Abstract B100: Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression
Authors: Ochiai Yosuke , Masaki Sunagawa , Malagola Ermanno , Kobayashi Hiroki , Wu Feijing , White Ruth A. , Zamechek Leah B. , Wang Timothy C. 
Published: January 2024

Abstract
Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer.
Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B100.