Normal Breast Biology Research Articles

Abstract 2136: Genetic ancestry dependent variability in stromal cells: An unexplored player in breast cancer disparity

Abstract Genetic ancestry dependent variability in cancer incidence, mutation patterns, response to chemotherapy, and outcome has been documented. While an association between social determinants of health and breast cancer disparity has already been established, there is emerging evidence for genetic ancestry dependent variability in normal breast biology impacting breast cancer biology and potentially outcome. Tumor biology studies in the context of genetic ancestry and disparity have often focused on intrinsic properties of tumor cells or tumor infiltrating immune cells. However, other stromal cell types have received very little attention. Through immunohistochemistry of breast tissues from healthy women of African and European ancestry and primary cell culturing system, we had previously demonstrated enrichment of a stromal cell population called PZP cells (PROCR+/ZEB1+/PDGFRA+) with mesenchymal stem-like and fibroadipogenic properties in the normal breast tissues of women of African ancestry. In this study, we used single nuclei ATAC-seq and/or RNA-seq to further characterize stromal fibroblasts in the breast tissues of women of Ashkenazi Jewish-European, European, Indigenous American, Hispanic-European, African, and Asian ancestry. Among eight fibroblast cell clusters generated from women of African and European ancestry, only three cell clusters overlapped between two groups. While Complement Factor D (CFD, also called adipsin) expression was observed in unique fibroblast clusters of African ancestry, the expression of Insulin-like Growth Factor 1 (IGF1) was enriched in clusters unique to European ancestry. Interestingly, previous studies have demonstrated African ancestry-specific genomic variants for CFD linked to cardiometabolic disorders. Additional genes that showed genetic ancestry dependent variability in expression within fibroblasts include ABCA10, ABCA9, ABCA8, NEGR1 (enriched in African ancestry), MMP16, MAGI1, KIAA1217, PTPRK and SEMA5A (enriched in European ancestry). NEGR1 (Neuronal Growth Regulator 1) is a trans-neural growth promoting factor, whereas PTPRK (Protein Tyrosine Phosphatase Receptor Type K) is a negative regulator of EGFR signaling. These results suggest genetic ancestry dependent variability in stromal-epithelial cell communications under normal and cancerous conditions. These genetic ancestry dependent differences could impact intracellular signaling networks in epithelial cells with consequential effects on cancer incidence, mutation patterns, drug sensitivity, and outcome. Citation Format: Poornima Bhat-Nakshatri, Hongyu Gao, Cihat Erdogan, Yunlong Liu, Harikrishna Nakshatri. Genetic ancestry dependent variability in stromal cells: An unexplored player in breast cancer disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2136.

Abstract P2-06-01: Intrinsic transcriptomic differences of luminal progenitors and mature luminal cells of the normal breast

Abstract Epithelial cells in the normal breast are organized hierarchically into stem, luminal progenitor (LP) and mature luminal cells (LC) with distinct gene expression patterns. However, whether these gene expression differences are cell-intrinsic, driven through LP-LC cell interactions or due to stromal-luminal cell interactions in the breasts of healthy women is incompletely understood. Furthermore, prior studies on these cell populations did not take genetic ancestry into consideration despite widely reported genetic ancestry-dependent differences in the normal breast biology and breast cancer susceptibility/outcome. We isolated LP and LC breast epithelial cells from core breast biopsies of healthy women representing European, African American and Latino ancestry (five each). After a brief propagation (five passages) under epithelial reprogramming assay conditions, transcriptomes and DNA methylomes were examined using RNA- and Methylcapture-sequencing. Differences in gene expression (FDR<0.05, fold change >2) in LC cells compared to LP cells were observed, with 1581 and 609 genes up- and down-regulated, respectively. These data were enriched for signaling networks associated with cell cycle control of chromosomal replication, mitotic roles of polo-like kinase (down-regulated genes), granulocyte adhesion and diapedesis and LXR/RXR signaling (up-regulated genes). Despite marked gene expression differences between LC and LP cells, only 14 upregulated genes and four down-regulated genes showed DNA hypo- and hyper-methylation, respectively, in LC cells compared to LP cells, suggesting a minimal role for DNA methylation in differential gene expression between the two cell types. Furthermore, of the 2190 differentially expressed genes, 7% (158 genes) were regulated by estrogen, suggesting that differences in hormone responsiveness contribute to differential gene expression in LP and LC cells. To determine relationships between genes differentially methylated and expressed in LC and LP cells and breast cancer, we used publicly available databases, including TCGA. Interestingly, all genes hypermethylated and expressed at lower levels in LC cells were similarly hypermethylated and expressed at lower levels in breast cancers compared to normal breast, suggesting that these genes are minimally altered in breast cancer. Furthermore, in LC compared to LP cells, of thegenes hypomethylated and expressed at higher levels, only four genes (AIM1L, PRSS27, PTK6 and UNC13D) showed a similar pattern in breast tumors compared to normal breast. Of these, PTK6 is significantly overexpressed in luminal and HER2+ breast cancers compared to basal-like breast cancers, suggesting that PTK6+ cells are the preferred cell-of origin of luminal breast cancers. Taken together, this is the first study to demonstrate that transcriptomic data of normal breast cells from healthy women can be used to further define cell-of-origin of tumors and identify cancer-specific gene expression changes in breast cancer. Citation Format: Fang Fang, Poornima Bhat-Nakshatri, Manjushree Anjanappa, Dough Rusch, Aaron Buechlein, Dave Miller, Kenneth P Nephew, Harikrishna Nakshatri. Intrinsic transcriptomic differences of luminal progenitors and mature luminal cells of the normal breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-06-01.

Abstract P1-03-11: Harnessing the distinctive properties of tumor-adjacent tissues to develop ethnicity-dependent biomarkers of breast cancer

Abstract Recent data demonstrating a correlation between lymph node positivity at the time of detection, and the probability of disease recurrence even decades post detection only solidifies the principle that the detection of breast cancer prior to lymph node metastasis can appreciably better clinical outcomes. Although radiologic methods have greatly improved early detection and remain the mainstay for detection, molecular assays to complement existing strategies will reduce number of false positives as well as enhance detection in cases that preclude conclusive diagnosis with radiologic techniques. Normal breast biology is routinely studied using tissues from reduction mammoplasty or normal tissues adjacent to tumor (NATs). However, studies have shown histologic abnormalities in reduction mammoplasty samples and DNA methylation and gene expression abnormalities in NATs due to “field” effects of the tumor. To interrogate the differences between normal breast and NATs as potential early detection markers, we created a tissue microarray (TMA) comprising breast tissues of 100 age-matched healthy women from the Komen Tissue Bank (KTB) and tumor-NAT pairs from 100 women (a total of 300 samples). Approximately 50% of women in each set were of African American (AA) ancestry and the remaining was of European decent. The TMAs was curated as such, because of our recent findings on ethnicity-dependent differences in breast stem-luminal progenitor-mature cell hierarchy. TMA was analyzed for ZEB1, an oncogenic transcription factor that is central to cell fate and stemness, and estrogen receptor alpha (ERα) and FOXA1, which are expressed predominantly in hormone-responsive mature luminal cells. ZEB1 expressing cells were localized to surrounding ductal structures of the normal breast, whereas ERα+ and FOXA1+ cells were located within the ductal compartment. KTB-normal of AA women contained significantly higher levels of ZEB1+ cells compared to KTB-normal of Caucasian women (CA). We observed only marginal increases in ZEB1+ cells in NATs or tumors of AA women. By contrast, in CA women, both NATs and tumors compared to KTB-normal contained higher levels of ZEB1+ cells. The unique localization pattern external to the ductal structures, as well as intrinsically higher expression in AA women suggest that ZEB1+ cells serve not only as stem cells from which cancers may originate but could also contribute to the microenvironment conducive for ductal tumor progression leading to aggressive and early onset of breast cancer as observed in AA women. Conversely, KTB-normal of AA showed modestly higher FOXA1 expression compared to CA women, and further, FOXA1 levels were declined in NATs of AA but not CA women. ERα levels did not change in any of our analyses, pointing to the specificity of ethnicity-dependent changes in this TMA. We also noted ethnicity-dependent variations in the levels of CD8+ T cells, PD-1+ immune cells and PD-L1+ cells but not CD68+ macrophages in NATs, suggesting distinctive immune environment in NATs. This comprehensive approach will not only serve as a platform to develop tumor-adjacent “normal” tissues as molecular markers for early detection but also provides a molecular basis for aggressive breast tumor in AA women. Citation Format: Nakshatri H, Kumar B, Burney H, Cox ML, Jacobson M, Sandusky G, D'Souza-Schorey C, Storniolo AM. Harnessing the distinctive properties of tumor-adjacent tissues to develop ethnicity-dependent biomarkers of breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-03-11.

Abstract 4993: Breast epithelial cell lines from normal breast with luminal and intrinsic subtypes -enriched gene expression document inter-individual differences in differentiation cascade

Abstract Breast cancers are classified into five intrinsic subtypes based on gene expression profile. It is suggested that these intrinsic subtypes originate from specific developmental stage of breast epithelial cell hierarchy, stem-progenitor-mature cell. However, normal breast epithelial cell lines representing these intrinsic subtypes are yet to be created. Using normal breast tissues of ancestry-mapped Caucasian, African American, and Hispanic women and a primary cell culturing system that allows growth of normal epithelial cells of different developmental stages including estrogen receptor-positive mature luminal cells, we created 15 human telomerase-immortalized breast epithelial cell lines. These cells formed acini on a matrigel and ductal structures on 3-dimensional collagen or hydrogel, indicating that these cell lines have retained characteristics of normal breast epithelial cells. RNA sequencing and PAM50 intrinsic subtype clustering algorithms were used to identify the intrinsic subtypes of the immortalized cell lines together with two well characterized “normal” breast epithelial cell lines MCF10A and HMEC as well as luminal breast cancer cell line MCF-7. Unlike MCF10A and HMEC, which are enriched for basal-like gene expression pattern, our cell lines classified into luminal A, basal, and normal-like subtypes. This was also reflected in the immunofluorescence staining with basal marker KRT14 and luminal marker KRT19. Few of these cell lines were dual positive for KRT14 and KRT19, but in varying proportions. Cell lines representing claudin-low subtypes were also created, which are phenotypically (CD201+/EpCAM-) different from the above cell lines. Cell lines showed inter-individual differences in stemness/differentiation capabilities and variable basal activity of signaling molecules such as NF-kB, AP-1 and pERK, which is consistent with, possibly reflecting, recent discoveries of genetic variations in gene regulatory regions among general population that contribute to widespread differences in gene expression/signaling under “normal” state. As majority of breast cancers are believed to originate from luminal progenitor cells, which are well represented our cell lines, these cell lines are ideal to delineate the impact of inter-individual and ethnic differences in normal breast biology on breast cancer initiation and progression as well as to determine whether cell-type-origin instead of genomic aberration drives intrinsic subtype-enriched gene expression patterns in breast tumors. Citation Format: Brijesh Kumar, Mayuri Prasad, Manjushree Anjanappa, Poornima Nakshatri, Natascia Marino, Anna Maria Storniolo, Xi Rao, Sheng Liu, Jun Wan, Yunlong Liu, Harikrishna Nakshatri. Breast epithelial cell lines from normal breast with luminal and intrinsic subtypes -enriched gene expression document inter-individual differences in differentiation cascade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4993.

Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids.

The development of in vitro culture systems quantitatively and qualitatively recapitulating normal breast biology is key to the understanding of mammary gland biology. Current three-dimensional mammary culture systems have not demonstrated concurrent proliferation and functional differentiation ex vivo in any system for longer than 2 weeks. Here, we identify conditions including Neuregulin1 and R-spondin 1, allowing maintenance and expansion of mammary organoids for 2.5 months in culture. The organoids comprise distinct basal and luminal compartments complete with functional steroid receptors and stem/progenitor cells able to reconstitute a complete mammary gland in vivo. Alternative conditions are also described that promote enrichment of basal cells organized into multiple layers surrounding a keratinous core, reminiscent of structures observed in MMTV-Wnt1 tumours. These conditions comprise a unique tool that should further understanding of normal mammary gland development, the molecular mechanism of hormone action and signalling events whose deregulation leads to breast tumourigenesis.

Abstract P1-06-05: The impact of ethnicity on normal breast biology

Abstract The incidence of/mortality from the most aggressive triple negative breast cancer (TNBC) is higher in African American- (AA) than Caucasian (CA)-women. In contrast, breast cancer in Hispanic-women is generally less aggressive. There is an ongoing debate as to whether AA-women have an increased incidence of TNBCs that have poor outcome or there are unique biological factors in AA-women that promote aggressive biology. Through unique approaches, we provide evidence for distinct biology in the normal breasts of CA-women, AA-women, and Hispanic-women. Using resources from the Komen Normal Tissue Bank and a primary cell culturing system that enabled propagation of normal epithelial cells of different lineages including mature-luminal, luminal-progenitor, and stem cells from different ethnic groups, we have identified a subpopulation of CD44high/CD24- cells that are unique to AA-women (p=0.0001). This AA-women-specific subpopulation expressed higher levels of Collagen 3A1, Collagen 5A2, CTNNB1 (β-Catenin of Wnt), FOXC2, and ZEB1 compared to common CD44+/CD24+ subpopulation in every breast. Gene expression pattern in the AA-specific CD44high/CD24- population showed marked similarity to gene expression pattern in the recently described PROCR+ multi-potent mammary stem cells. Indeed, the breast epithelial cells of AA-women were enriched for PROCR-positive stem cells compared to CA-women or Hispanic-women (p=0.015). In contrast to cells from CA- and AA-women, cells isolated from the healthy breast of Hispanic-women displayed mostly differentiated features as they were enriched for CD49f-/EpCAM+ and CD271-/EpCAM+ mature cells. Cells from CA-women, AA-women and Hispanic-women are currently being immortalized to determine the cell types that are preferentially immortalized in each of these ethnic groups. These results suggest ethnicity-dependent differences in Wnt, extracellular matrix, and epithelial to mesenchymal transition (EMT) signaling in normal breast epithelial cells and differences in the proportion of cells that are susceptible to immortalization/transformation. This work is supported by Susan G. Komen for the Cure to HN (SAC110025). Citation Format: Nakshatri H, Anjanappa M, Bhat-Nakshatri P. The impact of ethnicity on normal breast biology. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-05.

Abstract 2049: Characterization of a novel BRCA1-EGR1 interaction

Abstract The tumor suppressor protein BRCA1 orchestrates DNA repair and cell cycle arrest following DNA damage, with transcriptional regulation integral to these processes. We have identified a novel, DNA-damage responsive, interaction between BRCA1 and the zinc-finger transcription factor EGR1. EGR1 is rapidly and transiently expressed in response to a wide variety of stress stimuli and growth factors, and like BRCA1, decreased expression of EGR1 correlates with tumor formation in mammary cell lines and tissues. The stress response gene, NDRG1, was identified as a transcriptional target of BRCA1 by microarray analysis and subsequent RQ-PCR validation. NDRG1 is a known suppressor of breast cancer metastasis and we have observed a significant reduction of NDRG1 expression in breast cancer cell lines (relative to normal cell lines). Promoter analyses identified EGR1 binding motifs within the NDRG1 proximal promoter region, and EGR1 was confirmed as the transcription factor of interest by promoter assays. Downregulation of either BRCA1 or EGR1 (using a siRNA approach) resulted in a reduction in both basal and DNA-damage induced NDRG1 promoter activity, suggesting a co-regulatory mechanism. Indeed, recruitment of BRCA1 and EGR1 to the NDRG1 promoter was confirmed by ChIP assays. GST-fused BRCA1 deletion constructs showed that EGR1 specifically interacts with a C-terminal region of BRCA1 (which contains both BRCT domains). However, following DNA damage, we observed EGR1 interaction along the entire length of the BRCA1 protein, suggesting that both BRCA1 and EGR1 reside within a larger transcriptional complex upon DNA damage. In agreement with this, siRNA knockdown of either BRCA1 or EGR1 in breast cell lines (i) induced endogenous DNA damage signalling, (ii) increased intracellular ROS generation and (iii) upregulated numerous stress-response genes. To investigate transcriptional regulation downstream of this interaction we performed analyses of publically available ChIP-Seq data for both BRCA1 and EGR1, and identified a list of genomic locations (corresponding to gene promoters) at which both proteins localise. We will use these findings, in combination with our own ChIP-Seq experiments performed in the non-tumorigenic mammary cell line, 184A1, to investigate the roles of this novel complex in the maintenance of genomic stability and within normal breast biology. Citation Format: Naomi A. Dickson, Nyree T. Crawford, Paul B. Mullan. Characterization of a novel BRCA1-EGR1 interaction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2049. doi:10.1158/1538-7445.AM2015-2049

Acquired convergence of hormone signaling in breast cancer: ER and PR transition from functionally distinct in normal breast to predictors of metastatic disease.

Cumulative exposure to estrogen (E) and progesterone (P) over the menstrual cycle significantly influences the risk of developing breast cancer. Despite the dogma that PR in the breast merely serves as a marker of an active estrogen receptor (ER), and as an inhibitor of the proliferative actions of E, it is now clear that in the breast P increases proliferation independently of E action. We show here that the progesterone receptor (PR) and ER are expressed in different epithelial populations, and target non-overlapping pathways in the normal human breast. In breast cancer, PR becomes highly correlated with ER, and this convergence is associated with signaling pathways predictive of disease metastasis. These data challenge the established paradigm that ER and PR function co-operatively in normal breast, and have significant implications not only for our understanding of normal breast biology, but also for diagnosis, prognosis and/or treatment options in breast cancer patients.

Gene expression profiles of breast biopsies from healthy women identify a group with claudin-low features

BackgroundIncreased understanding of the variability in normal breast biology will enable us to identify mechanisms of breast cancer initiation and the origin of different subtypes, and to better predict breast cancer risk.MethodsGene expression patterns in breast biopsies from 79 healthy women referred to breast diagnostic centers in Norway were explored by unsupervised hierarchical clustering and supervised analyses, such as gene set enrichment analysis and gene ontology analysis and comparison with previously published genelists and independent datasets.ResultsUnsupervised hierarchical clustering identified two separate clusters of normal breast tissue based on gene-expression profiling, regardless of clustering algorithm and gene filtering used. Comparison of the expression profile of the two clusters with several published gene lists describing breast cells revealed that the samples in cluster 1 share characteristics with stromal cells and stem cells, and to a certain degree with mesenchymal cells and myoepithelial cells. The samples in cluster 1 also share many features with the newly identified claudin-low breast cancer intrinsic subtype, which also shows characteristics of stromal and stem cells. More women belonging to cluster 1 have a family history of breast cancer and there is a slight overrepresentation of nulliparous women in cluster 1. Similar findings were seen in a separate dataset consisting of histologically normal tissue from both breasts harboring breast cancer and from mammoplasty reductions.ConclusionThis is the first study to explore the variability of gene expression patterns in whole biopsies from normal breasts and identified distinct subtypes of normal breast tissue. Further studies are needed to determine the specific cell contribution to the variation in the biology of normal breasts, how the clusters identified relate to breast cancer risk and their possible link to the origin of the different molecular subtypes of breast cancer.

Stat5: From Breast Development to Cancer Prognosis, Prediction, and Progression

Signal transducer and activator of transcription (STAT) proteins transmit signals from extracellular polypeptides, through transmembrane receptors, directly to target gene promoters in the nucleus resulting in genetic reprogramming of the target cell. There are seven STAT genes in mammals (1, 2, 3, 4, 5A, 5B, and 6) derived phylogenetically from an ancestral gene (Stat93E) that first appeared in Drosophila. STAT proteins are latent transcription factors activated by tyrosine phosphorylation following recruitment to ligand-activated receptor complexes. Tyrosine phosphorylation allows tail-to-tail dimerizationleadingtotheiraccumulationwithinthenucleus.KnockoutofSTAT genes in mice has established their key roles in the distinct responses elicitedbyspecificpolypeptidehormoneligands,withfidelityinresponses being transmitted, in part, through specificity of binding of each STAT Src-homology 2 (SH2) domain to phosphotyrosine-containing motifs located within ligand-activated receptor complexes. Stat5 is encoded by two highly homologous genes—5A and 5B—which, along with Stat3, are clustered on chromosome 17q in humans (chromosome 11 in mice). Gene targeting studies in mice demonstrated interplay between Stat5 and Stat3 in normal breast biology: Stat5A is required for terminal mammary gland differentiation and lactogenesis, while Stat3 is required for breast involution after weaning. Stat5B-deficient mice did not demonstrate a breast phenotype. Constitutively activated STAT proteins, in particular Stat3 and Stat5, have been demonstrated to directly contribute to oncogenesis, in part, by stimulating proliferation and preventing apoptosis in various types of tumor cells. The role for Stat3 and Stat5 in the pathogenesis of human breast cancers is becoming increasingly recognized. There is evidence of increased STAT protein binding in the nuclei of breast cancer compared with the normal tissue of benign lesions. Several studies have shown the role of Stat3 as a prognostic factor in breast cancer. In addition, previous studies have shown an association between Stat5 nuclear localization/ phosphorylation and improved survival. These previous studies that suggest the prognostic significance of Stat5 were conducted in cohorts that previously received some form of adjuvant therapy in a percentage of patients. There have been other published studies that allude to the predictive significance of Stat5. In a study by Yamashita et al, patients with Stat5–positive primary breast tumors had a significantly improved response to endocrine therapy. In the current article by Peck et al accompanying this editorial, a comprehensive study of the utility of Stat5 is comprehensively delineated, including its role as a prognostic marker in the absence of any systemic therapy, in the progression from normal to in situ through invasive ductal cancer and nodal metastases, and finally, as a predictive marker of endocrine resistance. Firstly, the role of Stat5 as a prognostic marker was clearly defined in this article. In two independent cohorts of patients who did not receive adjuvant systemic therapy, phosphorylated and nuclear localized Stat5 (NUC-pYStat5) was found to be an independent marker of outcome in patients with lymph node–negative breast cancer with a total of close to 500 tumor specimens examined. Patients with reduced NUC-pYStat5 expression had approximately a 2.5-fold increase risk of disease recurrence and approximately a 2.4-fold risk of dying of breast cancer. This is the first publication, to our knowledge, that comprehensively addressed the role of NUC-pYStat5 as an independent marker of prognosis in patients with untreated node-negative breast cancer. Secondly, Peck et al define the role of Stat5 in cancer progression. Levels of NUC-pYStat5 quantitated by automated quantitative analysis (AQUA), decreased as the disease progressed from normal to in situ, to invasive, and then to nodal metastases. Thirdly, and perhaps most importantly, Peck et al found that the absence of detectable NUC-pYStat5 in tumors of patients treated with antiestrogen therapy, was associated with poor breast cancer–specific survival. Multivariate analysis showed that patients with the lowest levels of NUC-pYStat5 had nearly a seven-fold increased risk of dying from breast cancer despite antiestrogen therapy. Collectively, these consistent observations strongly suggest that low levels of NUC-pYStat5 are not only a poor prognostic factor that is associated with cancer progression, but also predicts for failure to respond to antiestrogen treatment. This final observation is critically important. Close to one third of patients with estrogen receptor– positive breast cancers fail to respond to antiestrogen therapy as a consequence of either inherent or acquired resistance. Many markers including human epidermal growth factor receptor 2 (HER2) suggest cross-talk between several pathways. However, most of these pathways, including HER2, are poor predictive markers of response to antiestrogen therapy. In contrast, NUC-pYStat5 may add to selecting patients who are unlikely to respond to antiestrogen therapy even though their tumors express estrogen receptor. In conclusion, this work supports the role of Stat5 signaling in cancer progression, and provides strong evidence for this marker as a JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L S VOLUME 29 NUMBER 18 JUNE 2

Loss of annexin A1 expression in human breast cancer detected by multiple high-throughput analyses

To test the efficacy of combined high-throughput analyses (HTA) in target gene identification, screening criteria were set using >fivefold difference by microarray and statistically significant changes ( p < 0.01) in SAGE and EST. Microarray analysis of two normal and seven breast cancer samples found 129 genes with >fivefold changes. Further SAGE and EST analyses of these genes identified four qualified genes, ERBB2, GATA3, AGR2, and ANXA1. Their expression pattern was validated by RT-PCR in both breast cell lines and tissue samples. Loss of ANXA1 in breast cancer was further confirmed at mRNA level by Human Breast Cancer Tissue Profiling Array and at protein level by immunohistochemical staining. This study demonstrated that combined HTA effectively narrowed the number of genes for further study, while retaining the sensitivity in identifying biologically important genes such as ERBB2 and ANXA1. A distinctive loss of ANXA1 in breast cancer suggests its involvement in maintaining normal breast biology.

Mitogenic Properties of Insulin-Like Growth Factors I and II, Insulin-Like Growth Factor Binding Protein-3 and Epidermal Growth Factor on Human Breast Epithelial Cells in Primary Culture

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) are growth factors implicated in mammary gland development and are believed to be involved in breast cancer. However, the interactions between components of the IGF system and breast epithelial cells, which give rise to breast cancer, are not well understood. We have investigated the mitogenic properties of IGF-I, IGF-II, IGF binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast epithelial cells (HBEC) in primary culture. We show that, under serum-free conditions, HBEC are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of HBEC growth in primary culture and also associated with breast cancer, appear to stimulate HBEC in a synergistic manner. IGFBP-3 inhibits the stimulation by IGF-I, IGF-II and IGF-I plus EGE In addition, it appears that IGFBP-3 has an inhibitory effect on HBEC growth that is IGF-independent. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on HBEC growth in primary culture. Characterizing the role of the IGF system in normal breast biology is significant because the system has been implicated in breast cancer and a number of the anti-estrogens used in treatment are believed to function through the IGF system.

Dipeptidyl peptidase IV expression identifies a functional sub-population of breast fibroblasts.

The immunocytochemical distribution of the cell-surface enzyme dipeptidyl peptidase IV (DPP IV) has been studied in the human breast at the light and ultrastructural level. The presence of the enzyme was demonstrated on the cell membranes of interlobular fibroblasts, whilst intralobular fibroblasts were DPP-IV-negative. A fluorograph, after immunoprecipitation of 35S-methionine-labelled proteins of fibroblasts from primary breast cultures with an anti-serum to DPP IV, demonstrated a band at 135 kDa consistent with the presence of the enzyme. The clear delineation of 2 functionally distinct subpopulations of breast fibroblasts was maintained in benign fibro-adenomas and cystosarcoma phyllodes, both tumour types having growth characteristics of intralobular stroma. This observation has important implications for both normal breast biology and for breast carcinogenesis.