Abstract 25: Neuroligin 4X: A neural cell adhesion molecule, in breast cancer
Authors: Henderson Henry J. , Balasubramanyam Karanam , Samant Rajeev , Vig Komal , Singh Shree R. , Yates Clayton , Bedi Deepa 
Published: August 2018

Abstract
Purpose: Neuroligins are neural cell adhesion molecules that are implicated in heterotopic cell adhesion. Our previous studies using a selection from a combinatorial random peptide library against breast and pancreatic cancer cell lines identified several peptides mimicking neuroligin (NLGN-1, 3 and NLGN4X respectively). In this study, we investigated the expression, relevance and functional significance of Neuroligin 4X in human breast cancer.
Methods: NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia were analyzed. The correlation between NLGN4X levels and clinicopathologic parameters were established within Oncomine datasets. As a proof-of- principle, we evaluated survival by generating Kaplan–Meier plots using publicly available microarray datasets. To examine the expression of NLGN4X, immunohistochemistry of breast cancer tissue arrays was conducted. Additionally, flow cytometry and immunofluorescence staining were performed to investigate NLGN4X expression in MDA-MB-231, MCF-7, and MCF-7 treated with TGFβ. To observe the effect of NLGN4X gene knockdown, MDA-MB-231 cells were transfected with NLGN4X-specific siRNA. NLGN4X gene expression was analyzed by RT-PCR, western blot and flow cytometry. Post transfection, wound healing and cell viability assays were performed to determine the effect of NLGN4X knockdown on migration and proliferation. Apoptotic outcomes were examined through detection of caspase activation and Annexin V-FITC methods by using flow cytometry.
Results: NLGN4X showed abundant expression in breast cancer tissues. The evaluation of bioinfomatic datasets revealed that NLGN4X expression was higher in triple negative breast cancer (TNBC) and in metastatic tissues. Interestingly, high NLGN4X expression correlated with a decrease in relapse free-survival in TNBC. RT-PCR, flow cytometry and immunofluorescence validated that NLGN4X expression was high in MDA-MB-231 as well as MCF-7 TGFβ, which suggests that NLGN4X is associated with the mesenchymal phenotype. Knockdown of NLGN4X expression by siRNA significantly decreased cell proliferation(P&lt;0.001) and migration(P&lt;0.05) in MDA-MB-231 in conjunction with the induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry.
Conclusion: Our findings suggest that NLGN4X could represent a novel biomarker and therapeutic target. This is the first study to link the expression of neuronal cell adhesion molecules, neuroligins, to breast cancer. We intend to continue the investigation of the role of NLGN4X to elucidate the mechanistic role of this adhesion protein in breast cancer progression and metastasis.
Citation Format: Henry J. Henderson, Karanam Balasubramanyam, Rajeev Samant, Komal Vig, Shree R. Singh, Clayton Yates, Deepa Bedi. Neuroligin 4X: A neural cell adhesion molecule, in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 25.