Abstract 4671: Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression
Authors: Elsehemy Ahmed A. , Selvadurai Hayden , Ortin-Martinez Arturo , Pokrajac Nenad , Mamatjan Yasin , Rowland Katherine , Aldape Kenneth , Dirks Peter , Wallace Valerie 
Published: August 2019

Norrin is an atypical WNT ligand that binds the Frizzled-4 (FZD4) and Low-density lipoprotein receptor-related protein (LRP5/6) receptor complex to activate canonical WNT/ β-catenin signaling. Norrin/FZD4 signaling is involved in the regulation of vasculature in several tissues including the retina and for blood-brain barrier function. The role of Norrin in cancer is not very well characterized. Here, we show that NDP, the gene encoding for Norrin, is expressed in a wide range of cancer types, with a particular enrichment in glioblastoma (GBM) and lower grade glioma (LGG). Kaplan-Meier survival analysis of publicly available datasets revealed a significant correlation between NDP expression and survival in GBM, LGG and neuroblastoma. To investigate the function of NDP in GBM, we performed a set of NDP and FZD4 gain and loss of function experiments in patient-derived GBM stem cell (GNS) lines. Recently Achaete-scute homolog 1 (ASCL1) expression was shown to stratify GNS lines into two cohorts with different tumorigenic, proliferation and differentiation dynamics. Surprisingly, we found that NDP manipulation resulted in opposite effects in ASCL1hi versus ASCL1lo lines. NDP inhibited proliferation and sphere formation in ASCL1lo lines through the WNT/ β-catenin pathway, while it stimulated proliferation and sphere formation in ASCL1hi lines independently of the WNT/β-catenin pathway. The NDP/FZD4 effects on growth in both GNS subtypes were recapitulated in xenografts, confirming effects of this pathway on tumor progression in vivo. To gain insight into the cellular and molecular effects of NDP/FZD4 on GNS growth we performed immunocytochemistry (ICC) and RNA-Seq analyses in GNS cells with NDP knockdown. In parallel with our in vitro and in vivo observations, we found that NDP/FZD4 signaling affects GNS proliferation index and cell cycle kinetics and the expression of cell cycle regulatory genes in both GNS subtypes, but in opposite ways. RNA-Seq analysis identified cell cycle regulatory genes and gene sets that are commonly affected by NDP in both GNS types. Interestingly, the analysis also identified several genes and genesets unique to each GNS type, consistent with the divergence of NDP functions between GNS subtypes. Collectively, our results uncover novel functions of NDP in regulating GBM stem cell progression, and that NDP functions in GBM stem cells stratify with ASCL1 expression. Additionally, our results highlight the significance of precision medicine in targeting tumor subtypes based on the molecular signature.

Citation Format: Ahmed A. Elsehemy, Hayden Selvadurai, Arturo Ortin-Martinez, Nenad Pokrajac, Yasin Mamatjan, Katherine Rowland, Kenneth Aldape, Peter Dirks, Valerie Wallace. Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression [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 4671.