Abstract P6-08-02: Disruption of the estradiol-regulated NTN1-UNC5A dependence receptor signaling axis causes a hybrid basal/luminal molecular phenotype in estrogen receptor-positive breast cancer cells
Authors: Padua MB , Bhat-Nakshatri P , Anjanappa M , Hao Y , Liu Y , McElyea K , Sandusky G , Althouse S , Perkins S , Nakshatri H 
Published: March 2017

Luminal subtype of breast cancers that express the estrogen receptor alpha (ERα) represents approximately two-thirds of all breast cancer cases. ER+ tumors tend to have the most favorable prognoses when treated with endocrine therapy. However, a relapse or endocrine therapy resistance is often seen in ER+ breast tumors. UNC5A belongs to the dependence receptor family which can mediate two different intracellular signals: cell survival, differentiation or migration when engaged with its ligand (such as Netrin-1; NTN1) or cell death/apoptosis in the absence of the ligand. Here we demonstrate that, depending upon the cell type, UNC5A and NTN1 are estradiol (E2)-inducible genes. Using shRNA or CRISPR knockdown strategies, we show that the disruption of the NTN1-UNC5A signaling axis in ER+ (MCF7 and T-47D) cells generates a mixed basal-like/luminal phenotype with stem cell-like characteristics. RNA-seq of UNC5A knockdown cells showed deregulated expression of several E2-target genes in both cell lines. Moreover, knockdown of UNC5A resulted in increased cell proliferation, and elevated expression of the E2-inducible anti-apoptotic, BCL2. Furthermore, the expression of ΔNp63 was enhanced in UNC5A knockdown cells. ΔNp63 is a TP53 family transcription factor that promotes breast epithelial stem cell maintenance and basal-like breast cancer. Accordingly, UNC5A knockdown cells displayed cancer stem cell phenotype as evident from ~3-fold increase in the number of CD44+/CD24+, CD44+/EPCAM+ and ITGA6+/EPCAM+ subpopulation compared with control cells. In addition, the expression of NTN4, a pro-angiogenic and lymphangiogenic factor, was increased upon UNC5A knockdown. In vivo, UNC5A knockdown cells implanted in nude mice were able to form tumors in the mammary fat pad independent of E2 supplementation and were able to colonize and develop into overt metastasis in multiple organs such as lungs, ovaries and adrenal glands. Consequently, analysis of mammary fat pad tumors from animals that received UNC5A knockdown cells revealed an increased expression of PECAM1 (CD31), a marker for endothelial cells used to evaluate tumor angiogenesis. In contrast to UNC5A, knocking down NTN1, decreased the expression of BCL2 and TP63 in both cell lines. Thus, knockdown of UNC5A resulted in deregulated expression of E2-regulated genes, E2-independent and anti-estrogen-resistant growth in vitro, and E2-independent tumor formation in xenograft models. Consistent with results of in vitro studies, analysis of tissue samples from breast cancer patients (n=196) revealed that lower expression of UNC5A is associated with lower overall survival (P &lt; 0.05). Thus, loss or mutational inactivation of UNC5A could lead to unrestricted E2:ERα signaling and anti-estrogen resistant growth while simultaneously enabling ERα-positive luminal breast cancer cells to acquire basal-like and cancer stem cell-like features.

Citation Format: Padua MB, Bhat-Nakshatri P, Anjanappa M, Hao Y, Liu Y, McElyea K, Sandusky G, Althouse S, Perkins S, Nakshatri H. Disruption of the estradiol-regulated NTN1-UNC5A dependence receptor signaling axis causes a hybrid basal/luminal molecular phenotype in estrogen receptor-positive breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-02.