and survivin overexpression. Taken together, this underscores the need for BRCA1-IRIS-specific therapy and strongly suggests that BRCA1-IRIS and/or signaling loops activated by it could be rational therapeutic targets for advanced TNBCs.
doi:10.1186/s13058-014-0512-9
PMCID: PMC4322455  PMID: 25583261
11.  Circulating tumor cells in newly diagnosed inflammatory breast cancer 
Introduction
Circulating tumor cells (CTCs) are an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. The prognostic value of a CTC count in newly diagnosed IBC has not been established. The aim of this study was to assess the prognostic value of a baseline CTC count in patients with newly diagnosed IBC.
Methods
This retrospective study included 147 patients with newly diagnosed IBC (77 with locally advanced and 70 with metastatic IBC) treated with neoadjuvant therapy or first-line chemotherapy during the period from January 2004 through December 2012 at The University of Texas MD Anderson Cancer Center. CTCs were detected and enumerated by using the CellSearch system before patients were started with chemotherapy.
Results
The proportion of patients with ≥1 CTC was lower among patients with stage III than among patients with metastatic IBC (54.5% versus 84.3%; P = 0.0002); the proportion of patients with ≥5 CTCs was also lower for stage III than for metastatic IBC (19.5% versus 47.1%; P = 0.0004). Patients with fewer than five CTCs had significantly better progression-free survival (PFS) (hazard ratio (HR) = 0.60; P = 0.02) and overall survival (HR = 0.59; P = 0.03) than patients with five or more CTCs. Among patients with stage III IBC, there was a nonsignificant difference in PFS (HR = 0.66; 95% confidence interval (CI), 0.31 to 1.39; P = 0.29) and OS (HR = 0.54; 95% CI, 0.24 to 1.26; P = 0.48) in patients with no CTCs compared with patients with one or more CTCs. In multivariate analysis, CTC was prognostic for PFS and OS independent of clinical stage.
Conclusions
CTCs can be detected in a large proportion of patients with newly diagnosed IBC and are a strong predictor of worse prognosis in patients with newly diagnosed IBC.
doi:10.1186/s13058-014-0507-6
PMCID: PMC4318180  PMID: 25572591
12.  Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis 
Introduction
Real-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.
Methods
CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.
Results
In total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.
Conclusion
This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0508-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0508-5
PMCID: PMC4318479  PMID: 25572662
13.  A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation 
Introduction
The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes.
Methods
We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance.
Results
We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein–coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype–independent overall survival differences.
Conclusion
Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0510-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0510-y
PMCID: PMC4322558  PMID: 25572802
14.  The use of the Gail model, body mass index and SNPs to predict breast cancer among women with abnormal (BI-RADS 4) mammograms 
Introduction
Mammography screening results in a significant number of false-positives. The use of pretest breast cancer risk factors to guide follow-up of abnormal mammograms could improve the positive predictive value of screening. We evaluated the use of the Gail model, body mass index (BMI), and genetic markers to predict cancer diagnosis among women with abnormal mammograms. We also examined the extent to which pretest risk factors could reclassify women without cancer below the biopsy threshold.
Methods
We recruited a prospective cohort of women referred for biopsy with abnormal (BI-RADS 4) mammograms according to the American College of Radiology’s Breast Imaging-Reporting and Data System (BI-RADS). Breast cancer risk factors were assessed prior to biopsy. A validated panel of 12 single-nucleotide polymorphisms (SNPs) associated with breast cancer were measured. Logistic regression was used to assess the association of Gail risk factors, BMI and SNPs with cancer diagnosis (invasive or ductal carcinoma in situ). Model discrimination was assessed using the area under the receiver operating characteristic curve, and calibration was assessed using the Hosmer-Lemeshow goodness-of-fit test. The distribution of predicted probabilities of a cancer diagnosis were compared for women with or without breast cancer.
Results
In the multivariate model, age (odds ratio (OR) = 1.05; 95% confidence interval (CI), 1.03 to 1.08; P < 0.001), SNP panel relative risk (OR = 2.30; 95% CI, 1.06 to 4.99, P = 0.035) and BMI (≥30 kg/m2 versus <25 kg/m2; OR = 2.20; 95% CI, 1.05 to 4.58; P = 0.036) were significantly associated with breast cancer diagnosis. Older women were more likely than younger women to be diagnosed with breast cancer. The SNP panel relative risk remained strongly associated with breast cancer diagnosis after multivariable adjustment. Higher BMI was also strongly associated with increased odds of a breast cancer diagnosis. Obese women (OR = 2.20; 95% CI, 1.05 to 4.58; P = 0.036) had more than twice the odds of cancer diagnosis compared to women with a BMI <25 kg/m2. The SNP panel appeared to have predictive ability among both white and black women.
Conclusions
Breast cancer risk factors, including BMI and genetic markers, are predictive of cancer diagnosis among women with BI-RADS 4 mammograms. Using pretest risk factors to guide follow-up of abnormal mammograms could reduce the burden of false-positive mammograms.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0509-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0509-4
PMCID: PMC4311477  PMID: 25567532
15.  Standardized uptake value of 18F-fluorodeoxyglucose positron emission tomography for prediction of tumor recurrence in breast cancer beyond tumor burden 
Introduction
18F-fluorodeoxyglucose positron emission tomography (FDG-PET) can reveal the metabolic activity of malignant tumors. Recent advances gained from molecular studies suggest that tumor biology can be a good predictor of prognosis in breast cancer. We compared the ability of maximum standardized uptake values (SUVmax) derived by FDG-PET with tumor burden in predicting tumor recurrence for patients with breast cancer.
Methods
496 patients with breast cancer who underwent preoperative FDG-PET between April 2004 and May 2009 were retrospectively identified. SUVmax was obtained by FDG-PET, and the cutoff point was defined using a time-dependent receiver operating characteristic curve for recurrence-free survival (RFS). The primary endpoint was RFS.
Results
In multivariate analysis for RFS, SUVmax carried independent prognostic significance (hazard ratio, 2.39; 95% confidence interval, 1.20 to 4.76; P = 0.012). When the patients were classified into four groups according to the combined factors of tumor size (≤2 cm versus >2 cm) and SUVmax (<4 versus ≥4), RFS differed significantly (P < 0.001). Similarly, SUVmax had prognostic value in combination with nodal status (negative versus positive) or stage (I versus II and III) (P < 0.001 and P = 0.001, respectively). In hormone receptor–positive disease, SUVmax remained a significant prognostic factor for RFS based on multivariate analysis.
Conclusions
Our results highlight the prognostic value of FDG-PET in prediction of tumor relapse for patients with breast cancer. Particularly in patients with hormone receptor–positive disease, the tumor metabolic information provided by FDG-PET is more significantly correlated with prognosis than tumor burden.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0502-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0502-y
PMCID: PMC4308858  PMID: 25551703
16.  Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence 
Breast Cancer Research : BCR  2014;16(6):3417.
Introduction
Elafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that elafin is downregulated during breast and ovarian tumorigenesis.
Methods
We examined elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of elafin in luminal breast cancer cell lines, wild-type or M25G elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells.
Results
Elafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to elafin expression in the normal mammary epithelium. However, the presence of elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to elafin expression in the normal fallopian tube. In ovarian tumors, elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of elafin or elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner.
Conclusions
Elafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0497-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0497-4
PMCID: PMC4326485  PMID: 25551582
17.  Outcomes of children exposed in utero to chemotherapy for breast cancer 
Introduction
The incidence of breast cancer diagnosed during pregnancy is expected to increase as more women delay childbearing in the United States. Treatment of cancer in pregnant women requires prudent judgment to balance the benefit to the cancer patient and the risks to the fetus. Prospective data on the outcomes of children exposed to chemotherapy in utero are limited for the breast cancer population.
Methods
Between 1992 and 2010, 81 pregnant patients with breast cancer were treated in a single-arm, institutional review board–approved study with 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC) in the adjuvant or neoadjuvant setting. Labor and delivery records were reviewed for each patient and neonate. In addition, the parents or guardians were surveyed regarding the health outcomes of the children exposed to chemotherapy in utero.
Results
In total, 78% of the women (or next of kin) answered a follow-up survey. At a median age of 7 years, most of the children exposed to chemotherapy in utero were growing normally without any significant exposure-related toxicity or health problems. Three children were born with congenital abnormalities: one each with Down syndrome, ureteral reflux or clubfoot. The rate of congenital abnormalities in the cohort was similar to the national average of 3%.
Conclusions
During the second and third trimesters, pregnant women with breast cancer can be treated with FAC safely without concerns for serious complications or short-term health concerns for their offspring who are exposed to chemotherapy in utero. Continued long-term follow-up of the children in this cohort is required.
Trial registration
ClinicalTrials.gov Identifier: NCT00510367. Other Study ID numbers: ID01-193, NCI-2012-01578. Registration date: 31 July 2007.
doi:10.1186/s13058-014-0500-0
PMCID: PMC4303207  PMID: 25547133
18.  Toca-1 is suppressed by p53 to limit breast cancer cell invasion and tumor metastasis 
Breast Cancer Research : BCR  2014;16(6):3413.
Introduction
Transducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis. Since metastatic breast tumors frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression.
Methods
Normal mammary epithelial cells (HBL-100, MCF10A) and breast cancer cell lines expressing wild-type (WT) p53 (DU4475, MTLn3) were treated with camptothecin or Nutlin-3 to stabilize p53 to test effects on Toca-1 mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assays were performed to identify p53 binding site in Toca-1 gene. Stable silencing of p53 and Toca-1 were performed in MTLn3 cells to test effects on invadopodia and cell invasion in vitro, and tumor metastasis in vivo.
Results
We observed that breast cancer cell lines with mutant p53 have high levels of Toca-1 compared to those with WT p53. Stabilization of WT p53 led to further reduction in Toca-1 mRNA and protein levels in normal breast epithelial cells and breast cancer cells. ChIP assays revealed p53 binding within intron 2 of toca1, and reduced histone acetylation within its promoter region upon p53 upregulation or activation. Stable silencing of WT p53 in MTLn3 cells led to increased extracellular matrix degradation and cell invasion compared to control cells. Interestingly, the combined silencing of p53 and Toca-1 led to a partial rescue of these effects of p53 silencing in vitro and reduced lung metastases in mice. In human breast tumors, Toca-1 levels were high in subtypes with frequent p53 mutations, and high Toca-1 transcript levels correlated with increased risk of relapse.
Conclusions
Based on these findings, we conclude that loss of p53 tumor suppressor function in breast cancers leads to upregulation of Toca-1, and results in enhanced risk of developing metastatic disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0503-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0503-x
PMCID: PMC4332744  PMID: 25547174
19.  Cancer progression by breast tumors with Pit-1-overexpression is blocked by inhibition of metalloproteinase (MMP)-13 
Introduction
The POU class 1 homeobox 1 transcription factor (POU1F1, also known as Pit-1) is expressed in the mammary gland and its overexpression induces profound phenotypic changes in proteins involved in cell proliferation, apoptosis, and invasion. Patients with breast cancer and elevated expression of Pit-1 show a positive correlation with the occurrence of distant metastasis. In this study we evaluate the relationship between Pit-1 and two collagenases: matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13), which have been related to metastasis in breast cancer.
Methods
We began by transfecting the MCF-7 and MDA-MB-231 human breast adenocarcinoma cell lines with the Pit-1 overexpression vector (pRSV-hPit-1). Afterward, the mRNA, protein, and transcriptional regulation of both MMP-1 and MMP-13 were evaluated by real-time PCR, Western blot, chromatin immunoprecipitation (ChIP), and luciferase reporter assays. We also evaluated Pit-1 overexpression with MMP-1 and MMP-13 knockdown in a severe combined immunodeficiency (SCID) mouse tumor xenograft model. Finally, by immunohistochemistry we correlated Pit-1 with MMP-1 and MMP-13 protein expression in 110 human breast tumors samples.
Results
Our data show that Pit-1 increases mRNA and protein of both MMP-1 and MMP-13 through direct transcriptional regulation. In SCID mice, knockdown of MMP-13 completely blocked lung metastasis in Pit-1-overexpressing MCF-7 cells injected into the mammary fat pad. In breast cancer patients, expression of Pit-1 was found to be positively correlated with the presence of both MMP-1 and MMP-13.
Conclusions
Our data indicates that Pit-1 regulates MMP-1 and MMP-13, and that inhibition of MMP-13 blocked invasiveness to lung in Pit-1-overexpressed breast cancer cells.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0505-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0505-8
PMCID: PMC4305241  PMID: 25527274
20.  Gene variations in oestrogen pathways, CYP19A1, daily 17β-estradiol and mammographic density phenotypes in premenopausal women 
Introduction
High mammographic density is an established breast cancer risk factor, and circulating oestrogen influences oestrogen-regulating gene expression in breast cancer development. However, less is known about the interrelationships of common variants in the CYP19A1 gene, daily levels of oestrogens, mammographic density phenotypes and body mass index (BMI) in premenopausal women.
Methods
Based on plausible biological mechanisms related to the oestrogen pathway, we investigated the association of single nucleotide polymorphisms (SNPs) in CYP19A1, 17β-estradiol and mammographic density in 202 premenopausal women. DNA was genotyped using the Illumina Golden Gate platform. Daily salivary 17β-estradiol concentrations were measured throughout an entire menstrual cycle. Mammographic density phenotypes were assessed using a computer-assisted method (Madena). We determined associations using multivariable linear and logistic regression models.
Results
The minor alleles of rs749292 were positively (P = 0.026), and the minor alleles of rs7172156 were inversely (P = 0.002) associated with daily 17β-estradiol. We observed an 87% lower level of daily 17β-estradiol throughout a menstrual cycle in heavier women (BMI >23.6 kg/m2) of rs7172156 with minor genotype aa compared with major genotype AA. Furthermore, the rs749292 minor alleles were inversely associated with absolute mammographic density (P = 0.032). Lean women with rs749292 minor alleles had 70 to 80% lower risk for high absolute mammographic density (>32.4 cm2); Aa: odds ratio (OR) = 0.23 (95% CI 0.07 to 0.75). Lean women with rs7172156 minor homozygous genotype had OR 5.45 for high absolute mammographic density (aa: OR = 5.45 (95% CI 1.13 to 26.3)).
Conclusion
Our findings suggest that two SNPs in CYP19A1, rs749292 and rs7172156, are associated with both daily oestrogen levels and mammographic density phenotypes. BMI may modify these associations, but larger studies are needed.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0499-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0499-2
PMCID: PMC4303212  PMID: 25522654
21.  Breast-related effects of selective estrogen receptor modulators and tissue-selective estrogen complexes 
A number of available treatments provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis. However, as breast safety is a major concern, new options are needed, particularly agents with an improved mammary safety profile. Results from several large randomized and observational studies have shown an association between hormone therapy, particularly combined estrogen-progestin therapy, and a small increased risk of breast cancer and breast pain or tenderness. In addition, progestin-containing hormone therapy has been shown to increase mammographic breast density, which is an important risk factor for breast cancer. Selective estrogen receptor modulators (SERMs) provide bone protection, are generally well tolerated, and have demonstrated reductions in breast cancer risk, but do not relieve menopausal symptoms (that is, vasomotor symptoms). Tissue-selective estrogen complexes (TSECs) pair a SERM with one or more estrogens and aim to blend the positive effects of the components to provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis without stimulating the breast or endometrium. One TSEC combination pairing conjugated estrogens (CEs) with the SERM bazedoxifene (BZA) has completed clinical development and is now available as an alternative option for menopausal therapy. Preclinical evidence suggests that CE/BZA induces inhibitory effects on breast tissue, and phase 3 clinical studies suggest breast neutrality, with no increases seen in breast tenderness, breast density, or cancer. In non-hysterectomized postmenopausal women, CE/BZA was associated with increased bone mineral density and relief of menopausal symptoms, along with endometrial safety. Taken together, these results support the potential of CE/BZA for the relief of menopausal symptoms and prevention of postmenopausal osteoporosis combined with breast and endometrial safety.
doi:10.1186/bcr3677
PMCID: PMC4076629
22.  Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis 
Introduction
Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer.
Methods
We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed.
Results
Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform.
Conclusions
Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0501-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0501-z
PMCID: PMC4303208  PMID: 25516216
23.  Mammary cancer initiation and progression studied with magnetic resonance imaging 
Introduction
Previous work from this laboratory demonstrated that magnetic resonance imaging (MRI) detects early murine mammary cancers and reliably differentiates between in situ and invasive cancer. Based on this previous work, we used MRI to study initiation and progression of murine mammary cancer, and monitor the transition from the in situ to the invasive phase.
Methods
In total, seven female C3(1) SV40 Tag mice were imaged every two weeks between the ages of 8 to 23 weeks. Lesions were identified on T2-weighted images acquired at 9.4 Tesla based on their morphology and growth rates. Lesions were traced manually on MR images of each slice. Volume of each lesion was calculated by adding measurements from individual slices. Plots of lesion volume versus time were analyzed to obtain the specific growth rate (SGR). The time at which in situ cancers (referred to as ‘mammary intraepithelial neoplasia (MIN)’) and invasive cancers were first detected; and the time at which in situ cancers became invasive were recorded.
Results
A total of 121 cancers (14 to 25 per mouse) were identified in seven mice. On average the MIN lesions and invasive cancers were first detected when mice were 13 and 18 weeks old, respectively. The average SGR was 0.47 ± 0.18 week-1 and there were no differences (P >0.05) between mice. 74 lesions had significantly different tumor growth rates before and after ~17 weeks of age; with average doubling times (DT) of 1.88 and 1.27 weeks, respectively. The average DT was significantly shorter (P <0.0001) after 17 weeks of age. However, the DT for some cancers was longer after 17 weeks of age, and about 10% of the cancers detected did not progress to the invasive stage.
Conclusions
A wide range of growth rates were observed in SV40 mammary cancers. Most cancers transitioned to a more aggressive phenotype at approximately 17 weeks of age, but some cancers became less aggressive. The results suggest that the biology of mammary cancers is extremely heterogeneous. This work is a first step towards use of MRI to improve understanding of factors that control and/or signal the development of aggressive breast cancer.
doi:10.1186/s13058-014-0495-6
PMCID: PMC4303211  PMID: 25510596
24.  Bone marrow stromal antigen 2 expressed in cancer cells promotes mammary tumor growth and metastasis 
Introduction
Several innate immunity genes are overexpressed in human cancers and their roles remain controversial. Bone marrow stromal antigen 2 (BST-2) is one such gene whose role in cancer is not clear. BST-2 is a unique innate immunity gene with both antiviral and pro-tumor functions and therefore can serve as a paradigm for understanding the roles of other innate immunity genes in cancers.
Methods
Meta-analysis of tumors from breast cancer patients obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets were evaluated for levels of BST-2 expression and for tumor aggressiveness. In vivo, we examined the effect of knockdown of BST-2 in two different murine carcinoma cells on tumor growth, metastasis, and survival. In vitro, we assessed the effect of carcinoma cell BST-2 knockdown and/or overexpression on adhesion, anchorage-independent growth, migration, and invasion.
Results
BST-2 in breast tumors and mammary cancer cells is a strong predictor of tumor size, tumor aggressiveness, and host survival. In humans, BST-2 mRNA is elevated in metastatic and invasive breast tumors. In mice, orthotopic implantation of mammary tumor cells lacking BST-2 increased tumor latency, decreased primary tumor growth, reduced metastases to distal organs, and prolonged host survival. Furthermore, we found that the cellular basis for the role of BST-2 in promoting tumorigenesis include BST-2-directed enhancement in cancer cell adhesion, anchorage-independency, migration, and invasion.
Conclusions
BST-2 contributes to the emergence of neoplasia and malignant progression of breast cancer. Thus, BST-2 may (1) serve as a biomarker for aggressive breast cancers, and (2) be a novel target for breast cancer therapeutics.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0493-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0493-8
PMCID: PMC4308845  PMID: 25499888
25.  Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor β/Smad signaling and associates with favorable breast cancer disease outcomes 
Introduction
This study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor β (TGFβ) pathway, which induces potent migration and invasion responses in breast cancer cells.
Methods
We surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFβ/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFβ-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging–based matrix degradation assay, respectively.
Results
Relatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFβ through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFβ/Smad signaling pathway.
Conclusion
BCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFβ/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3’s molecular mechanism of action and highlight BCAR3 as a novel TGFβ/Smad antagonist in breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0476-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0476-9
PMCID: PMC4311507  PMID: 25499443

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