Soluble E-cadherin-CXCL1-CXCR2 Axis as a Therapeutic Vulnerability in Inflammatory Breast Cancer Brain Metastasis.

Background: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer with rapid onset and a strong propensity to spread to distant organs. Five-year overall survival (OS) rates remain poor, in part because of the high risk of brain metastasis: 19% of patients with IBC have brain metastases within the first 2 years after diagnosis. Our hypothesis for this study was that soluble E-cadherin (sEcad), an 80-kDa extracellular proteolytic fragment of full-length E-cadherin - a tumor promoter in IBC, is crucial for driving brain metastasis in IBC. Methods: We analyzed serum sEcad levels in from 348 IBC patients by ELISA. Four IBC cell lines [ER–/HER2+ (MDA-IBC3; SUM190) and ER–/HER2– (SUM149; BCX010)], human brain microvascular endothelial cells, and immortalized human astrocytes were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Mass spectrometry and Bio-ID-based proteomics assays, and RNA sequencing were used to identify sEcad-interacting proteins and potential mechanisms. In vivo, we studied tumor growth and brain metastasis in mice by injecting IBC cells into the mammary fatpad or tail vein, respectively, of SCID/Beige mice. Results: In IBC patients, higher serum sEcad levels correlated with poorer OS (p=0.02), earlier development of metastasis (p=0.006), and development of brain metastasis (p=0.04). On multivariable analysis, sEcad independently predicted OS (hazard ratio [HR]=2.07 [95% CI 1.19-3.60], p=0.01). In vitro, sEcad overexpression in IBC cell lines promoted anchorage-independent growth, migration, invasion, and resistance to anoikis. In vivo, sEcad-overexpressing SUM149 cells promoted primary tumor growth (p=0.007). Mice injected with sEcad-overexpressing MDA-IBC3 cells also had higher incidence of brain metastasis (100% vs 50%, p=0.03), metastatic burden (p=0.02) and number of metastases per mouse (p=0.0009), and had worse OS (p=0.0016), and brain metastasis-free survival (p=0.04), relative to controls. We further found that sEcad increased cancer cell adhesion to brain endothelial cells (p=0.01) and promoted induction of reactive astrocytes (as identified by high glial fibrillary acidic protein levels) in vitro and in vivo. Mechanistically, mass spectrometry and Bio-ID assays identified X-linked inhibitor of apoptosis protein (XIAP), a potent inhibitor of apoptotic cell death, as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Further analysis showed that sEcad bound to the BIR2 domain of XIAP. XIAP is the most potent and best-defined anti-apoptotic IAP family member, and it could induce NF-κB activation to inhibit tumor cell apoptosis Gene set enrichment analysis of RNA-seq profiling data showed activation of NF-kB signaling and downregulation of apoptotic pathways in the sEcad-overexpressing SUM149 cells compared with controls. Immunoblotting revealed that sEcad enhanced XIAP expression, activated NF-κB signaling, and inhibited cleavage of caspase-3 in IBC cells. Conclusions: We found that higher serum sEcad correlates with development of brain metastases and independently predicts poor OS in patients with IBC. We further found that sEcad promotes tumor growth and brain metastasis, perhaps via activation of XIAP/NF-κB signaling in breast cancer cells and promotion of endothelial cell adhesion and reactive astrocytosis in the brain microenvironment. These findings uncover a novel and crucial role for sEcad in brain metastasis and provide new insights and potential therapeutic targets for patients with metastatic IBC. Citation Format: Xiaoding Hu, Yun Xiong, Emilly S. Villodre, Juhee Song, Ganiraju C Manyam, Moises J Tacam, Jing Wang, Chandra Bartholomeusz, Debu Tripathy, Naoto T. Ueno, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-08.

Background: Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of primary breast cancer. Although considered rare, IBC accounts for 10% of breast cancer-related deaths owing to its rapid proliferation and strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC remain elusive. Through transcriptome profiling we identified Decorin (DCN) as being significantly altered in metastatic IBC cell sublines. DCN is a secreted, small leucine-rich proteoglycan known to function as a tumor/metastasis suppressor by inhibiting several signaling pathways including EGFR, TGFβ, and c-MET. However, whether or how DCN regulates IBC tumorigenesis or metastasis is unknown. The aim of this study was to investigate the function and mechanism of DCN in IBC tumorigenesis and metastasis. Methods: Three IBC cell lines [ER-/HER2+ (MDA-IBC3, SUM190) and ER-/HER2- (SUM149)] were used. DCN gene expression in clinical samples was analyzed from publically available datasets and the IBC Consortium dataset. DCN was stably expressed in IBC cell lines by using lentiviral vectors. For in vivo studies, DCN-overexpressing stable cell lines were injected into cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. Reverse phase protein array analysis was used for proteomic profiling. Protein-protein interactions were analyzed by reciprocal immunoprecipitation of exogenous or endogenous proteins. Results: DCN expression was significantly lower in breast cancer samples than in normal breast (p<0.0001), in more aggressive breast cancer subtypes (p<0.0001), and in metastatic tumors relative to primary tumors (p<0.0001). High DCN expression correlated with improved overall survival (p<0.0001) and relapse-free survival (p=0.0003). In vitro, DCN overexpression in IBC cell lines inhibited colony formation (MDA-IBC3, p=0.0115; SUM149, p=0.0068), migration (SUM149, p=0.0165), invasion (SUM149, p=0.0159), and primary and secondary mammosphere formation (primary: MDA-IBC3, p=0.0209; SUM149, p=0.0232; secondary: MDA-IBC3, p=0.01; SUM149, p=0.0031). In vivo, DCN overexpression in MDA-IBC3 cells inhibited primary tumor growth (p=0.0092) and reduced skin invasion (89.9% DCN control vs 33.3% DCN-overexpressed, p=0.017, Chi-square test). Our proteomics data in DCN-overexpressing SUM149 and MDA-IBC3 cells showed downregulated EGFR and E-cadherin protein levels. Mechanistically, DCN reduced expression of E-cadherin and EGFR and reduced phosphorylation of EGFR in IBC cells. Exogenous and endogenous co-immunoprecipitation experiments showed direct physical binding between DCN and E-cadherin proteins in all IBC cell lines. Moreover, overexpression of DCN downregulated E-cadherin via protein instability rather than decreased E-cadherin mRNA expression. E-cadherin knockdown in IBC cells decreased, whereas its overexpression increased, activation of EGFR signaling without affecting DCN expression. Finally, restoring E-cadherin in DCN-overexpressing IBC cell lines rescued the inhibitory effect of DCN on EGFR signaling. Conclusions: We found that DCN inhibited tumorigenesis and skin invasion in IBC via its direct interaction with and stabilization of E-cadherin and its suppression of EGFR signaling. Our findings provide new insights and a novel mechanism for IBC pathobiology that may be therapeutically targetable. Future studies will determine the role of DCN in IBC metastasis and the detailed mechanism of DCN-mediated suppression of tumorigenesis and metastasis in IBC. Citation Format: Xiaoding Hu, Emilly Schlee Villodre, Richard Larson, Omar M. Rahal, Xiaoping Wang, Savitri Krishnamurthy, Debu Tripathy, Naoto T Ueno, Wendy A Woodward, Bisrat Godefay Debeb. Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-06.

Background: Brain metastasis is a frequent site of relapse in patients with inflammatory breast cancer (IBC) - a rare, highly aggressive and metastatic variant of breast cancer. We have discovered that soluble E-cadherin (sEcad), an 80-kDa proteolytic fragment of full-length E-cadherin, correlated with increased risk of brain metastasis and death in patients with metastatic IBC. Additionally, we demonstrated that sEcad promotes brain metastasis growth and progression in HER2+ and triple-negative IBC brain metastasis models. However, how sEcad promotes brain metastatic progression is unknown. We hypothesize that sEcad promotes the production of specific cytokines, which, upon extracellular release, promote the activation of astrocytes, priming the brain microenvironment for metastatic growth. Methods: Stable overexpression of sEcad in IBC cell lines (MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) was achieved using lentiviral vectors. We injected MDA-IBC3-sEcad and control cells (tail-vein) and SUM149-sEcad and control cells (intracardiac) into SCID/Beige mice to assess brain metastasis burden and survival in mice. Human cytokine array was used to examine conditioned medium from sEcad high and control cells. Clinical datasets were used to compare expression and percent risk of brain relapse. Mice were treated with brain-permeable CXCR2 inhibitor in both IBC brain metastasis models. Multiplex quantitative imaging was used to visualize cells of the brain metastatic microenvironment. Results: Higher serum sEcad levels were significantly associated with reduced OS, earlier metastasis onset, and increased brain metastasis incidence. sEcad is an independent predictor of OS on multivariate analysis (hazard ratio [HR]=2.07 [95% CI 1.19-3.60], p=0.01). Treatment of astrocytes with recombinant sEcad increased reactive astrocytosis, in vitro and in vivo. Cytokine array analysis showed increased levels of pro-inflammatory cytokine CXCL1, DKK1 and CXCL8 in conditioned medium from sEcad-overexpressing IBC cells compared with control cells, which was validated by ELISA. In patient samples, CXCL1, CXCL8 and CXCR2, the receptor for CXCL1 and CXCL8, were expressed higher in brain metastasis compared to other metastases. Additionally, patients with high CXCL1/CXCL8 or CXCR2 expression had reduced brain metastasis relapse. Inhibition of CXCR2 decreased sEcad-mediated induction of reactive astrocytes. Treatment of mice bearing MDA-IBC3-sEcad and SUM149-sEcad brain metastases with the brain permeable CXCR2 inhibitor reduced number of brain metastasis, metastasis burden and prolonged survival of mice. Multiplexed immunofluorescence staining showed a significant reduction of reactive astrocytes in brain metastasis lesions treated with the CXCR2 inhibitor. Conclusion: Our findings underscore that sEcad drives brain metastasis by promoting an inflammatory brain microenvironment via a targetable CXCL1/CXCL8-CXCR2 axis. Targeting this axis presents a promising therapeutic strategy to effectively block brain metastasis in aggressive breast cancers. Citation Format: Xiaoding Hu, Yun Xiong, Emilly S Villodre, Juhee Song, Maria Stenkamp, Natalie Fowlkes, Elizabeth Leigh, Jeffery, Savitri Krishnamurthy, Junjie Chen, Wendy A Woodward, Debu Tripathy, Bisrat G Debeb. Targeting CXCL1-CXCR2 axis blocks brain metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS14-08.

Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and is highly metastatic. The prognosis of patients with IBC is poor, and effective standard therapies for IBC are limited because the molecular mechanisms underlying the pathogenesis of IBC remain unknown. We recently found that tazarotene-induced gene 1 (TIG1) expression is significantly higher in IBC cell lines than in non-IBC cell lines. In both IBC and non-IBC data sets, estrogen receptor-negative/HER2−negative samples had significantly higher expression of TIG1 than did other clinical subtypes (estrogen receptor-positive/HER2−negative and HER2−positive). Therefore, we hypothesized that TIG1 plays an important role in the malignant process of IBC. In these studies, we determined the biological function of TIG1 in IBC cells and elucidated the molecular mechanism by which TIG1 regulates the invasiveness of IBC cells. Methods: TIG1 expression in SUM149 and KPL-4 IBC cells was stably knocked down, and the effects of this knockdown on in vitro cell proliferation, migration, and invasion were analyzed. The effects of restoring TIG1 expression on TIG1-silencing IBC cells were also examined. To determine the tumorigenic activity of TIG1 in vivo, TIG1 stable-knockdown SUM149 cells and control shRNA-transfected cells were implanted into the mammary fat pads of athymic nude mice, and tumor growth was monitored. The receptor tyrosine kinase Axl, a potential functional partner of TIG1, was identified using DNA microarray analysis. The interaction between TIG1 and Axl in IBC cells was examined using immunoprecipitation and confocal microscopy assays. The signaling pathway in IBC cells in which TIG1 participates was also investigated. Results: Knockdown of TIG1 expression in IBC cells reduced their proliferation, migration, and invasion in vitro. Also, silencing of TIG1 dramatically inhibited IBC tumor growth in a xenograft model. Moreover, restoring TIG1 expression rescued the proliferation, motility, and invasiveness of TIG1-silenced IBC cells. Most importantly, we identified Axl as a functional partner of TIG1 by showing that TIG1 interacted with and stabilized Axl in IBC cells. TIG1 regulated the invasiveness of IBC cells through mediation of the Axl signaling pathway. In SUM149 cells, TIG1 depletion decreased Axl expression, which led to downregulation of expression of matrix metalloproteinase-9, a molecule required for Axl-mediated invasion, and inactivation of nuclear factor-kB, ultimately leading to decreased invasiveness of IBC cells. Conclusion: Our results identified TIG1 as an oncogenic gene that contributes to the tumorigenic and metastatic properties of IBC. Our data also linked TIG1 with the key tumorigenic gene Axl in IBC cells. Further studies designed to establish TIG1 as a therapeutic target in the treatment of patients with IBC are under way. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-01.

Inflammatory breast carcinoma (IBC) is the most aggressive form of breast cancer. The hallmark of IBC is regional extension into dermal lymphatics as tumor emboli causing breast edema and erythema. Pathologic characteristics of IBC include high grade, negative hormone receptor status and overexpression of HER2 and E-cadherin. The latter is the most attractive therapeutic target in IBC. In preclinical studies the histone deacetylase inhibitor, romidepsin targeted E-cadherin, affecting tumor emboli and increasing taxane sensitivity. Rationale: In vitro studies show that histone deacetylase inhibitors (HDACi) with taxanes provide synergy to enhance cell death. HDACi alter expression of AIRH1, a regulator of autophagy, typically silenced in breast cancer. In vitro treatment with HDACi induces expression of AIRH1, resulting in enhanced cell death with taxanes. In vitro studies of IBC have demonstrated the utility of HDACi and romidepsin in IBC cell lines. SAHA and romidespin, HDACis, inhibited self-renewal of IBC tumor spheroids from IBC cell lines. This trial combines romidepsin with a taxane proven in metastatic breast cancer to explore whether the combination will be effective in IBC. Design: This is a phase I trial to assess the safety of romidepsin plus nab-paclitaxel in patients with recurrent or metastatic IBC. The maximum tolerated dose (MTD) of romidepsin + weekly nab-paclitaxel was determined to define the dose for the phase II trial. Secondary objectives included describing the adverse event profile and assessing the overall response rate (ORR) and Clinical Benefit Rate (CBR). This study employed a 3+3 design. DLTs included febrile neutropenia or non-hematologic grade 3 or 4 toxicities. Patients were treated with nab-paclitaxel 100 mg/m2 iv with romidepsin, 7 mg/m2 iv (1st cohort) and 10 mg/m2 iv (2nd cohort), on days 1, 8, 15 of a 28 day cycle. Results: Nine patients were treated. The median age was 52. Three patients were treated in the first cohort. Two patients showed progressive disease (PD). One patient has had stable disease (SD) over 10 cycles and continues treatment. DLT was not reached at 7 mg. Toxicities related to romidepsin included neutropenia, anemia and fatigue. Six patients were treated in the 2nd cohort. Grade 3 hypophosphatemia, a DLT, was reached. One patient had complete response (CR). One patient had SD; four patients had PD. Toxicities related to romidepsin were anemia, neutropenia, GI upset, edema, hyperglycemia, fatigue, hypophosphatemia, pruritis, dry mouth, and increased lab values. The overall response rate (ORR) was 33% (3/9). The table below shows results. CohortResponse# Prior TherapiesMetastatic Sites# Cycles on study1PD2pleura, nodes51PD0lung31SD2lung, nodes102PD2pleura, nodes, soft tissue42PD0liver, nodes, bone22CR2 chemotherapy, 2 endocrinebone, nodes72SD0nodes52PD0lung,liver, nodes22PD0liver, bone, nodes2 Conclusions: This phase I trial shows that romidepsin and nab-paclitaxel are well-tolerated in patients with advanced IBC. The MTD and recommended dose of romidepsin is 10 mg/m2 with nab-paclitaxel 100 mg/ m2 days 1, 8, 15 of a 28 day cycle. A phase II trial is planned in recurrent HER negative IBC patients. Citation Format: Avery TP, Jaslow R, Basu-Mallick A, Zibelli A, Fellin F, Cristofanilli M. A phase I study of romidepsin in combination with nab-paclitaxel in patients with metastatic HER-2 negative inflammatory breast cancer (IBC). [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 P4-13-18.

Background: Brain metastasis is a frequent and lethal site of relapse in inflammatory breast cancer (IBC), a rare, aggressive, and highly metastatic breast cancer subtype. We identified soluble E-cadherin (sEcad), an 80-kDa extracellular fragment of full-length E-cadherin, as being significantly associated with increased risk of brain metastasis and decreased overall survival (OS) in patients with metastatic IBC. Functional studies further indicate that sEcad promotes brain metastasis progression in both HER2+ and triple-negative IBC models. However, the underlying mechanism remains poorly understood. Methods: Stable overexpression of sEcad in IBC cell lines [MDA-IBC3 (ER-/HER2+) and SUM149 (ER-/HER2-)] was achieved using lentiviral transduction. PDIA4 knockdown was performed in the sEcad-overexpressing cells via shRNA. To evaluate brain metastasis and survival, MDA-IBC3-sEcad cells were injected via tail vein and SUM149-sEcad cells via intracardiac route into SCID/Beige mice. Mice were treated with 35G8, a blood-brain barrier-permeable PDIA4 inhibitor, administered subcutaneously. In silico analysis was performed using multiple independent breast cancer patient datasets. Results: High serum sEcad levels in IBC patients correlated with poorer OS (=0.02) and earlier development of metastasis (p=0.006). Overexpression of sEcad in IBC cell lines enhanced cell proliferation, and colony formation, migration, invasion, and reduced cell death in vitro. Mice injected with sEcad-overexpressing SUM149 and MDA-IBC3 cells had significantly higher tumor growth rates compared to controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, mass spectrometry and Bio-ID assays identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. PDIA4 expression was markedly elevated in sEcad-overexpressing cells in vitro and in vivo. Knockdown of PDIA4 in sEcad-overexpressing cells significantly reduced migration, invasion and survival. In silico analysis shows that PDIA4 is highly expressed in aggressive ER-negative, basal-like, and HER2+ tumors (p < 0.0001), which have increased brain metastasis risk. PDIA4 levels are also higher in brain metastases than matched primary tumors (GSE125989, P=0.011). Treatment with the brain-permeable PDIA4 inhibitor 35G8 significantly decreased brain metastasis burden in SUM149-sEcad models, including a decrease in brain metastasis incidence (p = 0.05) and the number of brain metastatic lesions (p = 0.03). Conclusion: Our findings uncover a novel mechanism by which sEcad drives brain metastasis in IBC through its interaction with PDIA4. Inhibiting PDIA4 with the BBB-permeable compound 35G8 reduced brain metastatic burden, highlighting the sEcad-PDIA4 axis as a potential therapeutic target. Citation Format: X. Hu, I. Longa Rizzo, E. Schlee Villodre, T. Phi, K. Tesfamariam, J. Song, Y. Gong, K. Savitri, W. A. Woodward, B. G. Debeb. Therapeutic Inhibition of the sEcad-PDIA4 Axis Suppresses Brain Metastasis in Inflammatory Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-09-25.

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.

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

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis and lower survival rate. IBC is characterized by peculiar clinical and biological features and resistance to standard treatments. IBC has shown the capacity to spread early through lymphatic channels and blood vessels causing the specific inflammatory signs. Currently, there are no specific therapeutic options for IBC patients because of very few molecular alterations associated with IBC. Thus, there is an unmet need to identify distinguished features of IBC that differentiate the disease from non-inflammatory breast cancer (nIBC). Metabolomic analysis of cancer cells holds the promise to identify unique chemical fingerprints of important cellular processes. In this study we investigated the global biochemical profiles of IBC and nIBC cells. METHODS: Three IBC cell lines, SUM190, SUM149, FC-IBC-02, and three nIBC cell lines, MCF-7, MDA-MB-231, MDA-MB-468, with five replicates for each cell line, were analyzed in this study. Metabolomic profiling analysis was performed by Metabolon, Inc (Durham, NC) on the gas chromatography/mass spectroscopy and ultrahigh performance liquid chromatography/mass spectroscopy platforms as previously described (Proc. Natl. Acad. Sci. U. S. A. 108, 3270-3275, 2011). A total of 347 metabolites were detected. Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (p≤0.001), as well as those approaching significance (0.001<p<0.05). RESULTS: Comparison of global biochemical profiles for IBC and nIBC cell lines revealed numerous statistically changed metabolites at a p-value threshold of p<0.001. Statistical comparisons also revealed a clear metabolic separation of samples due to cell line differences. Several biochemical pathways emerged as being commonly altered across all IBC cell lines compared to all nIBC cell lines. Notable observations include alterations in inflammatory signaling, energy metabolism, and cell proliferation. Signatures of inflammation were elevated and inflammatory signaling pathways showed activation in IBC cell lines. In energy metabolism, the cellular energy for IBC cells appeared to be derived from amino acid catabolism to a greater extent than in nIBC cells. Furthermore, fatty acids was significantly elevated in IBC cell lines, that may indicate a reduced cellular proliferation and increased glycogen storage in IBC cells. Moreover, an altered methylation and glutathione metabolism were shown in IBC cell lines. CONCLUSIONS: Our study is the first to demonstrate difference of metabolic profiles between IBC and nIBC cells. Future studies comparing global biochemical profiles in tissue biopsy and blood from IBC and nIBC patients may help to confirm the relevance of these findings in cell lines. Metabolic targets may provide more effective and specific therapeutic strategy for the treatment of IBC patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-05.

Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer known for its rapid progression and high metastatic potential without known distinctive drivers. Currently, there are no FDA-approved targeted therapies specifically for IBC patients, highlighting the urgent need for novel and effective treatments. Through our investigation of metastatic xenograft IBC sublines, we have identified soluble E-cadherin (sEcad), an extracellular proteolytic fragment of full-length E-cadherin, as a protein associated with IBC tumor progression. We hypothesize that sEcad promotes IBC tumorigenesis by suppressing ferroptosis. Methods: MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) IBC cell lines were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Cell death for ferroptosis was measured by propidium iodide staining using flow cytometry. Mass spectrometry and Bio-ID-based proteomics assays were used to identify sEcad-interacting proteins and potential mechanisms. For in vivo studies, control and sEcad -overexpressing SUM149 and MDA-IBC3 cells were injected into the cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Serum sEcad levels from IBC patients (n=301) were analyzed by ELISA. Results: High serum sEcad levels in IBC patients correlated with poorer OS (p=0.02) and earlier development of metastasis (p=0.006). Overexpression of sEcad in IBC cell lines enhanced cell proliferation and colony formation in vitro. Mice injected with sEcad-overexpressing SUM149 and MDA-IBC3 cells had significantly higher tumor growth rates compared to controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, mass spectrometry and Bio-ID assays identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Additionally, sEcad increased the expression of PDIA4, an enzyme that plays a role in cellular redox regulation and protein folding. PDIA4 has been shown to regulate key players of ferroptosis such as ATF4, SLC7A11. Our results showed that, compared to controls, sEcad-overexpressing IBC cells displayed significant resistance to ferroptosis inducers, including RSL3, FIN56, and sulfasalazine. Knockdown of PDIA4 in sEcad high-expressing cells significantly increased their sensitivity to ferroptosis-promoting drugs. Moreover, we found that PDIA4 regulates ATF4 to impact ferroptosis-mediated cell death in IBC cells. Conclusions: Our study shows that sEcad affects ferroptosis through PDIA4, impacting tumorigenesis in IBC. These findings uncover a novel and pivotal role for sEcad in IBC tumor growth and progression, providing new insights and potential therapeutic targets for IBC patients. Citation Format: Xiaoding Hu, Yun Xiong, Emilly Villodre, Juhee Song, Debu Tripathy, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-03.

Vitamin D is a known regulator of breast cancer cell proliferation, apoptosis, migration, invasion and differentiation in vitro. Recent studies have suggested a preventative role for vitamin D in breast cancer development and suggested a possible therapeutic application of vitamin D for patients with various forms of breast cancer. Inflammatory breast cancer (IBC) is a highly aggressive and phenotypically unique form of breast cancer that has a very poor prognosis. IBC invades the dermal lymphatics of the breast as tumor emboli early in the course of the disease. Because of the invasive nature of IBC, novel therapeutics are needed desperately. In the current study we examined the effect of the active form of vitamin D, calcitriol, treatment on the aggressive IBC phenotype. Herein we demonstrate that although the vitamin D receptor (VDR) is present in both IBC and non-IBC cell lines, the effect of vitamin D treatment is significant only on the IBC cells. SUM149 IBC cells showed increased protein concentration in response to 24h of calcitriol exposure; likely mediated by an increase in protein synthesis as opposed to increased cellular proliferation. In addition, treatment with 100nM calcitriol showed a significant decrease in SUM149 migration (67.8% decrease, P=0.030), invasion (43.9% decrease, P=0.015), and tumor spheroid size (69.4% decrease, P=0.018) compared to nontreated control groups. Finally, calcitriol treatment of SUM149 cells led to significantly fewer IBC experimental metastases as compared to control. Our study demonstrates that calcitriol treatment of SUM149 affected several of the processes important for IBC metastasis but had little effect on MDA-MB-231 cells. Therefore, calcitriol treatment may have the potential to decrease the rate and incidence of metastasis in IBC patients.

Inflammatory breast cancer (IBC) is an extremely aggressive and rare form of cancer that disproportionally affects African American and younger women. While IBC represents 1-5% of breast cancers, it accounts for 10% of all breast cancer deaths annually in the United States. We have previously shown that the metabolic characteristics of IBC, specifically in the triple negative (TN) and inflammatory breast cancer cell line SUM149, are significantly altered from those of normal breast cancer cells. We have shown previously that RhoC, a member of the Ras superfamily of GTPases, contributes to the metastatic IBC phenotype and acts as a regulator of the metabolite N-acetyl aspartate (NAA) in SUM149 cells. NAA is the second most abundant metabolite in the brain (only exceeded by glutamate) and has been used for the diagnosis of neurodegenerative disorders such as the fatal genetic disorder Canavan’s disease, which is characterized by toxic NAA levels due to a deleterious mutation in aspartoacyclase(ASPA). ASPA is the enzyme involved in the catabolism of NAA into aspartate and acetate. NAA has previously been considered a brain specific metabolite and the fundamental role of NAA outside of the central nervous system, especially in the context of cancer, remains elusive but highly intriguing. We have previously shown that NAA levels are significantly higher in the IBC-derived TN SUM149 cells than TN cancer cell line MDA-MB-231. Interestingly, and independently, recent studies have also implicated high levels of tumoral NAA with significantly worse survival rates in ovarian cancer patients. The synthesis of NAA from acetyl-CoA and aspartate is catalyzed by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. Knockdown of RhoC in SUM149 cells using shRNA significantly decreases both NAT8L expression and NAA metabolite levels. To further understand RhoC’s role in the modulation of NAA, we generated RhoC and RhoA knockout cell lines using CRIPSR-Cas9 in a set of inflammatory and noninflammatory breast cancer cell lines. RNA-seq analysis of these show that SUM149 RhoC knockout cells produce the largest amount of differentially regulated genes when compared to wild-type cells. The differentially regulated genes include many of the key genes involved in NAA associated pathways. Furthermore, metabolic studies reveal that the most downregulated metabolites in the SUM149 RhoC knockout cell line are N-acetyl derivates including NAA, N-acetylglutamate(NAG), and N-acetylaspartylglutamate (NAAG). Overexpression and siRNA cell lines for both NAT8L and ASPA have been generated to further elucidate the altered metabolism seen in these cells. Additional molecular studies are ongoing to determine the specific role of NAA in the adapted metabolic pathways of inflammatory breast cancer that may shed light on IBC’s increased metastatic potential and decreased survival. Citation Format: Laura E. Goo, Joel A. Yates, Andrew C. Little, Daniel Kremer, Ilya Kovalenko, Christopher R. Oliver, Trisha Westerhof, Zhifen Wu, Nathalie Vandecan, Liwei Bao, Peter J. Ulintz, Costas A. Lyssiotis, Sofia D. Merajver. Effects of the small GTPase RhoC on inflammatory breast cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4363.

Background: Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer and accounts for a significant 10 % of breast cancer deaths in the USA owing to its aggressive proliferation and metastasis, and a lack of effective therapeutic options. Unraveling the underlying mechanisms of growth and metastasis of this aggressive disease could lead to effective therapeutic strategies for an improved outcome in IBC patients. We recently generated in vitro and in vivo IBC models for brain metastasis studies [Debeb et al. JNCI, 2016] and observed an upregulation of Lipocalin 2 (LCN2), a small, secreted iron-trafficking protein which plays a significant role in immune and inflammatory responses and the promotion of malignant progression. The purpose of this study was to investigate the function of LCN2 in IBC tumorigenesis and metastasis. Methods: Stable knockdown (KD) of LCN2 in IBC cell lines was achieved with lentiviral vectors. Proteomic and gene expression profiling were performed using RPPA and Affymetrix Clariom D microarray. For in vivo studies, control and LCN2 KD IBC cells were transplanted into the cleared mammary fat pad of SCID/Beige mice. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. LCN2 gene expression levels in clinical samples were analyzed from the IBC Consortium as well as public data sets. LCN2 serum levels in IBC patients were measured using ELISA and were correlated with clinicopathological variables and outcome data. Results: LCN2 gene expression is higher in IBC versus non-IBC patients (p=0.00036), independently of the molecular subtypes, and higher in more aggressive (TNBC and HER2+) than hormone receptor-positive subtypes (p<0.00001). LCN2 expression in patient tissues is correlated with reduced overall survival (p<0.00001) and metastasis-free survival (p=0.04) in non-IBC; however, LCN2 was not associated with overall survival in IBC patient serum samples. LCN2 expression was also significantly higher in IBC cell lines, in their culture media, and in brain metastasis sublines compared to non-IBC cell lines (p=0.004). In IBC cell lines, LCN2 KD reduced proliferation, colony formation, migration, and cancer stem cell properties. In vivo silencing of LCN2 in SUM149 cells inhibited primary tumor growth (p=0.001)and resulted in a well-differentiated tumor histology. Additionally, SUM149 LCN2 KD significantly reduced skin invasion/recurrence (LCN2 control vs LCN2 KD: 88 % vs 25 %, p=0.01) suggesting LCN2 is a mediator of tumorigenesis. Analysis of proteomics data showed changes in major signaling pathways including PI3K-Akt signaling and EGF/EGFR signaling pathways. Mechanistically, LCN2 depletion in SUM149 abrogated EGF-induced EGFR phosphorylation and ERK activation. Conclusions: Our findings suggest that LCN2 may drive IBC tumor progression and skin invasion/recurrence potentially via the EGFR signaling pathway.Future studies will determine the role of LCN2 in metastasis and pinpoint the detailed mechanisms of LCN2-mediated IBC tumorigenesis and recurrence. Citation Format: Villodre ES, Larson R, Hu X, Stecklein SR, Gomez K, Finetti P, Krishnamurthy S, Ivan C, Su X, Ueno NT, Van Laere S, Bertucci F, Tripathy D, Vivas-Mejía P, A Woodward W, Debeb BG. Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-03.

Background: Inflammatory breast cancer (IBC) is a rare (1-2%) but lethal form of primary breast cancer responsible for 10% of breast cancer-related deaths in the United States. IBC tumors grow aggressively and have a high risk of metastasis, particularly to the brain. Unraveling the pathways involved in IBC aggressiveness and metastasis could reveal new therapeutic targets. We recently identified NDRG1 (N-Myc downstream regulated gene1) in a newly isolated subline of MDA-IBC3 cells as a top candidate protein associated with enhanced propensity for brain metastasis. Although the metastasis suppressor properties of NDRG1 are widely described, its function in the pathobiology of IBC is unknown. Here we investigated the function of NDRG1 in tumorigenesis and metastatic progression to brain in IBC. Methods: NDRG1 gene expression was analyzed in large publically available breast cancer clinical datasets and the IBC Consortium dataset, and median expression level was used to stratify patients into NDRG1-high and NDRG1-low groups. Tissue microarrays of IBC patient samples were immunostained with anti-NDRG1 antibody. Stable knockdown (KD) of NDRG1 in IBC cell lines (HER2+: MDA-IBC3; TNBC: SUM149, BCX010) was obtained by lentiviral gene transfer. For in vivo studies, control and NDRG1 KD IBC cell lines were transplanted into cleared mammary fat pads of SCID/Beige mice and tumor growth monitored via caliper measurements. Reverse phase protein array analysis was used for proteomic profiling. Results: In several independent cohorts of patients with breast cancer (including The Cancer Genome Atlas), NDRG1 mRNA expression was significantly higher in ER-negative and high-grade breast tumors (p<0.0001) and was associated with poor overall survival (p=0.0003), relapse-free survival (p<0.0001), and metastasis-free survival (p=0.0012). NDRG1 expression was higher in more aggressive, basal-like and HER2+ subtypes than in hormone receptor-positive subtypes (p<0.0001), and was also higher in IBC vs. non-IBC tumors (p=0.007, IBC consortium dataset). Immunostaining showed NDRG1 was frequently expressed in IBC patient tumors (83%) and was associated with worse overall survival (OS, p=0.0129) and disease-specific survival (DSS, p=0.013). On multivariate analysis, NDRG1 was an independent prognostic factor for poor OS [HR 2.5 (95%CI, 1.3-4.7), p=0.0047] and DSS [HR 2.6 (95%CI 1.3-5.1), p=0.006]. Notably, NDRG1 expression was higher in patients with brain metastasis vs. primary tumors (GSE43837, p=0.018) and correlated with shorter time to development of brain metastasis (GSE2034, p=0.026). NDRG1-high sublines had enhanced propensity for brain metastasis as shown by higher incidence (100% vs. 44% for NDRG1-low sublines, p=0.01) and increased brain metastasis burden (p=0.0008). Invitro, silencing NDRG1 in IBC cell lines significantly reduced colony formation, migration, invasion, and cancer stem-like properties. In vivo, silencing of NDRG1 inhibited primary tumor growth of SUM149 (p<0.0001) and MDA-IBC3 (p=0.0426) xenografts. Proteomics analysis showed that NDRG1 correlated positively with activation of the EGFR/STAT3 signaling pathway. Mechanistically, NDRG1 knockdown in IBC cell lines reduced EGFR expression and suppressed phosphorylation of EGFR and STAT3, whereas NDRG1 overexpression increased EGFR expression and enhanced EGFR-STAT3 activation. Conclusions: We showed that high NDRG1 was associated with worse clinical outcomes in patients with IBC and that NDRG1 drives tumor progression in IBC potentially via activation of the EGFR-STAT3 signaling pathway. Targeting the NDRG1-EGFR axis may be a novel therapeutic approach in IBC and other aggressive breast cancers. Additional investigations are underway to determine the detailed mechanisms of NDRG1-mediated IBC tumorigenesis and brain metastasis Citation Format: Emilly Schlee Villodre, Xiaoding Hu, Richard Larson, Bedrich L Eckhardt, Yun Gong, Lei Huo, Juhee Song, Savitri Krishnamurthy, Nuhad Ibrahim, Naoto T Ueno, Debu Tripathy, Wendy A Woodward, Bisrat G Debeb. Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-10.

Purpose: Inflammatory breast cancer (IBC) is the most lethal, aggressive variant of breast cancer characterized by rapid progression, local and distant metastases, younger age of onset, and poor overall survival. Despite advances in multimodal breast cancer care, no IBC specific therapy is available clinically, demonstrating a critical need to identify novel therapeutics that target the distinct biology of IBC. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and have been implicated as potential therapeutic targets. Although the oncogenic or tumor-suppressor functions of a large number of miRNAs have been described in various solid tumors including breast cancer, the role of miRNAs in the biology of IBC is poorly understood. Herein, we investigated miRNAs differentially expressed in IBC, particularly miR-200 family miRNAs as potential therapeutic targets in IBC. Methods: MicroRNA microarray profiling was conducted in IBC and non-IBC cell lines, and microarray results were validated using qRT-PCR. MirZip lentiviral based mirRNA inhibition was used to stably knockdown miRNAs and the effects of specific miRNA knockdown on proliferation, EMT, colony and mammosphere formation was evaluated. For tumor progression and metastasis in vivo experiments, we injected 500,000 SUM149 miR-200a, miR-141 knockdown and control (N=15 for each group) cells into the cleared mammary fatpad of SCID/Beige mice. We also validated the expression of miR-200 miRNAs in IBC patient samples. Results: Differential expression of mirRNAs was observed in IBC vs. non-IBC cell lines with a significantly higher expression of all miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-479) in IBC cell lines. These results were validated using qRT-PCR, with the IBC cell lines showing a >1,000-fold higher expression of miR-200s vs non-IBC aggressive cell lines (P<0.0001). Moreover, two known targets of the miR-200 family, Zeb1 and Zeb2, were significantly downregulated while E-cadherin was upregulated in the IBC samples. Importantly, these observations were validated in IBC patient samples which showed a significantly higher expression of all miRNA 200s compared to the normal breast tissues (P<0.001). In vitro, miR-200a and miR-141 knockdown significantly reduced both colony and mammosphere formation in the triple negative SUM149 cells (P<0.001) but did not have a significant effect on the HER2+ IBC cell lines SUM190 and MDA-IBC-3 (P>0.05) while it upregulated the EMT markers N-cadherin, Fibronectin, TGFβ, and Smad4 and reduced E-cadherin expression compared to transduced controls. In vivo, miR-200 knockdown resulted in an increase in tumor progression vs. controls (P<0.05, Day 63) while metastasis studies are undergoing. Conclusions: These data suggest that miR-200 family microRNAs play an important role in IBC tumor progression and altering miR-200 levels could be an efficacious approach to target IBC. Citation Format: Bisrat G. Debeb, Lara Lacerda, Richard Larson, Li Li, Wei Xu, Lei Huo, Caimiao Wei, Savitri Krishnamurthy, James Reuben, Naoto Ueno, Thomas Buchholz, Wendy A. Woodward, Morgan Welch Inflammatory Breast Cancer Research Program and Clinic. The miR-200 family as a potential therapeutic target in inflammatory breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3051. doi:10.1158/1538-7445.AM2013-3051