Inflammatory Breast Cancer Xenograft Research Articles

Abstract 5986: Significance of LIF/LIFR axis in the progression of inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare form of locally progressed breast cancer. Even though it is uncommon, IBC accounts for 7% of breast cancer deaths. There is a critical need for novel therapeutic targets to develop new therapeutics. IBC is more common in young women. Altered levels of growth factors, and cytokine signaling are suspected to play a role in the progression of IBC. Leukemia inhibitory factor (LIF) is the most pleiotropic member of the interleukin-6 family of cytokines. LIF and its receptor LIFR have been linked to the progression of many cancers including triple negative breast cancer. We recently developed a first-in-class inhibitor targeting LIFR named EC359. However, the role of the LIF/LIFR axis in IBC progression remains unknown. In this study, we examined the significance of LIFR and the effectiveness of LIFR inhibitor EC359 in treating IBC. Methods: The effect of LIFR inhibitor EC359 on cell survival, colony formation, and apoptosis was evaluated using three well-established IBC model cells (SUM149, SUM190PT, and KPL4). Mechanistic studies were conducted using Western blotting, CRISPR KO, reporter assays, and RT-qPCR. KPL4 cell-based xenografts were used to test the efficacy of EC359 in vivo. Results: Our results using Western blotting confirmed increased expression of LIF and LIFR in IBC cells compared to normal and ER+ breast cancer cells. Treatment with EC359 significantly decreased IBC cell viability, long term colony formation ability with an IC50 of ~10-20 nM. Similarly, knock out of LIFR significantly reduced the progression of IBC model cells in cell viability and colony formation assays. Further EC359 treatment promoted apoptosis of IBC cells. Mechanistic studies confirmed that EC359 treatment substantially reduced LIF/LIFR downstream signaling including STAT3, AKT, and mTOR signaling. We also tested the utility of EC359 in treating IBC in vivo using KPL4 xenografts. EC359 (5mg/kg/day) is highly efficacious in reducing IBC xenograft tumor growth in vivo. IHC analyses of tumors confirmed decreased cell proliferation as measured by Ki67 staining. Conclusion: Collectively, these studies demonstrated the therapeutic benefit of using EC359 to target the LIF/LIFR axis and opened the door for the development of new treatment approaches. Citation Format: Bianca Romo, Zenaida Fuentes, Lois Randolph, Bindu Santhamma, Hareesh Nair, Christoforos Thomas, Ratna Vadlamudi, Suryavathi Viswanadhapalli. Significance of LIF/LIFR axis in the progression of inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5986.

Metadherin Regulates Inflammatory Breast Cancer Invasion and Metastasis.

Inflammatory breast cancer (IBC) is one of the most lethal subtypes of breast cancer (BC), accounting for approximately 1-5% of all cases of BC. Challenges in IBC include accurate and early diagnosis and the development of effective targeted therapies. Our previous studies identified the overexpression of metadherin (MTDH) in the plasma membrane of IBC cells, further confirmed in patient tissues. MTDH has been found to play a role in signaling pathways related to cancer. However, its mechanism of action in the progression of IBC remains unknown. To evaluate the function of MTDH, SUM-149 and SUM-190 IBC cells were edited with CRISPR/Cas9 vectors for in vitro characterization studies and used in mouse IBC xenografts. Our results demonstrate that the absence of MTDH significantly reduces IBC cell migration, proliferation, tumor spheroid formation, and the expression of NF-κB and STAT3 signaling molecules, which are crucial oncogenic pathways in IBC. Furthermore, IBC xenografts showed significant differences in tumor growth patterns, and lung tissue revealed epithelial-like cells in 43% of wild-type (WT) compared to 29% of CRISPR xenografts. Our study emphasizes the role of MTDH as a potential therapeutic target for the progression of IBC.

Geometric tumor embolic budding characterizes inflammatory breast cancer.

Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli especially within dermal lymphatics. The explanation remains a mystery. This study combines experimental studies with two different IBC xenografts with image algorithmic studies utilizing human tissue microarrays (TMAs) of IBC vs non-IBC cases to support a novel hypothesis to explain IBC's sina qua non signature of florid lymphovascular emboli. In the human TMAs, compared to tumor features like nuclear grade (size), mitosis and Ki-67 immunoreactivity which show that IBC is only modestly more proliferative with larger nuclei than non-IBC, what really sets IBC apart is the markedly greater number of tumor emboli and distinctly smaller emboli whose numbers indicate geometric or exponential differences between IBC and non-IBC. In the experimental xenograft studies, Mary-X gives rise to tight spheroids in vitro which exhibit dynamic budding into smaller daughter spheroids whereas Karen-X exhibits only loose non-budding aggregates. Furthermore Mary-X emboli also bud dramatically into smaller daughter emboli in vivo. The mechanism that regulates this involves the generation of E-cad/NTF1, a calpain-mediated cleavage 100kDa product of 120kDa full length membrane E-cadherin. Inhibiting this calpain-mediated cleavage of E-cadherin by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides that both penetrate the cell membrane and mimic either the cleavage site or the binding site on E-cadherin, inhibits the generation of E-cad/NTF1 in a dose-dependent manner, reduces spheroid compactness and decreases budding. Since E-cad/NFT1 retains the p120ctn binding site but loses the α-and β-catenin sites, promoting its 360° distribution around the cell's membrane, the vacilating levels of this molecule trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of emboli seen in IBC.

Florid Lymphovascular Invasion of Inflammatory Breast Cancer is Due to Geometric Embolic Budding

The sina qua non of inflammatory breast cancer (IBC) is numerous tumor emboli especially within overlying dermal lymphatics. The explanation remains a mystery. In human studies comparing IBC with non‐IBC, although common tumor parameters such as Ki‐67 index, mitotic count and nuclear size show a close overlap, there is a dramatic exponential difference in embolic density in IBC v non‐IBC. Using contrasting properties of two IBC xenografts, Mary‐X and Karen‐X, this study offers a novel explanation and proposed mechanism to explain the very high embolic numbers in IBC. The explanation is geometric budding of emboli. Mary‐X exhibits florid lymphovascular invasion in vivo which give rise to high numbers of CTCs and pulmonary metastases whereas Karen‐X lacks these features. Mary‐X also gives rise to compact spheroids in vitrowhich exhibit dramatic budding whereas Karen‐X exhibits only loose non‐budding aggregates. Furthermore Mary‐X emboli also bud into daughter emboli in vivo. The mechanism that regulates the compactness of the spheroids as well as the emboli involves the generation of E‐cad/NTF1, a calpain‐mediated cleavage product of membrane E‐cadherin. Inhibiting the generation of E‐cad/NTF1 by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides both reduces spheroid compactness and decreases budding. Since E‐cad/NFT1 retains the p120ctn binding site but loses both the β‐catenin and α‐binding sites, promoting its 360° distribution around the cell’s membrane, the varying levels of expression of this truncated molecule may trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of IBC tumor emboli seen in patients.

Decorin-mediated suppression of tumorigenesis, invasion, and metastasis in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited tumor growth and metastasis in IBC xenograft mouse models. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6β1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 1235: EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. We have previously reported that mediators of inflammation, such as COX-2, promote the growth of Triple-Negative receptor (TN) IBC xenografts; therefore, inflammation in TN-IBC has a unique opportunity as a therapeutic strategy. Eicosapentaenoic acid (EPA), a non-toxic omega-3 fatty acid with anti-inflammatory properties, has partially reduced tumor growth in pre-clinical models of TN-IBC. Therefore, our goal is to develop a novel non-toxic approach that enhances EPA efficacy against TN-IBC in combination with targeted therapy. Methods and Results: Using a high-throughput, siRNA screen (939 genes) in the TN-IBC cell line SUM149PT, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a target that modulates the sensitivity of TN-IBC cells to EPA treatment. To determine the clinical relevance of EPHA2, we interrogated a meta-analysis of breast cancer mRNA expression data sets, and found that high EPHA2 tumor expression was significantly correlated with poor overall survival in TN-IBC patients, compared to low EPHA2 expressing tumors (P = 0.01). We observed no significant correlations to other breast cancer subtypes. Similar findings were observed in vitro were EPHA2 expression predominantly occurred in the TN-IBC subtypes (19 of 30) among 49 breast cancer cell lines. Gain/loss-of-expression studies were performed to functionally validate EPHA2 as a synergistic combinational target with EPA in two EPHA2-expressing TN-IBC models, SUM149PT and BCX010, using proliferation and apoptosis assays in vitro and established tumor xenografts in vivo. EPHA2 gene silencing significantly reduced cell growth and induced apoptosis in combination with EPA when compared with untreated control and monotherapy in vitro (P < 0.05) and in vivo (P < 0.001), while vector-induced EPHA2 expression reversed cell growth reduction and apoptosis induction following combination treatment with EPA in vitro (P < 0.05). To translate our findings to the clinic, we validated that dasatinib, a small molecule inhibitor of EPHA2, in combination with EPA significantly enhanced cell death of SUM149PT and BCX010 cells in vitro when compared to non-treated and monotherapy (P < 0.05). Finally, using membrane fluidity assessment and reverse-phase protein array (300 antibodies), we determined that combination treatment efficacy depended on EPA/EPHA2 inhibition-mediated increase in cell membrane rigidity (P < 0.001, compared to monotherapy), which subsequently inhibited receptor tyrosine kinase signaling activity, potentially resulting in induction of apoptosis. Conclusions: Our preclinical findings provide a rationale for the development of a phase 1 clinical trial investigating combination EPA and EPHA2-inhibitors in patients with EPHA2-positive TN-IBC. Citation Format: Angie M. Torres-Adorno, Heidi Vitrac, Yuan Qi, Yiwen Yang, Peiying Yang, Bedrich L. Eckhardt, Naoto T. Ueno. EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2017-1235

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

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

Abstract P6-14-05: A novel link between anti-apoptotic signaling, NFκB, and SMAD7 in IBC pathobiology

Abstract Background: Inflammatory breast cancer (IBC) has the highest lethality amongst all subtypes of breast cancer and develops rapid therapeutic resistance. High NFκB activation has been identified as a distinct molecular determinant in IBC pathobiology; however, the precise sequence of its activation and functional consequence in IBC remains unknown. Our previous work identified increased expression of the X-linked inhibitor of apoptosis protein (XIAP) due to altered translation in IBC, while other studies have noted a crosstalk between XIAP and NFκB. We hypothesized that XIAP drives NFκB activation in IBC promoting therapeutic resistance and tumorigenesis. Methods: NFκB phosphorylation, nuclear translocation, and target gene expression were evaluated in triple-negative SUM149 IBC cells with targeted overexpression or knockdown of XIAP. Using specific point mutants, we assessed the domain and mechanism of XIAP-mediated NFκB activation in IBC. We evaluated proliferation and viability in 2D and 3D culture of SUM149 cells treated with JSH-23, a small molecule inhibitor of NFκB nuclear translocation. We monitored the effects of XIAP overexpression or knockdown on in vivo tumorigenicity in IBC xenograft models by measuring tumor growth and NFκB signaling. IHC analysis of XIAP and NFκB was performed on tumor microarrays containing both non-IBC and IBC. Results: Knockdown of XIAP significantly decreased NFκB activation in IBC cells. Domain analysis revealed the necessity of the BIR1 domain of XIAP and TAB1:IKKβ complex formation in activating NFκB. NFκB antagonism inhibited proliferation of cells and sensitized therapy-resistant, XIAP overexpressing cells to targeted therapy. Loss of XIAP inhibited tumor growth of SUM149 tumor cells, correlating with decreased ALDH activity and varied epithelial-mesenchymal characteristics in these cells, while overexpression of XIAP significantly enhanced tumor growth of SUM149 cells. Further analysis revealed altered SMAD7 expression in XIAP knockdown cells, revealing crosstalk between XIAP, NFκB, and TGFβ signaling in IBC. IHC analysis of XIAP expression in invasive non-IBC tumors correlated with triple-negative status as well as increased grade and stage of tumors. In IBC tumors, XIAP expression associated with increased NFκB. Conclusions: In summary, our studies reveal that XIAP expression is necessary for NFκB activation in IBC and is critical for IBC development and progression. This study provides a novel insight into how an anti-apoptotic protein may regulate survival signaling and disease progression and may guide further research into innovative inhibitors of this interaction. Citation Format: Myron K Evans, Scott J Sauer, Amy J Aldrich, Joseph Geradts, Peter Vermeulen, Luc Dirix, Steven Van Laere, Gayathri R Devi. A novel link between anti-apoptotic signaling, NFκB, and SMAD7 in IBC pathobiology [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 P6-14-05.

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 P6-12-11: Celecoxib inhibits the growth of IBC tumors by suppressing the regulation of cancer stem-like cells by nodal

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with no known molecular targets for treatment. Although erythema is commonly associated with IBC, the molecular mechanism of inflammation in the pathogenesis of IBC remains unknown. We have previously shown that EGFR is an emerging target in IBC (Zhang D. et al Clin Can Res 2009). As crosstalk between EGFR and COX-2 plays an important role in the inflammatory response in several cancers, including breast cancer, we hypothesized that COX-2 promotes the tumorigenesis and metastasis of IBC cells. Methods: Using clinically derived IBC and non-IBC tumor samples, a Spearman's Rank correlation coefficient analysis was performed to analyze the expression levels of COX-2 and EGFR in IBC and non-IBC. The levels of COX-2 metabolites, prostaglandins (PGs) PGE2 and PGF2α, were measured in IBC and non-IBC cell lines by HPLC/MS method. Cell migration and invasion assays were performed using SUM149 and KPL-4 IBC cell lines treated with PGs or the COX-2 inhibitor, celecoxib. We evaluated the epithelial to mesenchymal transition (EMT)-like phenotype in 3D culture of SUM149 cells treated with celecoxib, and the stem-like population by mammosphere formation, and CD44+/CD24− and aldefluor+ population by FACS. We treated preclinical IBC xenograft mice with celecoxib and measured tumor growth, PGs levels, and the expression of EMT protein markers. Nodal, a stem cell regulator and potential biomarker for breast cancer progression, was evaluated in IBC cells following treatment with celecoxib and recombinant Nodal or transfection with Nodal cDNA. Results: EGFR and COX-2 expression levels positively correlated within IBC, but not non-IBC tumors. Elevated levels of PGE2 and PGF2α were identified in multiple IBC cell lines suggesting that COX activity is elevated within IBC compared to non-IBC cells. PGs altered EMT protein markers and promoted cell migration and invasion, while Celecoxib inhibited EMT and migration and invasion in SUM149 and KPL-4 cells. Celecoxib treatment inhibited tumor growth in mice, and downregulated the expression of EMT protein markers, including Nodal. Celecoxib decreased the stem-like CD44+/CD24−, and aldefluor+ population and the formation of mammospheres. Exogenous Nodal mitigated the effects of celecoxib on cell migration and invasion and the stem-like population in SUM149 cells. Conclusion: We conclude that activation of the COX-2 inflammatory signaling pathway is critical in the development and progression of IBC. This study provides a novel insight into how inflammation may regulate cancer stem cells via Nodal, and will guide future research into the development of stem cell targeted therapies for IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-11.

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.

Inhibiting JAK2 for Inflammatory Breast Cancer

Abstract JAK2 inhibitors significantly diminished the proliferation of inflammatory breast cancer (IBC) cells in vitro and inhibited tumor growth in an IBC xenograft model.

Abstract P5-03-02: Targeting mRNA Translation to Enhance the Radiosensitivity of Inflammatory Breast Cancer Stem Cells

Abstract Purpose/Objective(s): Inflammatory breast cancer (IBC) is a highly aggressive and radiation resistant malignancy with a dismal prognosis despite multimodality therapy, including ionizing radiation. We have previously shown that the unique pathogenic properties of IBC result in part from over-expression of translation initiation factor eIF4G1, which is part of the eIF4F translation initiation complex, along with eIF4E and eIF4A. eIF4F is regulated by mTOR, providing a promising target for anti-cancer therapeutics. We demonstrated that protein synthesis is highly regulated during IR by the DNA-damage response (DDR) pathway through mTOR signaling. Many key proteins required for the DDR pathway are encoded by mRNAs that require high levels of the eIF4F complex and mTOR activity for their efficient translation. We hypothesized that upregulation of eIF4F in IBC plays a crucial role in the radio-resistance of disease. Materials/Methods: Experiments were conducted in IBC SUM149 cells. eIF4G1, eIF4E and eIF4A were silenced through the generation of stable cell lines that express tetracycline-inducible shRNAs. eIF4A was also inhibited using the pharmacologic investigational inhibitor DAMD-PatA. Radiation sensitivity in vitro was determined by cell survival assay. Tumor xenografts were generated by the injection of stable shRNA inducible cell lines into nude mice. IBC SUM149 cancer stem cells (CSC) from both in vitro and in vivo experiments were analyzed by a combination of cell surface marker analysis, mammosphere formation and Aldefluor assays. Results: We show that moderate inhibition by silencing of individual components (or by pharmacologic inhibition of eIF4A) of the eIF4F complex prevents IBC xenograft tumor growth and strongly enhances radiosensitivity. In contrast to results obtained for non-transformed breast epithelial cells, reducing the high levels of eIF4G1 in epithelial IBC cells in 2D cultures provides no enhancement in radiation sensitivity. Rather, SUM149 IBC cells harbor a substantial population of CSCs, which are the cells that are strongly dependent on high levels of eIF4G1, and which are selectively radio-sensitized as a result of eIF4G1-silencing. CSCs also require eIF4E and eIF4A activity in order to survive radiation treatment. We also demonstrate that silencing of eIF4G1 radio-sensitizes the stem cell population within IBC tumor xenografts. Radio-resistance of IBC cells is likely mediated by differential responses to the DDR in the stem-cell populations and by selective mRNA translation of proteins involved in the DDR pathway. Conclusions: Our results demonstrate that regulation of mRNA translation plays an important role in conferring radio-resistance to advanced breast cancers, particularly by allowing the survival of the CSC compartment. While inhibition of eIF4F enhances radiation sensitivity in non-transformed cells, this process is abrogated in IBC due to enrichment of a radiation resistant CSC population, demonstrating translational control of the breast cancer stem cell population, and providing a novel understanding of the role of the regulation of mRNA translation in radiation resistance of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-02.

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.

Abstract 569: Targeting inflammatory pathways as an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive type of advanced breast cancer with a poor long-term outcome and a high risk of recurrence. Although one of the most common clinical manifestations of IBC is erythema of the skin overlying the breast, whether inflammation is involved in IBC has not yet been tested by pathologic examination of IBC specimens. Therefore, whether inflammation pathways are involved in the pathogenesis of IBC is still not clear. A large body of evidence indicates that chronic inflammation is a risk factor for cancer. Nonsteroidal anti-inflammatory drugs have been reported to reduce the risk of developing some solid cancers, including breast cancer. Cyclooxygenase (COX)-2 is the inducible isoenzyme catalyzing the conversion of arachidonic acid to prostanoids. COX-2 upregulation has been linked to breast tumorigenesis or inflammatory diseases, and COX-2 overexpression increases the growth rate and invasiveness of breast cancer cells. We tested the hypothesis that the COX-2 pathway is involved in IBC tumor growth and metastasis. Methods and Results: We examined the expression level of COX-2 and the concentrations of its enzymatic products by mass spectrometry (LC/MS/MS). We also determined the effect of inhibition of the COX2 pathway on cell proliferation, migration, invasiveness, and epithelial-mesenchymal transition (EMT) in IBC both in vitro and in vivo. We found that levels of COX-2 and its enzymatic products prostaglandins (PG), such as PGE2 and PGF2α, were higher in IBC than in non-IBC cell lines. Both PGE2 and PGF2α markedly enhanced the migration and invasion of SUM149 IBC cells, suggesting that COX-2 and its metabolites may play an important role in IBC development and progression. We examined the expression levels of COX-2 and EGFR in 25 IBC patient biopsy samples and found that the expression of COX-2 was positively correlated with the levels of EGFR in these tissues. To illustrate how COX-2 and EGFR are regulated in IBC cells, we treated SUM149 cells with EGF. We found that EGF stimulation increased COX-2 levels. COX-2 expression was notably downregulated in EGFR shRNA stably transfected SUM149 cells, suggesting that the upregulation of COX-2 is mediated through EGFR signaling. Furthermore, a selective COX-2 inhibitor, celecoxib, dramatically inhibited IBC tumor growth in a SUM149 IBC xenograft model. These celecoxib-treated tumors exhibited high expression of the epithelial marker E-cadherin and low expression of the mesenchymal marker vimentin compared with untreated tumors, suggesting that celecoxib inhibits epithelial-mesenchymal transition. Conclusions: Our results suggest that inflammatory mediators indeed play an important role in IBC cells. Targeting inflammatory pathways may represent an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in 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 569. doi:1538-7445.AM2012-569

Over-expression of Translation Initiation Factor eIF4G Confers Robust Radioresistance to the Cancer Stem Cell Population in Inflammatory Breast Cancer

Over-expression of Translation Initiation Factor eIF4G Confers Robust Radioresistance to the Cancer Stem Cell Population in Inflammatory Breast Cancer

Abstract P5-06-05: Catalytic mTOR Inhibition with pp242 but Not Allosteric Inhibition with Rapamycin RAD001 Enhances the Radiosensitivity of Inflammatory Breast Cancer in an Animal Model

Abstract Purpose/Objective(s): Inflammatory breast cancer (IBC) is a highly aggressive and radiation resistant cancer that continues to have a dismal prognosis despite aggressive multimodality therapy, which includes ionizing radiation (IR). The PI3K/Akt/mTOR pathway is frequently dysregulated in human cancers, including IBC. mTOR (mammalian target of rapamycin) is a central regulator of protein synthesis, linking mRNA translation to the metabolic state of the cell, playing a key role in the signaling of malignant cell growth, proliferation, differentiation, migration, and survival. We have shown that mTOR activation following treatment with DNA damaging agents such as ionizing radiation (IR) is a primary protector of advanced breast cancer cells, including IBC cells, through selectively increased translation of mRNAs for survival and DNA repair genes, including survivin, PARP, and DNA repair enzymes, among other radio-protective mRNAs. Thus, treatment with an mTOR inhibitor, coupled with IR, would be expected to provide a synergistic ability to control IBC. Therefore we compared the cytotoxic effects of combining mTOR inhibition and radiation in an IBC xenograft model, using either the selective mTORC1/2 inhibitor pp242, or the partial mTORC1 inhibitor RAD001 (Everlimus), a rapamcyin analog. Methods: Experiments were conducted using SUM149 cells, a well-established model for IBC. Cells were treated with RAD001 or TORC1/2 inhibitor pp242 alone or in combination with increasing doses of radiation from 0-8 Gy. In vitro studies preformed included; cell survival assays, immunoblot analysis of key proteins, and 35S-methionine labeling to evaluate protein synthesis. In vivo studies were performed in a SUM149 xenograft nude mouse model; animals were treated with RAD001 or pp242 alone or in combination with IR. Results: We found that only the combination of IR and catalytic mTOR inhibition by pp242 led to a number of striking additive/synergistic effects that were not observed with the combination of IR and the partial mTORC 1 inhibitor RAD001. These effects included; decreased clonogenic survival, inhibition of both endogenous and radiation-induced Akt activation, greater inhibition of mTOR signaling to its downstream effectors, efficient inhibition of protein synthesis, and greater induction of apoptotic cell death, as indicated by induction of caspase-3 cleavage. Importantly, SUM149 IBC xenografts treated with pp242 and concurrent radiation treatment exhibited significantly greater tumor control and survival compared to radiation treatment alone or RAD001 with radiation. Conclusions: These studies demonstrate that targeted inhibition of mTORC1/2 catalytic activity synergizes with radiation therapy in a model system of IBC. Mechanistically, mTORC1/2 inhibition likely prevents radiation induced pro-survival signals mediated through constitutively active Akt, which is not blocked by Rapamycin and its analogs. Experiments examining whether inhibition by pp242 prevents the selective increase in translation of prosurvival mRNAs in IBC as a result of IR treatment will be presented. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-05.

The altered redistribution and accumulation of E‐cadherin as a dominant‐acting oncogene in inflammatory breast cancer (IBC)

E‐cadherin, an adhesion protein within lateral junctions, is thought to function as a tumor suppressor in breast carcinomas and promote metastasis when its expression is lost by mutation, promoter methylation or repression by mediators of EMT. However in IBC, E‐cadherin is overexpressed circumferentially which accounts for the formation of the lymphovascular embolus, a structure efficient at metastasis and drug resistance. Our studies with a human IBC xenograft, MARY‐X, revealed that E‐cadherin transcripts were less than MCF‐7 and MCF‐10A (E‐cadherin positive lines) but the protein levels were 100 fold greater. We found E‐cadherin overexpressed because of altered degradation, redistribution and trafficking. Since previous studies had shown that E‐cadherin lysosomal targeting was mediated by the hepatocyte GF‐tyrosine kinase substrate (Hrs) and that v‐src activation of Rab7 depleted E‐cadherin in EMT, we investigated whether these genes might be decreased in MARY‐X and found this so. Since other Drosophila proteins, eg., sec10 mediated E‐cadherin trafficking from recycling endosomes to plasma membranes, we observed the human equivalent, exoC5, to be increased in MARY‐X. siRNA Rab 7 knockdown in MCF‐7 produced an increase in the 120 KD band of E‐cadherin as well as E‐cadherin intermediates. This suggested that altered E‐cadherin redistribution may be contributing to its oncogenic role in IBC.

Tumor-infiltrating endothelial cells and endothelial precursor cells in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a specific type of breast tumor that generally has a poor prognosis, in spite of recent advances in treatment. In the present study, semiquantitative reverse transcriptase polymerase chain reaction examination of resected specimens showed that angiogenic factors, not lymphangiogenic factors, are overexpressed in IBC tumors, compared with non-IBC tumors. Immunohistochemical analysis of the specimens revealed a significantly higher population of tumor-infiltrating (TI) endothelial cells (ECs) or endothelial precursor cells (EPCs) in tumor-associated stroma of IBC specimens than in non-IBC specimens. In a previous study, we examined the phenotype of host cells in response to transplanted IBC cells, using an established human IBC xenograft model (WIBC-9) (Shirakawa et al., Cancer Res 2001;61:445-51). The data obtained in that study are consistent with the findings of the present study. To explore the therapeutic potential of blocking vascular endothelial growth factor (VEGF) and angiopoietin (Ang) pathways in IBC, established vectors encoding soluble Flt-1 (sFlt-1) and soluble Tie2 (sTie2) were injected directly into WIBC-9. Both vectors produced growth inhibition ratios of WIBC-9 that were significantly higher than those of a non-IBC xenograft (MC-5). Also, both vectors suppressed WIBC-9 lung metastases. The efficacy correlated with the number of TI ECs/EPCs, which was determined by fluorescence-activated cell sorting. These ECs/EPCs incorporated acetylated lipoprotein and were integrated within a HUVEC monolayer in vitro culture on day 5.