Abstract P3-01-10: Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis
Authors: Villodre Emilly Schlee , Hu Xiaoding , Larson Richard , Eckhardt Bedrich L , Gong Yun , Huo Lei , Song Juhee , Krishnamurthy Savitri , Ibrahim Nuhad , Ueno Naoto T , Tripathy Debu , Woodward Wendy A , Debeb Bisrat G 
Published: March 2020

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