Abstract P2-01-03: Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion
Authors: 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 
Published: May 2019

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