Models Of Inflammatory Breast Cancer Research Articles

Cyclin D1-Dependent Induction of Luminal Inflammatory Breast Tumors by Activated Notch3

Accumulating evidence suggests that Notch3 (N3) is involved in breast cancer development, but its precise contributions are not well understood. Here, we report that pregnant mice expressing an activated intracellular form of N3 (N3(IC)) exhibit a cyclin D1-dependent expansion of premalignant CD24(+) CD29(low) luminal progenitors with enhanced differentiation potential in vitro and in vivo. Parous mice developed luminal mammary tumors in a cyclin D1-dependent manner. Notably, mice expressing higher levels of N3(IC) exhibited tumors resembling inflammatory breast cancer that frequently metastasized. N3(IC)-induced tumors contained a large percentage of tumor-initiating cells, but these were reduced significantly in tumors derived from N3(IC) transgenic mice that were heterozygous for cyclin D1. After transplantation in the presence of normal mammary cells, N3(IC)-expressing tumor cells became less malignant, differentiating into CK6(+) CK18(+) CK5(-) alveolar-like structures akin to expanded luminal progenitors from which they were likely derived. Taken together, our results argue that activated N3 signaling primarily affects luminal progenitors among mammary cell subsets, with more pronounced levels of activation influencing tumor type, and provide a novel model of inflammatory breast cancer.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKβ in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 μM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+ IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-497) contains supplementary material, which is available to authorized users.

Inflammatory breast cancer (IBC): clues for targeted therapies

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2600-4) contains supplementary material, which is available to authorized users.

Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization

Although there is no standard treatment protocol for inflammatory breast cancer (IBC), multi-modality treatment has improved survival. In this study we profiled the NCI approved oncology drug set in a qHTS format to identify those that are efficacious in basal type and ErbB2 overexpressing IBC models. Further, we characterized the sensitivity of an acquired therapeutic resistance model to the oncology drugs. We observed that lapatinib-induced acquired resistance in SUM149 cells led to cross-resistance to other targeted- and chemotherapeutic drugs. Removal of the primary drug to which the model was developed led to re-sensitization to multiple drugs to a degree comparable to the parental cell line; this coincided with the cells regaining the ability to accumulate ROS and reduced expression of anti-apoptotic factors and the antioxidant SOD2. We suggest that our findings provide a unique IBC model system for gaining an understanding of acquired therapeutic resistance and the effect of redox adaptation on anti-cancer drug efficacy.

Abstract 5552: Manganese porphyrins in combination with ascorbate act as pro-oxidants and mediate caspase-independent cancer cell death.

Abstract Resistance to therapy-mediated apoptosis in inflammatory breast cancer (IBC), an aggressive and distinct subtype of breast cancer was recently attributed to increased antioxidant expression (superoxide dismutase [SOD]; reduced glutathione) and decreased accumulation of reactive oxygen species (ROS) resulting in redox adaptation and increased cell survival. Mn porphyrins (MnPs) are amongst the most potent synthetic mimics of the antioxidant, superoxide dismutase (SOD). In the present study, we demonstrate the unique ability of two Mn porphyrin (MnP)-based SOD mimics (MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+) to act as pro-oxidants when combined with ascorbate (vitamin C) and enhance ROS-mediated cell death. We identified that the ethyl species, MnTE-2-PyP5+, was less stable than its butoxyethyl analog, MnTnBuOE-2-PyP5+, resulting in greater potency in inducing cytotoxicity via radical-driven oxidative degradation in cellular models of IBC isolated from patient tumors and an isogenic derivative with acquired resistance to therapeutic apoptosis. The combination of MnP and ascorbate induced accumulation of mitochondrial superoxide and H2O2-derived radicals in both therapy-sensitive and therapy-resistant cell lines. High intracellular ROS resulted in decreases in both pro-survival signaling (pNF-κB, pERK1/2) and expression of X-linked inhibitor of apoptosis protein (XIAP), the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and Annexin V staining, administration of a pan-caspase inhibitor (QVD-OPh) did not reverse the observed cytotoxicity. Nuclear translocation of apoptosis inducing factor (AIF) was observed, suggesting a caspase-independent cell death following mitochondrial outer membrane permeabilization after treatment with this combination. In conclusion, this study provides the molecular basis of a novel MnP-based anti-cancer strategy and suggests the feasibility of using MnPs to sensitize cancer cells to many chemotherapies and radiotherapy that are dependent on potent induction of the oxidative stress response. Citation Format: Myron Evans, Artak Tovmasyan, Ines Batinic-Haberle, Gayathri Devi. Manganese porphyrins in combination with ascorbate act as pro-oxidants and mediate caspase-independent cancer cell death. [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 5552. doi:10.1158/1538-7445.AM2013-5552 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models

The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.

Abstract B69: Mesenchymal stem cells promote tumor-skin involvement in inflammatory breast cancer models

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive variant of breast cancer characterized by blocked breast lymphatics and clinically apparent involvement of the skin resulting in erythema, swelling and a high rate of resistance to therapy. Tumor-skin involvement in IBC patients, despite being a rare event, is an event that develops very rapidly, can spread throughout the upper body (chest and back) and ultimately results in breast cancer death. Very little is known about this event and how to prevent it. Mesenchymal stem cells (MSC) are multipotent progenitor cells which are found in normal tissues, including the bone marrow and adipose tissue. MSC exhibit a unique tropism for tumors, where they engraft, form tumor stroma, and alter the tumor microenvironment. MSC have been shown to increase the growth of certain cancers and the incidence of breast xenograft metastasis. Whether MSC directly modify the primary tumor microenvironment or the pre-metastatic niche remote from the primary tumor has not been addressed in IBC tumor models or patients. Methods and Results: Many IBC xenografts grow tumors but do not faithfully recapitulate the skin phenotype of IBC. In order to investigate whether direct exposure to MSC or paracrine exposure using the MSC-conditioned media (MSC-CM) would influence skin involvement and metastasis development and with this recapitulate the IBC phenotype in pre-clinical models, we performed two different in vivo experiments. In the first, we labeled SUM149 cells with luciferase reporter gene and then injected them into the cleared mammary fat pads of female immunocompromised mice (SCID/Beige) with or without 10% MSC (obtained from healthy donors). Transplants were allowed to grow until 300 mm3 in volume and then resected in a survival surgery. At resection time we found that there was a significant difference in the tumor-skin involvement (p=0.0189, Fisher's exact test) between the 0% MSC group (4/20) and 10% MSC group (11/18). Furthermore, we assessed metastasis development with live bioluminescence imaging and identified unexpected development of metastasis in the upper body skin plexus (31.6% vs. 58.8%, p = NS). In vitro, MSC co-culture is associated with increased EGFR signaling, and tumors in MSC co-injected animals had increased p-EGFR staining by IHC (70.6% vs. 50.0%, p =0.0455). In a second experiment, we cultured in vitro SUM149 cells (labeled with luciferase reporter gene) with or without MSC-CM for 5 days and then injected them into the cleared mammary fat pads of female immunocompromised mice (SCID/Beige), to determine whether the observed tumor-skin involvement was an effect of MSC secreted factors. Transplants were allowed to grow until 300 mm3 in volume and then resected in a survival surgery. MSC-CM increased significantly the tumor-skin involvement (p=0.0227, Fisher's exact test), between the control group (4/16) and MSC-CM group (9/13). We followed up metastasis development with live bioluminescence imaging after tumor resection and observed that several mice developed metastasis in the upper body skin plexus (20.0% vs. 27.2%, p=NS). Conclusion: Our studies show that MSC and MSC-CM increase tumor-skin involvement and metastasis development in an IBC pre-clinical model independent of growth rate. Understanding the role of MSC on the primary tumor microenvironment and the pre-metastatic niche is critical to further develop therapies that target and suppress the tumor-skin involvement and the rapid development of metastasis that IBC patients experience. Citation Format: Lara Lacerda, Daniel Smith, Bisrat Debeb, Travis Solley, Richard Larson, Wei Xu, Savitri Krishnamurthy, Naoto Ueno, Ann Klopp, Wendy Woodward. Mesenchymal stem cells promote tumor-skin involvement in inflammatory breast cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B69.

Abstract P4-06-01: JAK2/STAT3 activity in inflammatory breast cancer supports the investigation of JAK2 therapeutic targeting

Abstract Background: Inflammatory breast cancer (IBC) is a virulent subtype of locally advanced breast cancer that accounts for 1–5% of all breast cancers diagnosed in the United States. Conventional therapy for IBC (preoperative chemotherapy, mastectomy, radiation therapy) results in a median 5 year overall survival of 50%, which supports the need for investigation into the molecular distinctiveness of IBC, with the ultimate goal of developing effective therapeutic agents that target these molecular changes. Some of the distinctive molecular characteristics of IBC include the presence of a substantial proportion of CD44+/CD24- breast cancer cells that express stem cell-like characteristics, over-expression of E-cadherin, and extensive formation of tumor emboli. Examination of breast tumors and pre-clinical data suggests that the JAK2/STAT3 pathway is active in CD44+/CD24- breast cancer cells, and is stimulated by various mechanisms associated with IBC, such as high IL-6 levels. We investigated the role of JAK2/STAT3 activation in IBC as preclinical evidence to pursue JAK2 targeted therapy for IBC in a clinical setting. Methods: Using de-identified breast tissue specimens from both untreated and treated IBC patients obtained on a protocol approved by our institutional review board; we performed immunohistochemical (IHC) and multicolor immunofluorescence analysis to detect the presence of CD44+/CD24- cells and the fraction of activated phospho-STAT3 (pSTAT3). The number of cells positive for pSTAT3 was assessed pre and post-treatment. We also analyzed the status of CD44+/CD24- cells and pSTAT3 in SUM149 and SUM190 IBC cell lines, and an IBC xenograft model (IDC31) derived from primary tumor obtained from an IBC patient. We tested the efficacy of multiple different JAK2 inhibitors to inhibit the growth and viability of cell cultures and xenografts. Results: All IBCs were highly enriched in CD44+/CD24- cells, with approximately 80% of all cancer cells within tumors displaying this phenotype. About 85% of IBC cases show activation of pSTAT3, and a significant fraction (40%) of CD44+/CD24- cells were pSTAT3 positive. Interestingly, the fraction of pSTAT3+ cells was lower in the CD44+/CD24- cells in post treatment samples from triple negative but not from luminal tumors. JAK2 inhibition significantly decreased the proliferation of pSTAT3+ IBC cells lines in vitro and the growth of pSTAT3+ xenografts including the IBC model ICD31. Discussion: Specific molecular characteristics of IBC result in a unique and virulent disease course. Based upon a predominance of CD44+/CD24- breast cancer stem cells present in IBC, we investigated the JAK2/STAT3 pathway in IBC tissue specimens, cell lines and an IBC xenograft model. Our preclinical data demonstrates a highly active JAK2/STAT3 pathway in IBC which lends itself to exploration of the efficacy of a JAK2 inhibitor in the treatment of this disease. A phase I/II study is underway to evaluate combination ruxolitinib and chemotherapy for the primary treatment of triple negative IBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-01.

Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism

X-linked inhibitor of apoptosis protein (XIAP), the most potent mammalian caspase inhibitor, has been associated with acquired therapeutic resistance in inflammatory breast cancer (IBC), an aggressive subset of breast cancer with an extremely poor survival rate. The second mitochondria-derived activator of caspases (Smac) protein is a potent antagonist of IAP proteins and the basis for the development of Smac mimetic drugs. Here, we report for the first time that bivalent Smac mimetic Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells and significantly increases potency of TRAIL-induced apoptosis in TRAIL-sensitive SUM149 (triple-negative, EGFR-activated) cells, two patient tumor-derived IBC models. Birinapant has high binding affinity (nM range) for cIAP1/2 and XIAP. Using isogenic SUM149- and SUM190-derived cells with differential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) and another bivalent Smac mimetic (GT13402) with high cIAP1/2 but low XIAP binding affinity (K (d) > 1 μM), we show that XIAP inhibition is necessary for increasing TRAIL potency. In contrast, single agent efficacy of Birinapant is due to pan-IAP antagonism. Birinapant caused rapid cIAP1 degradation, caspase activation, PARP cleavage, and NF-κB activation. A modest increase in TNF-α production was seen in SUM190 cells following Birinapant treatment, but no increase occurred in SUM149 cells. Exogenous TNF-α addition did not increase Birinapant efficacy. Neutralizing antibodies against TNF-α or TNFR1 knockdown did not reverse cell death. However, pan-caspase inhibitor Q-VD-OPh reversed Birinapant-mediated cell death. In addition, Birinapant in combination or as a single agent decreased colony formation and anchorage-independent growth potential of IBC cells. By demonstrating that Birinapant primes cancer cells for death in an IAP-dependent manner, these findings support the development of Smac mimetics for IBC treatment.

New Therapeutic Targets in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer. Although recognized as a distinct clinical entity, there have been few advances in the development of pre-clinical models of IBC, and a lack of IBC-specific therapeutic targets translated into clinical utility to increase overall survival, which is currently 40 % at three years. By use of newly developed pre-clinical models of IBC and patient tumor tissues, E-cadherin, anaplastic lymphoma kinase (ALK), and HSP90 have been identified as targets relevant to IBC that are matched by therapeutics that are either currently in clinical trials or will be tested in clinical trials within the next year. These exciting results illustrate the advances that have been made in recent years in defining the molecular basis of IBC as a distinct disease and the significant strides made in identifying more effective strategies for treatment of patients with IBC.

XIAP Inhibition and Generation of Reactive Oxygen Species Enhances TRAIL Sensitivity in Inflammatory Breast Cancer Cells

We recently identified superoxide dismutase (SOD) overexpression and decreased induction of reactive oxygen species (ROS)-mediated apoptosis in models of inflammatory breast cancer (IBC) cells with acquired therapeutic resistance. This population of cells has high expression of X-linked inhibitor of apoptosis protein (XIAP), which inhibits both extrinsic and intrinsic apoptosis pathways. We therefore wanted to evaluate the effect of classical apoptosis-inducing agent TRAIL, a proapoptotic receptor agonist that selectively triggers death receptor (DR)-mediated apoptosis in cancer cells, in the IBC acquired resistance model. XIAP levels and subsequent inhibition of caspase activity inversely correlated with TRAIL sensitivity in our models of IBC. These include SUM149, a basal-type cell line isolated from primary IBC tumors and isogenic SUM149-derived lines rSUM149 and SUM149 wtXIAP, models of acquired therapeutic resistance with endogenous and exogenous XIAP overexpression, respectively. Inhibition of XIAP function using embelin, a plant-derived cell permeable small molecule, in combination with TRAIL caused a synergistic decrease in cell viability. Embelin treatment resulted in activation of extracellular signal-regulated kinase (ERK)1/2 and ROS accumulation, which correlated with downregulation of antioxidant protein SOD1 and consumption of redox modulator reduced glutathione in the XIAP-overexpressing cells. Simultaneous treatment with an SOD mimic, which protects against ROS accumulation, reversed the decrease in cell viability caused by embelin + TRAIL treatment. Embelin primes IBC cells for TRAIL-mediated apoptosis by its direct action on the anti-caspase activity of XIAP and by shifting the cellular redox balance toward oxidative stress-mediated apoptosis. Thus, ROS modulators represent a novel approach to enhance efficacy of TRAIL-based treatment protocols in IBC.

The genesis and unique properties of the lymphovascular tumor embolus are because of calpain-regulated proteolysis of E-cadherin

The genesis and unique properties of the lymphovascular tumor embolus are poorly understood largely because of the absence of an experimental model that specifically reflects this important step of tumor progression. The lymphovascular tumor embolus is a blastocyst-like structure resistant to chemotherapy, efficient at metastasis and overexpressing E-cadherin (E-cad). Conventional dogma has regarded E-cad as a metastasis-suppressor gene involved in epithelial-mesenchymal transition. However, within the lymphovascular embolus, E-cad and its proteolytic processing by calpain and other proteases have a dominant oncogenic rather than suppressive role in metastasis formation and tumor cell survival. Studies using a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated the equivalence of xenograft-generated spheroids with lymphovascular emboli in vivo with both structures demonstrating E-cad overexpression and specific proteolytic processing. Western blot revealed full-length (FL) E-cad (120 kDa) and four fragments: E-cad/NTF1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa) and E-cad/NTF4 (80 kDa). Compared with MARY-X, only E-cad/NTF1 was present in the spheroids. E-cad/NTF1 was produced by calpain, E-cad/NTF2 by γ-secretase and E-cad/NTF3 by a matrix metalloproteinase (MMP). Spheroidgenesis and lymphovascular emboli formation are the direct result of calpain-mediated cleavage of E-cad and the generation of E-cad/NTF1 from membrane-associated E-cad rather than the de novo presence of either E-cad/NTF1 or E-cad/CTF1. E-cad/NTF1 retained the p120ctn-binding site but lost both the β-catenin and α-binding sites, facilitating its disassembly from traditional cadherin-based adherens junctions and its 360° distribution around the embolus. This calpain-mediated proteolysis of E-cad generates the formation of the lymphovascular embolus and is responsible for its unique properties of increased homotypic adhesion, apoptosis resistance and budding.

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

Genomic Profiling of Pre-Clinical Models of Inflammatory Breast Cancer Identifies a Signature of Epithelial Plasticity and Suppression of TGFβ Signaling

Abstract Study background: Inflammatory Breast Cancer (IBC) is the most metastatic variant of breast cancer. Although IBC is recognized as a distinct variant of breast cancer, the molecular basis for the rapid progression of IBC remains largely undefined, in part due to the lack of preclinical models that recapitulate the human disease as well as a lack of comprehensive analysis of the preclinical models of IBC that are available. Methods: All available 7 pre-clinical models of IBC, including 2 new models, FC-IBC01 and FC-IBC02 developed from pleural effusion, were used to identify genes and molecular pathways that are selectively altered compared to non IBC breast tumor models. Laser capture micro dissection of biopsy tissue from core biopsy and skin punch biopsies were also analyzed by whole transcriptome analysis. Results: Whole transcriptome analysis defined 7 pre-clinical models of IBC as being within either the triple negative or ErbB2/Her-2 expressing subtypes, similar to the prevalence of these subtypes of breast cancer observed in IBC patients. Comparative analysis of the FC-IBC01, FC-IBC02 and Mary-X models of IBC demonstrated that each of these recapitulate the formation of tumor emboli with encircling lymphovasculogenesis. The majority (6/7) of the pre-clinical models of IBC express CDH1, which encodes for E-cadherin, which was associated with a loss of ZEB1, a transcriptional repressor of E-cadherin. The lack of ZEB1 expression was validated in a limited set of 4 skin punch biopsy samples from IBC patients that were isolated by laser capture micro-dissection, demonstrating concordance with loss of ZEB1 in pre-clinical models of IBC. Expression of other transcription factors involved in acquisition of a cancer stem cell phenotype, including Snai1, which encodes for Snail, SNAI2, which encodes for Slug and TWIST1, was retained in pre-clinical models of IBC. Maintenance of E-cadherin in pre-clinical models of IBC was associated with a loss of genes within the transforming growth factor beta (TGFβ signaling pathway, with expression of SMAD6, a known repressor of TGFβ. This is similar to a recent study reporting the persistence of E-cadherin and loss of TGFβ signaling in IBC patient tumors based on gene profiling of 3 independent data sets. Conclusion: The present studies provide first time comparison of gene signatures of 7 pre-clinical models of IBC, including our 2 newly developed pre-clinical models, FC-IBC01 and FC-IBC02, that recapitulate formation of tumor emboli with encircling lymphatic vessels, similar to that observed in biopsy tissues of IBC patients. We demonstrate that E-cadherin expression was associated with both loss of ZEB1 and diminished expression of multiple genes within the TGFβ signaling pathway, with retention of expression of transcription factors and surface markers consistent with maintenance of a cancer stem cell phenotype, as has been reported to be a characteristic of IBC tumors. Collectively, these observations provide first time characterization of the molecular signatures of all available pre-clinical models of IBC, and suggest that IBC has a signature of epithelial plasticity, with characteristics of their ability to undergo the mesenchymal to epithelial reverting transition. The loss of genes within the TGFβ signaling is also consistent with the tight cell: cell aggregation of IBC tumor cells within tumor emboli that exhibit “cohesive invasion”. The new pre-clinical models of IBC that recapitulate the human disease will serve as useful tools to accelerate our understanding of the molecular underpinnings and therapeutic targets of IBC as the most lethal form of breast cancer.

P4-03-06: Development and Comparative Characterization of Metastasis in Newly Developed Pre-Clinical Models of Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer. It is associated with a poor survival rate (40% 5-year survival) despite appropriate multidisciplinary care. For such an aggressive type of cancer, IBC has been understudied, in part due to the lack of adequate numbers of cell lines and mouse models that recapitulate the human disease. To expand our understanding of IBC, we have obtained all of the previously developed and characterized IBC cell lines and models including Mary-X, SUM149, SUM190, KPL-4, MDA-IBC-3 and have developed two new IBC models, designated as FC-IBC01 and FC-IBC02, using tumor cells derived from pleural effusion of IBC patients. Materials and Methods: Each of these IBC cell lines has been luciferase (LUC)-tagged, allowing the growth of orthotopic injection or subcutaneous implantation to be evaluated by bioluminescent imaging (BLI). Alternatively, the LUC-tagged IBC cells can by injected via either intra-cardiac or intravenous route of delivery, which promotes rapid tumor colonization, resulting in both visceral and skeletal metastasis. Growth of IBC tumors can then be monitored immediately using BLI, thus eliminating the lag time needed for the physical detection of palpable tumors. BLI imaging also allows for monitoring of the kinetics and location of development of metastatic lesions. Whole transcriptome analysis was performed on IBC cell lines and xenograft tissues to define the heterogeneity of IBC as a distinct variant of breast cancer Results: These models have allowed us to identify micro-metastatic foci in multiple sites distant from the IBC primary tumor in each of these models of IBC and allow the quantitation of anti-tumor and anti-metastatic effects of targeted therapeutics as single agents as well as the potential synergy of combinations of agents. As an example, injection of LUC-tagged IBC cell lines such as SUM149-Luc, into the left ventricle of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice allows the metastatic tumor burden to be monitored longitudinally by whole animal BLI, which can be validated at necropsy and by immunohistochemical analysis. Whole transcriptome analysis of pre-clinical models of IBC reflect the molecular subtypes observed in IBC patients, with the majority of IBC models being of the basal like, luminal B and Her2 amplified. Discussion: First time analysis of known and newly developed pre-clinical models of IBC allows a more complete analysis of IBC as a distinct variant of breast cancer. Furthermore, these approaches allow rapid evaluation of the promising targeted therapeutics identified based on whole transcriptome analysis of both IBC patient tumors and pre-clinical models developed from IBC patients. We believe that this extensive collection of LUC-tagged IBC cell lines is an invaluable tool for IBC research since the cell lines encompass the broad spectrum of IBC heterogeneity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-06.

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

P4-09-08: Secretory Leukocyte Protease Inhibitor as a Differential Diagnostic Biomarker of Tumor Emboli in Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of locally advanced breast cancer. Pathologically, IBC is characterized by the presence of nests of cells, defined as tumor emboli, that undergo lymphovascular invasion into the skin and chest wall and represent the metastatic lesion of IBC. Due to the distinct presentation of IBC, it is often misdiagnosed due to a lack of defined biomarkers that are specific for tumor emboli in biopsy samples of IBC patients. To date, the only validated marker that has shown to consistently identify IBC tumor emboli is the surface glycoprotein E-cadherin. Materials and Methods: With the goal of identifying genes specifically expressed by IBC tumor emboli, the present studies performed whole transcriptome analysis of tumor emboli from skin punch biopsy samples from IBC patients and tumor emboli from the Mary-X pre-clinical model of IBC which were isolated using laser capture microdissection. Results: Secretory leukocyte peptidase inhibitor (SLPI) was a gene identified as being highly expressed by tumor emboli. This gene was previously identified as a metastasis related gene, has been reported to be associated with poor prognosis in serous ovarian carcinoma and is implicated in mediating resistance to paclitaxel. Immunofluorescence staining and confocal microscopy revealed that SLPI is a robust marker of tumor emboli in skin punch biopsies from IBC patient and in Mary-X tumor emboli. SLPI differentially delineates between IBC tumor emboli and hair follicles present in the skin, which also stain with E-cadherin. An additional marker, Cytokeratin 15 (CK15), can be used in combination with SLPI to differentially identify the components of the skin that are CK15 positive, and IBC tumor emboli that reside within the skin, which are CK15 negative. Use of podoplanin antibodies, which stain lymphovascular endothelium, demonstrate that IBC tumor emboli that express SLPI protein, are completely encircled within the dermal lymphovasculature. Conclusions: These studies are among the first to identify SLPI as a potential biomarker of tumor emboli in skin punch biopsy samples from IBC patients and in pre-clinical models of IBC. Studies are ongoing to establish the molecular function and role of SLPI in mediating the survival and invasion of IBC tumor emboli. Collectively, these studies are the first to identify SLPI as a potential biomarker of IBC tumor emboli in skin punch biopsy samples. In addition to serving as a potential biomarker of tumor emboli, SLPI may also serve as a therapeutic target for development of approaches to eradicate IBC tumor emboli. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-08.

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

ErbB1/2 tyrosine kinase inhibitor mediates oxidative stress-induced apoptosis in inflammatory breast cancer cells

Overexpression of epidermal growth factor receptors (ErbB) is frequently seen in inflammatory breast cancer (IBC). Treatment with ErbB1/2-targeting agents (lapatinib) mediates tumor apoptosis by downregulating ErbB1/2 phosphorylation and downstream survival signaling. In this study, using carboxy-H(2)DCFDA, DHE, and MitoSOX Red to examine changes in hydrogen peroxide radicals, cytoplasmic and mitochondrial superoxide, respectively, we observed that GW583340 (a lapatinib-analog) increases reactive oxygen species (ROS) in two models of IBC (SUM149, SUM190) that are sensitive to ErbB1/2 blockade. This significant increase in ROS levels was similar to those generated by classical oxidative agents H(2)O(2) and paraquat. In contrast, minimal to basal levels of ROS were measured in a clonal population of GW583340-resistant IBC cells (rSUM149 and rSUM190). The GW583340-resistant IBC cells displayed increased SOD1, SOD2, and glutathione expression, which correlated with decreased sensitivity to the apoptotic-inducing effects of GW583340, H(2)O(2), and paraquat. The ROS increase and cell death in the GW583340-sensitive cells was reversed by simultaneous treatment with a superoxide dismutase (SOD) mimic. Additionally, overcoming the high levels of antioxidants using redox modulators induced apoptosis in the GW583340-resistant cells. Taken together, these data demonstrate a novel mechanism of lapatinib-analog-induced apoptosis and indicate that resistant cells have increased antioxidant potential, which can be overcome by treatment with SOD modulators.

Abstract 5232: Lymphovascular breast carcinoma tumoral emboli beget emboli through stem-cell initiated self-budding histogenesis

Abstract Previous studies in our laboratory have indicated that the lymphovascular embolus is not simply a cellular fragment that detaches from the main tumor passively but represents an active clonal selection for a stem cell phenotype exhibiting enhanced stem cell signaling and survival pathways. Lymphovascular emboli exhibit increased resistance to chemotherapy and patients with large numbers of lymphovascular emboli exhibit decreased disease-free survival and poorer prognosis. Florid lymphovascular emboli is the diagnostic signature of inflammatory breast cancer (IBC) and other aggressive metastasizing cancers. The emboli have been presumed to form as a result of lymphovascular invasion but this event is thought to be rare and therefore would not alone explain the large number of emboli observed in IBC and other highly metastatic carcinomas. The reasons why some patients exhibit large numbers of lymphovascular emboli whereas other patients exhibit only rare numbers remain unknown. We wanted to specifically address this question and thus carried out both animal and in vitro imaging studies with our human xenograft model of inflammatory breast cancer, MARY-X, which exhibits florid emboli in mice and gives rise to high density spheroids in vitro. We also carried out morphometric studies on the density of human lymphovascular emboli in 25 IBC and 250 non-IBC cases and subsequent laser capture microdissection. Animal and in vitro multicolor imaging studies using anti-E-cadherin and anti-podoplanin showed evidence of self-budding histogenesis within the lymphovascular spaces with one parent embolus giving rise to daughter emboli. Correspondingly, budding spheroidgenesis was observed in vitro. Density studies of tumoral emboli in the human cases showed their numbers distributed over an exponential rather than linear range. By both RT-PCR and IHC, emboli compared to their non-embolic invasive carcinoma areas exhibited five-ten fold higher stem cell markers including Stellar, H19, Rex-1, Nestin, CD133 and Aldehyde Dehydrogenase 1 (ALDH1) as well as stem cell transcriptional determinants including OCT4, SOX2 and Nanog, and stem cell signaling pathways, eg., Bmi-1, Hedgehog and Notch3. These studies, while not explaining the genesis of the initial embolus, show conclusively that emboli beget emboli through stem-cell initiated self-budding histogenesis. It is this later process which is the more important one because it accounts for the exponential numbers of emboli seen in the human cases which exhibit aggressive behavior. 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 5232. doi:10.1158/1538-7445.AM2011-5232