Inflammatory Breast Cancer Metastasis Research Articles

Abstract PO1-25-04: PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer

Abstract Background Inflammatory breast cancer (IBC) is a lethal subtype of breast cancer characterized by rapidly arising diffuse erythema and edema of the overlying skin that was thought to contribute to rapid metastasis. Dissecting the intricate cellular ecosystems within tumors and affected skin and their complex interplay may be pivotal in unraveling the aggressive nature of IBC. Methods Integrative analysis containing single-cell RNA sequencing, bulk RNA sequencing, and whole exome sequencing, was performed on paired tumor and skin samples from 17 treatment-naive IBC patients and 5 non-IBC patients from multiple centers. Meanwhile, paraffin sections and serum specimens from two centers were conducted to validate the omics results. Additionally, public single-cell transcriptomic data of non-IBC tumors and normal mammary glands, RNA microarray data from IBC cases, and the Cancer Genome Atlas (TCGA) database were utilized to validate the characteristics of IBC microenvironment. The regulatory mechanism was verified by in vitro experiments. Results We delineated the landscapes of IBC tumor and affected skin microenvironment at the single-cell level and demonstrated that the luminal progenitors (LP) cells highly expressing pleiotrophin (PTN) were most significantly enriched in IBC tumors compared to non-IBC tumors and normal mammary glands, in two independent cohorts. Moreover, the serum concentrations of PTN were significantly higher in IBC patients compared with non-IBC patients. Notably, PTN secreted by PTN+ LP cells interacted with Neuropilin 1(NRP1) receptor on endothelial tip cells in both tumor and affected skin of IBC. Moreover, in vitro assay demonstrated knocking down the expression of NRP1 on human umbilical vein endothelial cells significantly blocked PTN-induced migration, tube formation and sprouting, which might be independent of VEGFA. In addition, two immature phenotypes of PVL_MMP9 and PVL_CCL19 cells recruited by endothelial tip cells via PDGFB/PDGFRB axis were accumulated in affected skin of IBC, which might be associated with promotion of malignant cell metastasis. Finally, we revealed that malignant cells in affected skin of IBC had a higher ability of epithelial-mesenchymal transition than IBC intratumoral malignant cells and identified enhanced crosstalk between immature PVL cells and malignant cells in the skin affected with IBC , through TNFSF12-TNFRSF12A, TGFB-TGFBR1/TGFBR2, MDK-SDC1/4/NCL/(ITGA6+ITGB1). Conclusion We identified a unique, non-malignant epithelial cell subpopulation PTN+ LP cells, which induce massive angiogenesis via the PTN-NRP1 axis, recruit the immature phenotype PVL cells accumulated in affected skin of IBC thus promoting tumor progression. Our findings offer comprehensive insights into the pathogenesis of IBC and uncover a previously unknown, promising therapeutic target. Patient of discovery cohort details Clinical and pathology details for inflammatory breast cancer(IBC) and non-IBC patients analysed by single-cell RNA sequencing(scRNA-seq), bulk RNA sequencing(bulk RNA-seq) and whole exome sequencing(WES) in this study. Clinicalpathological feature of validation cohort Citation Format: Mengmeng Zhang, Kaiwen Zhou, Ting Liu, Filipa Lynce, Filipa Lynce, Wendy Chen, Qianjun Chen, Xiaoping Li, Zhongyu Yuan, Juan Xu, Zhenhai Cai, Jianping Guo, Nan Shao, Ying Lin. PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer [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 PO1-25-04.

A Subpopulation of Luminal Progenitors Secretes Pleiotrophin to Promote Angiogenesis and Metastasis in Inflammatory Breast Cancer.

Nonmalignant luminal progenitor cells expressing pleiotrophin promote angiogenesis by activating NRP1 and induce a prometastatic tumor microenvironment in inflammatory breast cancer, providing potential therapeutic targets for this aggressive breast cancer subtype.

Abstract GS5-08: Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer

Abstract 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.

Abstract P1-10-06: Carcinoma en Cuirasse Secondary to Locally Advanced Breast Carcinoma: a case report

Abstract Introduction & Objective This report highlights an unusual presentation of skin metastasis of inflammatory breast cancer that resembles a rare condition called Carcinoma en Cuirasse (CeC). CeC was first described by anatomist Alfred Velpeau in 1838 when he observed how the coalescing nodules and diffuse sclerodermoid induration found on the chest and abdomen in CeC resembled the medieval steel breastplate or a cuirasse. Case Presentation A 62-year-old female presented to the emergency department for evaluation of a palpable left breast mass and concurrent skin lesions for the past 5 months. Based on the erosion of the nipple, chest CT, as well as core needle and skin biopsy, a diagnosis of stage IV (cT4d, cN3, cM1) ER+, PR-, HER2/neu- advanced/inflammatory breast carcinoma was made. The patient underwent a 20-week course of neoadjuvant chemotherapy followed by 25 treatments of neoadjuvant external beam radiotherapy (EBRT) with a total dose of 5000cGy to the left breast and the regional lymph node due to disease progression after 4 cycles of neoadjuvant chemotherapy. She proceeded to bilateral modified radical mastectomy and bilateral lymph node involvement was found, and later disease progressed with skin involvement outside the EBRT treatment field. Additional 25 treatment of palliative radiotherapy was utilized with another 5000cGy, followed by 2 months of chemotherapy to provide better palliation and quality of life. Upon completion of therapy, a unique skin metastasis pattern was noticed. Shortly afterward the patient passed away from severe sepsis secondary to multiple wounds infected by drug-resistant Pseudomonas aeruginosa followed by Clostridium difficile colitis. Conclusion This case shows clinical characteristics of Carcinoma en Cuirasse and provides important considerations for more research to be done to highlight the unique pattern of skin metastasis following radiation and the potential use for radiation to treat chemotherapy-resistant skin metastasis from recurrent invasive breast carcinoma. Citation Format: Chung-Tang Liu, Keri Lanier. Carcinoma en Cuirasse Secondary to Locally Advanced Breast Carcinoma: a case report [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 P1-10-06.

Differential Gene Expression of fresh tissue and patient-derived explants' matricellular proteins augment inflammatory breast cancer metastasis: the possible role of IL-6 and MCP-1.

Matricellular proteins comprising matrisome and adhesome are responsible for structure integrity and interactions between cells in the tumour microenvironment of breast cancer. Changes in the gene expression of matrisome and adhesome augment metastasis. Since inflammatory breast cancer (IBC) is characterized by high metastatic behaviour. Herein, we compared the gene expression profile of matrisome and adhesome in non-IBC and IBC in fresh tissue and ex vivo patient-derived explants (PDEs) and we also compared the secretory inflammatory mediators of PDEs in non-IBC and IBC to identify secretory cytokines participate in cross-talk between cells via interactions with matrisome and adhisome. Fifty patients (31 non-IBC and 19 IBC) were enrolled in the present study. To test their validation in clinical studies, PDEs were cultured as an ex vivo model. Gene expression and cytokine array were used to identify candidate genes and cytokines contributing to metastasis in the examined fresh tissues and PDEs. Bioinformatics analysis was applied on identified differentially expressed genes using GeneMANIA and Metascape gene annotation and analysis resource to identify pathways involved in IBC metastasis. Normal and cancer fresh tissues and PDEs of IBC were characterized by overexpression of CDH1 and MMP14 and downregulation of CTNNA1 and TIMP1 compared with non-IBC. The secretome of IBC cancer PDEs is characterized by significantly high expression of interleukin 6 and monocyte chemoattractant protein-1 (CCL2) compared with non-IBC. Genes expressed by adhisome and matrisome play a significant role in IBC metastasis and should be considered novel target therapy.

Inflammatory Breast Cancer: The Cytokinome of Post-Mastectomy Wound Fluid Augments Proliferation, Invasion, and Stem Cell Markers.

Inflammatory breast cancer (IBC) is an aggressive phenotype with a high recurrence and low survival rate. Approximately 90% of local breast cancer recurrences occur adjacent to the same quadrant as the initial cancer, implying that tumor recurrence may be caused by residual cancer cells and/or quiescent cancer stem cells (CSCs) in the tumor. We hypothesized that wound fluid (WF) collected after modified radical mastectomy (MRM) may activate cancer cells and CSCs, promoting epithelial mesenchymal transition (EMT) and invasion. Therefore, we characterized the cytokinome of WF drained from post-MRM cavities of non-IBC and IBC patients. The WF of IBC patients showed a significantly higher expression of various cytokines than in non-IBC patients. In vitro cell culture models of non-IBC and IBC cell lines were grown in media conditioned with and/without WF for 48 h. Afterwards, we assessed cell viability, the expression of CSCs and EMT-specific genes, and tumor invasion. Genes associated with CSCs properties and EMT markers were regulated in cells seeded in media conditioned by WF. IBC-WF exhibited a greater potential for inducing IBC cell invasion than non-IBC cells. The present study demonstrates the role of the post-surgical tumor cavity in IBC recurrence and metastasis.

Abstract PD10-08: Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no targeted therapy has been approved specifically for this disease. There is a critical need for innovative treatment approaches for patients with IBC. There is a paucity of data on immune checkpoint inhibitors (ICIs) in IBC, yet a strong biological rationale exists to lay the groundwork for testing the efficacy of ICIs in IBC. We have previously shown that an anti–epidermal growth factor receptor (EGFR) antibody, panitumumab (sponsored by Amgen), combined with Abraxane (sponsored by Bristol Myers Squibb), and carboplatin followed by 5-fluorouracil, epirubicin, and cyclophosphamide achieves a very high pathologic complete response (42%) in patients with triple-negative receptor status (TN-IBC) (NCT01036087). We hypothesize that inducing a broad shift in the IBC tumor microenvironment (TME), a critical driver of the IBC clinical phenotype and metastasis, from an immunosuppressive to an immunoreactive phenotype can enhance the efficacy of ICIs. Here we report the impact of targeting EGFR on ICI effectiveness by modulating the immunosuppressive TME in TN-IBC. Methods: We examined the effects of panitumumab on components of the immune TME in IBC patient tissues collected before and after panitumumab treatment. Furthermore, we analyzed panitumumab effects in an IBC SUM149 humanized mouse model (SUM149-hu-NSG-SGM3) by using multiplex immunofluorescence staining (mIF) of single-cell RNA-sequencing (scRNA-seq) and multicolor flow cytometry. We examined the changes in chemokines in humanized mouse tissue after panitumumab treatment using a cytokine antibody array and enzyme-linked immunosorbent assay (ELISA). We also studied the mechanism of EGFR regulation of immunosuppressive chemokine expression via transcription factor EGR1 by using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. In addition, we tested the efficacy of panitumumab combined with anti-PD-L1 antibody (Bio X Cell, clone 29E.2A3) in reducing IBC tumor growth in a humanized IBC SUM149 mouse model. Results: IBC patients who achieved pathologic complete response after receiving panitumumab/neoadjuvant chemotherapy had increased levels of CD8+ T-cells and decreased levels of M2 macrophages and T-reg cells. In the humanized mouse model, panitumumab treatment reduced IBC tumor growth and increased immune response that favors an antitumor effect, as shown by scRNA-seq, flow cytometry, and mIF analyses. In addition, we observed that panitumumab treatment increased the expression of chemokines that function as a chemoattractant for CD8+ T-cells, including CXCL10, CCL4, and CXCL9. We also observed that panitumumab treatment reduced EGR1 expression, leading to the downregulation of chemokines involved in the recruitment of immunosuppressive M2 macrophages and T-reg cells, including CCL2, CCL20 CXCL5, and IL-8. Finally, panitumumab enhanced the reduction in tumor growth by anti-PD-L1 antibody treatment; furthermore, the combination of panitumumab and anti-PD-L1 antibody increased the presence of CD8+ T-cells and reduced the presence of T-reg cells and M2 macrophages more effectively than either treatment alone. Conclusion: Panitumumab treatment converts the immunosuppressive IBC TME to an immunoreactive status by modulating the global expression of chemokines by downregulating the EGR1 transcription factor. This modulation of the TME by panitumumab improves the inhibition of IBC tumor growth by an ICI. Our study is the first to demonstrate that targeting the EGFR/EGR1 axis remodels the IBC TME by regulating chemokine expression, thus boosting the antitumor immune response in IBC and response to ICI. Citation Format: Xiaoping Wang, Takashi Semba, Ganiraju C. Manyam, Jing Wang, Shan Shao, Francois Bertucci, Pascal Finetti, Savitri Krishnamurthy, Lan ThiHanh Phi, Troy Pearson, Jared K. Burks, Evan N. Cohen, James M. Reuben, Fei Yang, Hu Min, Nicholas Navin, Toshiaki Iwase, Yichao Shen, Xiang Zhang, Debu Tripathy, Naoto T. Ueno. Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-08.

Contralateral Axillary Metastasis in Patients with Inflammatory Breast Cancer.

Nearly one-third of patients with inflammatory breast cancer (IBC) present with de novo stage IV disease. There are limited data on frequency and clinical outcomes of contralateral axillary metastasis (CAM) in IBC with no consensus diagnostic and treatment guidelines. Frequency of synchronous CAM was calculated in unilateral IBC patients at a single center (10/2004-6/2019). Clinicopathologic variables, diagnostic evaluation, treatment received, and overall survival (OS) were assessed and compared. Of 588 unilateral IBC patients, 49 (8.3%) had synchronous CAM. Of these, 32 (65.3%) also presented with metastatic disease at another distant site. CAM was not associated with age, tumor laterality, breast cancer subtype, grade, or cN stage (p > 0.05). The sensitivity/specificity to detect CAM was as follows: mammography (18.2%/99.2%), ultrasound (92.3%/95.5%), PET (90.1/99.1%), and MRI (76.0%/98.6%). Following systemic therapy, 22 patients had contralateral axillary surgery, and 18 received adjuvant contralateral nodal radiation. On multivariable analysis including tumor receptor subtypes, patients with stage IV-isolated CAM has statistically similar survival to stage III patients (HR 1.37, 95% CI 0.70-2.69, p = 0.36). Patients with Stage IV non-CAM (HR 2.18, 95% CI 1.66-2.85, p < 0.001) and stage IV-CAM plus other distant metastasis (HR 2.57, 95% CI 1.59-4.16, p < 0.001) had higher risk of death (reference: stage III disease). CAM in IBC was diagnosed in 8.3% of patients at presentation and was best identified by ultrasound and PET. We recommend routine contralateral axillary ultrasound as part of staging for all IBC patients. Diagnosis of CAM is a key first step toward much-needed prospective clinical trials evaluating management and outcomes of CAM in IBC.

ASO Author Reflections: Metastasis of Inflammatory Breast Cancer to the Contralateral Axilla: A Finding Meriting Further Study.

ASO Author Reflections: Metastasis of Inflammatory Breast Cancer to the Contralateral Axilla: A Finding Meriting Further Study.

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.

Ultrastructural Analysis of Inflammatory Breast Cancer Cell Clusters in an Ex Vivo Environment Mechanically Mimicking the Lymph Vascular System.

Background:Inflammatory breast cancer (IBC) is a rare form of breast cancer with a poor prognosis. IBC is characterized by florid lymphovascular tumor emboli in the skin and the parenchyma of the breast. We hypothesized that the formation of these emboli/clusters plays a pivotal role in IBC metastasis and its rapid progression, and that their structure and function may be a key to identifying molecular biological differences between IBC and non IBC.Methods:Mechanical methods were used to mimic the lymph fluid viscosity by adding 2.25% of PEG8000 to the media. Clusters were obtained for IBC tumor cell lines (SUM149 and IBC-3), non IBC tumor cell lines (MDA-MB-231, MDA-MB-468, and MCF7), and a non-tumorigenic human mammary epithelial cell line (MCF10A). Clusters were analyzed by light microscopy, and then prepared for and observed by transmission electron microscopy (TEM).Results:Significant differences were seen between IBC and non IBC clusters. The TEM analysis revealed that IBC cells harbored numerous microvilli and microvesicles, both on the free outer surface and inside the cluster. Microvilli from IBC cell clusters were noted at higher density and were longer than those of non IBC cell clusters.Conclusions:IBC tumor cell clusters exhibited distinct ultrastructural features characterized by the presence of long, crowded microvilli and numerous microvesicles. These microvilli may play an important role in the biology and aggressiveness of IBC.

Infrared Microspectroscopy and Imaging Analysis of Inflammatory and Non-Inflammatory Breast Cancer Cells and Their GAG Secretome.

Glycosaminoglycans (GAGs)/proteoglycans (PGs) play a pivotal role in the metastasis of inflammatory breast cancer (IBC). They represent biomarkers and targets in diagnosis and treatment of different cancers including breast cancer. Thus, GAGs/PGs could represent potential prognostic/diagnostic biomarkers for IBC. In the present study, non-IBC MDA-MB-231, MCF7, SKBR3 cells and IBC SUM149 cells, as well as their GAG secretome were analyzed. The latter was measured in toto as dried drops with high-throughput (HT) Fourier Transform InfraRed (FTIR) spectroscopy and imaging. FTIR imaging was also employed to investigate single whole breast cancer cells while synchrotron-FTIR microspectroscopy was used to specifically target their cytoplasms. Data were analyzed by hierarchical cluster analysis and principal components analysis. Results obtained from HT-FTIR analysis of GAG drops showed that the inter-group variability enabled us to delineate between cell types in the GAG absorption range 1350–800 cm−1. Similar results were obtained for FTIR imaging of GAG extracts and fixed single whole cells. Synchrotron-FTIR data from cytoplasms allowed discrimination between non-IBC and IBC. Thus, by using GAG specific region, not only different breast cancer cell lines could be differentiated, but also non-IBC from IBC cells. This could be a potential diagnostic spectral marker for IBC detection useful for patient management.

ERBB4 promotes the progression of inflammatory breast cancer through regulating PDGFRA.

BackgroundThe regulatory roles of human epidermal growth factor receptor [erb-b2 receptor tyrosine kinase 4 (ERBB)] family in tumors was received widespread attention. Although ERBB4 was crucial regulator in metastasis of malignant tumors, the exact mechanism of ERBB4 in inflammatory breast cancer (IBC) remains unclarified.MethodsIn this study, we collected IBC tissues and cell lines, and explored the expression levels of ERBB4 and platelet-derived growth factor receptor alpha (PDGFRA) using real-time quantitative polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and western blot assays. Furthermore, cell viability with ERBB4 silencing in SUM149 cells was examined by MTT assay and cell migration and invasion were detected by transwell assay.ResultsThe data indicated thatERBB4abolishing dramatically depressed capacity of proliferation, migration and invasion of IBC cells. Moreover, PDGFRA was an important factor for the function of ERBB4 and PDGFRA overexpression could, at least, partly rescue the ERBB4 silencing-mediated inhibition in proliferation and metastasis of IBC cells.ConclusionsTake together, we verified the first time that ERBB4 promoted the progression of IBC through regulating PDGFRA. Thus, inhibition of ERBB4 might be a novel therapeutic candidate against IBC.

Orbital metastasis in inflammatory breast cancer.

Orbital metastasis in inflammatory breast cancer.

Abstract P3-01-06: Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis

Abstract 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&amp;lt;0.0001), in more aggressive breast cancer subtypes (p&amp;lt;0.0001), and in metastatic tumors relative to primary tumors (p&amp;lt;0.0001). High DCN expression correlated with improved overall survival (p&amp;lt;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.

Abstract PD7-02: Estrogen receptor β suppresses metastasis of inflammatory breast cancer by regulating signaling that promotes cytoskeleton remodeling

Abstract Inflammatory breast cancer (IBC) is the most lethal form of breast cancer that accounts for about 10% of breast cancer mortality annually in US. Poor prognosis is largely due to the high propensity of IBC tumors to develop distant metastasis. Owing to the complex metastatic process, the molecular basis of IBC aggressiveness is poorly understood and no specific therapeutic target has been identified. Among the few signaling pathways that differ between IBC and non-IBC is the Rho GTPase that is hyperactivated in many IBC tumors and is associated with cell motility in IBC cells. However, Rho GTPases are not druggable factors and the pathways that activate these proteins in IBC are not fully defined. Another difference is that up to 83% of IBC tumors lack estrogen receptor α (ERα) expression compared with other forms of breast cancer that are mostly ERα-positive. Despite the lack of ERα in the majority of IBC tumors, estrogen may still play a role in these cancers through pathways that involve ERβ. Our tissue staining reveals expression of ERβ in a significant number of IBCs that is reproduced in IBC cell lines. Furthermore, analysis of IBC datasets indicates correlation of receptor expression with good prognosis. We studied this association in preclinical models of IBC by knocking out ERβ in IBC cells. This promotes migration and invasion through cytoskeleton remodeling whereas re-expression of the receptor in knockout cells restores the cytoskeletal structure and migration to the levels of control cells. Consistent with increased migration, deletion of ERβ activates large gene networks of cell de-differentiation including cell surface receptor signaling that enhances Rho GTPase activity to promote the motile phenotype of IBC cells. In contrast, ligands that activate ERβ inhibit the same signaling that contributes to metastasis in IBC. Analysis of orthotopic xenograft models shows that IBC tumors lacking ERβ have higher propensity for metastasis compared with the ERβ-proficient tumors. Conversely, ERβ agonists decrease the metastatic potential of IBC tumors supporting the anti-metastatic activity of the receptor. Our findings point towards a role of ERβ in preventing distant metastases by inhibiting dissemination of IBC cells. This function combined with distinct expression indicates the potential of ERβ to represent a unique prognostic marker and therapeutic target that can be utilized to repress IBC metastasis and eliminate its associated mortality. Citation Format: Christoforos Thomas, Ilias Karagounis, Ratnesh Srivastava, Sushil Kumar, Noelle Francois, Fotis Nikolos, Igor Bado, Hann-Hsiang Chao, Constantinos Koumenis, Savitri Krishnamurthy, Naoto T Ueno, Rumela Chakrabarti, Amit Maity. Estrogen receptor β suppresses metastasis of inflammatory breast cancer by regulating signaling that promotes cytoskeleton remodeling [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 PD7-02.

Abstract P3-01-10: Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis

Abstract 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&amp;lt;0.0001) and was associated with poor overall survival (p=0.0003), relapse-free survival (p&amp;lt;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&amp;lt;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&amp;lt;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.

Abstract P1-03-06: Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics

Abstract Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (&amp;lt;3 passages or &amp;gt;6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with &amp;gt;6 passages or &amp;lt;3 passages of spheroid cultures manifested little to moderate skin lesions and developed tumor sizes of ~1 cm within 2-3 months while having a longer life span. Liver and lung metastases were observed in the breast fat pad of xenografted PDX tumor mice with one case of original HR+-IBC pleural effusion. 3D tumor spheroid/organoid cultures were successfully established from original IBC pleural effusion or/and IBC PDX tumor cultures. Spheroid generated from HR+-IBC pleural effusion showed phenotypic plasticity: the HR+-IBC spheroid cells display both tumor and macrophage cell characteristics and spheroid cultures can convert to adhesive cell phenotypes with addition of condition media. These spheroid/organoid cultures are used for evaluation of new drug strategies such as selectively targeting ER degraders (SERDS), CDK4/6 inhibitor, or Tyrosine kinase inhibitors for treatment of HR+-IBC or TN-IBC. Conclusions: Our results suggested 3D spheroid cultures and PDX tumor models from advanced IBC recapture aggressive features of the disease and can be an ideal model for the mechanism study of metastatic advanced IBC and preclinical testing of novel agents in IBC and endocrine resistant disease. Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics [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 P1-03-06.

Abstract P5-05-10: Estrogen receptor β suppresses metastasis of inflammatory breast cancer by regulating cell cytoskeleton and cytokine signaling

Abstract Inflammatory breast cancer (IBC) is the most lethal form of breast cancer that accounts for about 10% of breast cancer mortality annually in US. Poor prognosis is largely due to the high propensity of IBC tumors to develop distant metastasis that occurs directly from the gland epithelium and through lymphatic invasion in which dermal lymphatics are filled with tumor emboli. Owing to the complex metastatic process, the molecular basis of IBC aggressiveness is poorly understood, and no specific therapeutic target has been identified. Despite the lack of estrogen receptor α (ERα) in the majority of IBC tumors, estrogen may still play a role in these cancers through pathways that involve ERβ. Our tissue staining reveals expression of ERβ in more than 50% of IBCs that is reproduced in IBC cell lines. Furthermore, analysis of IBC datasets indicates correlation of receptor expression with good prognosis. We studied this association in preclinical models of IBC by knocking out ERβ in IBC cells. This promotes migration and invasion through cytoskeleton remodeling whereas re-expression of the receptor in knockout cells restores the cytoskeletal structure and migration to the levels of control cells. Consistent with increased migration, deletion of ERβ activates large gene networks of cell de-differentiation and cytokine synthesis that trigger tumor microenvironment responses to promote the motile phenotype of IBC cells. In contrast, ligands that activate the receptor inhibit signaling that contributes to metastasis in IBC. Analysis of an orthotopic xenograft model shows that IBC tumors lacking ERβ have higher propensity for metastasis compared with the ERβ-proficient tumors supporting the anti-metastatic activity of the receptor. Our findings point towards a role of ERβ in preventing distant metastases by inhibiting dissemination of IBC cells and maintaining the integrity of emboli. This function combined with distinct expression indicates the potential of ERβ to represent a unique prognostic marker and therapeutic target that can be utilized to repress IBC metastasis and eliminate its associated mortality. Citation Format: Thomas C, Karagounis I, Srivastava RK, Kumar S, Karar J, Chao H-H, Kazimierczak A, Bado I, Nikolos F, Leli N, Koumenis C, Krishnamurthy S, Ueno NT, Chakrabarti R, Maity A. Estrogen receptor β suppresses metastasis of inflammatory breast cancer by regulating cell cytoskeleton and cytokine signaling [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 P5-05-10.

Abstract P2-01-03: Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion

Abstract 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&amp;lt;0.00001). LCN2 expression in patient tissues is correlated with reduced overall survival (p&amp;lt;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.