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It is unknown why some patients with hormone receptor-positive (HR(+)) breast cancer present with more aggressive and invasive disease. Metastatic dissemination occurs early in disease and is facilitated by crosstalk between the tumor and tissue environment, suggesting that undefined host-intrinsic factors enhance early dissemination and the probability of developing metastatic disease. Here, we have identified commensal dysbiosis as a host-intrinsic factor associated with metastatic dissemination. Using a mouse model of HR(+) mammary cancer, we demonstrate that a pre-established disruption of commensal homeostasis results in enhanced circulating tumor cells and subsequent dissemination to the tumor-draining lymph nodes and lungs. Commensal dysbiosis promoted early inflammation within the mammary gland that was sustained during HR(+) mammary tumor progression. Furthermore, dysbiosis enhanced fibrosis and collagen deposition both systemically and locally within the tumor microenvironment and induced significant myeloid infiltration into the mammary gland and breast tumor. These effects were recapitulated both by directly targeting gut microbes using non-absorbable antibiotics and by fecal microbiota transplantation of dysbiotic cecal contents, demonstrating the direct impact of gut dysbiosis on mammary tumor dissemination. This study identifies dysbiosis as a pre-existing, host-intrinsic regulator of tissue inflammation, myeloid recruitment, fibrosis, and dissemination of tumor cells in HR(+) breast cancer.

Breast cancer is currently the second leading cause of cancer-related deaths and the most commonly diagnosed cancer among women in the Unites States. Localized breast cancer has a five-year survival rate of 99%, however the survival rate for patients with distant metastasis is 27%, despite improved screening and detection methods. Currently, there are few targeted therapies that specifically inhibit metastasis. Research performed by our lab and others has shown that inflammatory cytokines play a crucial role in promoting cancer growth and metastatic potential in cancer cells. While efforts have been made to develop FDA approved therapeutics for inflammatory cytokines, very few have successfully made it through clinical trials and several cytokines still lack effective clinical inhibitors. Our research has developed a stepwise approach towards designing and testing novel small molecule inhibitors (SMIs) against inflammatory cytokines. In silico computational screening of compound libraries towards specific inflammatory cytokines revealed potential molecules that interact with receptor binding regions and inhibit cytokine biological function. Top candidate inhibitors were analyzed for direct interaction with cytokines using differential scanning fluorimetry, fluorescent quenching, and Chemical Shift Pertubation NMR (CSP-NMR). Additionally, to quantitatively measure cytokine-receptor interference, a novel ligand-receptor binding assay was developed highlighting small molecule inhibitors that effectively prevent biological cytokine function. In vitro efficacy of the SMIs were analyzed by measuring inhibition of various signaling cascades activated by inflammatory cytokines using enzyme-linked immunosorbent assay (ELISA) and immunoblot assays. Inhibition of tumor cell proliferation, epithelial to mesenchymal transition (EMT), and cytokine-induced secretion of pro-tumorigenic molecules was also assessed. From these in silico and in vitro methods, SMI's are structurally modified with effective functional groups to increase efficacy of binding interaction and reduce toxicity for living systems. Finally, small molecule inhibitors that have been sufficiently tested for efficacy are then examined in vivo to evaluate toxicity and determine the maximum tolerated dose. SMI's that prove to be non-toxic are evaluated further in vivo by mouse mammary breast cancer models to analyze reduction of distant metastases. Our interdisciplinary team has designed, synthesized, and tested small molecule inhibitors (SMIs) to target inflammatory cytokines shown to promote metastasis in cancer with the long-term goal of understanding lethal breast cancer metastasis, as well as providing a novel FDA approved therapeutic for breast cancer patients. Citation Format: Cody L. Wolf, Clyde Pruett, Darren Lighter, Cooper McGrath, Amberlyn Nemeth, Joey Tuccinardi, Grace Coughlin, Samuel Randall, Joey Day, Matthew King, Lisa Warner, Don Warner, Cheryl L. Jorcyk. Designing novel small molecule inhibitors as possible targeted therapeutics to prevent breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1298.

Background: Pathologists reviewing breast tissue slides must identify the presence of many salient features within each slide, including invasive and in situ breast cancer as well as various forms of atypia. In breast pathology particularly, the large volume of slides poses significant challenges for workload management and pathologist productivity (Johnson et al. 2019). The shift to a digital workflow in pathology, augmented by machine learning algorithms, has the potential to increase the efficiency, and productivity of pathologists by identifying cancer and pre-cancerous lesions in digitized slides. While there has been extensive work using machine learning algorithms to detect breast cancer metastasis in lymph nodes (Liu et al. 2018; Steiner et al. 2018), almost no research has been done on using such systems to detect breast cancer in biopsies and excisions. Methods: We created and assessed Paige Breast Alpha, a machine learning system for the detection of breast cancer in hematoxylin and eosin (H&E) stained whole slide images (WSIs) of glass slides. The system is a binary classifier, intended to draw a pathologist’s attention to concerning features. Concerning features (the positive category) consisted of invasive breast cancer, in situ breast cancer, and various forms of atypia (atypical ductal hyperplasia, atypical lobular hyperplasia, etc. ). The deep learning system is based on the method proposed in Campanella et al. (2019). It learns directly from diagnosis using multiple instance learning, without the need for pixel-wise annotations. Paige Breast Alpha was trained on 17354 images from 3378 patients, and was assessed on 7921 images from 2443 patients. All slides were scanned on a Leica Aperio AT2. Results: For detecting invasive or in situ cancer at the part level, the system achieved an overall sensitivity of 97.3% and a specificity of 98.0% in biopsies and 96.1% sensitivity and 91.5% specificity in excisions. Each part had between 1—10 slides. Conclusions: We hypothesized that a machine learning system trained to detect predefined types of breast cancer and pre-cancerous lesions could be applied to a range of breast biopsy and resection WSIs to detect for the presence of these lesions. Herein we showed that the presence of these predefined features can be detected with high accuracy. Future studies are being initiated to assess the potential benefits of such a system when used by a pathologist. Additional systems are under development that would have the capability of subtyping the lesion present, in addition to acting as an overall binary classifier. Citation Format: Matthew G Hanna, Patricia Raciti, Ran Godrich, Adam Casson, Julian Viret, Donghun Lee, Matthew Lee, Alican Bozkurt, Jillian Sue, Belma Dogdas, Brandon Rothrock, Leo Grady, Christopher Kanan, Thomas Fuchs. Clinical-grade detection of breast cancer in biopsies and excisions using machine learning [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD6-03.

Metastasis represents the main route of cancer progression and is responsible for approximately 90% of all cancer-related deaths ([1][1]). Metastatic disease is generally considered incurable, emphasizing the need for novel biologically targeted pharmaceutical therapies. In this issue, Li and

Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Cancer associated fibroblasts (CAFs) are the majority of stroma in BC and critical players in BC malignancy. For example, CAFs are the main source of SDF-1, a prominent chemokine in the tumor microenvironment (TME) that also imparts stem cell-like characteristics to BC cells. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. Stem-like CTCs and clusters have a greater propensity to establish metastasis. We recently identified circulating CAFs (cCAFs) in blood from patients with BC and in spontaneous, syngeneic, and xenograft mouse models of BC. cCAFs not only circulate individually, but are also found in clusters with CTCs. In this study, we examine the role of CAFs in promoting egress of stem-like CTCs (cCSCs), determine the capacity of stem-like CTCs to cluster with CAFs, and evaluate the involvement of CTC/cCAF clustering in augmenting BC metastasis. Methods: Our model employs NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines: the non-metastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis, and we used BALB/c mice injected with syngeneic 4T1 or 67nR cells to evaluate cCAFs, CTCs, and cluster egress in preclinical models. Mice were sacrificed at specific time points, and cardiac blood was collected. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were stained for IF and cCAFs, CTCs, cCSCs, and clusters were enumerated. Tumors from CAF co-injected mice were evaluated for their stem cell-like phenotype and re-implanted in mice to evaluate tumorigenicity and metastasis. Results: In spontaneous, syngeneic, and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs co-clusters appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. Co-inoculation of CAFs with BC cells resulted in a significant increase in tumor progression, metastasis, and in a substantially higher number of both individual cells and clusters in circulation. Dissociated tumor cells from CAF co-injected tumors had a higher proportion of CD44+stem cell-like cells (CSCs), enhanced ALDH-1 expression, and enhanced mammosphere formation. CD44+ CSCs, individually and in clusters, are found early on in the circulation of mice injected with dissociated tumor cells from CAF co-injected tumors. Upon re-implantation of CAF co-injected dissociated tumor cells without CAFs, dissociated tumor cells showed enhanced tumorigenicity and malignancy. Conclusion: CAFs are highly motile and cCAFs precede CTCs into circulation and can do so independently of tumor cells. CAFs sustain egress of tumor cells by augmenting malignancy and stemness of BC cells. cCAF clusters with the highly metastatic stem cell-like subset of CTCs bolster metastatic colonization. Targeting primary CAF function and/or cCAF/cCSC co-clusters may provide novel avenues to abrogate BC metastasis. Citation Format: Sharma U, Miller P, Medina Saenz K, Picon-Ruiz M, Morata-Tarifa C, Spartz A, Troness B, Park DN, Seagroves TN, Slingerland JM, Lippman ME, El-Ashry D. Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis [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 PD9-10.

Duffy Antigen Receptor for chemokines (DARC) is known to regulate the biological activities of chemokines. It has been implicated in the progression of various diseases including different types of cancer, affecting survival, proliferation and metastasis. Genetic variants in the DARC genes are strongly associated with African ancestry and are linked to malaria resistance. There are at least two isoforms of DARC, which have not been appreciably investigated in these contexts, that may have differing chemokine activity functions. To fill this knowledge gap, we have been characterizing the impact of DARC gene variants on DARC isoform expression in specific tissue types in order to define relationships between the polymorphic DARC and its effect as an immune regulator in breast cancers. We hypothesize that DARC variants, which are known to impact expression of the gene on erythrocytes, also are associated with functional activity related to DARC’s control of metastatic potential in breast cancer. Specifically, we have determined that one DARC polymorphism which is well defined in ancestral groups (i.e. Duffy null - rs2814778; -541T>C) is associated with differential expression of DARC isoforms in epithelial cell types, including lymphoblast and breast tissues. We investigated the erythroid silencing Duffy Null allele of DARC using DNA sequencing and quantitated DARC isoform expression by real time PCR in breast cancer cell lines, ancestral cell lines and breast cancer tumor tissues. We will show that DARC isoforms have significantly variant expression among ancestral groups that is associated with the Duffy Null allele. We will also show that certain types of breast tumors have variable DARC expression that is also associated with this genotype. Additionally, we visualized the subcellular localization of DARC and known interacting chemokines breast cancer cells with IHC and IF confocal microscopy. Specifically, our results indicate breast tumors differentially express DARC in which may be affecting the chemokine pool and immune response within the tumor and its microenvironment. In our Hapmap ancestral populations, all DARC genotypes expressed the gene in lymphoblast, with significantly different levels of isoforms among African, African American and European individuals. Breast cancer cells also show different levels of specific DARC isoform mRNA levels and distinct levels and patterns of binding chemokines, related to tumor subtype origins (i.e. ER status). Based on these findings we conclude that DARC isoforms have differential expression levels in breast cancer cells within tumor and non-tumor regions that may drive both pro and anti tumorigenic events with varying chemokine binding or localizations during tumorigenesis of specific breast cancer subtypes. Different levels of isoforms in epithelial cells may contribute to its essential function as atypical chemokine receptor. We hypothesize that immune responses in breast cancer patients with Duffy Null genotypes will differ and may express more severe metastasis due to altered epithelial regulation of alternate DARC isoforms. Citation Format: Rupali Hire, Andrea Walens, Brianna Bennett, Kauthar Mumin, Hailey Campbell, Michael Lou, Michele Monteil, Elizabeth Howerth, Melissa Davis. Investigating the role of Duffy antigen receptor for chemokines (DARC) isoforms in breast cancer metastasis [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 P1-07-22.

[This article][1] ([1][2]) has been retracted at the request of the editors. Following an institutional review by Peter MacCallum Cancer Center (Victoria, Australia), the primary affiliation for several of the authors, it was not possible to confidently identify and assign the primary datasets to

Although there are several therapies available for the treatment of breast cancer, many patients have tumor recurrences that ultimately metastasize. There are several oncogenic estrogen receptor alpha (ERα) coregulators that promote breast cancer metastasis and targeting these coregulators could be a promising cancer therapeutic. One coactivator of ERα that is known to be upregulated in breast cancer and promote metastasis is proline-, glutamic acid-, and leucine-rich protein 1 (PELP1). PELP1 affects the migratory and invasive potential of breast cancer cells both in vitro and in vivo and modulates the expression of several genes involved in the epithelial to mesenchymal transition (EMT) including Snail, Twist and Zeb1. To elucidate the mechanism by which PELP1 promotes metastasis, we studied the proteins that interact with PELP1 and promote cancer metastasis. We identified a novel PELP1-interacting protein G9a (Bat8/KMT1C/EHMT2), a histone lysine methyltransferase known to overexpressed in aggressive breast cancer and promote invasion and metastasis. G9a contributes to the epigenetic silencing of tumor suppressor genes, interacts with Snail to promote EMT and its knockdown inhibits cell migration and invasion as well as lung metastasis. The objective of this study is to characterize the interaction of PELP1 and G9a in the promotion of breast cancer metastasis. The complex formation of PELP1 and G9a was confirmed by an in vivo immunoprecipitation and an in vitro binding assay. The region of PELP1 that binds to G9a was mapped further by PELP-GST deletions. To determine whether formation of the complex is necessary for PELP1's oncogenic functions including the promotion of metastasis, we developed a peptide to disrupt the complex formation. The sequence of the peptide has homology to both PELP1 and G9a with a TAT-sequence to enable cellular uptake. Cellular uptake was confirmed by fluorescent microscopy while biotin-avidin peptide pull-down assays confirmed PELP1-peptide binding. A peptide competition assay confirmed that the peptide could interrupt PELP1-G9a interaction. Treatment of two ER-positive, PELP1-overexpressing breast cancer cells (MCF7-PELP and ZR75-PELP) with the peptide significantly inhibited the PELP-1 mediated proliferation. Peptide treatment also substantially inhibited PELP1-mediated cell migration in Boyden chamber assays as well as decreased anchorage independence as seen by soft agar assay. Interestingly, disrupting PELP1-G9a interaction with the peptide also sensitized tamoxifen and letrozole resistant cells to the respective hormonal therapy. Mechanistic studies revealed that PELP1-G9a interactions play a critical role in epigenetic changes in therapy resistant cells. In conclusion, our studies identified a novel complex involved in breast cancer metastasis. The interaction of PELP1 and G9a is required for PELP1-mediated oncogenic properties and disruption of the complex by peptide treatment results in decreased metastatic potential and changes in epigenetic modifications. The novel peptide could be used for therapeutic targeting of breast cancer metastasis and therapy resistance. This work was supported by the NIH grant CA095681 and CPRIT pre-doctoral fellowship RP101491. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-10.

We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice are regulated a distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. We also compared the activity of these compounds against tasquinimod, a small molecule targeting S100A9 only. However, tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. In line with these data, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, this compound eliminated the cytotoxicity caused by cyclophosphamide and animals were free of cancer. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process and our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Thomas Albers, Maria Ouzounova, Charlie Weeks, Riley Rodier, Ahmet K. Korkaya, Khaled A. Hassan. Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis [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 1100.

Metastases are responsible for the vast majority of deaths due to breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor prognosis. We are employing patient-derived xenograft (PDX) models of TNBC to identify drivers of metastasis. Tumor samples are obtained from the breast tumors of patients with TNBC and engrafted immediately into the humanized mammary fat pads of immune-compromised mice. Lentiviral transduction was employed to express bioluminescent and fluorescent markers in two independent PDX models of TNBC. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain, and lymph nodes. Dynamic and reversible shifts in the epithelial-to-mesenchymal transition (EMT) were observed as tumors metastasized to lung and were re-passaged to recipient mouse mammary fat pads (MFPs). Lung metastases were isolated using bioluminescence imaging and lung metastasis gene expression signatures were generated. Metastasis signatures from two independent PDX models were compared to identify genes that were commonly deregulated in lung metastases relative to corresponding mammary tumors. Comprehensive gain-of-function screens were then conducted in vivo to identify functional drivers of TNBC metastasis. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver in this screen. CEACAM5 mRNA and protein levels were elevated in lung metastases relative to corresponding MFP tumors in mice. In addition, we demonstrated that CEACAM5 expression was upregulated in the lung metastases of breast cancer patients, and its expression inversely correlated with patient survival. Our data indicate that the metastatic function of CEACAM5 is to promote growth of breast tumors in the lung by inducing MET (mesenchymal-to-epithelial transition). Citation Format: Emily Powell, Jiansu Shao, Hector Picon, Christopher Bristow, Zhongqi Ge, Caitlin Grzeskowiak, Michael Peoples, Frederick Robinson, Sabrina Jeter-Jones, Christopher Schlosberg, Fei Yang, Yun Wu, Ignacio Wistuba, Stacy Moulder, Fraser Symmans, Kenneth Scott, John Edwards, Han Liang, Timothy Heffernan, Helen Piwnica-Worms. A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis [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 5172.

Introduction: Retrospective studies have suggested that anesthesia maintained with intravenous propofol during oncological surgery is associated with better outcome than anesthesia with volatile anesthetics. Here, we determine the effects of anesthetics on breast cancer lung metastasis in an animal model. Methods: A primary tumor is generated in 6-8 week old Balb/c female mice by orthotopic implantation of 4T1 mouse breast cancer cells expressing firefly luciferase, into the mammary gland. When the primary tumor volume reaches 500 mm3, it is resected under three-hour anesthesia either with 2.7% inhaled sevoflurane or a 150 mg/kg intraperitoneal injection of propofol. A control group does not receive surgery or anesthesia. After surgery, lung metastasis is tracked and quantified with noninvasive bioluminescent imaging (IVIS). Two weeks later, the mice are euthanized, and the lungs are harvested for macroscopic and histological analyses, which include counting metastatic nodules, and CD11b/CD31 overlay staining. As a step towards understanding the mechanism, we examine the effect of propofol and sevoflurane on angiogenesis. An in vivo matrigel plug assay is used to evaluate the effect of anesthetics on blood vessel formation. Balb/c mice are administered 2.7% sevoflurane or 150 mg/kg propofol 1 hour each week for three weeks. A control group receives no anesthetic. Blood vessels are visualized by perfusion of TRITC labeled dextran. No surgery is done in this experiment. Results: In the 4T1 syngeneic model, surgical resection of the primary tumor under propofol anesthesia significantly reduced spontaneous lung metastasis in comparison with sevoflurane. Using IVIS imaging, similar levels of bioluminescence in the lung were observed in control and propofol treated mice two weeks after surgery. In contrast, mice anesthetized with sevoflurane had 1.5-fold higher levels (p Conclusion: We used a mouse model to recapitulate the human clinical situation in which the choice of anesthestic given during cancer surgery may be a risk factor for cancer metastasis. Our results show that the volatile anesthetic sevoflurane preferentially promotes metastasis of breast cancer cells in the lung. This may be related to the ability of sevoflurane to promote angiogenesis. The 4T1 mouse model successfully recapitulated the differential effects of anesthetic agents on breast cancer development seen in clinic, and further deepen our understanding of the mechanism behind that phenomenon. Citation Format: Ru Li, Yujie Huang, Hengrui Liu, James P. Dilger, Jun Lin. Comparing volatile and intravenous anesthetics in a mouse model of breast cancer metastasis [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 2162.

The purpose of this study is to investigate the mechanism by which the cholesterol metabolite, 27-hydroxycholesterol (27HC) promotes breast cancer metastasis. Breast cancer is the most commonly diagnosed cancer and among the leading causes of death in women in the United States. Elevated cholesterol is identified as a major risk factor of breast cancer onset and recurrence, while cholesterol-lowering drugs are associated with a good prognosis. Previous work has found that many of the effects of cholesterol on breast cancer progression are due to the actions of its primary metabolite, 27HC. 27HC is a ligand of both the estrogen receptor (ER) and liver X receptor (LXR). Intriguingly, the pro-metastatic effects of 27HC require the presence of myeloid-immune cells. This cell type has been implicated in suppressing acquired immunity, allowing cancer cells to escape immune-surveillance. Therefore, we hypothesize that 27HC suppresses the immune system to promote metastasis. To elucidate the immunomodulatory capacity of 27HC, we co-cultured vehicle- or 27HC-treated bone marrow-derived macrophages (BMDMs) with activated, CFSE-prelabeled T cells. As a readout of immune activation, we measured the resulting proliferation of T cells by flow cytometry. In support of our hypothesis, we found that 27HC-treated BMDMs inhibited T cell expansion in a dose dependent manner. In order to determine the mechanisms by which 27HC-treated BMDMs suppress T cell proliferation, we first evaluated the relative contributions of two receptors known to bind 27HC: the ERs and LXRs. To this end, BMDMs were treated with various combinations of pharmacologic agonists or antagonists of the ERs or LXRs. These treated BMDMs were subsequently co-cultured with activated T cells to study their impact on T cell proliferation. Interestingly, a combination treatment of suboptimal doses of an ER antagonist and LXR agonist moderately reduced T cell expansion, similar to 27HC. Therefore, our results suggest that 27HC affects the microenvironment of breast cancer by altering the activity of antigen-presenting cells through the modulation of the ER and LXR. This in turn reduces the expansion of T cell function, ultimately resulting in tumor progression. Our ongoing work is aimed at further elucidating the crosstalk between the ERs and LXRs and identifying the downstream targets of 27HC involved in mediating this immune-suppressive phenotype. Our work is of considerable importance given the prevalence of metastatic breast cancer and that the cholesterol axis is highly amenable to therapeutic targeting. This work was supported by the National Cancer Institute of the National Institutes of Health (R00CA172357, E.R.N.). Citation Format: Liqian Ma, Amy E. Baek, Erik R. Nelson. Mechanisms by which 27-hydroxycholesterol promotes breast cancer metastasis [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 2133.

Background: Tumor progression is an evolutionary process associated with the accumulation of somatic genomic alterations. Even though metastasis is a fundamental process and a challenging issue for the progress in targeted treatment, the molecular drivers of cancer metastasis remains poorly understood. Despite the advance in high throughput sequencing and the characterization of hundreds of breast tumor genomes, it is still unclear whether cancer genomes evolve by neutral processes or whether genetic alterations that favorable tumor progression are selected under pressures of cancer therapy or both. To gain insights into the molecular processes driving breast cancer metastasis and to identify targetable genetic events associated with early and late stages of the disease, we studied the mutational profiles of a series of paired primary breast tumors and subsequent metastases collected as part of the ESOPE clinical Trial. Methods: Matched primary breast tumors, first metastases and germline DNAs were obtained from 31 patients, divided in 10 therapy-naïve synchronous primary cancers and metastases and 21 metachronous primary tumors and metastases that undergone first line adjuvant treatments. On Illumina platform, WES was performed and three variant callers were used in parallel to predicted variations. After annotation, variants with low 1000Genome frequency and absent in germline samples, were validated by IGV visualization and then considered as somatic variants. To identify significantly mutated genes, we used the Mutation Significance (MutSigCV) tool and the Cancer Gene Census list of relevant genes. Analysis of mutational process signatures was also performed though DeconstructSigs R packages. Results: Synchronous and metachronous metastases globally exhibited similar mutation rates, however a significant difference was observed in the number of accumulated private mutations in both types of metastases. With few exceptions, our data indicate that the genomic profiles of metastases mainly diverge late in breast cancer progression, with relatively few private mutations in the primary tumors. In contrast, an increase of somatic variants was found in the metachronous metastases as compared to their primary tumors of origin. Most of driver mutations were shared by primary tumors and metastases but additional drivers were highlighted in the metastatic lesions, such as variants of PIK3CA, IGF1R, CDH1, NF1 as well as ESR1. Moreover, recurrent mutated genes were also identified in metastases that have not been previously described (FDR Citation Format: Keltouma Driouch, Zakia Tariq, Sylvain Baulande, Virginie Raynal, Virginie Bernard, Francois-Clement Bidard, Vanessa Benhamo, Ivan Bieche, Brigitte Sigal, Rosette Lidereau, Paul Cottu. Genomic evolution of synchronous and metachronous breast cancer metastasis revealed from a prospective clinical trial [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 3440.

The major challenges for breast cancer includes therapy resistance and metastasis to distant organs. Chronic inflammation has been intimately linked with these processes. We have observed increased level of CXCR2 ligands, inflammatory immunosuppressive microenvironment, and increased metastasis in chemotherapy resistant cells. CXCR2 and its ligands have been shown to be the primary mechanism for neutrophils recruitment to the sites of primary tumors and metastases. However, their precise roles in therapy resistance and metastasis remains unclear. The aim of this project is to investigate the role of neutrophils in chemotherapy resistance and metastasis in breast cancer. In this report, we investigated the mechanisms and putative role of neutrophils in chemotherapy resistance and metastasis. We evaluated the expression level of the factors critical for neutrophil recruitments, such as interleukin (IL)-17 and its receptors (IL-17R), granulocyte colony stimulating factor (GCSF), CXCR2 and its ligands in primary tumors and metastases established from parents and chemotherapy resistant cells. The frequency of neutrophils and T-helper 17 (Th17) cells were analyzed using immunohistochemistry. Chemotherapy-resistant cell lines express higher levels of GCSF, IL-17, and IL-17R compared to Cl66 parent cells. We observed higher expression of IL-17R, CXCR2, and CXCR2 ligands in metastatic lesions compared to primary tumors. Furthermore, there were more recruitments of neutrophils and Th17 cells in resistant tumors than parent tumors. In addition, treatment of tumor cells with IL-17 significantly increased cellular proliferation together with CXCL1 and CXCL5 expression in a concentration-dependent manner. Moreover, we observed higher expression of TGFβ 2 and Th17 cell priming factors, IL-6 and IL-23, in neutrophils co-cultured with therapy resistant cells compared to the control suggesting a feed forward loop. In addition, we observed that IL17 treatment of tumor cells did not increase their tolerance to chemotherapy drug, indicating its role in establishing immune suppressive microenvironments. Together our data demonstrate an IL17-CXCR2 ligands axis induces protumorigenic inflammation, facilitating therapy resistance and metastasis through recruited neutrophils. Citation Format: Lingyun Wu, Bhawna Sharma, Rakesh K. Singh. Role of IL-17-CXCR2 axis in neutrophil recruitment facilitating breast cancer metastasis and therapy resistance [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 132.

The establishment of breast cancer metastases at sites distant from the primary lesion site is predicated on the ability of breast cancer cells to mobilize from the primary tumor, survive in circulation, and colonize in a suitable and permissive metastatic niche. While quantitation of individual CTCs has prognostic value in breast cancer patients with metastatic disease, and elevated counts of CTCs may associate with poor outcome in early breast cancer, recent evidence from mouse models indicates that CTCs in clusters have a greater capacity to produce metastases than individual CTCs and new studies in breast cancer suggest that the presence of CTC clusters in patients can be prognostic. We have found in breast cancer patients, clusters of circulating cells that include CTCs, immune cells, and stromal cells found in the tumor microenvironment, specifically cancer associated fibroblasts (CAFs). Like CTC and circulating CAF (cCAF) counts, the incidence and number of CTC/cCAF clusters increases with disease stage. While the clinical significance of circulating complexes of CTCs and stromal cells remains to be fully elucidated, the recent identification of cCAFs in the peripheral blood of patients with metastatic breast cancer, traveling individually or in clusters with CTCs emphasizes the potential importance of stromal cells as circulating mediators of cancer metastasis. Preclinical murine models of breast cancer metastasis provide opportunities to observe the composition, dynamics, and behavior of these circulating CTC/stromal cell complexes, and to interrogate mechanisms driving their mobilization and clustering. Identifying the cellular components in these circulating complexes, and understanding the mechanisms by which they facilitate breast cancer metastasis, may provide novel therapeutic avenues for the treatment or prevention of metastatic breast cancer. Citation Format: El-Ashry D. Circulating complexes of stromal and tumor cells in breast cancer metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr ES8-3.

Introduction: Obesity with associated inflammation is now recognized as a risk factor for breast cancer and increased incidence of distant metastases. However, the link between obesity and breast cancer progression remains poorly understood. There is growing evidence that sphingosine-1-phosphate (S1P), a pleiotropic bioactive sphingolipid metabolite enriched both in blood and lymphatic fluid is involved in inflammation, obesity, and breast cancer progression. Our hypothesis is that obesity increases levels of S1P in both tumor and its microenvironment, which play a role in obesity-induced inflammation and breast cancer metastasis. The aim of this study is to test this hypothesis in in vitro and in vivo as well as patient settings. Methods: Levels of sphingolipids including S1P in serum from breast cancer patients were quantified. Orthotopically-implanted E0771 syngeneic breast cancer and MMTV-PyMT transgenic breast cancer mouse models were used. Mice were fed with normal or high-fat diet (HFD). FTY720 was administered orally (1 mg/kg/day). To examine pre-metastatic niche formation, a mouse model utilizing tail vein injection of E0771 cells was used. In this model, mice were treated with conditioned media from E0771 breast cancer cells overexpressing SphK1 (K1-CM) or that from E0771 cells cultured with the vector control (CT-CM), prior to tail vein injections of naive E0771 cells. S1P levels were determined by electrospray ionization-tandem mass spectrometry. Results: We found that obesity significantly increased S1P levels in serum from breast cancer patients. In animal breast cancer models, HFD upregulated expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, and its receptor S1PR1 in syngeneic and spontaneous breast tumors. HFD also significantly increased S1P in breast tumors and in the tumor interstitial fluid, which is a component of the tumor microenvironment and bathes cancer cells in the tumor. Targeting the SphK1/S1P/S1PR1 axis with FTY720/fingolimod attenuated obesity-induced key pro-inflammatory cytokines, macrophage infiltration, and tumor progression. In addition, S1P produced by tumor SphK1 primed lung pre-metastatic niches, increased macrophage recruitment into the lung, and induced IL-6 and signaling pathways important for lung metastatic colonization. FTY720 suppressed HFD-induced lung IL-6 and macrophage infiltration as well as S1P-mediated signaling pathways and dramatically reduced formation of metastatic foci. In tumor bearing mice, FTY720 also suppressed obesity-related inflammation, S1P signaling, pulmonary metastasis, and prolonged survival. Conclusion: Our results highlight a critical role for circulating S1P produced by tumor and the SphK1/S1P/S1PR1 axis in obesity-related inflammation, metastatic niche formation and breast cancer metastasis and suggest that targeting the SphK1/S1P/S1PR1 axis would be a useful therapeutic for obesity promoted metastatic breast cancer. Citation Format: Nagahashi M, Yamada A, Aoyagi T, Huang W-C, Terracina KP, Hait N, Allegood JC, Tsuchida J, Nakajima M, Katsuta E, Milstien S, Wakai T, Spiegel S, Takabe K. Targeting the SphK1/S1P/S1PR1 axis that connects obesity, chronic inflammation, and breast cancer metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-06.

Breast cancer mortality is primarily due to metastatic lesions rather than primary tumors, yet relatively little is known regarding the mechanisms of metastatic breast cancer, making it difficult to identify patients who are at risk for metastatic disease. Our hypothesis suggests that inherited germline mutations contribute to metastatic disease and that these single nucleotide polymorphisms (SNPs) could be used to predict outcome in breast cancer patients. To investigate the effect of inherited SNPs on metastasis, we used a mouse genetics approach comparing strains with high (FVB) and low (MOLF) metastatic phenotypes and identified Arntl2, a circadian rhythm transcription factor, as a gene whose differential expression predicted outcome in breast cancer patients. To identify SNP differences in Arntl2 between MOLF and FVB, we performed whole genome sequencing of MOLF and compared it to the FVB genome. Overlapping the data with DNase hypersensitivity sites revealed 10 SNPs in the predicted promoter of Arntl2. To test the causative role of the SNPs on Arntl2 expression in vivo, metastatic cell lines were engineered using the CRISPR-Cas9 approach to specifically replace the FVB Arntl2 promoter with that of MOLF. In agreement with our hypothesis, substitution of the MOLF promoter reduced Arntl2 transcript levels and subsequently decreased lung metastases in orthotopic implantation assays. In vitro pulldown experiments with strain-specific promoter probes revealed potential differential binding of chromatin modifier proteins, demonstrating the significance of the SNPs in regulating Arntl2 transcription. Finally, analysis of SNPs associated with Arntl2 expression in a cohort of Chinese breast cancer patients revealed significant correlation of Arntl2 expression with overall survival, validating this gene as a marker in humans. Since Arntl2 is a transcription factor, current studies are focused on identifying Arntl2-regulated genes to investigate downstream pathways involved in metastasis. This study has important implications regarding the role of circadian rhythm in cancer progression and provides a potential mechanism to explain the increased risk of breast cancers in nightshift workers. Furthermore, this provides the first evidence that transcriptional control elements can be engineered using CRISPR-Cas9 to establish the causative role of SNPs in inherited susceptibility to cancer metastasis. Citation Format: Ngoc-Han Ha, Kent Hunter. Polymorphisms in the arntl2 promoter affect breast cancer metastasis susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3050. doi:10.1158/1538-7445.AM2017-3050

Bone metastasis is a devastating and fatal complication of breast cancer for which we lack effective therapies. Thus, identifying therapies that effectively limit metastases will significantly reduce comorbidities and improve long-term survival. Recently we demonstrated that the p38MAPK pathway sustains the pro-tumorigenic senescence-associated secretory phenotype (SASP) and targeting this pathway limits the tumor-promoting capabilities of senescent cells and cancer-associated fibroblasts (CAFs). Because we found that a significant percentage of p38MAPK-dependent SASP factors are expressed in the stroma associated with breast cancer lesions, we asked whether targeting p38MAPK could limit primary and metastatic breast cancer growth. While p38MAPK inhibition modestly limited primary tumor growth, we found that its inhibition significantly reduced breast cancer bone metastases by specifically targeting the stromal compartment. Further, p38MAPK inhibition was as effective as paclitaxel at limiting tumor growth in the bone but in contrast to paclitaxel, which failed to protect from cancer-induced bone loss, p38MAPK inhibition also protected against devastating bone loss. This contrasts our p38MAPK approach from zoledronic acid, which limits bone loss but fails to slow tumor growth in already engrafted tumors. Analysis of the mechanism(s) responsible for this reduced metastasis suggests that p38MAPK inhibition targets reactive and/or senescent osteoblasts within the bones of animals harboring metastatic lesions. Because we find that senescent osteoblasts are present in human bone, we postulate that they promote metastatic outgrowth and thus p38MAPK inhibition limits the pro-metastatic activities of these cells. Finally, we will present recent data from our preclinical model that demonstrates that inhibition of the p38MAPK pathway can drastically reduce metastasis from the primary site. We propose that p38MAPK is an important stromal-specific therapy for breast cancer metastasis to the bone. Citation Format: Sheila A. Stewart, Bhavna Murali, Qiaho Ren, Xinming Su, Kevin Flannagan, Xianmin Luo, Jasmine Sponagel, Yujie Fu, Elise Alspach, Kathlees Leahy, Roberta Faccio, Kathleen Weilbaecher, Joseph Monahan. Inhibition of stromal p38MAPK abrogates breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2017-2963

Brain metastasis is a devastating, late-stage event affecting approximately 10-30% of breast cancer patients. However, it is not well understood how breast cancer cells migrate across the blood-brain barrier (BBB) and invade into the brain. Syndecans (Sdcs) are cell surface heparan sulfate proteoglycans (HSPGs) that have previously been linked to breast cancer progression and metastasis. Preliminary findings from our lab indicate that Sdc1 may be involved in breast cancer metastasis to the brain. In clinical samples from breast cancer patients with brain metastases, 67% of the brain metastasis samples stained positive for Sdc1 expression. In an experimental mouse model of metastasis, we found that silencing expression of Sdc1 in MDA-231 breast cancer cells greatly reduced metastasis specifically to the brain, with no difference in lung or bone metastases. Additionally, silencing expression of Sdc1 in MDA-231 cells resulted in a reduction in cancer cell migration across an in vitro BBB transwell model system. These findings prompted us to investigate the mechanism through which Sdc1 facilitates breast cancer cell migration across the BBB. Since BBB endothelial cell junctions have been reported to be disrupted in metastasis to the brain, immunofluorescence was performed to examine localization of junction proteins in our BBB model following addition of conditioned medium (CM) from MDA-231 non-silenced (NS1) control and Sdc1KD cells. We observed disruption in PECAM-1 localization at BBB endothelial cell junctions upon addition of CM from MDA-231 NS1 cells with markedly less disruption occurring upon treatment with CM from Sdc1KD cells. These results suggest that paracrine factors secreted from MDA-231 cells may facilitate breast cancer cell migration across the BBB by affecting PECAM-1 localization on BBB endothelial cells. By performing a cytokine array using CM from MDA-231 cells, we determined that Sdc1KD CM contained lower levels of IL-8 than MDA-231 NS1 cell CM. These findings were confirmed by ELISA, qRT-PCR, and multiplex analysis. We then went on to treat endothelial cells in the xCELLigence in vitro BBB model system with IL-8 and observed a sufficient decrease in cell index readings, suggesting that IL-8 affects BBB barrier permeability. Taken together, our results suggest that Sdc1 supports breast cancer cell migration across the BBB through a signaling mechanism involving IL-8 and PECAM-1. Elucidating this mechanism will allow for the development of therapeutic strategies to combat breast cancer cell metastasis to the brain. [S.M.W. and M.S. contributed equally to this work.] Citation Format: Sierra Mosticone Wangensteen, Megan Sayyad, Madhavi Puchalapalli, Megan Sullivan, Jamie Singh, Briana Ratchford, Jayda Abrams, Majid Jahromi, Bin Hu, Michael Idowu, Jennifer Koblinski. Syndecan-1 mediates breast cancer metastasis to the brain through IL-8 and PECAM-1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3058. doi:10.1158/1538-7445.AM2017-3058

Metastasis is the cause of 90% of cancer-related deaths. Selective mRNA translation has been found to be crucial for breast cancer development, progression and metastasis; however, there is a poor biological and mechanistic understanding of the role of translation in these processes. Our work and others have pioneered an understanding of translational regulation in breast cancer. We have previously shown that overexpression of initiation factor eIF4GI in advanced breast cancer cells selectively increases the translation of mRNAs encoding survival, cell cycle inhibition, and DNA damage and repair proteins, among other DNA damage-protective mRNAs. eIF4G consists of three family members: eIF4GI, eIF4GII, and the poorly studied eIF4G homolog, DAP5, the subject of this study. DAP5 lacks the N-terminal domain for eIF4E and PABP binding. DAP5 is therefore suspected to promote eIF4E-independent or IRES-driven translation of mRNAs involved in cell stress and oncogenesis, but its role in metastasis hasn’t been investigated. We analyzed the NCI human tissue genome cancer atlas (TCGA) and found that higher DAP5 mRNA expression is strongly associated with estrogen receptor negative breast cancer metastasis with lower overall survival. We then engineered a highly metastatic breast cancer cell line MDA-MB-231TR to express dox-inducible shRNA to DAP5 or a non-silencing (NS) control. Tumors were developed in clinically relevant orthotopic mouse models, with and without DAP5 silencing by addition of dox to the drinking water. Remarkably, reduction of DAP5 expression by ~70% had no impact on primary tumor growth but fully eliminated metastasis to the lung. Genome-wide transcriptome and translatome analysis of DAP5 and in vivo ultraviolet-crosslinking and high-throughput sequencing (HITS-CLIP) identified specific mRNAs that interact directly with DAP5. A significant fraction of DAP5 mRNA targets are involved in cell death and survival, cell proliferation, cell mobility, DNA repair and translation initiation and do not have IRESs, suggesting a novel role for DAP5 in a non-classical translation initiation process upon cellular stress and metastasis. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis by a unique mechanism for metastasis-specific translation initiation and provides new concepts for therapeutic strategies involving translational regulation. Citation Format: Columba de la Parra, Amandine Alard, Amanda Ernlund, Robert J. Schneider. Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2017-4481

Our major goal is to determine the role of Chromodomain-helicase-DNA-binding protein 7 during breast cancer (BC) metastasis. This protein is an ATP-dependent nucleosome remodeling factor and is critical for embryonic development in both animal models and human patients. Our most recent studies provide direct evidence suggesting its novel role in repressing BC metastasis. We found that this protein, but not the mRNA, is highly expressed in a low-metastatic breast cancer cell line (Mcf-7), while its expression is much reduced in metastatic cell lines (MDA-MB-468 and MDA-MB-231). Furthermore, this protein is predominantly localized in the cytoplasm of 468 and 231 cells, in contrast to its nuclear localization in Mcf-7 cells. In the cytoplasm of 468 and 231 cells, this protein is colocalized with a lysosome marker, suggesting that in high metastatic BC cells, this protein is exported to the cytoplasm to be degraded in lysosomes. Blocking the activity of lysosomes increased expression of this protein in high metastatic BC cell lines. This result suggests a mechanism accounting for the reduced expression of this protein in high metastatic BC cell lines. To further support the potential clinic relevance of our results, this protein is localized in the nucleus of almost all duct epithelial cells in normal breast tissues; however, in a large portion of epithelial cells of breast tumors, this protein is localized in the cytoplasm. Through reporter and mutagenesis analysis, we identified the nuclear exporting signal (NES) sequence responsible for exporting this protein into cytoplasm. In the next set of experiments, we attempted to determine the function of this gene in BC metastasis. We found that knocking down expression of this gene in 468 cell significantly increased their migration and invasion. We next applied the CRISPR genome editing technique to mutate the critical amino acids within the NES region in the endogenous gene locus and found that the amount of protein in the nucleus is significantly increased compared to wild type cells. Migration/invasion of these cells was also dramatically decreased. At the molecular level, we found that forced expression of this protein in nucleus reduced expression of epithelial markers and increased expression of epithelial-mesenchymal-transition markers. We are now applying ChIP-Seq and RNA-Seq approaches to determine direct regulatory network of this protein in 231 cells. We will further test whether this protein acts through modulating the epigenetic status of the enhancers/promoters to regulate expression of its target genes to repress BC metastasis. In summary, our data collectively suggest that Chromodomain-helicase-DNA-binding protein 7 is a novel epigenetic regulator to repress BC metastasis. To the best of our knowledge, our study represents the first to address the activity of this protein in BC. Note: This abstract was not presented at the meeting. Citation Format: Kenneth Jiao, Yin Peng, Lizhong Wang, Runhua Liu. A novel mechanism for regulation of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2017-2403

Introduction: We have previously described motile sperm domain-containing protein 2 (MOSPD2), a protein which up until recently no function was ascribed to, as a key regulator of monocyte migration in-vitro. In this study, the role of MOSPD2 in promoting breast cancer migration and metastasis in-vitro and in-vivo was assessed. Experimental procedure: The prevalence of MOSPD2 was evaluated by IHC in tissue microarray layered with cores, representing different stages of invasiveness in breast cancer. MOSPD2 abundance was scored according to the staining intensity on a scale from 0 to 3. MOSPD2 expression was silenced in MDA-231 breast cancer cell line using lentiviral particles for sh-RNA or CRISPR-CAS9 (CRISPR). Cells were then tested by a trans-well assay for migration towards EGF in vitro as well as for lung dissemination following orthotopic or systemic inoculation in vivo. For mechanism studies, EGF-induced signaling events were analyzed. Results: Within normal adjacent tissue (NAT), 82% of the samples had no staining and the remaining 18% percent displayed an intensity of 1. In the tissue cores with in-situ carcinoma pathology, 79% of the samples had no staining intensity while the remaining 21% only scored 1 or 2. However, analysis of invasive and metastatic cancer demonstrated higher frequency in score of 2 and increased staining intensity up to score of 3 compared with NAT and in-situ carcinoma. Thus, the percent of combined scores 2 and 3 for invasive lobular carcinoma, invasive ductal carcinoma and metastatic invasive ductal carcinoma were 63%, 77% and 81%, respectively. MOSPD2-silenced cells, either by sh-RNA or by CRISPR, were severely impaired in their ability to migrate in-vitro towards EGF. Following EGF activation of CRISPR-Control MDA-231 cells, all tested proteins down-stream to the EGFR pathway, including AKT and p38, became phosphorylated. However, in CRISPR-MOSPD2 silenced cells, phosphorylation of all tested proteins excluding p38 were markedly inhibited. Moreover, activation with IGF of CRISPR-MOSPD2 MDA-231 cells did not hinder AKT phosphorylation. In vivo, SCID mice injected orthotopically or systemically with CRISPR-MOSPD2 silenced MDA-231 cells displayed significantly reduced number of metastases in the lungs relatively to mice injected with CRISPR-Control treated cells. Conclusions: We suggest that MOSPD2 promotes metastasis of breast cancer cells and is a potential target for the treatment of metastatic breast cancer. Citation Format: Itzhak Mendel, Yaniv Salem, Niva Yacov, Oshrat Propheta-Meiran, Eyal Breitbart. MOSPD2, a newly characterized protein, promotes breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4892. doi:10.1158/1538-7445.AM2017-4892

TGFβ-activated kinase 1 (TAK1), a serine/threonine kinase from the MAPKKK family, is regulated by different cytokines such as IL1, TGFβ, TNFα and BMPs, and it is therefore a key player in the cellular responses induced by changes in the microenvironment. TAK1 regulates cell survival, differentiation and inflammatory responses by activating other intracellular kinases such as p38, JNK or IKK. TAK1 activation is thought to influence the progression of several types of cancer, including lymphomas and pancreatic, colon, liver and breast cancer. In this study, we aimed to investigate the mechanisms by which TAK1 regulates breast cancer progression. (5Z)-7-Oxozeanol is a compound that inhibits the kinase activity of TAK1. Treatment of mice injected with MDA-MB-231 cells with (5Z)-7-Oxozeanol containing nanoparticles reduced metastasis formation, while growth of the primary tumor was not affected. TAK1 involvement in metastasis was confirmed by xenotransplantation experiments done with MDA-MB-231 cells overexpressing wild type or dominant negative forms of TAK1. Using these stable cell lines, we confirmed that TAK1 is required for p38 activation induced by several cytokines. Interestingly, cells cocultured with macrophages showed a TAK1-dependent increase in the expression levels of IL1α and IL1β, which can lead to TAK1 activation themselves. These cytokines were also detected in cancer cells that colonized the mouse lung, suggesting that the lung microenvironment could promote the initiation of a positive feedback loop by cancer cells, promoting the growth of metastatic lesions. Disruption of this positive autocrine feedback loop may be a potential therapeutic approach to suppress breast cancer metastasis to the lung. Citation Format: Oihana Iriondo, Mostafa Elhodaky, Yarong Liu, Grace Lee, Julie E. Lang, Pin Wang, Min Yu. TAK1 mediated IL1 expression as autocrine signaling to promote breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4857. doi:10.1158/1538-7445.AM2017-4857

Due to the advance in technologies for rare cell isolation, circulating tumor cells (CTC) have recently received vast interest. During tumor progression tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred as CTCs. These CTCs disseminate to other organs and a subset of these cells will be able to form metastasis. The growing interest for CTC is confronted with the difficulty associated with their isolation and characterization. To address this challenge, our lab has recently optimized the ex vivo culture condition and was able to establish CTC lines from breast cancer patients. These CTC lines constitute a unique cell population in the metastatic process and gave us a rare opportunity to investigate the signal pathways involved in each step of the metastatic cascade. To assess the metastatic potential of breast cancer patient-derived CTC lines we utilized an experimental mouse model for metastasis by injecting CTCs directly into the left ventricle of the heart in female immuodeficient NSG mice. The ability of 4 CTC lines to arrest and colonize in organ was monitored by bioluminescence imaging once every 2 weeks for 5 months. CTC lines were capable of generating brain, lung, bone and ovary metastases. Most of those organs are common sites of metastases in breast cancer patients. Two patient-derived CTC lines have a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in multiple organs such as bone, lung and Ovary. These mice remained brain-metastases free for up to 8 months. However, two other patient-derived CTC lines demonstrated the brain as preferential site of metastasis despite their overall low metastatic potential. Interestingly, of 4 breast cancer patients where CTC lines were generated, one developed a metastatic brain tumor and her CTC line has the highest risk of brain metastases in our mouse model. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. We isolated metastatic variants corresponding to a subpopulation of CTCs with a preferential tropism for the brain, lung and bone. Gene expression analysis (RNA-seq) of these variants identified potential gene signatures of breast cancer metastasis. The pathways with the highest enrichment scores were glioblastoma multiform, ceramide biosynthesis and PCP pathway for the brain metastatic variants and were interferon and mTOR signaling for the lung metastatic variants. Next we used a method for assaying chromatin accessibility (ATAC-seq) and identified potential regulatory regions mediating the organ tropism in breast cancer. Together our data provide the evidence of a promising role of CTC as an early prognostic factor in metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancers. Citation Format: Remi Klotz, Thomas Amal, Alan Wang, Matthew Mackay, Kathleen Heller, Lin Li, Maxwell Serowoky, Grace Lee, Jane Han, Andrew Smith, Min Yu. Understanding breast cancer metastasis through circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2017-1045

This abstract was not presented at the symposium.

This abstract was not presented at the symposium.

Background: Metastasis is the primary cause of breast cancer mortality. Interactions between cancer cells and non-cancer cells of the tumor microenvironment (TME) are pivotal in governing tumor initiation, progression and metastasis, and cancer associated fibroblasts (CAFs) are critical orchestrators of these interactions. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic breast cancer. We established CAF cell lines from dissociated luminal A, ER- Her-2 amplified, and triple-negative/basal-like (TN) breast tumors. We demonstrated that “aggressive” CAFs differentially secrete miRNAs that contribute to ER-negativity, activated growth factor signaling, and induction of EMT in breast cancers compared to “indolent” CAFs. We hypothesized that a hierarchy exists within CAFs regarding their ability to facilitate tumor progression and metastasis. Here we demonstrate that CAFs derived from aggressive TN breast tumors differ from those derived from more indolent Luminal A breast tumors in secretion of cytokines and chemokines that can confer differential effects on the behavior of breast cancer cells. We also demonstrate that “aggressive” CAFs more potently facilitate tumor progression and metastasis than “indolent” CAFs. We additionally evaluated if “aggressive” and “indolent” CAFs differ in their ability to mobilize CTCs and circulating CAFs into circulation. Methods: Conditioned media (CM) from “aggressive” and “indolent” CAFs was analyzed for chemokine/cytokine expression. Luminal A breast cancer cells (MCF-7) or primary tumor cells from an aggressive TN tumor (DT28) were injected into the mammary fat pad of 6-8 week old female NSG mice, either alone or in combination with CAF19-I or CAF23-A. Tumor progression was monitored and mice were examined for metastasis at necropsy. Tissues were harvested for histology and blood was collected by cardiac puncture. Plasma was analyzed for cytokine/chemokine expression and blood was processed for enumeration of circulating tumor cells (CTCs) and cCAFs. Results: “Aggressive” CAF CM had significantly higher levels of a number of factors, including IL-8, SDF-1, and CXCL1, compared to “indolent” CAF CM. MCF-7 cells co-injected with “aggressive” CAFs formed tumors much faster than those co-injected with the “indolent” CAFs or without CAFs. While DT28 cells readily form tumors and metastasize in the NSG model, fewer DT28 cells do not form metastases in the timeframe that this same lower number of DT28 cells co-injected with “aggressive” CAFs demonstrated robust tumor growth and developed metastases in liver and pancreas. DT28 cells co-injected with “indolent” CAFs did not exhibit metastases. Conclusion: The data presented here further demonstrate that there is a hierarchy within CAFs regarding their ability to facilitate tumor growth and metastasis, and that this may largely be mediated by secreted soluble factors. “Aggressive” CAFs may retain their programmed role in circulation and accelerate metastasis more than “indolent” CAFs. We suggest that targeting CAFs in situ and in circulation and disrupting their interactions with breast cancer cells could provide novel strategies to combat breast cancer and breast cancer metastasis. Citation Format: Sharma U, Miller P, Speransky S, Medina-Saenz K, Ferrer P, Lippman M, El-Ashry D. A hierarchy of cancer associated fibroblasts in situ and in circulation promote breast cancer metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-18.

Extracellular microRNAs (miRNAs) that can be detected in the circulation are novel mediators of intercellular communication and are considered emerging biomarkers for human diseases. Using cell-secreted extracellular vesicles (e.g., exosomes) as vehicles, miRNAs secreted by cancer cells can travel to and enter various types of niche cells in primary and pre-metastatic tumor microenvironments. Upon entering niche cells, miRNAs regulate gene expression to prepare the niche for cancer progression. Our lab focuses on defining the roles of breast cancer secreted miRNAs in adapting local and distal niche cells and tissues in cancer progression and metastasis. Through de novo sequencing and PCR of circulating small RNAs in the pre-treatment sera of stage II-III breast cancer patients we identified circulating miRNAs as biomarkers for cancer progression to metastatic disease. Subsequent mechanistic studies revealed important functions of breast cancer secreted miRNAs in various aspects of cellular behaviours. Our lab is currently exploring additional mechanisms through which breast cancer-derived, extracellular miRNAs contribute to the multifaceted reprogramming of non-cancerous cells in the tumor microenvironment as well as novel therapeutic strategies targeting cancer-derived extracellular miRNAs for their function in cancer-host communication. Citation Format: Wang SE. A role of extracellular miRNA in breast cancer metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr BS2-1.

NEDD9 has been characterized as a metastasis-promoting gene in various cancer cells including breast. We previously reported that NEDD9 promotes malignant phenotypes of breast cancer cells through distinct and non-overlapped domains. For example, the FAT (Focal Adhesion Targeting) domain of NEDD9 promotes cancer cell growth, while the SH-domain facilitates cell migration. These results suggest that NEDD9 promotes tumor metastasis by enhancing dissemination and growth in the tumor-host microenvironments through distinct and non-overlapped domains. Thus, targeting functions of NEDD9 is a promising approach for breast cancer therapies. In order to further characterize NEDD9-mediated breast cancer growth, we performed yeast-two hybrid (Y2H) screening to identify proteins that associate with the FAT domain of NEDD9. Using the FAT domain constructed in pGBKKT7 (Clonetech, CA) as a bait to screen library of human fibroblast (Clonetech, CA), we identified several proteins that associate with the domain. They are small GTPases (i.e. RAB11a and ARF4), cytoskeletal proteins (i.e. Nexilin), and cytosolic proteins (i.e. HAX-1). Among of these potential partner proteins, we focused on the interaction between NEDD9 and HAX-1 in breast cancer cells. Co-immunoprecipitation assays confirm the molecular complex of NEDD9-HAX-1 in both SK-Br3 and SUM149 cells. Importantly, p130cas, which harbors similar domain structures with NEDD9, was not precipitated with NEDD9, suggesting a specific interaction between NEDD9 and HAX-1. Given the fact of NEDD9 as a key metastasis promoting gene, these results suggest that NEDD9-HAX-1 plays a key role breast cancer metastasis by facilitating growth in microenvironments. While the biological function are not clear at present, previous studies demonstrated that HAX-1 localizes in mitochondria in breast cancer cells, Indeed, we demonstrated that NEDD9 was found in both cytosol and mitochondria fractions in malignant breast cancer cell MDA-MB-231, but not non-metastatic HCC38. These results suggest the presence of NEDD9-HAX1 complex in mitochondria and this complex may facilitate breast cancer metastasis. In addition to HAX-1, several mitochondrial proteins such as EFG1, DCTN6, and MMADHC were found in the Y2H screening system as described above. These results suggest that NEDD9 facilitates breast cancer metastasis through regulating multiple pathways including signaling pathways and mitochondrial functions, thus serving as a promising therapeutic target for cancer patients including breast. The view expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S.Government. Citation Format: Iida J, Dorchak J, Slavik J, Clancy R, Cutler ML, Shriver CD. NEDD9 promotes breast cancer metastasis by regulating mitochondrial functions [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-02.

This abstract was not presented at the symposium.

Our studies to date have demonstrated that retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo, associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator (“suicide”) gene therapy in a variety of cancer models. Clinical testing of RRV-mediated gene therapy using the yeast cytosine deaminase (CD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug, 5-fluorouracil (5-FU), is currently underway in multi-center clinical trials being conducted in the United States for patients with recurrent high-grade glioma. In the present study, our goal has been to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers to the CNS, which occur 5-10 times more frequently than primary brain tumors. Moreover, brain metastases are often accompanied by neurological deficits, which deprive patients of their quality of life. In particular, brain-metastatic breast cancer frequently arises from highly aggressive, treatment-refractory, ‘triple-negative' (ER(-), PR(-), HER2(-)) cells, is associated with a dismal prognosis of 4-6 months, and as treatment options are largely palliative, is the main cause of death in half of these patients. Hence this condition represents an unmet medical need. We first examined the replication kinetics of RRV expressing the GFP reporter gene (AC3-emd) in human and murine breast cancer cell lines by flow cytometry. The replication kinetics of AC3-emd in the triple-negative human breast cancer cell line MDA-MB-231-BR, and in the murine breast cancer cell line JC, showed robust replication activity over time at both MOI = 0.01 and 0.1, resulting in high levels of transduction within 1-2 weeks. Next, we tested in vitro cytotoxicity by MTS assay after 5-FC treatment of 231BR and JC cells that had been fully transduced with RRV expressing the CD prodrug activator gene (clinical-grade vector AC3-yCD2, also designated “Toca 511”). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% even after exposure to 0.1 mM 5-FC treatment, and complete cell killing was observed after 6 days (231-BR) or 4 days (JC) exposure to 1 mM 5-FC treatment. We then examined in vivo replication kinetics of AC3-emd in intracranial 231-BR xenograft models. Intratumoral injection of 10e6 AC3-emd on Day 7 after tumor implantation was confirmed to achieve >80% transduction efficiency by flow cytometry on Day 10 post-injection. In survival studies, animals treated with AC3-yCD2+5-FC prodrug activator gene therapy showed statistically significant (p Citation Format: Akihito Inagaki, Sara Collins, Suzanne Matsuura, Noriyuki Kasahara. Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A43.

The role of TGFβ in promoting mammary cell EMT and breast cancer metastasis was well established. However, the involvement of long non-coding RNA (lncRNA) in this process remains elusive. Here we demonstrated that mouse lncRNA BB179049 and its human ortholog WDFY3-AS2 were significantly increased upon TGFβ-induced EMT. WDFY3-AS2 was frequently elevated in invasive, metastatic and triple-negative breast cancer. Depletion of WDFY3-AS2 abrogated TGFβ-induced EMT and breast cancer metastasis. WDFY3-AS2 positively regulates STAT3 and WDFY3 through interaction with hnRNP-R. These data indicate that WDFY3-AS2 could play a pivotal role in TGFβ-induced EMT and metastasis and serves as a critical therapeutic target in aggressive breast cancer. Citation Format: Edward Richards, Sridevi Challa, Yajuan Li, Jennifer Permuth-Wey, Marilyn Bui, Domenico Coppola, Thomas Sellers, Jin Cheng. LncRNA WDFY3-AS2 contributes to the EMT and metastasis in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 998.

WASF3 is overexpressed in high-grade breast cancer and promotes invasion and metastasis but does not affect proliferation. The HER2/ERBB2/NEU gene is also frequently overexpressed in breast cancer and has been shown to promote invasion and metastasis in these tumors. Here we show that WASF3 in present in the HER2 immunocomplex and suppression of WASF3 function leads to suppression of invasion even in the presence of HER2 expression. Overexpression of both HER2 and WASF3 in non-metastatic MCF7 breast cancer cells promotes invasion and metastasis more significantly than either gene alone. HER2 forms homodimers as well as heterodimers with other HER family members and we now show that the ability of WASF3 to promote invasion is highly dependent on the HER2/HER3 heterodimer. The engagement of WASF3 with the HER2/HER3 complex facilitates its phospho-activation and transcriptional upregulation, which is facilitated by HER2/HER3 activation of JAK/STAT signaling. In breast cancer cells overexpressing HER2, therefore, WASF3 is specifically required to facilitate the invasion/metastasis response. Targeting WASF3, therefore, could be a potential therapeutic approach to suppress metastasis of HER2-overexpressing breast tumors. Citation Format: yong teng, Wenhu Pi, John Cowell. Critical role and mechanism of WASF3 in HER2/HER3 regulation of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 687.

With a woman's lifetime risk of developing invasive breast cancer at one in eight, breast cancer remains the most common cancer and the second leading cause of cancer death in women. Despite the success of frontline endocrine therapies, relapse with resistant metastatic disease is common, and triple negative disease remains without a targeted therapy. Importantly, breast cancer associated mortality is most often due to the metastatic spread of the disease. Therefore, there continues to be a need for studies that may lead to the development of new chemopreventive strategies and/or lifestyle changes that reduce breast cancer metastasis. In this regard, both obesity and elevated cholesterol are risk factors for metastatic relapse, while patients receiving cholesterol-lowering drugs such as HMG-CoA reductase inhibitors (statins) display an increased relapse-free survival time. Therefore, clinical observations strongly implicate cholesterol in the metastatic progression of, although the underlying mechanisms remain unknown. Thus, we initiated a series of studies to evaluate the mechanisms by which cholesterol promotes breast cancer metastasis. Using murine models of mammary cancer, we find that elevated cholesterol does indeed increase metastasis. Further, statins attenuated the metastatic effects of a high fat diet, implicating cholesterol as a primary mediator of this pathology. Importantly, the pro-metastatic effects of cholesterol required the presence of CYP27A1, the enzyme responsible for its conversion to 27-hydroxycholesterol (27HC). We have recently shown that 27HC is an endogenous ligand for the both the estrogen receptors (ERs) and liver X receptors (LXRs). Interestingly, however, the metastatic effects of 27HC were also prevalent in ER-negative models. While LXR activation did have a modest effect on epithelial to mesenchymal transition (EMT), we find that the robust pro-metastatic effects of 27HC are mediated in a cancer cell-extrinsic manner. Specifically, we find that 27HC alters myeloid cell recruitment and function at distal metastatic sites, and thereby facilitates cancer cell colonization and growth by modulating the host environment. In summary, our data strongly suggest that by altering the host immune system, 27HC is the down-stream mediator of the effects of obesity and cholesterol on breast cancer metastasis. These results provide additional support for development of specific CYP27A1 inhibitors and immune-modulatory therapies for the prevention and treatment of breast cancer metastasis. This work was supported by an NIH-NCI grant to ERN (4R00CA172357-03). Citation Format: Amy Eun Joo Baek, Erik Russell Nelson. A cholesterol metabolite acts on the host to increase breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A14.

Regulation of mRNA translation and degradation is critical in the control of cell growth and survival. Translational control is often exploited by human cancers and is crucial to cancer development and progression. Translational control processes include mRNA degradation, mRNA surveillance, RNA gene silencing, and translational repression. These processes share several mRNA processing enzymes (mRNPs), all observed to co-localize in discrete cytoplasmic foci referred to as processing bodies (P-bodies). Specifically, it has been shown that colorectal cancer cells promote the assembly of P-bodies to control the translation of specific mRNAs using TGF-β/Smad signaling. TGF-β is a known driver of the epithelial to mesenchymal transition (EMT). This process contributes to the invasive and metastatic potential of breast cancer cells. Syk is an essential protein tyrosine kinase in immune cells but is also present in non-hematopoietic cells such as mammary epithelial cells. Syk appears to have more diverse roles that have not yet been elucidated. What has been observed, however, is lost of Syk expression in invasive breast carcinoma tissue / cell lines and that the reintroduction of Syk into metastatic breast cancer cells suppresses tumor growth and metastasis. We collaborated with Dr. Andy Tao's lab, which specializes in mass spectrometry technologies to identify potential Syk substrates in breast cancer cells. Among these substrates were proteins associated with P-bodies and involved in mRNA metabolic processes. These interactions lead us to hypothesize that Syk co-localizes with P-bodies. To investigate this we used fluorescent microscopy and biochemical methods. We induced P-body formation using several different stimuli that included MG132, Sodium Arsenite, and TGF-β and we observed Syk co-localizing within P-bodies in several cell models. Moreover, we observed tyrosine phosphorylation occurring in P-bodies in a Syk-dependent manner. Interestingly, we found that Syk-expressing cells displayed decreased average number of P-bodies when expose to oxidative stress. Apparently, the presence of Syk enhances P-body clearance, possibly influencing the integrity of P-body function. These findings prompted us to investigate the physiological role of P-bodies in breast cancer cells. We discovered that TGF-β induction of P-bodies increases with EMT. However, once cells are transformed, P-body formation is loss. Moreover, mRNA decaying enzyme and P-body component, Dcp1a decreases in more metastatic cells. When Dcp1a is over-expressed, which results in aberrant P-bodies, EMT is blocked. These findings suggest that the formation of P-bodies contribute to the EMT process. We are now investigating the relationship between Syk's role in the disassembly of P-bodies and its role as a tumor suppressor in breast cancer cells. We are also interested in understanding the mechanistic role for Syk in P-bodies and mRNA metabolism Citation Format: Shana D. Hardy, Robert L. Geahlen. Investigating the role of Syk in TGF-β induced P-bodies and 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 4972. doi:10.1158/1538-7445.AM2015-4972

Metastasis is a complex multistep process, responsible for as much as 90% of cancer-related deaths, yet obtaining successful treatment for these patients remains an elusive challenge. It has long been recognized that the tyrosine kinase receptor ErbB-2 and the signal transducer and activator of transcription 3 (Stat3), two major players in the breast cancer (BC) scenario, are involved in BC metastatic dissemination through a mechanism where Stat3 acts as a downstream effector of ErbB-2 action. In addition, we and others have also disclosed the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. On the other hand, microRNAs are short non-coding endogenous RNAs with regulatory functions. In particular, high levels of microRNA-21 (miR-21), a well-known oncomiR, have been reported to actively promote invasion and metastasis in BC cell lines and tissues. Here, we describe a novel hierarchical interaction between Stat3, ErbB-2 and miR-21, underlying the metastatic phenotype of ErbB-2-positive BC. We disclosed that Stat3 acts as an upstream regulator of ErbB-2 expression and function. In a panel of cell lines corresponding to different BC subtypes we found that Stat3 induced ErbB-2 expression at the transcriptional level through its recruitment to response elements (called GAS) at the ErbB-2 promoter. Furthermore, we demonstrated that Stat3 co-opted NErbB-2 function, recruiting it as a coactivator, to assemble a transcriptional complex at the GAS sites of the miR-21 promoter, leading to miR-21 up-regulation. We showed that the increase in miR-21 levels resulted in the downregulation of the metastasis suppressor protein PDCD4, a well known miR-21 target. In order to assess the physiological relevance of our molecular findings, we developed an in vivo model of ErbB-2-overexpressing metastatic BC, in which Stat3 activation was inhibited by transfection with a Stat3 dominant negative variant (Stat3Y705F) or its expression was silenced by siRNAs. We demonstrated through reconstitution assays that ErbB-2 and miR-21 were necessary downstream mediators of Stat3-induced metastases development. Furthermore, we explored the clinical significance of our findings in a cohort of ErbB-2-positive primary invasive BC patients and demonstrated that Stat3 and ErbB-2 nuclear co-expression was associated with low PDCD4 expression levels, and that this correlated with the presence of nodal metastases. Our present results in experimental models and in the clinic shed light on the molecular mechanisms underlying BC metastasis and highlight targeting either Stat3 or NErbB-2 as novel therapeutic strategies for ErbB-2-positive BC patients. #: 15-A-2849-AACR Citation Format: Leandro Venturutti, Lucía V. Romero, Alejandro J. Urtreger, María F. Chervo, María F. Mercogliano, Rosalía I. Cordo Russo, Matías G. Pereyra, Gloria Inurrigarro, María C. Díaz Flaqué, Victoria Sunblad, Juan C. Roa, Pablo Guzmán, Elisa Bal de Kier-Joffe, Eduardo H. Charreau, Roxana Schillaci, Patricia V. Elizalde. Stat3 and ErbB-2 interaction in 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 2265. doi:10.1158/1538-7445.AM2015-2265

New therapeutic targets that can inhibit cancer metastasis are desperately needed. To address this issue, we have leveraged a computational approach and performed an unbiased screen for therapies that can inhibit an epithelial-to-mesenchymal transition (EMT) gene expression profile, a phenotype that is believed to promote metastases. This yielded predictions of drugs that can both promote and inhibit an EMT transcriptional profile. Following up on these predictions, we confirmed that these drugs can induce or repress an EMT in a range of breast cancer cell lines. Furthermore, we discovered that they do so by altering the fluidity of the plasma membrane. An increase in plasma membrane fluidity is necessary and sufficient to induce and maintain an EMT state, increased motility, and increased mammosphere forming ability. Therapies that decrease fluidity can inhibit metastasis in xenograft mouse models. Finally, we found that human cancers can regulate their membrane fluidity through cholesterol homeostasis pathways. 41% of human breast cancers have alterations in cholesterol regulatory pathways, and are associated with a 9 year loss in time to distant metastasis. These data demonstrate that plasma membrane fluidity is a cellular attribute that is leveraged by human tumors to increase metastatic capacity, and that it can, in principle, be targeted by therapeutics to prevent metastasis. Citation Format: Jeffrey T. Chang. EMT and breast cancer metastasis driven by plasma membrane fluidity. [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 4984. doi:10.1158/1538-7445.AM2015-4984

Breast cancer is the most frequent cancer and the second leading cause of cancer mortality in women. The vast majority of breast cancer mortality is due to metastatic disease since the primary tumor can be relatively easily surgically resected. More comprehensive understanding of biology of metastasis is therefore clearly warranted to unveil novel metastasis-associated molecules and cellular processes that might be targeted for clinical intervention. Previously we demonstrated that like cancer incidence, metastatic progression has a significant inherited component. Using mouse complex trait mapping strategies we have been isolating metastasis susceptibility genes from backcross analysis. However, studies suggest that many of these QTL peaks are the result of the contribution of multiple genes, suggesting that causal genes are not being identified. To increase our ability to detect causal genes we have implemented a new integrated strategy. Whole genome sequencing of appropriate high or low metastatic mouse strains is performed to identify variants, which are then filtered for those within DNAse hypersensitive sites (DHS), based on the hypothesis that most inherited phenotypes are due to expression differences rather than missense variants. The genes associated with polymorphic DHS are then screened through mouse and human tumor expression databases to identify those genes associated with development of metastatic disease. This strategy was able to identify genes previously associated with metastatic breast cancer in both mouse and humans and new genes have also been validated. The results further suggest that inherited variation in cellular mediated immune response may be an important contributor to metastatic disease. Citation Format: Anjali Shukla, Ling Bai, Howard Yang, Anthony Doran, Ying Hu, Thomas Geiger, Maxwell Lee, Thomas Keane, Kent W. Hunter. Integrating SNPs, epigenetics and transcriptomics to better understand the inherited predisposition to 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 4138. doi:10.1158/1538-7445.AM2015-4138

We investigated the efficacy of tumor-targeting Salmonella typhimurium A1-R in the perioperative period to eradicate micrometastatic disease. Fifty female BALB/c mice were inoculated in the mammary fat pad with metastatic murine mammary adenocarcinoma (4T1-RFP (3×105/mouse), and divided into five groups. Control group (n = 10) with no treatment; surgery group (n = 10) in which the tumor was completely resected at day 21 after cell inoculation; Salmonella typhimurium A1-R (A1-R) (n = 10) in which the primary tumor was treated with A1-R alone at day 10 after cell inoculation; postoperative-administration of A1-R (n = 10) in which A1-R was administrated only after the primary tumor was resected; perioperative administration of A1-R (n = 10) in which A1-R was administrated before and after the primary tumor was resected. Metastatic tumor burden was assessed by quantitative fluorescence imaging of metastasis. Efficacy was assessed by metastatic burden and survival. The study showed that excision of the primary tumor was associated with increased systemic metastatic burden, and postoperative metastases exhibited increased proliferation (p < 0.05). A1-R alone treatment inhibited metastasis compared to untreated control and surgery only (p Citation Format: Yong Zhang, Nan Zhang, Robert M. Hoffman, Ming Zhao. Salmonella typhimurium A1-R inhibits surgically induced breast cancer metastasis. [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 712. doi:10.1158/1538-7445.AM2014-712

The development of breast cancer resistance to endocrine therapy results from altered cellular plasticity leading to the emergence of a hormone independent tumour. We have previously shown that the endocrine resistant phenotype is poorly differentiated and has greater metastatic potential when compared with an endocrine sensitive phenotype. The underlying mechanism of how an ER positive, endocrine sensitive tumour can turn on these adaptive mechanisms remains unresolved. We hypothesise that cellular reprogramming can occur as a result of long-term exposure to endocrine treatment which manifests clinically as tumour recurrence. Our aim was to identify the mechanism of breast cancer cellular reprogramming in a clinical context. In this study we adopted a global approach to define transcriptional networks significantly elevated in the breast cancer metastatic setting. Using RNAseq we determined the gene expression profile of three ER positive patients who developed tumour recurrence on tamoxifen treatment. RNAseq was performed on primary tumours, axillary node metastases and subsequent distant metastases. Following bioinformatic analysis, we defined the genes that were significantly elevated in the metastatic tumours. In the metastases, genes clustered away from those in the primary and node, with 209 genes uniquely elevated in the metastatic context, suggesting altered gene expression in response to endocrine therapy. Pathway analysis of genes significantly elevated in the metastatic setting included developmental pathways (WNT signalling and TGFbeta), growth factor signalling pathways (MAPkinase), cell adhesion molecules, junction adherens and pathways in cancer. Further analysis provided a list of clinically relevant transcriptional networks which may facilitate tumour cell de-differentiation. Transcription factors including Myc, SMAD2, ESR, TCF3, CUX1 and CLOCK were identified which potentially drive reprogramming in response to endocrine treatment. We interrogated this data to identify downstream targets of this ‘de-differentiation’ network for new predictive markers of response to endocrine treatment. Bioinformatic analysis identified PAWR, an inhibitor of progenitor cell expansion as a potential SMAD2 target. In a xenograft model of endocrine resistance, PAWR expression was found to be reduced in metastatic tissue from lung, liver and bone compared with the primary tumour. In human breast cancer patients PAWR significantly associated with a good response to endocrine treatment and luminal A status (p = 0.0008), establishing PAWR as a predictor of good response to endocrine therapies. We have identified a transcriptional network which may drive a de-differentiated phenotype following endocrine treatment. Data presented here proposes a mechanism by which apparently less aggressive Luminal A type tumours may fail endocrine treatment and develop subsequent recurrence. This represents a fundamental shift in how we approach the development of resistance to endocrine therapy, establishing a number of biomarkers which will allow tracking of response to endocrine therapy or allow accurate early prediction of those who will respond to treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-01.

The ability to measure biochemical and molecular processes underlies progress in breast cancer biology and treatment. These assays have traditionally been performed by analysis of cell culture or tissue samples. More recently, functional and molecular imaging allows in vivo assay of biochemistry and molecular biology that is highly complementary to tissue-based assay. Molecular imaging can serve and number of roles, from characterization of pre-clinical models, to testing the ability of novel drugs to interact with their intended targets, to characterizing entire burden of disease for patients with breast cancer, to helping direct therapeutic choices and assign their efficacy. This talk will review basic principals of molecular imaging, with an emphasis on those methods of greatest translational potential. The session will discuss the use of pre-clinical models of breast disease to help test and validate new molecular imaging methods, and in turn, the use of molecular imaging to characterize the disease process in the animal models. The session will then review the current state of molecular imaging in breast cancer patients, including methods in routine clinical use, those undergoing advanced clinical trials, and those in early-phase testing. The session is designed for a broad audience, ranging from breast cancer translational scientists to practicing physicians caring for breast cancer patients. References 1. Vernon AE, Bakewell SJ, Chodosh LA. Deciphering the molecular basis of breast cancer metastasis with mouse models. Rev Endocr Metab Disord. 2007 Sep;8(3):199–213. 2. Mankoff DA. Molecular imaging as a tool for translating breast cancer science. Breast Cancer Res. 2008;10 Suppl 1:S3. 3. Specht JM, Mankoff DA. Advances in molecular imaging for breast cancer detection and characterization. Breast Cancer Res. 2012 Mar 16;14(2):206. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr BS3-2.

Breast cancer is the most common cancer in women in the US. Metastasis is responsible for a majority of these deaths in spite of significant improvements in diagnosis and treatments. Recently it was reported that under conditions of asthma, metastasis to the lung is increased. Patients with pulmonary inflammatory illnesses demonstrate higher levels of chitinase-3-like-1 protein (CHI3L1) in their lungs and in circulation. We have shown that there are elevated levels of CHI3L1 in circulation with increased tumor growth, higher levels of metastasis to the lung with shorter survival rates in mice with asthma. However, the direct role of CHI3L1 in promoting metastasis is not clearly delineated. It is also unknown if exposure to high levels of CHI3L1 previous to tumorigenesis directly predisposes to accelerated metastasis. We hypothesize that CHI3L1-induced pulmonary inflammation generates the proper environment for recruiting circulating breast cancer cells, thus increasing the rate of metastasis to the lung. To determine if inflammation associated with CHI3L1 in the lung alters the pulmonary environment to attract circulating breast tumor cells and accelerate metastatic growth, pulmonary inflammation is induced by either ragweed allergen sensitization or by locally expressing CHI3L1 in the lung using AAV-CC10-CHI3L1 vector. The role of CHI3L1 in pulmonary metastasis is delineated in CHI3L1 over-expressing lung epithelia by assessing the effects on the host immune system and the downstream effects. The studies proposed here will provide a greater understanding of the role of CHI3L1 in enhancing metastasis and the tissue-specific molecular signals that act to exacerbate metastasis under conditions of chronic inflammation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1393. doi:1538-7445.AM2012-1393

Breast cancer metastasis is the major cause for mortality of patients with invasive breast cancer. Metastasis involves multiple steps including cancer cell invasion through basement membrane, intravasation, survival in the circulation, extravasation and colonization of distant organs. Identification of the signaling pathways through which prometastatic factors promote cancer cell migration and invasion is key to develop effective therapeutics for breast cancer. Mixed lineage kinase 3 (MLK3) is a MAP3K involved in activating each of the three major MAPK signaling pathways. The MLK3-JNK-AP-1 signaling axis has been shown to be required in breast cancer cell migration and invasion. Herein, we demonstrate that the prometastatic factors, CXCL12 and HGF, signal through MLK3-JNK to induce phosphorylation of the focal adhesion scaffold, paxillin on Ser 178, which is required for migration and invasion of human breast cancer cells. Furthermore, we show that MLK3 regulates the phosphorylation of paxillin at Tyr 118 and promotes the interaction of FAK with paxillin, which can be blocked by inhibition of MLK3. To further understand the role of paxillin phosphorylation at S178, a nonphosphorylatable paxillin mutant S178 A was generated. Expression of S178A blocks Y118 phosphorylation of paxillin in breast cancer cells. Silencing of mlk3, inhibition of JNK, or expression of the nonphosphorylatable paxillin S178A increases focal adhesion numbers and increases Rho activity, suggesting that the MLK3-JNK-paxillin axis limits Rho activity in breast cancer cells. Furthermore, MLK3 silencing is sufficient to prevent formation of metastases in a mouse xenograft model of spontaneous breast cancer metastasis. Our findings have identified a novel role of MLK3 in regulation of focal adhesion dynamics and cytoskeletal remodeling in the process of breast cancer cell migration and suggest that MLK3 may be a potential therapeutic target for metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2161. doi:1538-7445.AM2012-2161

In a previous study, our group has shown that the expression of caveolin-1 (CAV1) in normal tissue surrounding a primary breast cancer is a powerful prognostic indicator of subsequent metastatic disease. Whilst reports linking expression of CAV1 within breast tumour cells to clinical outcome have not led to any clear conclusions, our finding of a strong positive correlation between loss of CAV1 expression in breast tumour stroma and poor prognosis (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1486. doi:1538-7445.AM2012-1486

Metastasis and drug resistance are two major challenges in cancer therapies. The occurrence of these two events requires cancer cells to gain new mutations or change their expression profiling, or both, adapt to new environment (i.e. distal organs or drug treatments). These changes likely follow Darwin's evolutionary theory: heterogeneous of cancer cells are generated randomly and more aggressive cells are selected by the tissue environment. Stable inheritance of genome and epigenetic states could delay metastasis and drug resistance. Trimethylated lysine 4 in histone H3 (H3K4me3) is the epigenetic marker for transcriptionally active promoters and the binding site for transcriptional activators. Removing the methyl groups from trimethylated H3K4 on gene promoter regions is a common epigenetic strategy for gene silencing. The JARID1 family members are to date the only known proteins responsible for catalyzing demethylation of trimethylated H3K4. Therefore they are essential in cell differentiation and development. Even though JARID1A (RBP2) was implicated in tumorigenesis of certain cancer types, its exact roles are still poorly understood. I hypothesized that RBP2 contributes to changes of epigenetic states during the occurrence of metastasis and drug resistance. Consistent with my hypothesis, I found that RBP2 is highly expressed in metastatic breast cancer cells and Herceptin tolerant population. Loss of RBP2 decreased lung metastatic gene signature and increase drug sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-389. doi:1538-7445.AM2012-LB-389

Utilizing a cross-species expression profiling strategy that combines microarray techniques and metastatic cell lines of human and mouse origins, 22 up-regulated and 12 down-regulated genes were identified as an essential genetic program in highly-metastatic cell lines. The correlation of all gene expression levels and metastasis potential was confirmed by RT-PCR in a set of human and mouse cancer cells. Among these genes, Forkhead-Box Q1 (Foxq1) is a transcription factor with a function in metastasis that has not yet been reported upon. We showed that ectopic expression of Foxq1 increased cell migration and invasion in vitro; significantly enhanced lung metastatic capabilities of mammary epithelial cells in vivo and resulted in a marked epithelial to mesenchymal transition (EMT). In contrast, knockdown of Foxq1 expression led to the opposite change in vitro and in vivo. Neither ectopic expression nor knockdown of Foxq1 had significant effects in cell proliferation and colony formation in vitro. Additionally, we showed that Foxq1 repressed E-cadherin expression by binding to the E-box in its promoter region. Finally, we found that the expression of Foxq1 is regulated by TGF-β1, and that knockdown of Foxq1 blocked EMT induced by TGF-β1 treatment at morphological and molecular levels. Our study highlights the feasibility of cross-species expression profiling as a strategy in identifying metastasis-related genes. It also reveals that the inducement of EMT is a potential mechanism for the novel metastasis-promoting function of Foxq1 in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3441. doi:10.1158/1538-7445.AM2011-3441

Brain is a common site of breast cancer metastasis and is associated with significant neurologic morbidity, decreased quality of life, and greatly shortened survival. However, the molecular and cellular mechanisms underpinning brain colonization by breast carcinoma cells are poorly understood. In this study, we used 2D-DIGE (Difference in Gel Electrophoresis) proteomic analysis followed by LC-tandem mass spectrometry to identify the proteins differentially expressed in brain-targeting breast carcinoma cells (MB231-Br) compared with parental MDA-MB-231 cell line. Between the two cell lines, we identified 12 proteins consistently exhibiting greater than 2-fold (p This work was supported in part by Award Number 1I01BX000609 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development to VVG and AHA National SDG 0830287N to OVG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5124. doi:10.1158/1538-7445.AM2011-5124

A recent report identified six microRNAs with repressed expression in highly metastatic MDA-MB-231 breast cancer cell derivatives (miRs 335, 126, 206, 122a, 199a*, and 489). Expression of all six miRs was consistently decreased in cells that produced aggressive lung and bone metastases, as compared to parental cells, indicating that metastatic colonization is exacerbated by the concurrent loss of multiple miRs from this set. With the hypothesis that target genes involved in metastatic progression are regulated by cooperative activity of these six microRNAs, we performed a computational analysis to identify transcripts having multiple binding sites targeted by the collective set of miRs. Using miR-AT!, a new web-based computational tool developed in our laboratory, we identified 767 target transcripts with multiple sites, and we characterized the biological processes and pathways associated with the target genes. Among cellular processes enriched with genes from this set are regulation of cell cycle, cytoskeleton organization, and regulation of cell adhesion. Importantly, perturbation of these processes was recently noted as a common feature among all metastatic tumors. We have identified subsets of the six miRs that are predicted to regulate the genes in each of these critical cellular processes. Additionally, we analyzed signaling pathways downstream of the collective set of target genes to identify key regulatory molecules where the networks converge. The top scoring nodes were cyclin D1 and c-myc, suggesting that dysregulation of target genes due to the collective loss of the microRNAs may have a focused effect on the activity of these two key genes. Access to miR-AT! is available at www.mir-at.org. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2069.

Steroid receptor coactivator-1 (SRC-1) is a member of the p160 SRC family, which also contains SRC-2 (TIF2/GRIP1) and SRC-3 (AIB1, p/CIP, ACTR, RAC3, TRAM-1). These molecules can interact with nuclear hormone receptors and certain other transcription factors to promote gene expression. In breast cancer, SRC-1 expression positively correlates with HER2 expression, disease recurrence and resistance to endocrine therapy. Our previous work has demonstrated that genetic ablation of SRC-1 in mice significantly suppresses breast cancer metastasis without affecting primary tumor initiation and growth in the MMTV-PyMT (polyoma middle T) mouse model. However, the mechanisms by which SRC-1 promotes breast cancer metastasis are unclear. To address the role of SRC-1 overexpression in breast cancer metastasis and mechanism, we generated MMTV-hSRC1 transgenic mice and found that MMTV-hSRC1 virgin mice exhibited normal mammary gland development. To take an advantage of the avian virus-mediated oncogene expression system, we crossed MMTV-hSRC1 mice to MMTV-tva mice and generated MMTV-hSRC1;MMTV-tva bi-transgenic mice. Intraductal injection of RCAS-PyMT avian viruses induced more metastasis in these bi-transgenic mice than in MMTV-tva mono-transgenic mice, suggesting that SRC-1 overexpression promotes lung metastasis. We also performed studies using MMTV-PyMT;SRC1−/− (KO) and MMTV-PyMT;SRC1+/+ (WT) cell lines derived from primary mouse mammary tumors. KO cells showed much lower migration and invasion capabilities than WT cells. KO cells also adhered much slowly to fibronectin and had reduced levels of active FAK, c-Src and Rac1. Mechanistic analyses revealed that SRC-1 was associated with a promoter region of integrin α5 with C/EBP and AP-1 binding sites and the integrin α5 gene might be a direct target of SRC-1. Accordingly, knockdown of integrin α5 in WT cells reduced cell adhesion and migration. Therefore, one of the pathways for SRC-1 to promote breast cancer cell migration and metastasis is to up-regulate integrin α5, leading to super activation of FAK, c-Src and Rac1. Our study provides a possibility to inhibit SRC-1-promoted breast cancer metastasis by targeting the integrin α5 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5114.

Background: Breast cancer mortality is largely attributable to the development of systemic, hematogenous metastatic disease. Although nearly 10-15% of patients have an aggressive form disease that metastasizes within three years after initial diagnosis, the clinical manifestations of metastatic disease can appear ten or more years later. To decrease the risk for the emergence of metastatic tumors, nearly 80% of the patients are treated with adjuvant chemotherapy. The clinical benefit is a 3-10% increase in 15-yr survival, depending upon patient age at diagnosis. Currently established prognostic criteria such as the histopathological grade of the tumor or tumor size do not successfully predict systemic metastatic potential. An enrichment of cancer stem cells in the primary tumor has been shown to be a poor prognostic marker in patients. Epithelial to Mesenchymal Transition (EMT) in breast cancer cells have been shown to give rise to cells that have cancer stem cell like properties. Both of these cells types have been shown to be highly invasive. We have identified invasion markers that can predict EMT, drug and radio resistance in breast cancer cells. In this study we are using biomarkers for cancer stem cells (CD44+/CD24−) and markers of invasion (Mena/Mena11a) to identify cancer initiating cells in Fine Needle Aspiration (FNA). We correlate these ratios with a microanatomical structure called Tumor Microenvironment of Metastasis (TMEM) comprising of a direct apposition of an endothelial cell, a cancer cell and a stromal macrophage in the breast cancer tissue. TMEM has been previously reported to strongly correlate with metastasis. Hypothesis: The percentage of CD44+/CD24− expression can be used to estimate the number of cancer stem cells in FNA samples. The ratio of Mena to Mena11a protein can be used as a measurement of tumor cell response to the microenvironment and its metastatic phenotype. Measuring the stem cell biomarkers and Mena isoforms in cells collected by FNA biopsy using FACS and combining that with the assessment of TMEM density we will be able to develop a simple, quick and inexpensive method of determining metastatic potential of an individual tumor. Methods: FNA was conducted on lumpectomy or mastectomy tissues using fine needles, quality of FNA determined by smear staining. Cells were enzymatically digested, fixed and permeabilized. They were then be subjected to flowcytometry analysis using antibodies against stem cell and invasion markers. Rest of the tumor was processed for histopathology and TMEM analysis. Statistical analysis was performed to identify correlation. Results: Our initial results show that there is a strong correlation between CD44+/CD24− cells in the FNA with TMEM scores. We also report a very high correlation with can also be predicted by the ratios of different splice variants of Mena. This method can be easily used in the clinical setting and applied to a number of different biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4219.