Neuropilins (NRPs) are transmembrane proteins involved in vascular and nervous system development by regulating angiogenesis and axon guidance cues. Several published reports have established their role in tumorigenesis. NRPs are detectable in tumor cells of several cancer types and participate in cancer progression. NRP2 is also expressed in endothelial and immune cells in the tumor microenvironment and promotes functions such as lymphangiogenesis and immune suppression important for cancer progression. In this review, we have taken a comprehensive approach to discussing various aspects of NRP2-signaling in cancer, including its regulation, functional significance in cancer progression, and how we could utilize our current knowledge to advance the studies and target NRP2 to develop effective cancer therapies.

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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.

Background: Prolonged ablation of androgen receptor pathway in prostate cancer leads to the emergence of therapy resistant neuroendocrine-like (NE-like) aggressive lineage. This NE development is associated with chromatin reprogramming underlying epigenetic alterations and changes in transcriptional network. Clinically, NE development follows a poorer progression with multifaceted therapeutic challenges. Current lack of effective understanding of this NE trans-differentiation process limits the stratification of therapeutic window with further worsened patient management. The following study aims to identify the sequential molecular players responsible for therapy resistant NE like development. Objective: The primary objective of this study is to understand how chromatin modifications along with epigenetic regulation dictates the establishment of NE trans-differentiation process in Prostate Cancer in response to Androgen Receptor blockade therapy. Methods: We generated various NE-like cell lines either by genetic modification or therapeutic selection. We performed RNA Seq, ATAC Seq and acetylated Histone (H3K18 and H3K27) ChIP Seq in our developed NE and adenocarcinoma cell lines. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during adenocarcinoma to NE transformation. Overlapping RNA seq results further suggested newly transcribed neuronal genes with higher degrees of promoter accessibility. Transcription Factor scanning analysis among newly active gene promoters revealed preferential binding of Pax5, whose expression has been validated to be selectively occurring in NE lineage unlike adenocarcinoma. Further analysis of Pax5 promoter site suggested that changes in 5-hydroxymethylation pattern during adenocarcinoma to NE transformation guided the recruitment of PBX1 at Pax5 promoter. Validating NE patient expression in silico supported that PBX1/Pax5 expressions are selective in NE lineage development. Conclusions: Our study concludes that specific chromatin alterations guide the recruitment of PBX1 to upregulate Pax5 to maintain a therapy resistant neuronal surrounding in NE-like prostate cancer development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Chromatin modifications guide Pax5 dependent gene expression in NE like 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 LB037.