Abstract 3611: PAUF induces migration of human pancreatic cancer cells exclusively via the TLR4/MyD88/NF-κB signaling pathway

Abstract Background: Pancreatic adenocarcinoma up-regulated factor (PAUF) is a cancer-secreted protein which is overexpressed on pancreatic cancer (PC) cells and promotes tumor growth in an autocrine way. However, the receptor of PAUF on PC cells has yet to be identified. Meanwhile, it is known that PAUF, as a tumor microenvironment (TME) modulator, plays a role in immune evasion and suppression through toll-like receptors notably TLR4 which are mainly expressed on immune cells. Previous studies show that TLR4 is also highly expressed in PC tumor tissues, compared to normal tissues, and activation of TLR4 signaling pathway induces the migration and invasion of PC cells. Objective: This study aimed to investigate if TLR4 expressed on cancer cells can act as a receptor for PAUF to mediate its tumor promoting effects. Methods and Results: We tested the TLR4 expression in six PC cell lines and one normal pancreatic cell line. And confirmed that TLR4 is expressed in all PC cell lines at variable levels, but not in the normal pancreatic cell line.Next, we found that a chemical TLR4-specific inhibitor TAK-242 significantly reduced the migration of PC cells with high TLR4 expression (BxPC-3), but it did not impact the migration of PC cells with low TLR4 expression (Panc-1). This experiment suggests that some endogenous ligands of TLR4 may have mediated PC cell migration.Using TLR4 overexpressed and knockout PC cell lines, we showed that treatment with recombinant PAUF increased the migration and invasion of TLR4 overexpressed PC cells, but not in the TLR4 knockout cells. Likewise, treatment of anti-PAUF antibody reduced the migration and invasion of TLR4 overexpressed PC cells, but not in the TLR4 knockout cells. These results shows that PAUF’s migration and invasion promoting effects of PC cells is TLR4 dependent.To further understand the molecular mechanism of PAUF-induced metastasis (migration/invasion)-promoting effects via TLR4, we conducted a series of in vitro studies including immunoprecipitation (IP), western blot analysis, flow cytometry, and luciferase reporter assay. We demonstrated that PAUF activates TLR4/MyD88 signaling pathway, but not TLR4/TRIF pathway. Conclusions: In conclusion, for the first time this study demonstrates that TLR4 expressed on PC cell surfaces functions as a receptor of PAUF to mediate its metastasis-promoting effects, which are exclusively through the MyD88/NF-κB signaling pathway. This study also suggests TLR4 as a potential biomarker for identification of optimal patients, and a new therapeutic target to treat PC. Anti-PAUF antibody used in this study is currently evaluated by clinical trials in France, Spain, and US FDA. Citation Format: Fen Jiang, So Eun Youn, Da Eun Hong, Tae Heung Kang, Hye Yun Won, Yun Yong Park, Sang Seok Koh. PAUF induces migration of human pancreatic cancer cells exclusively via the TLR4/MyD88/NF-κB signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3611.