Inflammatory Breast Cancer Cell Line SUM149 Research Articles

Inflammatory Breast Cancer: The Secretome of HCMV+ Tumor-Associated Macrophages Enhances Proliferation, Invasion, Colony Formation, and Expression of Cancer Stem Cell Markers.

Inflammatory breast cancer (IBC) is a highly aggressive phenotype of breast cancer that is characterized by a high incidence early metastasis. We previously reported a significant association of human cytomegalovirus (HCMV) DNA in the carcinoma tissues of IBC patients but not in the adjacent normal tissues. HCMV-infected macrophages serve as “mobile vectors” for spreading and disseminating virus to different organs, and IBC cancer tissues are highly infiltrated by tumor-associated macrophages (TAMs) that enhance IBC progression and promote breast cancer stem cell (BCSC)-like properties. Therefore, there is a need to understand the role of HCMV-infected TAMs in IBC progression. The present study aimed to test the effect of the secretome (cytokines and secreted factors) of TAMs derived from HCMV+ monocytes isolated from IBC specimens on the proliferation, invasion, and BCSC abundance when tested on the IBC cell line SUM149. HCMV+ monocytes were isolated from IBC patients during modified radical mastectomy surgery and tested in vitro for polarization into TAMs using the secretome of SUM149 cells. MTT, clonogenic, invasion, real-time PCR arrays, PathScan Intracellular Signaling array, and cytokine arrays were used to characterize the secretome of HCMV+ TAMs for their effect on the progression of SUM149 cells. The results showed that the secretome of HCMV+ TAMs expressed high levels of IL-6, IL-8, and MCP-1 cytokines compared to HCMV- TAMs. In addition, the secretome of HCMV+ TAMs induced the proliferation, invasion, colony formation, and expression of BCSC-related genes in SUM149 cells compared to mock untreated cells. In addition, the secretome of HCMV+ TAMs activated the phosphorylation of intracellular signaling molecules p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK in SUM149 cells. In conclusion, this study shows that the secretome of HCMV+ TAMs enhances the proliferation, invasion, colony formation, and BCSC properties by activating the phosphorylation of p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK intracellular signaling molecules in IBC cells.

Vasculogenic mimicry-associated ultrastructural findings in human and canine inflammatory breast cancer cell lines

BackgroundHuman inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most lethal mammary cancers. An exacerbated angiogenesis and the existence of vasculogenic mimicry (VM) are hallmarks of these tumors. The information regarding VM and ultrastructural characteristics of mammary cell lines is scant.MethodsIn this study, IBC cell line SUM149 and IMC cell line IPC-366 in adherent (2D) and non-adherent (3D) (mammospheres, cancer stem cells) conditions were analyzed by transmission and scanning electron microscopy (TEM and SEM, respectively).ResultsThe TEM revealed round to oval shape cells with microvilli on the surface, high numbers of peroxisomes in close apposition to lipid droplets and some extracellular derived vesicles. The TEM and the SEM mammospheres revealed group of cells clumping together with a central lumen (resembling a mammary acini). The cells joint are tight junctions and zonula adherens. By SEM two cell morphologies were observed: spherical and flattened cells. There was evidence endothelial-like cells (ELCs), which is characteristic for this disease, showing several or unique cytoplasmic empty space. ELCs were more frequent in 3D than in 2D culture conditions and contained Weibel-Palade cytoplasmic bodies, which are exclusive structures of endothelial cells.ConclusionsBoth cell lines, IPC-366 and SUM-149, shared ultrastructural characteristics, further supporting canine IMC as a model for the human disease. To the best of our knowledge, this is the first study that demonstrate the morphological differentiation of cultured cancer stem cells from cancer epithelial cell lines into endothelial-like cells, confirming the vasculogenic mimicry phenomenon from an ultrastructural point of view.

Abstract 3731: A novel heat shock protein 90-targeted photosensitizer (HS-201) enables enhanced tumor-specific photodynamic therapy of inflammatory breast cancers

Abstract Background: Photodynamic therapy (PDT) is an effective local anti-cancer modality applied for the treatment of early stage disease and palliation of advanced disease, and could be used in managing inflammatory breast cancer (IBC). Although PDT can be focused by limiting the area of near infrared (nIR) light exposure, normal tissue injury due to the photosensitizer (PS) uptake by non-tumor tissues remains a barrier to broader applicability. We reported that selective delivery of a compound to malignant cells by exploiting their heat shock protein 90 (Hsp90) activity could be used for imaging breast cancers. This work was extended by the development of an Hsp90 targeted PS (HS201), in which a well characterized PS, verteporfin (VP), has been chemically tethered to a small molecule Hsp90 inhibitor and assessed the tumor-specificity and antitumor efficacy of HS201-based PDT (HS201-PDT). Methods & Results: HS201 was taken up by human breast cancer cell lines, including IBC in vitro more readily than VP, resulting in increased tumor killing in vitro after exposure to a laser with a 690 nm wavelength. The in vivo selectively of HS201 uptake in tumor tissue was confirmed in both human breast cancer xenograft models and spontaneous tumors from HER2-transgenic mice. HS201 accumulation in the tumor was dramatically enhanced after the first laser exposure due to the feed forward increase in Hsp90 expression in the treated tumor cells, and thus a repeated laser exposure was conducted to enhance antitumor effects. This strategy enhanced antitumor effects, as MDA-MB-231 tumors implanted into SCID-beige mice responded to HS201-PDT but not to VP-PDT. Human xenograft breast cancer cell lines, including the IBC cell lines SUM149 and KPL-4, as well as non-IBC cell line BT474M1 and the patient-derived breast cancer xenograft HCI-013, were also tested in SCID-beige mice and demonstrated significantly suppressed tumor growth by HS201-PDT. Conclusions: HS201 showed selective uptake and longer retention by tumor cells, including IBC, especially when the tumors were exposed to local laser treatment. Due to the advantage of this feed-forward control of HS201 distribution, HS201-PDT demonstrated superior anti-tumor effects compared to VP-PDT against various human breast cancer xenografts in mice. These results suggest that HS201-PDT may be used in managing IBC, and may have an important role in anti-cancer therapy. Citation Format: Kensuke Kaneko, Takuya Osada, Philip F. Hughes, Timothy A. Haystead, Michael A. Morse, Herbert K. Lyerly. A novel heat shock protein 90-targeted photosensitizer (HS-201) enables enhanced tumor-specific photodynamic therapy of inflammatory breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3731.

Abstract 3445: Functional interaction between SEPT9_v1 and EGFR in triple-negative and inflammatory breast cancer

Abstract The poor prognosis for patients with inflammatory breast cancer (IBC) emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. Importantly, 20-40% of IBC cases are triple-negative breast cancers (TNBC) in which targeted hormone therapy is not effective. Interestingly, approximately 1/2 of cases of IBC overexpress the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors involve in cell proliferation, migration, and cytoskeleton remodeling. Recently, SEPT9, a member of a large family of cytoskeletal proteins important in diverse biological processes, was found to interact with EGFR. Dysregulation of SEPT9 isoforms have been associated in different cancer types, including breast cancer. To date, no studies have looked at the potential pro-oncogenic role of SEPT9 isoforms in IBC or TNBC. We will examine the functional relationship between SEPT9_v1, cytoskeleton remodeling and EGFR signaling in the IBC cell lines SUM149, SUM190 and TNBCs. Our hypothesis is that SEPT9_v1 contributes to oncogenesis by regulating the activity of ErbB receptors in IBC and/or TNBC. To test our hypothesis, generation of stable cell lines with knockdown or overexpression of SEPT9_v1 using lentiviral and delivering vectors will be developed. Establishment of 2D and 3D cultures of the cell lines will be performed to analyze if the acquisition of pro-oncogenic phenotypes and the effects on cytoskeleton dynamics are SEPT9_v1 and/or EGFR dependent. To assess these phenotypes, we will use cell proliferation assays using alamar blue to determine the viability of cells over time, in vitro transwell migration, invasion assays and fluorescent microscopy to determine if there is aberrant morphology or cytoskeleton remodeling. We will determine the specific effect of SEPT9_v1 overexpression and knockdown to the activation of the EGFR signaling. Stability of ErbB receptors upon SEPT9_v1 overexpression or knockdown will be also measured by protein stability assays and by looking directly in the plasma membrane of cells with fluorescent microscopy. Downstream activation of the EGFR pathway will be assessed by Western blot analysis of activated effector proteins. Co-immunoprecipitation analysis will be performed to test if there is a specific interaction between EGFR and SEPT9_v1. Our preliminary data shows that approximately 50% of breast cancer cell lines overexpress SEPT9_v1 mRNA when compared to a panel of human mammary epithelial cell lines. Also, by WB analysis we determined that SUM149 expresses two isoforms, SEPT9_v1 and SEPT9_v3. This work will provide new insights into the functional relationship between SEPT9_v1 and EGFR and their contribution to the aggressive metastatic phenotype in IBC. Furthermore, it will help us to identify new potential therapeutic targets for the better management of this disease. Citation Format: Amanda Agosto, Jose G. Cirino, Kimberleve Rolón, Gabriel E. Figueroa, Gabriela Martinez, Katixa Montañez-Negrón, Keishla Rodríguez-Mártir, Xavier Bittman, Esther A. Peterson. Functional interaction between SEPT9_v1 and EGFR in triple-negative and inflammatory breast cancer [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 3445.

Abstract PD6-06: Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients

Abstract Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [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 PD6-06.

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars.

Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%–30% in case of women with stage III–IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model.

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs.

Canine cell line, IPC-366, as a good model for the study of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is an aggressive type of cancer with poor survival in women. Inflammatory mammary cancer (IMC) in dogs is very similar to human IBC and it has been proposed as a good surrogate model for study the human disease. The aim was to determine if IPC-366 shared characteristics with the IBC cell line SUM149. The comparison was conducted in terms of ability to grow (adherent and nonadherent conditions), stem cell markers expression using flow cytometry, protein production using western blot and tumorigenic capacity. Our results revealed that both are capable of forming long-term mammospheres with a grape-like morphology. Adherent and nonadherent cultures exhibited fast growth in vivo. Stem cell markers expressions showed that IPC-366 and SUM149 in adherent and nonadherent conditions has mesenchymal-like characteristics, E-cadherin and N-cadherin, was higher in adherent than in nonadherent cultures. Therefore, this study determines that both cell lines are similar and IPC-366 is a good model for the human and canine disease.

The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells.

BackgroundInflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC.MethodsCell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models.ResultsCEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines.ConclusionsCEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0694-4) contains supplementary material, which is available to authorized users.

Abstract P6-14-06: CCR5 antagonists suppresses the migration and invasion of human inflammatory breast cancer cells

Abstract Background: Inflammatory breast cancer (IBC) is a rare and highly metastatic variant of breast cancer with a poor prognosis and lower survival rate. The disease has a peculiar pattern of early recurrence to soft tissue, bone and central nervous system irrespective of molecular subtype. Currently, there are no specific therapeutic options for IBC patients, particularly there are no therapeutic agents aimed at controlling metastatic spread. Chemokine CCL5 and its receptor CCR5 play a significant role in breast cancer progression and metastasis. We have recently shown that CCR5 promotes breast cancer invasiveness and metastatic potential, and CCR5 inhibition by the FDA approved CCR5 antagonists Maraviroc and Vicriviroc reduced in vivo metastasis in a basal-like breast cancer model (Cancer Res 2012;72:3839-3850). Here, we examined the expression of CCR5, as well as the effects of CCR5 inhibition on the migration and invasion in vitro in human IBC cells. Methods: IBC cell lines SUM149, SUM190, and FC-IBC-02 derived from pleural effusion fluid of an IBC patient were examined for the CCR5 expression by immunofluorescence staining. The effects of CCR5 antagonists (Maraviroc and Vicriviroc) on cell migration and invasion in vitro were examined using matrigel-coated Boyden chamber assay in FC-IBC-02 cells, which are triple-negative, basal-like, cancer stem cell phenotype, and rapidly developed primary tumors and metastasis in vivo (Breast Cancer Res Treat 2013;140:23–33). Cells were treated by Maraviroc and Vicriviroc at 100 µM. Results: CCR5 was expressed at low positive component comprising only about 5-7 % of the total cell population in SUM 149 cells. CCR5 expression was not detected in SUM190 cells. CCR5 was expressed at significantly higher levels with a higher percentage of positive population (50-60%) in FC-IBC-02 cells compared with SUM149 cells. FC-IBC-02 cells have a relatively high percentage of CCR5 positive cells in suspension culture. CCR5 inhibition by both CCR5 antagonists Maraviroc and Vicriviroc significantly inhibited the migration and invasion of IBC cells in vitro. Maraviroc and Vicriviroc demonstrated effective agents in controlling migrations (55% and 60% relative inhibition). Conclusions: CCR5 is highly expressed in human IBC cells. CCR5 antagonists demonstrated the inhibition of migration and invasion of IBC cells. Further studies are warranted to determine the effects of CCR5 antagonists in combination with standard treatment in the ability to control IBC progression and metastasis. These results may suggest a potential antimetastatic role for CCR5-inhibitors in IBC. Citation Format: Zhaomei Mu, Xuanmao Jiao, Richard G Pestell, Massimo Cristofanilli. CCR5 antagonists suppresses the migration and invasion of human inflammatory breast cancer cells [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 P6-14-06.

AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor (EGFR), HER2, and HER3: preclinical activity in HER2 non-amplified inflammatory breast cancer models.

IntroductionEpidermal growth factor receptor (EGFR) overexpression has been associated with prognostic and predictive value in inflammatory breast cancer (IBC). Epidermal growth factor receptor 2 (HER2) overexpression is observed at a higher rate in IBC compared with noninflammatory breast cancer. Current clinically available anti-HER2 therapies are effective only in patients with HER2 amplified breast cancer, including IBC. AZD8931 is a novel small-molecule equipotent inhibitor of EGFR, HER2, and HER3 signaling. In this study, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel using preclinical models of EGFR-overexpressed and HER2 non-amplified IBC cells.MethodsTwo IBC cell lines SUM149 and FC-IBC-02 derived from pleural effusion of an IBC patient were used in this study. Cell growth and apoptotic cell death were examined in vitro. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. AZD8931 was given by daily oral gavage at doses of 25 mg/kg, 5 days/week for 4 weeks. Paclitaxel was subcutaneously injected twice weekly.ResultsAZD8931 significantly suppressed cell growth of IBC cells and induced apoptosis of human IBC cells in vitro. Significantly, we showed that AZD8931 monotherapy inhibited xenograft growth and the combination of paclitaxel + AZD8931 was demonstrably more effective than paclitaxel or AZD8931 alone treatment at delaying tumor growth in vivo in orthotopic IBC models.ConclusionAZD8931 single agent and in combination with paclitaxel demonstrated signal inhibition and antitumor activity in EGFR-overexpressed and HER2 non-amplified IBC models. These results suggest that AZD8931 may provide a novel therapeutic strategy for the treatment of IBC patients with HER2 non-amplified tumors.

Abstract B40: Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties

Abstract Introduction: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and is characterized by rapid progression and dismal outcome. Although there is a growing literature describing the role of macrophages in non-IBC, the role of macrophages in IBC is unclear. The aims of the present study were to: 1) assess macrophages infiltration within the tumor tissues of non-IBC and IBC patients; 2) isolate and profile cytokines of macrophages drained from breast tumor axillary tributaries of non-IBC and IBC patients and 3) test whether major cytokines secreted by tumor draining macrophages induce motility of the IBC cell line SUM149. Methods: Tumor-infiltrating macrophages were measured by immunohistochemical (IHC) techniques using CD14 and CD68 monoclonal antibodies. Blood drained from axillary vein tributaries was collected from non-IBC and IBC patients during breast cancer surgery and total mononuclear cells isolated by density gradient centrifugation using Histopaque. The percentage of monocytes/macrophages (CD14+) in the total isolated leukocytes was calculated by flow cytometry using fluorochrome-labeled monoclonal antibodies (APC-CD14 and PerCP-CD3). CD14+ population was separated from total mononuclear cells using an immuno-magnetic bead separation technique. CD14+ cells were seeded overnight in appropriate culture media at 37°C in a humidified CO2 incubator and conditioned media were collected and cytokines profiled. To test whether cytokines secreted by CD14+ enhances motility and invasion of SUM149 cells, we used wound healing and invasion assays. Results: Our results revealed that there was an increase in the number of infiltrated macrophages (CD14+ cells) in IBC tumor tissues and axillary tributaries drained blood as compared to non-IBC. Cytokine profiling of IBC-CD14+ cells revealed a significant increase in tumor necrosis factor-alpha (TNF-α); monocyte chemoattractant protein-1 (MCP-1) also known as CC-chemokine ligand 2 (CCL-2); interleukin-10 (IL-10) and interleukin-8 (IL-8) as compared to non-IBC CD14+ cells. TNF-α, IL-8 and IL-10 cytokines significantly increased motility and invasion of SUM149 cells. Conclusion: IBC cancer tissues are characterized by high infiltration of monocytes/macrophages that secrete cytokines that may contribute to dissemination of IBC cells. Citation Format: Mona Mostafa Mohamed, Eslam A. El-Ghonaimy, Mohamed A. Nouh, Robert J. Schneider, Bonnie F. Sloane, Mohamed El-Shinawi. Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B40.

Abstract 1600: Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells.

Abstract Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths although contributing to a small percentage (∼1%) of total breast cancer cases each year. The hallmarks of IBC are skin involvement and a high propensity to metastasize. It is thought that a small population of stem-like cancer cells is responsible for the metastatic potential and treatment resistance to therapies such as radiation therapy in IBC. In this study we explored the radiosensitization of several breast cancer cell lines in vitro with simvastatin and the mechanism for this. Methods: In order to determine cell proliferation, breast cancer cell lines SUM149, SUM159 and MCF-7 were grown in 96 well plates with different concentrations of simvastatin for one week. In order to determine the effect of simvastatin in stem-like cancer cells, cells were seeded in serum free, growth factor enriched, anchorage independent, treated with simvastatin for 24 hours and mammosphere formation was quantified a week later. In addition, SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and in stem cell enriching anchorage independent cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated using Sigmaplot 8.0 and t-test was used to compare surviving fraction (SF) of groups. Finally, cell lysates were obtained from cultures untreated and treated with simvastatin and radiation and analyzed through western blot. Results: Simvastatin significantly decreased cancer cell proliferation in vitro in all cell lines (p<0.002). Cell lines SUM 159 and MCF 7 grown in mammosphere enriched media showed significant decreased mammosphere formation when grown in the presence of simvastatin (p<0.05). Moreover, simvastatin radiosensitized all cell lines in both monolayer and anchorage independent clonogenic assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p<0.0001) and in stem cell enriching cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p<0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p<0.001) than in stem cell enriching cultures. Combination of simvastatin and radiation therapy decreased protein expression of phospho-rho A, VEGF-A, and phospho-ras. Conclusions: IBC remains a difficult disease to treat with a high rate of local recurrence after radiation therapy. Statins have been shown to increase tumor free survival in breast cancer. Herein we show simvastatin targets breast cancer stem-like cells resulting in radiosensitizing of IBC in vitro. Citation Format: Adam R. Wolfe, Lara Lacerda, Richard Larson, Bisrat G. Debeb, Wei Xu, Naoto T. Ueno, Wendy A. Woodward. Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1600. doi:10.1158/1538-7445.AM2013-1600

In Vitro Assessment of the Inflammatory Breast Cancer Cell Line SUM 149: Discovery of 2 Single Nucleotide Polymorphisms in the RNase L Gene

Background: Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer. The mechanism of IBC carcinogenesis remains unknown. We sought to evaluate potential genetic risk factors for IBC and whether or not the IBC cell lines SUM149 and SUM190 demonstrated evidence of viral infection.Methods: We performed single nucleotide polymorphism (SNP) genotyping for 2 variants of the ribonuclease (RNase) L gene that have been correlated with the risk of prostate cancer due to a possible viral etiology. We evaluated dose-response to treatment with interferon-alpha (IFN-α); and assayed for evidence of the putative human mammary tumor virus (HMTV, which has been implicated in IBC) in SUM149 cells. A bioinformatic analysis was performed to evaluate expression of RNase L in IBC and non-IBC.Results: 2 of 2 IBC cell lines were homozygous for RNase L common missense variants 462 and 541; whereas 2 of 10 non-IBC cell lines were homozygous positive for the 462 variant (p= 0.09) and 0 of 10 non-IBC cell lines were homozygous positive for the 541 variant (p = 0.015). Our real-time polymerase chain reaction (RT-PCR) and Southern blot analysis for sequences of HMTV revealed no evidence of the putative viral genome.Conclusion: We discovered 2 SNPs in the RNase L gene that were homozygously present in IBC cell lines. The 462 variant was absent in non-IBC lines. Our discovery of these SNPs present in IBC cell lines suggests a possible biomarker for risk of IBC. We found no evidence of HMTV in SUM149 cells. A query of a panel of human IBC and non-IBC samples showed no difference in RNase L expression. Further studies of the RNase L 462 and 541 variants in IBC tissues are warranted to validate our in vitro findings.

Abstract PD03-06: Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with post-mastectomy radiation

Abstract Among women with non-inflammatory triple-negative and triple-negative inflammatory breast cancer (IBC), the 5-yr actuarial rates of local failure after radiation are 11%–35% and 45%, respectively, in part influenced by the contribution of radioresistant cancer stem cells to these cancers. Herein we explored the radiosensitization of breast cancer stem-like cells in vitro and examined the influence on local control after post-mastectomy radiation (PMRT) among IBC patients taking statins. SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and stem cell enriching anchorage independent clonogenic cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated and t-test was used to compare surviving fraction (SF) of groups. p < 0.05 was considered significant. Clinical information was extracted from the charts of 534 Stage III IBC patients treated with neoadjuvant systemic therapy, mastectomy and PMRT from 1970–2011. Clinical characteristics between statin and no statin cohorts were compared using the chi-square statistic. Local regional recurrence-free survival (LRFS) was determined using the Kaplan Meier method and compared with log rank. Simvastatin radiosensitized all cell lines in both types of clonogenic culture assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p < 0.0001) and in anchorage independent cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p < 0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p < 0.001) than in stem cell enriching cultures. In a retrospective clinical study of 534 IBC patients, 84 patients used a statin. Median follow-up time for the entire cohort was 2.6 yrs. As expected, statin users were older (80% vs 44% over 50, p < 0.001) and were more often obese than non-statin users (59% vs 42%, p < 0.001). Receptor status was balanced between these groups. Statin users were more often pathologically node-negative (p < 0.01) and had fewer grade III tumors (p < 0.04). On univariate analysis actuarial 3-yr LRFS was higher among statin users, 89% vs 75%, p = 0.009. On multivariate analyisis TNBC (HR = 0.369, p < 0.0001), lymphatic invasion (HR = 0.430, p = 0.0007), neoadjuvant pathological response (HR = 0.242, p = 0.006) and statin use (HR = 0.422, p = 0.03) were independently associated with higher LRFS. Patients with IBC and triple negative non-IBC breast cancer have the highest rates of local failure and no available known radiosensitizers. Here we report significant improvement in local control after PMRT among statin users with IBC and significant radiosensitization across triple-negative and IBC cell lines of multiple subtypes using simvastatin. Clinical value in patients without hypercholesterolemia remains to be established. These encouraging data suggest simvastatin may be an appropriate radiosensitizing agent for clinical trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-06.

Abstract LB-387: EZH2 knockdown suppresses the growth of human inflammatory breast cancer cells both in vitro and in vivo in xenograft models

Abstract PURPOSE: Inflammatory breast cancer (IBC) is a rare but very aggressive type of advanced breast cancer with a poor prognosis. Thus, there is an urgent need for novel IBC therapeutics. Enhancer of zeste homolog 2 (EZH2) is of the catalytic subunit of the polycomb repressive complex 2, which silences the expression of its target genes by generating trimethylation of lysine 27 on histone H3 (H3K27Me3). EZH2 is overexpressed in several types of cancer including IBC and its overexpression correlates with a poor prognosis in IBC patients. Here, we investigated the molecular basis by which EZH2 promotes IBC. METHODS: IBC cell lines SUM149 and FC-IBC-02 which were developed from pleural effusion of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. MTS and soft-agar colony formation assays were used for anchorage-dependent and -independent cell growth in vitro, respectively. xCELLigence system was used to determine the cell migration and invasion. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. RESULTS: EZH2 was expressed at higher levels in all tested human IBC cell lines when compared with non-transformed human mammary epithelial cells. Knockdown of EZH2 caused a significant reduction in the levels of H3K27Me3 in human IBC cell lines. Notably, knockdown of EZH2 expression significantly suppressed both anchorage-dependent and -independent growth of human IBC cells in vitro due to induction of apoptosis. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the growth IBC cells in vivo in an orthotopic IBC model. CONCLUSIONS: EZH2 knockdown suppresses the growth of human IBC cells in vitro and in vivo in an orthotopic IBC model in immunodeficient mice. In addition, EZH2 knockdown inhibits the invasion of human IBC cells. Together, we conclude that EZH2 contributes to IBC by promoting proliferation and invasion of human IBC cells. These results suggest that targeting EZH2 may provide a novel therapeutic strategy for the treatment of patients with IBC. 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-387. doi:1538-7445.AM2012-LB-387

Alternate Estrogen Receptors Promote Invasion of Inflammatory Breast Cancer Cells via Non-Genomic Signaling

Although Inflammatory Breast Cancer (IBC) is a rare and an aggressive type of locally advanced breast cancer with a generally worst prognosis, little work has been done in identifying the status of non-genomic signaling in the invasiveness of IBC. The present study was performed to explore the status of non-genomic signaling as affected by various estrogenic and anti-estrogenic agents in IBC cell lines SUM149 and SUM190. We have identified the presence of estrogen receptor α (ERα) variant, ERα36 in SUM149 and SUM190 cells. This variant as well as ERβ was present in a substantial concentration in IBC cells. The treatment with estradiol (E2), anti-estrogenic agents 4-hydroxytamoxifen and ICI 182780, ERβ specific ligand DPN and GPR30 agonist G1 led to a rapid activation of p-ERK1/2, suggesting the involvement of ERα36, ERβ and GPR30 in the non-genomic signaling pathway in these cells. We also found a substantial increase in the cell migration and invasiveness of SUM149 cells upon the treatment with these ligands. Both basal and ligand-induced migration and invasiveness of SUM149 cells were drastically reduced in the presence of MEK inhibitor U0126, implicating that the phosphorylation of ERK1/2 by MEK is involved in the observed motility and invasiveness of IBC cells. We also provide evidence for the upregulation of p-ERK1/2 through immunostaining in IBC patient samples. These findings suggest a role of non-genomic signaling through the activation of p-ERK1/2 in the hormonal dependence of IBC by a combination of estrogen receptors. These findings only explain the failure of traditional anti-estrogen therapies in ER-positive IBC which induces the non-genomic signaling, but also opens newer avenues for design of modified therapies targeting these estrogen receptors.

Monocytes conditioned media stimulate fibronectin expression and spreading of inflammatory breast cancer cells in three-dimensional culture: A mechanism mediated by IL-8 signaling pathway

BackgroundInflammatory breast cancer (IBC) is the most aggressive form of breast cancer characterized by invasion of carcinoma cells into dermal lymphatic vessels where they form tumor emboli over expressing adhesion molecule E-cadherin. Although invasion and metastasis are dynamic processes controlled by complex interaction between tumor cells and microenvironment the mechanisms by which soluble mediators may regulate motility and invasion of IBC cells are poorly understood. The present study investigated the effect of media conditioned by human monocytes U937 secreted cytokines, chemokines and growth factors on the expression of adhesion molecules E-cadherin and fibronectin of human IBC cell line SUM149. Furthermore, cytokines signaling pathway involved were also identified.ResultsU937 secreted cytokines, chemokines and growth factors were characterized by cytokine antibody array. The major U937 secreted cytokines/chemokines were interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1/CCL2). When SUM149 cells were seeded in three dimensional (3D) models with media conditioned by U937 secreted cytokines, chemokines and growth factors; results showed: 1) changes in the morphology of IBC cells from epithelial to migratory spindle shape branched like structures; 2) Over-expression of adhesion molecule fibronectin and not E-cadherin. Further analysis revealed that over-expression of fibronectin may be mediated by IL-8 via PI3K/Akt signaling pathway.ConclusionThe present results suggested that cytokines secreted by human monocytes may promote chemotactic migration and spreading of IBC cell lines. Results also indicated that IL-8 the major secreted cytokine by U937 cells may play essential role in fibronectin expression by SUM149 cells via interaction with IL-8 specific receptors and stimulation of PI3K/Akt signaling pathway.

Abstract P5-05-05: Tazarotene-Induced Gene 1(TIG1) Is a Novel Tumorigenic Gene of Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of advanced breast cancer. Effective standard therapies for IBC are limited and the molecular mechanisms underlying IBC are largely unknown. In a comparison of samples from patients with IBC and non-IBC, our laboratory identified TIG1 (tazarotene-induced gene 1), also known as retinoic acid receptor responder 1 (RARRES1) as the gene with the largest difference in expression between the two groups. TIG1 is specifically expressed in IBC cell lines (SUM149 and KPL-4). In addition, TIG1 has been identified as a top druggable gene for triple-negative breast cancer. Of particular interest is that TIG1 is significantly expressed in IBC ER-/HER2- patient samples when compared with non-IBC ER-/HER2- patient samples (p = 0.084).Therefore, we hypothesize that TIG1 plays an important role in the malignant process of IBC and it promotes the tumorigenesis and metastasis of IBC. In these studies we determined the tumorigenic and metastatic activities of TIG1 in IBC. Methods: TIG1 was stably knocked down in the highly TIG1-expressing IBC cell line SUM149 and the effects of knockdown on cell proliferation, migration, invasion, and growth in three-dimensional culture were analyzed in vitro. To evaluate the tumorigenic activity of TIG1 in vivo, TIG1 stable-knockdown SUM149 cells and control shRNA-transfected cells were implanted into the mammary fat pads of athymic nude mice, and tumor growth was monitored. To investigate the underlying mechanism of TIG1- mediated tumorigenesis and metastasis of IBC, the gene expression profiles of control siRNA-transfected and TIG1 siRNA-transfected SUM149 cells were compared using cDNA microarray analysis. Results: Suppressing TIG1 significantly inhibited the proliferation, migration, and invasion of IBC cells in vitro. SUM149 cells exhibit a mesenchymal-like phenotype when grown in three-dimensional culture. However, knocking down TIG1 reversed the mesenchymal-like phenotype to the epithelial phenotype in three-dimensional culture, suggesting that TIG1 might have metastatic activity in IBC cells. TIG1 knockdown dramatically inhibited IBC tumor growth in a xenograft model. Several differentially expressed genes between control siRNA-transfected and TIG1 siRNA-transfected SUM149 cells, including Decorin, GBP1, and VAV3 were identified using cDNA microarray analysis. These genes have been reported to affect a number of biological processes, including proliferation, metastasis, inflammatory response, and angiogenesis. We will focus on these candidates and determine the molecular mechanism by which TIG1 may function as a tumorigenic gene in IBC. Conclusion: Our data demonstrate that TIG1 promotes the tumorigenesis and metastasis of IBC and it may contribute to the development and aggressiveness of IBC. Our long-term goal is to develop TIG1 as a therapeutic target for this extremely aggressive type of breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-05.

Abstract 1461: Zoledronate inhibits proliferation and invasion of inflammatory breast cancer cells

Abstract Introduction: Inflammatory breast cancer (IBC) is aggressive, as approximately one third of patients present with distant metastases. Bisphosphonates are often administered to breast cancer patients for cancer-induced osteolysis. Recent evidence suggests bisphophanates may also have anti-tumor properties. This study examined the effects of the bisphosphonate, zoledronate (ZA), on the proliferation and invasive capabilities of highly aggressive IBC cell lines. Methods: The IBC cell line SUM149 was cultured utilizing both adherent and serum-free, non-adherent cancer stem cell enriching conditions. An MTT assay was used to quantify proliferation and mammosphere formation of cells. Adherent cell growth was measured at days 1-4 following 0, 0.1, 1, 5, 10, 50 and 100uM zelodronate treatment. Mammosphere formation was measured at 10 days following 0, 0.01, 0.1, 1, 10, and 100uM ZA treatment. Modified Boyden chambers were employed to assess the ability of both adherent and non-adherent cells to invade to SDF-1 (100ng/mL) following 24 hour 1uM ZA treatment. Protein expression was assessed using immunofluorescent staining, protein arrays and Western blotting. Results: ZA treatment with <5uM did not inhibit SUM149 adherent cell growth at any of the measured time points. ZA concentrations ≥10uM significantly inhibited SUM149 adherent cell growth at day 4 (p≤0.05). SUM 149 mammosphere formation was significantly inhibited following 10uM ZA treatment (p≤0.001). However, significantly more SUM149 non-adherent cells invaded to SDF-1 compared to adherent cells (p≤0.001). SUM 149 non-adherent cell invasion was inhibited by 24 hour 1uM ZA treatment (p≤0.001). SUM 149 non-adherent cells expressed significantly increased levels of the epithelial-mesenchymal transition (EMT) associated proteins smooth muscle actin (SMA), fibronectin, and N-Cadherin. The increased expression of SMA, fibronectin, and N-Cadherin observed in non-adherent cells returned to levels comparable to adherent cells following 24 hour 1uM ZA treatment. Conclusions: ZA inhibited the growth of IBC cells grown under adherent and non-adherent conditions. SUM 149 non-adherent cells had much greater invasive potential than adherent cells did, and their invasive capability was inhibited following low dose ZA treatment. Interestingly, ZA inhibited up-regulation of EMT associated proteins SMA, fibronectin, and N-Cadherin in SUM 149 non-adherent cells. These results indicate that some IBC cells grown under cancer stem cell enriching conditions have the potential to undergo EMT and invade. ZA may also inhibit metastases through novel mechanisms. 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 1461.