Invasive Ductal Carcinoma Cells Research Articles

P13: Features of proteasome system functioning in the tumor and microenvironment cells of breast cancer

Background The tumor microenvironment plays an important role in the progression of cancer and may be regulated by proteolysis, including the proteasomes. The proteasomes modify the biologically important molecules involved in the pathogenesis and progression of a variety of malignancies, including breast cancer (BC). The aim of this study was to investigate the features of the subunit composition of proteasomes in the tumor and its microenvironment of breast cancer. Material and Methods The material for investigation was samples of tumor tissue of invasive ductal breast cancer. The study was conducted to estimate the distribution of the total pool of proteasome, proteasomes activator PA700, immune proteasome forms containing LMP7 and/or LMP2 subunit in tumor cells and stromal component using immunofluorescence. Furthermore, there was evaluated the distribution of the proteasome in the tumor. The expression of immune proteasomes and proteasomes activators in the cells was studied by immunofluorescence labeling of the cells by antibodies to immune proteasome subunits and cell markers. Fluorescence was analyzed by using a fluorescence microscope DM RXA2 (“Leica”, Germany) and confocal microscope TCS SP (“Leica”, Germany). The specificity of the primary antibodies was confirmed by check samples, at which the reaction was carried out only with the second antibody. No cross reaction between the first and second antibodies was tested by incubation of each primary antibodies with the opposing second antibodies. In addition, there was carried out labeling the cell nuclei by reagent Hoechst 33,342. Results It was found that the tumor cells comprise immune proteasomes, also PA700 and PA28αb activators. Activator PA28αb and immune proteasomes are localized in the cytoplasm of tumor cells, whereas α1, 2, 3, 5, 6, 7 subunits and PA700 activator detected in the cytoplasm and in the nuclei of tumor cells. Availability of α1, 2, 3, 5, 6, 7 subunits in the nuclei of tumor cells shows the expression of constitutive proteasome subunits. Stromal cells are characterized by a high ratio of the α1, 2, 3, 5, 6, 7 subunits to the immune LMP2 subunit as compared to cells of invasive ductal carcinoma. This means that the pool of proteasome in tumor cells of invasive ductal cancer is enriched by the immune proteasomes as compared to stromal cells. Thus, samples of invasive ductal breast cancer contain predominantly tumor cells enriched by immune proteasomes, activators PA700 and PA28αb. The presence of proteasomes in stromal component indicates that the tumor microenvironment also has active processes of proteolysis, with involving proteasome system. Probably the processes occurring in the stromal component contribute to the output of the proteasome into the extracellular space, which is confirmed by other researchers about the existence of circulating proteasome pools and their further participation in dissemination of cancer. This work was supported by the Russian Foundation for Basic Research (Grant numbers 13-04-00169 ).

Abstract 3250: Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis

Abstract Cyclooxygenases (COX-1 and COX-2) catalyze the formation of prostaglandins and play a role in the pathogenesis of breast cancer. Prostaglandin E2 (PGE2), the chief COX product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways; and EP1 is coupled to calcium mobilization and PKC activation. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with any nuclear EP1 expression. Pharmacologic antagonism or reduction of EP1 expression increased metastatic capacity in a murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. Western blot analysis of total and subcellular fractions of EpH4 (normal, immortalized mammary epithelial) and malignant 410, 410.4 and 66.1 cell lines demonstrates that EP1 protein is present in the total and cytoplasmic fractions for all cell lines examined. Nuclear EP1 is detected in EpH4 and non-metastatic 410 cells; however, very little to no nuclear protein is detected in metastatic cell lines 410.4 and 66.1. The absence of nuclear EP1 in metastatic cell lines is consistent with the prognostic data that overall survival for women with tumors negative for nuclear EP1 was significantly worse. Previously, we determined that reduction of EP1 expression leads to increased metastatic capacity; therefore, we investigated the effect of EP1 overexpression on lung colonization in 410.4 and 66.1 cells via tail vein injection in a syngeneic murine model of breast cancer. In 410.4 cells, EP1 overexpression leads to a 57%-97% decrease in metastasis, and in 66.1 cells EP1 overexpression resulted in a 10% - 38% decrease in lung tumor burden compared to vector control mice. The inverse correlation between EP1 expression and metastatic capacity supports our hypothesis that EP1 functions as a metastasis suppressor. We explored potential mechanisms leading to the alteration in the metastatic behavior in response to manipulation of the expression of EP1. We previously published that an EP1 antagonist altered adhesion to laminin in 410.4 cells. Overexpression of EP1 altered the expression of several integrin receptors in 410.4 and 66.1 cell lines including integrins alpha-V, alpha-6 and alpha-3. Also, overexpression of EP1 led to altered adhesion of these cells to several extracellular matrices including fibronectin, collagen, laminin and fibrinogen. Alteration of the adhesive properties of these cells by EP1 could contribute to the metastasis suppressor function of the EP1 receptor. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Prostaglandin E2 receptor EP1 suppresses breast cancer metastasis. [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 3250. doi:10.1158/1538-7445.AM2015-3250

Abstract 4112: Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients

Abstract The majority of breast cancer related deaths are not due to the primary tumor, but rather to the dissemination of metastatic tumor cells from it to distant sites. Our lab has previously identified the tumor microenvironment of metastasis (TMEM) in mouse and human mammary tumors, sites where transendothelial migration, intravasation and dissemination occur. The constituent cells of TMEM are an endothelial cell, a perivascular TIE2-expressing macrophage (TEM) and an invasive Mena-over expressing tumor cell in direct contact. TMEM are present in human invasive breast tumors and the density of TMEM is positively associated with the risk of developing metastases. Using invasive ductal carcinoma cells of the breast obtained from patients by fine needle aspiration (FNA), we demonstrated that intravasation-directed transendothelial migration (iTEM) of these cancer cells requires macrophages and, depending on clinical subtype, involves either paracrine (macrophage CSF1-R; tumor cell EGFR), or both paracrine and autocrine (tumor cell EGFR + CSF1-R) signaling. Compared to the total population of primary breast cancer cells assayed, cells capable of transendothelial migration expressed relatively high MenaINV and low Mena11a levels, independently of clinical subtype. MenaINV and Mena11a are functionally distinct isoforms of Mena, a key regulator of motility and invasion. Depletion of MenaINV using siRNA showed that MenaINV is required for efficient macrophage-dependent iTEM of tumor cells. Furthermore, relative MenaINV expression correlated with the density of TMEM, which were previously shown to correlate with risk of metastasis in patients. From these observations we have identified the signaling pathways involved in macrophage-dependent tumor cell iTEM. Two novel small molecule inhibitors, Rebastinib (Tie2 inhibitor) and Altiratinib (c-MET inhibitor), have been identified that show great promise in preventing key steps in iTEM. Our data reveal targetable signaling events required for transendothelial migration of human breast cancer cells, and indicate that relative MenaINV levels and TMEM frequency are correlated prognostic markers of metastasis and therapeutic targets for most human breast cancers. Citation Format: Jeanine Pignatelli, Joan Jones, Xiaoming Chen, Bryan Smith, Daniel Flynn, John Condeelis, Maja Oktay. Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients. [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 4112. doi:10.1158/1538-7445.AM2015-4112

Abstract P3-04-05: Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth

Abstract Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than similar invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Additionally, we recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells. Among ILC-specific estrogen-regulated genes in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44), Wnt signaling genes were highly differentially expressed. The secreted ligand WNT4 was the most strongly estrogen-induced gene in ILC cells. The frizzled receptor FZD7 is also strongly induced in ILC cells, but only transiently induced in the ER-positive IDC cell line MCF-7. Among IDC cell lines, either WNT4 or FZD7 is over-expressed in ER-positive or ER-negative cells, respectively. Conversely, MM134 and SUM44 over-express both WNT4 and FZD7. Also, we identified an ILC-specific ER binding site at WNT4; located in intron 1, this site contains a predicted estrogen response element. Direct WNT4 regulation and parallel regulation of pathway genes suggests that ER controls a WNT4 signaling pathway in ILC cells. In samples from the Cancer Genome Atlas, WNT4 and FZD7 are each over-expressed in ER-positive ILC versus IDC; co-expression is also enriched only in ILC. These observations suggest that a WNT4 signaling pathway may be specifically active in ILC tumors. To assess whether WNT4 is necessary for estrogen-induced growth, we used siRNA to knock down WNT4. Using either of two siRNAs, WNT4 knockdown completely blocks estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, WNT4 knockdown abrogated estrogen-regulation of a subset of ER-target genes in MM134 cells; induction or repression was inhibited by WNT4 knockdown prior to estrogen treatment. Thus, a subset of estrogen-induced gene expression changes is mediated by WNT4 signaling. Though Wnt signaling typically acts via the canonical, β-catenin-dependent pathway, we observed that β-catenin signaling is dysfunctional in ILC cells. Additionally, WNT4 over-expression or recombinant protein cannot activate canonical Wnt signaling in breast cancer cell lines. This suggests that WNT4 signaling mediates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway. Wnt signaling pathway genes including WNT4 are uniquely regulated in ILC cell lines, and are over-expressed in ILC tumors, suggesting that a WNT4-driven pathway may be active specifically in ILC. WNT4 is necessary for estrogen-mediated growth in ILC cells, and likely signaling via a novel non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on identifying the signaling pathway controlled by WNT4 in order to identify novel therapeutic targets. Citation Format: Matthew J Sikora, Amir Bahreini, Caroline M Alexander, Steffi Oesterreich. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-05.

Differential cell adhesion of breast cancer stem cells on biomaterial substrate with nanotopographical cues.

Cancer stem cells are speculated to have the capability of self-renewal and re-establishment of tumor heterogeneity, possibly involved in the potential relapse of cancer. CD44+CD24−/lowESA+ cells have been reported to possess tumorigenic properties, and these biomarkers are thought to be highly expressed in breast cancer stem cells. Cell behavior can be influenced by biomolecular and topographical cues in the natural microenvironment. We hypothesized that different cell populations in breast cancer tissue exhibit different adhesion characteristics on substrates with nanotopography. Adhesion characterizations were performed using human mammary epithelial cells (HMEC), breast cancer cell line MCF7 and primary invasive ductal carcinoma (IDC) cells obtained from patients’ samples, on micro- and nano-patterned poly-L-lactic acid (PLLA) films. Topography demonstrated a significant effect on cell adhesion, and the effect was cell type dependent. Cells showed elongation morphology on gratings. The CD44+CD24−/lowESA+ subpopulation in MCF7 and IDC cells showed preferential adhesion on 350-nm gratings. Flow cytometry analysis showed that 350-nm gratings captured a significantly higher percentage of CD44+CD24− in MCF7. A slightly higher percentage of CD44+CD24−/lowESA+ was captured on the 350-nm gratings, although no significant difference was observed in the CD44+CD24−ESA+ in IDC cells across patterns. Taken together, the study demonstrated that the cancer stem cell subpopulation could be enriched using different nanopatterns. The enriched population could subsequently aid in the isolation and characterization of cancer stem cells.

Targeting cell-intrinsic and cell-extrinsic mechanisms of intravasation in invasive breast cancer.

The survival of breast cancer patients with metastatic disease has not markedly improved over recent decades, highlighting the need to better understand this process. In this issue of Science Signaling, Pignatelli et al. used freshly obtained invasive ductal carcinoma cells from patients to demonstrate the need for high abundance of the invasive isoform of the Mena protein (Mena(INV)) in cancer cells and colony-stimulating factor 1 (CSF-1)-mediated paracrine signaling in macrophages for efficient transendothelial migration and metastasis formation in all clinical breast cancer subtypes. Furthermore, the triple negative and HER2(+) subtypes, but not the ERPR(+)/HER2(-) subtype, had high CSF-1 receptor (CSF-1R) abundance and also partially used autocrine CSF-1/CSF-1R signaling for invasion. These data establish Mena(INV), CSF-1/CSF-1R, and macrophages as potential therapeutic targets for most human breast cancers.

Abstract 4755: Estrogen receptor mediates novel mechanisms of estrogen-induced growth and tamoxifen resistance in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing ∼10% of newly diagnosed breast tumors. Over 90% of ILC cases are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. Additionally, we have recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells. We focused on three putative mechanisms of endocrine response and resistance in ILC cells. First, we investigated the role of the Wnt signaling protein WNT4 in driving E2-induced growth. E2 induced ER binding at the WNT4 promoter and up-regulated expression specifically in ILC cells (versus IDC cells). As WNT4 has also been previously implicated in normal mammary gland growth and development, we hypothesize that ER-driven WNT4 expression of mediates E2-induced growth of ILC cells. Second, we investigated the role of the transcriptional repressor SNAI1 in ER-mediated gene expression and tamoxifen-resistance. SNAI1 gene expression was induced by both E2 and tamoxifen specifically in ILC cells, which paralleled unique patterns of E2-mediated gene repression in ILC cells. Thus, SNAI1 may mediate E2-driven gene repression and tamoxifen resistance. Third, our prior identification of ILC-specific ER-target genes and ER-mediated tamoxifen resistance suggested that novel co-factors may associate with ER in ILC. We hypothesize that identification of these co-factors would provide novel targets for therapy in ILC. Cell lines MDA MB 134VI and SUM44PE are used as in vitro models of ILC versus MCF-7 and T47D as models of IDC. siRNA-mediated knockdown of gene expression is utilized to assess the function of WNT4 and SNAI1. Cell proliferation and ER-target gene expression are assessed in both ILC and IDC models following knockdown. To identify novel ER co-factors in ILC, we co-immunoprecipitate ER with associated factors, and identify bound proteins by mass spectrometry. Preliminary experiments have demonstrated that knockdown of either WNT4 or SNAI1 can suppress the growth of ILC cells, and that this effect is specific to ILC cells. Additionally, Co-IP/mass spec identifies co-factors that are specifically associated with ER in ILC cells. These observations suggest that unique ER-mediated signaling pathways drive endocrine response and resistance in ILC cells, and that further understanding of these pathways may present novel therapeutic targets in ILC. Citation Format: Matthew J. Sikora, Amir Bahreini, Steffi Oesterreich. Estrogen receptor mediates novel mechanisms of estrogen-induced growth and tamoxifen resistance in invasive lobular carcinoma. [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 4755. doi:10.1158/1538-7445.AM2014-4755

Abstract 1112: Role of osteoprotegerin in inflammatory and invasive breast cancer

Abstract Inflammatory breast cancer (IBC) is a highly aggressive and angioinvasive form of breast cancer associated with a high incidence of early nodal and systemic metastasis. A proactive, complex and dynamic tumor microenvironment of IBC adds to the grim scenario of the disease by accumulating inflammatory and angiogenic growth factors and creating a niche for the growth and metastasis of tumor cells. The secretions from the primary inflammatory ductal carcinoma of the breast (SUM149PT), and highly invasive ductal carcinoma cells (SUM1315MO2) secrete unusually high levels of osteoprotegerin (OPG) as compared to those obtained from the primary human mammary epithelial cells (HMEC). OPG, which plays a pivotal role in bone remodeling by regulating osteoclast formation, is a multifaceted molecule, and has been found to be involved in multiple myeloma and prostate cancers. This prompted us to examine the role of OPG in the aggressive breast cancers, which has not been deciphered till date. For a better understanding of the OPG functions, apart from using adherent cells, we used in vitro ‘sphere culture’ as our working model. We studied the functional significance of OPG in aggressive breast cancer biology by utilizing well standardized techniques such as OPG staining (immunohistochemistry, immunofluorescence), OPG secretion (ELISA), angiogenesis (in vitro tube formation), proliferation (MTT), and cell cycle/aneuploidy analysis (PI staining). We demonstrated that (1) OPG secretion as well as protein level is upregulated in IBC (SUM149PT) and highly Invasive ductal carcinoma cells (SUM1315MO2), and their spheres compared to those of normal human mammary epithelial (HMEC); (2) OPG stained specifically in patient breast cancer tissue samples when compared to the uninvolved tissue from the same patient; (3) OPG rich secretions from IBC and highly invasive breast cancer were highly angiogenic; (4) OPG enriched culture medium induced proliferation of the control HMEC spheres; (5) the presence of OPG triggered the onset of aneuploidy in the control HMEC adherent cells as well as spheres. The functional studies showing the biological significance of OPG (i,e its effect on proliferation, angiogenesis, and aneuploidy) were confirmed using recombinant human OPG and OPG rich/depleted medium obtained from breast cancer cells. Overall our studies highlight the link between OPG and aneuploidy in aggressive breast cancer cells, and opens up avenues for new therapeutic targets for treating IBC. Citation Format: Sudeshna Goswami, NEELAM SHARMA-WALIA. Role of osteoprotegerin in inflammatory and invasive breast cancer. [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 1112. doi:10.1158/1538-7445.AM2014-1112

MiR-638 mediated regulation of BRCA1 affects DNA repair and sensitivity to UV and cisplatin in triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) represents 15 to 20% of all types of breast cancer; however, it accounts for a large number of metastatic cases and deaths, and there is still no effective treatment. The deregulation of microRNAs (miRNAs) in breast cancer has been widely reported. We previously identified that miR-638 was one of the most deregulated miRNAs in breast cancer progression. Bioinformatics analysis revealed that miR-638 directly targets BRCA1. The aim of this study was to investigate the role of miR-638 in breast cancer prognosis and treatment.MethodsFormalin-fixed, paraffin-embedded (FFPE) breast cancer samples were microdissected into normal epithelial and invasive ductal carcinoma (IDC) cells, and total RNA was isolated. Several breast cancer cell lines were used for the functional analysis. miR-638 target genes were identified by TARGETSCAN-VERT 6.2 and miRanda. The expression of miR-638 and its target genes was analyzed by real-time qRT-PCR and Western blotting. Dual-luciferase reporter assay was employed to confirm the specificity of miR-638 target genes. The biological function of miR-638 was analyzed by MTT chemosensitivity, matrigel invasion and host cell reactivation assays.ResultsThe expression of miR-638 was decreased in IDC tissue samples compared to their adjacent normal controls. The decreased miR-638 expression was more prevalent in non-TNBC compared with TNBC cases. miR-638 expression was significantly downregulated in breast cancer cell lines compared to the immortalized MCF-10A epithelial cells. BRCA1 was predicted as one of the direct targets of miR-638, which was subsequently confirmed by dual-luciferase reporter assay. Forced expression of miR-638 resulted in a significantly reduced proliferation rate as well as decreased invasive ability in TNBC cells. Furthermore, miR-638 overexpression increased sensitivity to DNA-damaging agents, ultraviolet (UV) and cisplatin, but not to 5-fluorouracil (5-FU) and epirubicin exposure in TNBC cells. Host cell reactivation assays showed that miR-638 reduced DNA repair capability in post UV/cisplatin-exposed TNBC cells. The reduced proliferation, invasive ability, and DNA repair capabilities are associated with downregulated BRCA1 expression.ConclusionsOur findings suggest that miR-638 plays an important role in TNBC progression via BRCA1 deregulation. Therefore, miR-638 might serve as a potential prognostic biomarker and therapeutic target for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0435-5) contains supplementary material, which is available to authorized users.

Comprehensive proteome quantification reveals NgBR as a new regulator for epithelial-mesenchymal transition of breast tumor cells.

Our previous publication showed that NgBR is highly expressed in human breast invasive ductal carcinoma. However, the roles of NgBR and NgBR-mediated signaling pathway in breast tumor cells are still unclear. Here, we not only demonstrated that the quantitative proteomics analysis is a powerful tool to investigate the global biological function of NgBR, but also revealed that NgBR is involved in the transition of breast epithelial cells to mesenchymal stem cells, which is one of the major mechanisms involved in breast cancer metastasis. These findings provide new insights for understanding the roles of NgBR in regulating breast epithelial cell transform during the pathogenesis of breast cancer.

Abstract P5-09-03: Endocrine response in invasive lobular carcinoma is characterized by unique estrogen-mediated gene expression and de novo tamoxifen resistance

Abstract Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, or ∼30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n = 107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ∼25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. Tam, 4OHT, and Bx acted as agonists for 38/107 genes, whereas ICI acted as an antagonist. All 38 genes were E2-repressed targets (e.g. CCNG2), suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-03.

Abstract B018: Evaluation of the expression of coding and noncoding transcripts in epithelial cells of invasive ductal breast cancer of patients with and without hormone receptor

Abstract Introduction: Breast cancer is the most common tumor among women and is responsible for a high mortality rate worldwide. Gene expression profiling is vastly used in cancer research, and in breast cancer in particular it is used for diagnostic and prognostic characterization. The most frequent subtype, invasive ductal carcinoma (IDC), is considered a heterogeneous group of tumors with different alterations at the molecular level. Perou et al. in 2000 initially described five groups (Luminal A, Luminal B, HER-enriched, normal and basal-like) which have distinct gene expression patterns with different clinical implications. The use of genomics tools has contributed greatly to the understanding of the biology of breast tumors and the identification of new biomarkers. In recent years, long transcripts named long non-coding RNAs (lncRNAs) have been identified that do not encode proteins and are derived from intronic and intergenic sequences. Analysis of lncRNAs has shown differences in transcript expression in specific tissues and some tumors, showing that this area is promising for a better comprehending of cancer. In this context, the present study aims to evaluate the expression of coding and non-coding transcripts, in epithelial tumor cells of invasive ductal carcinoma. Experimental procedure: Determination of gene expression was performed by hybridization to a customized microarray slide with 244,000 probes, of which 44,000 are directed against protein-coding transcripts and 200,000 against lncRNAs. RNA samples derived from laser microdissected epithelial cells (103 IDCs of all subtypes, characterized by immunohistochemical markers) was twice amplified, labeled and hydribized to the microarray slide. The microarrays were analyzed using GeneSpring GX 12.1 software. Probes close to the background, saturated, or with uncertain reading were eliminated, resulting in 23.215 probes for further analysis. Clustering of the samples was performed according to Perou′s intrinsic groups following the PAM50 UNC microarray database guidelines. Coding and non-coding probes were analyzed separately, searching for differentially expressed transcripts in hormone receptor positive and negative subgroups, using t-test (unpaired, with FDR correction) considering fold change ≥|2| and p≥0.05. We compared the lists searching for genes and non-coding anti-sense transcripts within the same locus and performed Pearson correlation considering sense and anti-sense (SAS) pairs with coefficient ≥|0.5| and p ≥ 0.05. Partial Results: Robustness of microarray data was shown by our ability to replicate clustering of molecular profiles using only PAM50 genes. We identified SAS pairs with inverted expression pattern within the same locus, associated with hormone receptor status in breast tumors. We found 2386 coding probes and 358 non-coding probes differentially expressed, of which 56 were from the same locus and 15 had inverted expression (Pearson correlation ≥|0.5|). We are performing qRT-PCR validation of two pairs of candidate genes (LCOR and RFWD2) and their non-coding transcripts on the opposite strand of the same locus, which were inversely expressed with greater correlation coefficient. Perspective: The influence of non-coding antisense transcripts on the sense transcript′s coding is not yet fully understood, but it is suggestive that there is a role in regulating the expression of the gene encoding protein. Citation Format: Mabel Fernandez Pinilla, Alex de calvalho Fiorini, Dirce Maria Carraro. Evaluation of the expression of coding and noncoding transcripts in epithelial cells of invasive ductal breast cancer of patients with and without hormone receptor. [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 B018.

Abstract B060: Invasive lobular carcinoma cells express unique estrogen-mediated genes and are de novo tamoxifen resistant

Abstract Invasive lobular carcinoma (ILC) represents ~10% of newly diagnosed breast tumors, or ~30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n=107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ~25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. 4OHT acted as an agonist for E2-induced genes including SNAI1 and MYC. Further, for the majority of E2-repressed genes assessed including FOXO1, 4OHT also repressed gene expression, suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Format: Matthew J. Sikora, Kristine L. Cooper, Amir Bahreini, Soumya Luthra, Uma R. Chandran, Guoying Wang, David J. Dabbs, Alana L. Welm, Steffi Oesterreich. Invasive lobular carcinoma cells express unique estrogen-mediated genes and are de novo tamoxifen resistant. [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 B060.

Abstract 3862: Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer.

Abstract Prostaglandin E2 (PGE2), the chief cyclooxygenase-2 enzyme (COX-2) product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways with EP1 coupled to calcium mobilization and PKC. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with EP1 expression. Pharmacologic antagonism and reduction of EP1 expression increased metastatic capacity in our murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. We have also identified the presence of a variant EP1 (EP1v) transcript. EP1v has been identified previously in murine mast cell line MC/9 and rat uterus but not in malignant cells. EP1v has a pattern of mRNA expression different than full-length EP1. Compared to the cell line 410, EP1v expression is slightly increased in 410.4 and decreased in 66.1 cell lines. In order to determine the underlying mechanism in which EP1 suppresses metastasis, a metastasis gene array was performed comparing gene expression in EP1-vector, and EP1-silenced 66.1 cell lines and several candidate genes were identified including Fn1, whose protein is altered during epithelial-to-mesenchymal transition. qPCR analysis of Fn1 expression in EP1-silenced and overexpression cell lines revealed an inverse relationship between EP1 and Fn1 expression. A decrease in EP1 expression led to a 4-5 fold increase in Fn1 expression; whereas, increased EP1 expression resulted in a 0.50 fold decrease in Fn1 expression. Like full length EP1, overexpression of EP1v also resulted in a decrease in Fn1 expression. Bioinformatic analysis of the EP1 gene identified several CpG islands. DNA methylation analysis revealed hypermethylation of the CpG island nearest to the promoter in normal, non-metastatic and metastatic murine mammary tumor cell lines. In the quest to identify a clinically relevant strategy to increase expression of this protective receptor, we treated 410.4 and 66.1 cells with demethylating agent 5-azacytidine which resulted in an increase in EP1 expression. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2013-3862

Abstract 5022: Early progenitor cells but not late luminal progenitor cells preserve in invasive breast carcinoma.

Abstract Background CD133 and CD117 represent markers of human breast tissue differentiation. CD133 is an early progenitor marker while CD117 represents late luminal progenitor marker (Lim, et al, Nature Medicine 2009). Morphologic expression of the two markers has not been well established in paraffin embedded tissue of human breast lesions. Design In this study, we evaluated CD133 and C117 expression in different human breast lesions. Group 1 consisted of 15 benign breast cases (5 reduction mammoplasty cases with no significant pathology and 10 fibroadenoma or fibrocystic changes). Group 2 (malignant group 2) included 13 breast cases with invasive ductal carcinoma (IDC). Group 3 (malignant group 3) was composed of 12 BRCA-mutant breast cases but only six cases had IDC. All sections from paraffin embedded tissue were immunohistochemically stained for CD133 (AC133 clone) and CD117 (monoclonal antibody). Expression of the two markers in normal breast glands adjacent to lesions (normal controls), benign lesions (benign controls) and IDC were evaluated and positive rate was calculated. Results Both CD133 and CD117 were expressed in normal breast glands (normal controls) of all three groups and benign breast lesions (Table 1). However, the expression of CD133 was detected in the majority of IDC lesions as compared to low positive percent of CD117 expression in both groups 2 and 3 (Table 1). Recombined tumor cases, based on the molecular luminal status, also showed 56 - 70% CD133 positivity but 0 - 20% CD117 positivity in IDC cells. Table 1. CD133 and CD117 in benign and malignant breast lesions Group 1, total Group 2, total Group 3, total CD133+ in normal 15/15 (100%) 13/13 (100%) 12/12 (100%) CD117+ in normal 15/15 (100%) 13/13 (100%) 11/12 (91%) Group 1, Benign Lesions Group 2, Invasive cancer Group 3 Invasive cancer CD133+ in lesions 10/10 (100%) 8/13 (61%) 4/6 (67%) CD117+ in lesions 9/10 (90%) 1/13 (8%) 1/6 (17%) Conclusions We show that normal breast glands expressed both CD133 and CD117, compatible with the presence of stem cell niche in normal breast ducts and acini. However our data suggest that in IDC, there is presence of early progenitor cells but not late luminal progenitor cells, regardless of the molecular luminal status, supporting the view that cancer stem cells exist in the invasive breast carcinoma. Citation Format: Kevin J. Zhang, Suling Liu, Nava Siegelmann-Danieli, Ping L. Zhang. Early progenitor cells but not late luminal progenitor cells preserve in invasive breast carcinoma. [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 5022. doi:10.1158/1538-7445.AM2013-5022

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA-MB-231 compared with ER+ cell lines MCF-7 and SK-BR-3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event-free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.

Podoplanin expression in cancer-associated fibroblasts correlates with VEGF-C expression in cancer cells of invasive ductal breast carcinoma

Podoplanin (D2-40) was shown to be expressed in cancer-associated fibroblasts (CAFs) of various malignancies. The study aimed at examining its impact on angiogenesis and lymphangiogenesis markers in invasive ductal carcinoma of the breast (IDC). The studies were performed on 104 archival cases of IDC using immunohistochemical technique. Podoplanin expression in CAFs correlated positively with cancer cell VEGF-C expression (r=0.19, p=0.0495) and intratumoral microvessel count (MVC) of CD31 positive vessels (r=0.30, p=0.0018), whereas negative correlations were observed with peritumoral MVC of D2-40 and Lyve-1 positive lymphatic vessels (r=-0.26, p=0.008 and r=-0.27, p=0.0058, respectively). Podoplanin expression in CAFs did not correlate with VEGF-A and VEGF-D expression in cancer cells, nor exerted any prognostic significance. Podoplanin expression in CAFs may have impact on angio- and lymphangiogenesis processes in IDC.

Abstract P6-04-20: Endocrine resistance in invasive lobular carcinoma cells parallels unique estrogen-mediated gene expression

Abstract Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, accounting for ∼30,000 cases annually in the US. However, ILC has been understudied as a breast cancer subtype. ILC-specific signaling pathways and responses to endocrine therapies are not well characterized. Recent retrospective analyses of luminal-type breast cancers suggest that ILC patients treated with endocrine therapy have poorer disease-free survival and overall survival than invasive ductal carcinoma (IDC) patients with similar biomarkers. We hypothesize that unique transcriptional control of estrogen receptor-alpha (ERα) by estrogens and anti-estrogens in ILC cells drive a differential response of ILC tumors to endocrine therapies. The ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of ILC tumors to examine responses to estradiol (E2) and the anti-estrogens tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI). We examined cell growth and expression of canonical ERα-regulated genes in response to E2. Cells were also treated with anti-estrogens +/− E2 to examine their ability to inhibit E2-induced growth. To establish a genome-wide profile of ERα-mediated gene regulation in ILC cells, ILC cells were subjected to gene expression microarray analysis following 0–24hrs treatment with 1nM E2. In parallel to these in vitro studies, in vivo models of ILC are being assessed for endocrine responsiveness, including MDA MB 134VI xenografts and the primary human tumor xenograft HCI-013. We observed that both MDA MB 134VI and SUM44PE are growth-induced by E2 treatment. However, both cell lines also present de novo resistance to Tam, 4OHT, and Bx. While ICI treatment completely blocked E2-induced growth in MDA MB 134VI, Tam, 4OHT, and Bx acted as partial agonists. Treatment with these compounds alone induced ∼25% growth relative to E2, and E2-induced growth could only be inhibited ∼75%. Similarly, SUM44PE cells are strongly growth-inhibited by ICI treatment, whereas growth is not reduced by Tam, 4OHT, or Bx treatment. Consistent with these observations, novel patterns of gene expression are induced by E2 treatment in ILC cells. E2-treatment induced canonical targets (e.g. GREB1, IGFBP4) in both ILC cell lines. However, in MDA MB 134VI cells only ∼35% of genes regulated by E2 at 24hrs overlap with those regulated in the IDC cell line MCF-7. Further, a subset of the overlapping genes is differentially regulated between cell types, including ESR1 (1.25-fold and 0.67-fold versus control in MDA MB 134VI and MCF-7, respectively), HOXC6, PMP22, and L1CAM. Examination of these observations in in vivo models is currently ongoing. These data are consistent with the hypothesis that E2 and anti-estrogens differentially regulate ERα-mediated gene expression in ILC cells versus IDC cells. The de novo resistance to tamoxifen observed in ILC cells may correlate with the worse outcomes in ILC patients observed in retrospective analyses. We hypothesize that elucidation of the mechanisms responsible for differential ERα regulation may reveal novel therapeutic targets for treating ILC patients and overcoming endocrine resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-20.

Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression

The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S (<3 mm from IDC), DCIS-S (<3 mm from DCIS) and breast cancer associated-normal stroma (BC-NS; >10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development.

Identification of copy number alterations associated with the progression of DCIS to invasive ductal carcinoma

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive ductal carcinoma (IDC). Annotation of the genetic differences between the two lesions may assist in the identification of genes that promote the invasive phenotype. Synchronous DCIS and IDC cells were microdissected from FFPE tissue and analysed by molecular inversion probe (MIP) copy number arrays. Matched IDC and DCIS showed highly similar copy number profiles (average of 83% of the genome shared) indicating a common clonal origin although there is evidence that the DCIS continues to evolve in parallel with the co-existing IDC. Four chromosomal regions of loss (3q, 6q, 8p and 11q) and four regions of gain (5q, 16p, 19q and 20) were recurrently affected in IDC but not in DCIS. CCND1 and MYC showed increased amplitude of gain in IDC. One region of loss (17p11.2) was specific to DCIS. IDC-specific regions include genes with previous links to breast cancer progression and potential therapeutic targets such as AXL, SPHK1 and PLAUR.