Abstract LB035: Understanding the mechanism of neuropilin2 upregulation in advanced prostate cancer
Authors: Bodas Sanika , Islam Ridwan , Bhattacharya Sreyashi , Mishra Juhi , Das Dipanwita , Muders Michael , Dutta Samikshan , Teply Benjamin A. , Datta Kaustubh 
Published: June 2022

Abstract
Background: Prostate cancer management involves administering Androgen Deprivation Therapy (ADT). Patients with advanced form of the disease become resistant to ADT and progress to a lethal phenotype called Castration Resistant Prostate Cancer (CRPC). Neuropilin-2 is upregulated in advanced prostate cancer. It has been implicated in decreasing the overall survival of such patients by inducing therapy-resistance.
Significance: Understanding how NRP2 is differentially expressed will uncover molecular pathways that can be targeted to develop a specific treatment strategy for better management of patients.
Experimental Design: We checked NRP2 expression in different prostate cancer cell lines {LNCaP, C4-2 and C4-2 P53/RB1 null cells (DKD cells)} and the level of methylation of the NRP2 promoter in these cells by pyrosequencing. We demethylated the NRP2 promoter by treatment with 5’Azacytidine and checked NRP2 mRNA expression. We analyzed the NRP2 promoter activity under demethylated conditions by transfecting cells with a demethylated NRP2 promoter construct using a GLuc dual-luciferase assay. Using Biobase we found DNA sequences for binding of transcription factors within the NRP2 promoter. RNA-Seq, RT-PCR was performed to identify potential transcription factors of NRP2(highly expressed in DKD cells). To validate SOX2 as a transcription factor for NRP2, we tested its binding to NRP2 promoter by ChIP-q-PCR. Requirement of SOX2 in transcribing NRP2 mRNA in C4-2 and DKD cells was tested by both knocking down or ectopically expressing SOX2. To determine whether demethylation of NRP2 promoter is a prerequisite for SOX2 binding, SOX2 was overexpressed in LNCaP cells following 5’Azacytidine and monitored the change in the NRP2 expression.
Results: NRP2 expression was low in LNCaP cells. It steadily increased in C4-2 cells with a steep rise in the DKD cells. Pyrosequencing showed a high level of DNA methylation in LNCaP cells compared to C4-2 cells. On treatment with 5’Azacytidine, NRP2 mRNA expression was upregulated in the LNCaP cells and it significantly rose in the C4-2 cells. Our dual-luciferase assay showed us that in C4-2 cells, the promoter activity of NRP2 upon demethylation went up causing a significant rise in Luciferase expression. RNA-seq and RT-PCR analysis showed high SOX2 levels in DKD cells indicating that SOX2 might play a role in elevating NRP2 expression in these cells. ChIP q-PCR indicated enriched binding of SOX2 in the NRP2 promoter in DKD cells. On silencing SOX2 in DKD cells, the expression of NRP2 was decreased. Analogous to this finding, overexpressing SOX2 in C4-2 cells caused significant rise in NRP2 expression, whereas in LNCaP cells, there was no change. Demethylation of NRP2 promoter and simultaneous overexpression of SOX2 in LNCaP lead to a significant increase in NRP2 mRNA suggesting the requirement of demethylated NRP2 promoter for SOX2 function.
Conclusion: The differential expression of NRP2 in advanced prostate cancer is a two-step process of initial demethylation of the DNA in the NRP2 promoter region, followed by binding of SOX2
Citation Format: Sanika Bodas, Ridwan Islam, Sreyashi Bhattacharya, Juhi Mishra, Dipanwita Das, Michael Muders, Samikshan Dutta, Benjamin A. Teply, Kaustubh Datta. Understanding the mechanism of neuropilin2 upregulation in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB035.