Models Of Inflammatory Breast Cancer Research Articles

Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of canine inflammatory mammarycancer

BackgroundIndole-3-carbinol, derived from Cruciferous vegetables is an estrogen receptor antagonist considered a preventive agent that is naturally present in diet. There are no previous studies on its effects in human inflammatory breast cancer or canine inflammatory mammary cancer that is the most aggressive type of breast cancer.MethodsThe aim of this study was to analyze the effect of indole-3-carbinol on a SCID mice xenograft model of canine inflammatory mammary cancer, using equivalent human oral dose as a preventive therapy in humans for 3 weeks.ResultsIndole-3-carbinol treatment decreased tumor proliferation and increased apoptosis, although tumor embolization and liver metastasis were observed in some animals. There was a characteristic subpopulation of lipid-rich cells and increased contents of select steroid hormones in tumor homogenates and serum.ConclusionsOur data reveal for the first time that the ingestion of indole-3-carbinol, as administered, diminishes proliferation and increases apoptosis of tumor cells in an experimental model of inflammatory breast cancer, although this effect could not be enough to avoid the appearance of tumor embolization and metastasis. Future clinical trials will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved.

Abstract B49: Profiling of GLI antagonists in phenotypic models for effects on inflammatory breast cancer cell growth

Abstract Our objective was to assess GLI pathway antagonists for activity in inflammatory breast cancer (IBC) phenotypic models. Introduction: IBC is one of the most lethal forms of breast cancer and accounts for ~15% of all breast cancer deaths. IBC affects younger patients and is diagnosed in minorities at higher rates than in other racial and ethnic groups1. African-American women with IBC appear to have worse survival regardless of inflammatory status, stage, socioeconomic position, tumor, or treatment characteristics2,3. IBC is characterized by rapid progression, is highly invasive and is not usually detected by mammograms or ultrasounds. The presence of tumor emboli (TE) is a hallmark of the disease in which distinctive clusters of tumor cells migrate collectively and drive lymphatic invasion and metastasis. Activation of GLI1, the terminal effector of hedgehog (Hh) signaling, via gene amplification, has been linked to tumorigenesis, and invasiveness in many cancers including our studies on IBC4. Targeting at the level of GLI1 with small molecules has been effective with some demonstrating activity in vivo. Experimental procedures: In our studies we have used a tiered approach of phenotypic and functional assays to evaluate a panel of GLI antagonists for effects on IBC cell growth, in particular assessing their effects in models that more closely model or predict tumor growth in vivo including; migration, anchorage independent growth (AIG), mammosphere formation and TE assays. Results: Using our previously described quantitative cell proliferation assay5 we first identified a subset of GLI antagonists that effected IBC proliferation and down-regulated GLI1 transcriptional activity. We have now used high content imaging to simultaneously measure nuclear count, morphology, cell integrity, and mitochondrial health in live cells. We have identified GLI antagonists that show an anti-proliferative response but have no effect on nuclear size suggesting a lack of general cytotoxicity. In contrast, other antagonists had dramatic effects on nuclear area and nuclear texture indicative of cytotoxicity. We further examined their effects on colony formation using the AIG assay. SUM149 cells were treated with GLI inhibitors and effects on colony size and number noted. All three GLI antagonists tested reduced the size of the colonies with one also giving a significant reduction in number of colonies formed. Recently we have optimized an in vitro TE assay and are using this to screen GLI antagonists for effects on TE formation. Conclusion: we have identified a subset of GLI antagonists by phenotypic profiling for those with effects in cancer-specific functional assays that will be prioritized for testing in vivo. Funded in part by DOD/CDMRP IDEA (BC121850) (KPW). 1 Anderson, W. F., Schairer, C., Chen, B. E., Hance, K. W. & Levine, P. H. Epidemiology of inflammatory breast cancer (IBC). Breast disease 22, 9-23 (2006). 2 Schlichting, J. A., Soliman, A. S., Schairer, C., Schottenfeld, D. & Merajver, S. D. Inflammatory and non-inflammatory breast cancer survival by socioeconomic position in the Surveillance, Epidemiology, and End Results database, 1990-2008. Breast Cancer Research and Treatment 134, 1257-1268 (2012). 3 Yang, R. et al. A comprehensive evaluation of outcomes for inflammatory breast cancer. Breast cancer research and treatment 117, 631-641 (2009). 4 Thomas, Z. et al. Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104, 1575-1586, doi:10.1038/bjc.2011.133 (2011). 5 Williams, K. P. et al. Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Letters 337, 77-89 (2013). Citation Format: Helen Oladapo, Scott Sauer, Michael Tarpley, Gayathri Devi, Kevin P. Williams. Profiling of GLI antagonists in phenotypic models for effects on inflammatory breast cancer cell growth. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B49.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including (13)C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Abstract P5-04-02: The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation

Abstract Purpose: Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the two most aggressive types of breast cancer whose molecular mechanisms remain unclear, representing a challenge for the development of effective targeted therapies. Single agent targeted therapies are of limited effectiveness in these types of breast cancer. Therefore, we aim to identify new combination therapeutic candidates for these aggressive diseases by comprehensive genomic screening. Experimental Design: We screened kinome siRNA libraries with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor [pimasertib], and genome-wide functional mRNA expression with the histone deacetylase (HDAC) type I inhibitor [entinostat] in TNBC and IBC cell lines. We evaluated the relationship between targets of interest and breast cancer patient survival using the IBC consortium database composed of breast cancer patient samples with clinical follow up. After identifying the targets, we assessed the combinational synergistic effect and its mechanism via cytotoxicity assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, western blotting, and mammary fat pad xenograft mouse models. Results: We identified that knock-down of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, enhanced the anti-proliferative effect of pimasertib. We observed that entinostat induced the expression of Noxa, a pro-apoptotic BH3-only member of the Bcl-2 family that is known to bind and degrade Mcl-1. Interestingly, in a breast cancer patient cohort (N = 389), high Mcl-1/low Noxa co-expression was associated with poorer survival outcomes than low Mcl-1/high Noxa co-expression (P = 0.0038). We found that combination with pimasertib and entinostat enhanced the inhibition of tumor cell proliferation (P < 0.001) compared with entinostat or pimasertib alone. We also observed significant in vivo tumor growth inhibition in both IBC (SUM190, P < 0.0001) and TNBC (SUM149, P < 0.05) xenograft models. Specifically, TNBC and IBC cell lines that overexpressed Noxa after treatment with entinostat were observed to be selectively sensitive to combination treatment with pimasertib. The synergistic antitumor activity of the entinostat- pimasertib combination was due to increased expression of Noxa, which induced the degradation of Mcl-1, resulting in the induction of mitochondrial cell death. Conclusion: Our data provide evidence that entinostat has enhanced antitumor effect in combination with pimasertib, resulting in the induction of apoptosis by Noxa-mediated Mcl-1 degradation. These findings provide a novel preclinical rationale for developing a clinical trial based on combinatorial HDAC and MEK inhibition therapy for TNBC and IBC with high Mcl-1 expression. Citation Format: Torres-Adorno AM, Lee JJ, Kogawa T, Bartholomeusz C, Pitner MK, Ordentlich P, Lim B, Tripathy D, Ueno NT. The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-02.

Steroid Tumor Environment in Male and Female Mice Model of Canine and Human Inflammatory Breast Cancer.

Canine inflammatory mammary cancer (IMC) shares clinical and histopathological characteristics with human inflammatory breast cancer (IBC) and has been proposed as a good model for studying the human disease. The aim of this study was to evaluate the capacity of female and male mice to reproduce IMC and IBC tumors and identify the hormonal tumor environment. To perform the study sixty 6–8-week-old male and female mice were inoculated subcutaneously with a suspension of 106IPC-366 and SUM149 cells. Tumors and serum were collected and used for hormonal analysis. Results revealed that IPC-366 reproduced tumors in 90% of males inoculated after 2 weeks compared with 100% of females that reproduced tumor at the same time. SUM149 reproduced tumors in 40% of males instead of 80% of females that reproduced tumors after 4 weeks. Both cell lines produce distant metastasis in lungs being higher than the metastatic rates in females. EIA analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors. Serum steroid levels were lower than those found in tumors. In conclusion, IBC and IMC male mouse model is useful as a tool for IBC research and those circulating estrogens and intratumoral hormonal levels are crucial in the development and progression of tumors.

Photodynamic therapy as an effective therapeutic approach in MAME models of inflammatory breast cancer

Photodynamic therapy (PDT) is a minimally invasive, FDA-approved therapy for treatment of endobronchial and esophageal cancers that are accessible to light. Inflammatory breast cancer (IBC) is an aggressive and highly metastatic form of breast cancer that spreads to dermal lymphatics, a site that would be accessible to light. IBC patients have a relatively poor survival rate due to lack of targeted therapies. The use of PDT is underexplored for breast cancers but has been proposed for treatment of subtypes for which a targeted therapy is unavailable. We optimized and used a 3D mammary architecture and microenvironment engineering (MAME) model of IBC to examine the effects of PDT using two treatment protocols. The first protocol used benzoporphyrin derivative monoacid A (BPD) activated at doses ranging from 45 to 540mJ/cm(2). The second PDT protocol used two photosensitizers: mono-L-aspartyl chlorin e6 (NPe6) and BPD that were sequentially activated. Photokilling by PDT was assessed by live-dead assays. Using a MAME model of IBC, we have shown a significant dose-response in photokilling by BPD-PDT. Sequential activation of NPe6 followed by BPD is more effective in photokilling of tumor cells than BPD alone. Sequential activation at light doses of 45mJ/cm(2) for each agent resulted in >90% cell death, a response only achieved by BPD-PDT at a dose of 360mJ/cm(2). Our data also show that effects of PDT on a volumetric measurement of 3D MAME structures reflect efficacy of PDT treatment. Our study is the first to demonstrate the potential of PDT for treating IBC.

A Preclinical Model of Inflammatory Breast Cancer to Study the Involvement of CXCR4 and ACKR3 in the Metastatic Process

Inflammatory breast cancer (IBC) is an aggressive and invasive tumor, accounting for 2.5% of all breast cancer cases, and characterized by rapid progression, regional and distant metastases, younger age of onset, and lower overall survival. Presently, there are no effective therapies against IBC and a paucity of model systems. Our aim was to develop a clinically relevant IBC model that would allow investigations on the role of chemokine receptors in IBC metastasis. Primary cultures of tumor cells were isolated from pleural exudates of an IBC patient and grown as spheres or monolayers. We developed a human xenograft model where patient-derived IBC cells, stably transduced with lentiviral vectors expressing fluorescent and bioluminescent markers, were inoculated directly into the left ventricle of mice. Our in vivo data show that these IBC cells (FC-IBC02A) are able to seed and proliferate into various organs, including brain, lungs, lymph nodes, and bone, closely replicating the metastatic spread observed in IBC patients. Moreover, cells were able to generate tumors when grafted in the mammary fat pad of mice. RT-PCR and microscopy studies revealed expression of both CXCR4 and ACKR3 receptors in FC-IBC02A cells. Furthermore, CXCL12 (the endogenous chemokine ligand of these receptors) induced transendothelial migration of these cells and stimulated signaling pathways involved in cell survival and migration - an effect reduced by CXCR4 or ACKR3 antagonists. This new model can be used to develop chemokine-based pharmacological approaches against the IBC metastatic process. This work also provides the first evidence of ACKR3 expression in IBC cells.

Abstract 3232: CEP-37440, a highly selective and potent dual inhibitor of ALK and FAK1 inhibits the proliferation of inflammatory breast cancer cells

Abstract Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. Due to its propensity to rapidly metastasize, IBC is responsible for a disproportionate number of breast cancer-related deaths; 15% of all breast cancer deaths result from this disease. Approximately 30% of IBC patients have distant metastases at time of diagnosis, in contrast to 5% of patients with non-IBC breast cancers. Currently, there is no adequate adjuvant therapy to reduce the risk of recurrence and mortality in IBC patients. We recently found that FAK1, also known as PTK2, is amplified, up-regulated and phosphorylated (active) in some inflammatory breast cancer cells [1]. The focal adhesion kinase FAK1 is a cytoplasmic tyrosine kinase that localizes to focal adhesions, and controls a number of cell pathways including proliferation, viability and survival. Activated FAK1 is absent or low in normal tissue and benign neoplasms and up-regulated in invasive and metastatic tumors. In this study, we investigated a new dual FAK1/ALK inhibitor, CEP-37440, in vitro and in vivo using preclinical models of IBC. We found that 300 nM CEP-37440 was able to decrease the proliferation of the triple negative FC-IBC02 cell line and, 1000 nM CEP-37440 was able to complete inhibit the proliferation of these cells. SUM190 showed a 50% decreased in proliferation when these cells were treated with 1000 nM CEP-37440. Genes involved in functions such as cellular growth and proliferation, cell cycle, cell death and survival and cellular movement and cell-to cell signaling and interaction were differentially regulated by CEP-37440 in sensitive IBC cell lines. Among them, TGFβ1 and MMP3 were down-regulated and DKK3, CAV1 and TFPI2 were up-regulated by CEP-37440. In FC-IBC02 orthotopic breast cancer xenograft models, CEP-37440 was able to reduce the growth of the primary tumor and inhibit the development of spontaneous metastases in brain. In conclusion, CEP-37440 was highly effective in reducing the proliferation in vitro and in vivo of IBC cells that expressed high levels of phospho-FAK (Tyr397).

Abstract 680: Identification of GLI1 antagonists for breast cancer therapy

Abstract Introduction: The transcription factor, glioma-associated oncogene homolog 1 (GLI1) is a downstream marker for hedgehog (Hh) pathway activation. Recently, Hh-GLI has emerged as a central pathway target in several human cancers (breast, leukemia, lung etc.) with elevated GLI1 in breast cancer having been linked to poor survival outcomes. A number of GLI targeted inhibitors have been shown to inhibit Hh/GLI signaling with some having efficacy in tumor models. We have previously demonstrated that siRNA downregulation of GLI1 expression in inflammatory breast cancer reduced proliferation and migration[1]. Further, we previously demonstrated the feasibility of employing a high throughput (HT) approach to profile drugs in dose response in cell line panels [2]. In this study, we have taken a comprehensive approach to profile a panel of GLI antagonists (including GANTS, HPIs, ATO and JK184) in a range of breast cancer cell lines with varying GLI1 expression levels to identify those with effects on growth. Experiment procedure: to assess cell proliferation, we employed an automated quantitative high throughput (qHT) approach to profile compounds on three phenotypic subtypes of breast cancer cell line models: triple negative breast cancer (TNBC), inflammatory breast cancer, and Claudin low breast cancer cell lines. High-content imaging of nuclear count utilizing Hoechst staining was also used as an alternative measure of proliferation. Quantitative PCR and Western blotting was used to assess the effects of the inhibitors on the GLI1 expression. Results: A subset of GLI antagonists were identified as decreasing cell growth and viability in all the cell lines. Conclusion: Several agents showed efficacy in in vitro BC cancer models demonstrating that GLI inhibitors may be a valid therapeutic approach for targeting GLI-dependent BC cancers. [1] Z. Thomas, W. Gibson, J. Sexton, K. Aird, S. Ingram, A. Aldrich, H. Lyerly, G. Devi, K. Williams, Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104 (2011) 1575-1586. [2] K.P. Williams, J.L. Allensworth, S.M. Ingram, G.R. Smith, A.J. Aldrich, J. Z Sexton, G. R Devi, Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Lett. 337 (2013) 77-89. Funded in part by DOD/CDMRP IDEA W81XWH-13-1-0141 award (KPW); NIH U54CA156735 (KPW); Duke Cancer Institute - Cancer and Environment Initiative P3917733 sub-award (GRD); W81XWH-13-1-041 subcontract (GRD) and National Cancer Institute training grant T32CA009111 (SJS). Citation Format: Helen Oladapo, Jodie M. Fleming, Kezia Addo, Mike Tarpley, Ben Ehe, Shalonda Ingram, Scott Sauer, Gayathri Devi, Kevin P. Williams. Identification of GLI1 antagonists for breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2015-680

Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

Abstract P6-14-01: A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in both HER2-overexpressing inflammatory and non-inflammatory breast cancer cells through FOXO3-mediated Bim1 expression

Abstract Background: Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in 15%-20% of breast cancers and is associated with shorter survival and worse clinical outcome. Inflammatory breast cancer (IBC), a very aggressive subtype of advanced breast cancer, accounts for approximately 2% of all breast cancers and 8%-10% of all breast cancer-related deaths in the United States. Recent study represented that about 60% of IBCs overexpress HER2. Although there are effective HER2-targeted agents, patients with HER2+ breast cancer often have intrinsic and acquired resistance to the anti-HER2 agents that are currently approved by the U.S. Food and Drug Administration. Therefore, novel combination strategies are needed to treat HER2+ breast cancers that develop drug resistance. To this end, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/epidermal growth factor receptor dual tyrosine kinase inhibitor, in HER2+ inflammatory breast cancer (IBC) and non-IBC breast cancer cells. Methods: We assessed the combinational synergistic effect and its mechanism via CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. Results: We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited tumor cell proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in both IBC and non-IBC xenograft mouse models (SUM190 and BT474, P < 0.001). The synergistic antitumor activity of the entinostat-lapatinib combination was due to downregulation of phosphorylated Akt, which induced the transcriptional activity of FOXO3, resulting in the induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistant HER2+ cells to the trastuzumab-lapatinib combination and enhanced the anti-proliferation effect compared with single-agent with lapatinib or combination treatment with lapatinib and trastuzumab. Conclusion: Taken together, our data provide evidence that entinostat has enhanced antitumor effect in combination with the HER2-targeted reagent lapatinib and results in the induction of apoptosis by FOXO3-mediated Bim1 expression. Our findings justify conducting a clinical trial (clinicaltrial.gov, NCT01434303) of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2+ IBC or non-IBC that is resistant to trastuzumab-based treatment. Citation Format: Jangsoon Lee, Chandra Bartholomeusz, Gabriel N Hortobagyi, Peter Ordentlich, Naoto T Ueno. A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in both HER2-overexpressing inflammatory and non-inflammatory breast cancer cells through FOXO3-mediated Bim1 expression [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 P6-14-01.

Abstract P3-06-39: Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer

Abstract Recent studies suggest that anaplastic lymphoma kinase (ALK) could serve as a therapeutic target for inflammatory breast cancer (IBC) and that strategies targeting ALK should be considered for evaluation in clinical trials. Reverse phase protein arays revealed activation of the ALK receptor tyrosine kinase and biochemically-linked downstream signalling molecules in pre-clinical models of IBC. ALK genetic abnormalities have been reported in 80% (20/25) of IBC patients with a high prevalence of ALK alterations in basal-like IBC (Robertson F. et al., 2013). However, ALK protein expression has not been studied in human IBC samples. Immunohistochemical detection of the ALK protein is increasingly being used as a screening tool to test samples for ALK rearrangements. The biological premise is that the genetic abnormality of the ALK gene leads to overexpression of the ALK protein and therefore overactivity of the ALK tyrosine kinase; since this kinase is the target of crizotinib, it makes sense to assess the drug target directly. Formalin fixed paraffin embedded tissue samples from pre-treatment surgical biopsies of 79 consecutive IBC patients were immunohistochemically (IHC) tested for ALK protein over-expression using a validated IHC test (NCL-ALK, clone 5A4) with a sensitivity of 93% and a specificity of 100% when the Vysis ALK-FISH break apart test is used as a gold standard. Stained tissue sections were evaluated using the IHC histoscore proposed by Thunnissen E. et al., 2012, assessing both the proportion of tumor cells showing cytoplasmic staining and the staining intensity. Cytoplasmic staining of appendiceal ganglion cells was used as an on-slide external positive control; weak to moderate cytoplasmic staining of macrophages was used as an internal positive control. In 75 of the 79 tissue samples none of the tumor cells showed any ALK immunoreactivity. One tumor showed moderate cytoplasmic staining in a few cells (<1%, score 2+); ALK (2p23) FISH showed no rearrangement. This tumor was hormone receptor negative and HER2 negative. Three tumors showed minimal cytoplasmic staining in a few cells (<1%, score 1+). These results demonstrate that ALK protein overexpression is not a feature of IBC. Although genetic abnormalities of ALK without protein overexpression are not excluded, our results show that ALK IHC cannot be used to identify IBC patients eligible for enrollment in clinical trials evaluating ALK targeted therapeutics. Citation Format: Cecile Colpaert, Melike Marsan, Peter Vermeulen, Luc Dirix, Steven Van Laere, Inflammatory Breast Cancer International Consortium. Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-39.

Abstract P5-01-04: In vivo lymphatic imaging of a human inflammatory breast cancer model

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive but poorly understood type of breast cancer. IBC constitutes only appoximately 5% of all newly diagnosed breast cancers, yet responsible for 8-10% of breast cancer-related deaths. Unfortunately, there are no definitive molecular or pathological, early diagnostic criteria for IBC. IBC grows in nests or sheets, spreading outward into the skin and eventually distant sites through dermal lymphatic vessels; therefore, one of the key pathological characteristics of IBC is dermal lymphatic invasion by tumor emboli, which can lead to obstruction of the lymphatic drainage possibly causing the clinical inflammatory features of diffuse erythema, rapid breast enlargement and edema as indicated by peau d’orange (orange peel) appearance covering at least a third of the breast surface. However, relatively little is known about the role of lymphatic function in IBC growth and metastasis. The purpose of this study was to non-invasively and longitudinally image changes of lymphatic drainage patterns in mice bearing the triple negative, human IBC SUM149 cells, which were stably transfected with iRFP gene reporter (iRFP-SUM149) in order to better monitor tumor growth and metastasis. Methods: Eight to ten weeks old female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were housed and fed special sterilized pelleted food and sterilized water. Two weeks after feeding special diets, mice were subcutaneously injected with iRFP-SUM149 cells in the left hindlimb or orthotopically in the left inguinal mammary fat pad (MFP). Fluorescence images were acquired immediately after and for up to 10 mins after i.d. injection of indocyanine green (ICG) using a custom-built NIRF imaging system. To achieve a greater magnification, a macrolens was used. Results: Our NIRF imaging data showed gradual changes of lymphatic vessels around the tumor. In mice with an orthotopic tumor, ICG accumulation was observed in the distal peritumoral region at up to 3 weeks p.i., indicative of the start of tumor obstruction of normal lymphatic drainage. As tumors grew, the greater extent of lymph flow, but not all, was obstructed by a tumor, resulting in dermal backflow and thus staining more lymphatic capillaries and eventually rerouting of lymphatic drainage was detected due to complete obstruction of lymph flow by a tumor. Similar to an orthotopic model, mice with a s.c. tumor also showed altered lymphatic drainage patterns during tumor growth. Interestingly, extravasation of ICG into iRFP-SUM149 was detected at 3 weeks p.i., due to leaky lymphatic vessels. A similar lymphatic phenotype has also been observed in IBC patients. Previously, we have imaged atypical tortuous lymphatics and altered lymphatic drainage in the arm and affected breast of a patient with IBC using clinical NIRF imaging developed in our team to image as deep as 3-5 cm with a microdose of ICG. Conclusions: Longitudinal, non-invasive imaging of lymphatic functional and architectural changes provides a new method to dynamically monitor lymphatic response to IBC growth and metastasis. Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Eva M Sevick-Muraca. In vivo lymphatic imaging of a human inflammatory breast cancer model [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 P5-01-04.

Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. In vivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Abstract A01: Pharmacological approaches to metastatic disease using an Inflammatory Breast Cancer model

Abstract Goal of Study: The lymphovascular embolus is a marker of breast cancer aggressiveness, recurrence, metastatic progression and therapeutic failure. This is especially reflective in inflammatory breast cancer (IBC) which presents as florid intravasation in situ of the lymphovasculature by tumor emboli. This study utilizes a novel 3-dimensional (3D) in vitro and in vivo model of IBC to systematically and strategically develop pharmacological approaches in the treatment of both IBC and metastatic disease. Background: Intravasation, the rate-limiting step of metastasis, is extremely important in tumor progression and expansion, yet the mechanism underlying intravasation is still poorly understood. The consequence of intravasation is the resultant lymphovascular embolus – the key defining feature of metastasis. This is most prevalent in IBC, the most lethal form of breast cancer. Clinical IBC features, erythema, peau d'orange (dimpling of thickened skin) and warmth are due to the IBC signature phenotype, namely extensive intravasation in situ of the lymphatic and blood vessels by tumor emboli. Nearly 100% of all women with IBC have lymph node involvement and approximately 25% of patients with IBC have distant metastases at diagnosis. The IBC xenograft model, MARY-X, has precisely captured the IBC phenotype, where the tumor emboli grow exclusively within the murine lymphatic and blood vessels and exhibit erythema of the overlying skin. MARY-X is an invaluable preclinical IBC model and an overall model of metastasis providing a means to validate and translate therapeutic strategies. MARY-X, in vitro, is a cellular derivative of primary tumor explants. These tumor cells form tight, compact aggregates of cells termed “MARY-X spheroids”. Comparable to human IBC emboli, a persistent, over-expression of an intact E-cadherin/α, β-catenin axis mediates the compaction of both in vitro and in vivo MARY-X spheroids and tumor emboli, respectively. These comparative architectural features (i.e. mimics the in vivo metastasis) of the spheroid of MARY-X provide an in vitro model with tractable in vivo applications. Research Strategy: By exploiting the MARY-X in vitro 3-D model, high throughput (HTP) screening is performed to systematically probe the factors involved in maintenance of tumor emboli architecture as it pertains to sensitivity/resistance to therapeutics in an overall effort to develop efficacious therapeutics for treatment of IBC and metastatic disease. Additionally, MARY-X spheroids co-cultured with bone marrow derived cells (BMDCs) in matrigel overlays are employed to assess targeting of mediators of crosstalk between tumor spheroid/embolus soluble factors (e.g. cytokines, exosomes) and the microenvironment. Soluble factors identified as mediators (i.e. targets) of metastatic progression, that showed the most promise in the HTP screen, are further validated in the in vivo preclinical model, MARY-X. A significant decrease in vessel density of the MARY-X tumor will be indicative of successful blocking of this ‘crosstalk’ i.e. lymphovasculogenesis or the formation of lymphovascular emboli (i.e. intravasation) – the defining feature of metastatic disease. Preliminary Data and Conclusions: Preliminary data show that dissolution of the spheroid/embolus augment apoptotic effects of chemo/irradiation therapy. Suggesting that apoptosis resistance of lymphovascular emboli may be independent of traditional apoptotic mechanisms. Therefore, therapy evasion, in part, is due to formation and maintenance of the architectural features of the lymphovascular embolus. In addition, abrogation of ‘crosstalk’ has proven to hinder the establishment of lymphovascular emboli. Citation Format: Mary L. Alpaugh, Emmanuel Theodorakis. Pharmacological approaches to metastatic disease using an Inflammatory Breast Cancer model. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A01.

Abstract C42: Quantitative high-throughput efficacy profiling of hedgehog/GLI pathway antagonists in inflammatory breast cancer

Abstract The primary objective of this study was to profile a panel of GLI pathway antagonists in inflammatory breast cancer (IBC) cell models for effects on cell growth and viability. Introduction: IBC, representing ∼5% of breast cancers in the USA, is a unique form of locally advanced breast cancer characterized by rapid progression and a highly invasive nature intrinsic to the tumor. IBC is not easily detected by mammograms or ultrasounds with detection typically after the cancer has spread. IBC patients have a 5 year survival rate of less than 50%, significantly less than patients with non-IBC breast cancer (85%), and IBC accounts for ∼15% of all breast cancer deaths. IBC affects young, African-American, Arab-American and American-Indian women at a higher rate than in other groups[1; 2; 3]. A recent analysis of SEER data showed an increase in IBC incidence associated with decreasing socioeconomic position[4]. Despite polychemotherapy regimens, women with IBC continue to have worse survival outcomes than non-IBC breast cancer patients and current strategies for targeting IBC are limited. The downstream transcription factor GLI1, an essential marker for hedgehog (Hh) pathway activation, has emerged as a therapeutic target for several cancers including breast cancer. We have demonstrated that siRNA downregulation of GLI1 expression in IBC reduced proliferation and migration[5]. Experimental Procedures: We employed a high throughput (HT) approach to profile a panel of GLI antagonists in an IBC model (SUM149, a basal type). To assess drug effects on proliferation we utilized an automated and validated 384-well cell-based MTT assay [6]. Compounds were screened in a secondary assay using high-content imaging of nuclear count following Hoechst staining as an alternative measure of cell proliferation. Results: A panel of Hh/GLI pathway antagonists was assembled based on literature surveys and formatted in 384-well plates. To obtain pharmacological information from the screening data, we employed a titration-based screening paradigm, quantitative HTS (qHTS) where compounds were screened at multiple concentrations. A subset of GLI antagonists were identified as having effects on IBC cell proliferation. Conclusion: Several agents showed efficacy in in vitro IBC cancer models demonstrating that GLI inhibitors may be a valid therapeutic approach for targeting GLI-dependent IBC cancers. Funded in part by DOD/CDMRP IDEA W81XWH-13-1-0141(BC121850) (KP Williams) [1] W.F. Anderson, C. Schairer, B.E. Chen, K.W. Hance, P.H. Levine, Epidemiology of inflammatory breast cancer (IBC). Breast disease 22 (2006) 9-23. [2] W.A. Woodward, M. Cristofanilli, Inflammatory breast cancer. Seminars in Radiation Oncology 19 (2009) 256-265. [3] K.A. Hirko, A.S. Soliman, M. Banerjee, J. Ruterbusch, J.B. Harford, R.M. Chamberlain, J.J. Graff, S.D. Merajver, K. Schwartz, Characterizing inflammatory breast cancer among Arab Americans in the California, Detroit and New Jersey Surveillance, Epidemiology and End Results (SEER) registries (1988–2008). SpringerPlus 2 (2013). [4] J.A. Schlichting, A.S. Soliman, C. Schairer, M. Banerjee, L. Rozek, D. Schottenfeld, J.B. Harford, S.D. Merajver, Association of Inflammatory and Non-Inflammatory Breast Cancer with Socioeconomic Characteristics in the Surveillance, Epidemiology, and End Results Database, 2000-2007. Cancer Epidemiol. Biomark. Prev. (2011) DOI:10.1158/1055-9965.EPI-1111-0853. [5] Z. Thomas, W. Gibson, J. Sexton, K. Aird, S. Ingram, A. Aldrich, H. Lyerly, G. Devi, K. Williams, Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. Br. J. Cancer 104 (2011) 1575-1586. [6] K.P. Williams, J.L. Allensworth, S.M. Ingram, G.R. Smith, A.J. Aldrich, J. Z Sexton, G. R Devi, Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Lett. 337 (2013) 77-89. Citation Format: Helen Oladapo, Shalonda Ingram, Amy Stefanowicz, Gayathri Devi, Kevin Williams. Quantitative high-throughput efficacy profiling of hedgehog/GLI pathway antagonists in inflammatory breast cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C42. doi:10.1158/1538-7755.DISP13-C42

Abstract 4988: Clues for targeted therapies in inflammatory breast cancer (IBC)

Abstract Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development, and poor prognosis. The peculiar clinical presentation of IBC characterized by breast erythema and swelling is caused by the invasion of aggregates of tumor cells (tumor emboli) into the dermal lymphatics, causing an obstruction of the lymph channels. Although, tumor emboli are also occasionally demonstrated in non-IBC tumors, they are more frequently detected and more numerous in IBC. Tumor emboli expressed cell-cell adhesion molecules that maintain the tumor cells together. The high risk of disease recurrence in soft tissue and lymph nodes shortly after completion of multidisciplinary treatment particularly in patients with residual disease supports the hypothesis that these tumor emboli are critically related to the development of micro metastatic disease. We 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 in suspension that were strongly positive for E-cadherin, β-catenin, and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. The maintenance of cell-cell adhesions allow the migration of tumor cells through the lymphatic and blood vessels as clusters. FC-IBC02 cells shown a partial or incomplete epithelial-mesenchymal transition (EMT); these cells express some epithelial markers (EpCAM, E-cadherin) and also mesenchymal markers (VIM, FN1, Snai2, Twist1). Furthermore, circulating tumor cells (CTC) from IBC patients are present in the blood as single cells and clusters supporting the collective migration of tumor cells in IBC. FC-IBC02 cells were highly metastatic when injected in the fat mammary pad of SCID mice and these cells were able to produce brain metastasis in mice by intracardiac or intra-peritoneal 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 2.5-7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6-4 copies in IBC cells. FAK1 is amplified, upregulated and phosphorylated (active) in inflammatory breast cancer. Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. Our results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC. We are currently evaluating an ALK/FAK inhibitor using the novel established IBC model. Citation Format: Sandra V. Fernandez, Fredika M. Robertson, Sankar Addya, Zhaomei Mu, Massimo Cristofanilli. Clues for targeted therapies in inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4988. doi:10.1158/1538-7445.AM2014-4988

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.

In vivo lymphatic imaging of a human inflammatory breast cancer model.

Background: Inflammatory breast cancer (IBC) remains the most aggressive type of breast cancer with the greatest potential for metastasis and as a result, the highest mortality rate. IBC cells invade and metastasize through dermal lymphatic vessels; however, it is unknown how lymphatic drainage patterns change during IBC growth and metastasis. Herein, we non-invasively and longitudinally imaged lymphatics in an animal model of IBC using near-infrared fluorescence (NIRF) imaging.Materials and methods: Mice were imaged in vivo prior to, and up to 11 weeks after subcutaneous or orthotopic inoculation of human IBC SUM149 cells, which were stably transfected with infrared fluorescence protein (iRFP) gene reporter (SUM149-iRFP), following intradermal (i.d.) injection of indocyanine green (ICG).Results: Fluorescence images showed well-defined lymphatic vessels prior to SUM149-iRFP inoculation. However, altered lymphatic drainage patterns including rerouting of lymphatic drainage were detected in mice with SUM149-iRFP, due to lymphatic obstruction of normal lymphatic drainages caused by tumor growth. In addition, we observed tortuous lymphatic vessels and extravasation of ICG-laden lymph in mice with SUM149-iRFP. We also observed increased and dilated fluorescent lymphatic vessels in the tumor periphery, which was confirmed by ex vivo immunohistochemical staining of lymphatic vessels.Conclusions: Our pre-clinical studies demonstrate that non-invasive NIRF imaging can provide a method to assess changes in lymphatic drainage patterns during IBC growth and metastasis.

Abstract P2-05-04: Comparative expression profiling of patient samples and preclinical models of inflammatory breast cancer reveals gene signatures of epithelial plasticity and suppression of TGFb signaling

Abstract Introduction: Genome-wide expression profiling of samples from patients with and without Inflammatory Breast Cancer (IBC) has revealed novel insights into the biology of IBC. The present study was undertaken to compare these novel insights with data obtained from all available preclinical IBC models including 2 new models that we have recently developed that recapitulate the characteristics of IBC including retention of E-cadherin, formation of tumor emboli and encircling lymphoangiogenesis Materials and Methods: Five replicates of 7 preclinical IBC models (SUM149, SUM190, FC-IBC-01, FC-IBC-02, MDA-IBC-03, KPL-4, and Mary-X) were profiled using Affymetrix HGU133plus2 GeneChips. Using a nearest shrunken centroid algorithm, each expression profile was classified according to an IBC-specific signature identified in patient samples. Available expression profiles were further queried for expression patterns related to Epithelial-to-Mesenchymal Transition (EMT), TGFβ-signaling and IBC-specific patterns of transcription factor activation. Results: Application of our IBC-specific signature (posterior probabilities exceeded 0.50 in at least 4/5 replicates) revealed that out of 7 pre-clinical models of IBC, 3 of these robustly classified as IBC (FC-IBC-01, FC-IBC-02, and KPL-4). All preclinical IBC models were characterized by retention of E-Cadherin expression, absence of ZEB1 expression, attenuated expression of specific components of the TGFβ pathway (TGFβR2, SMAD3, SMAD7, and TGFβ1), and ambiguous activation patterns of several transcription factors involved in regulating cellular plasticity and cell fate decisions (Up in IBC: NR4A2, RARB/RXRA, PTX3, GSC2, and ZEB1; Down in IBC: SOX10, PAX5, and SMAD2). For each of the molecular alterations described above, Z-scores greater than 2 were achieved in at least 4/5 replicates. Conclusions: The observations that we have made using IBC patient tumor samples with regards to EMT, cell plasticity and TGFβ-signaling are corroborated in pre-clinical models of IBC using current analytic approaches, despite the fact that expression patterns of the majority of preclinical models of IBC deviate from the IBC-specific expression patterns observed in patient samples. Our data suggest that despite their highly invasive nature, IBC cancer cells retain an epithelial cell phenotype characterized by E-cadherin expression and loss of ZEB1 which appears to be mediated by, amongst others, attenuated TGFβ-signaling. This study strengthens our hypothesis that cancer cells from IBC exhibit cohesive invasion, and invade as a unit, possibly explaining the presence of florid tumour emboli which is a primary characteristic observed in IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-04.