Background. It has been reported that signaling from the nerve growth factor (NGF) pathway associated with peripheral nerves is able to contribute to perineural invasion (PNI) of pancreatic cancer (PC). Nevertheless, the underlying mechanism by which NGF leads to PNI remained poorly understood. Methods. Western blotting was employed to determine NGF level in PC and paracarcinoma tissues and in PC cell lines as well as pancreatic ductal epithelial cells. MiaPaCa‐2 and CFPAC‐1 cells were treated with 100 ng/ml of NGF or the NGF inhibitor Tanezumab for 24 h, CCK‐8 and Transwell assays were employed to test cell proliferation, invasion, and migration, respectively. TrkA expression was knocked down in MiaPaCa‐2 and dorsal root ganglion (DRG) cells treated with NGF to determine its effect on the Warburg effect. To reveal that the NGF‐TrkA signaling pathway was closely associated with PC PNI, in vitro neuroinvasion model was established by using MiaPaCa‐2 cells via coculturing DRG cells in Matrigel. Further, exosomes were extracted from PC cells and identified by examining the levels of specific markers for exosomes. Then RT‐qPCR was applied to test miR‐21‐5p level in tumor derived exosomal (TDE‐miR‐21‐5p). RIP assay was performed to validate NGF and miR‐21 binding ability in MiaPaCa‐2 cells. Rescue experiments were performed by using coprocessing of Tanezumab and miR‐21‐5p mimic on MiaPaCa‐2 cells, followed by coculture with DRG cells. Subsequently, we used a model of neuroinvasion in nude mice to assess the effect of NGF in vivo on tumor nerve invasion as well as on nociceptive transmission. Results. NGF level was preeminently higher in PC tissues and cell lines than in paracarcinoma tissues and normal pancreatic epithelial cell lines. NGF promoted MiaPaCa‐2 and CFPAC‐1 cell invasion and migration, while Tanezumab treatment showed the opposite results. Besides, NGF binding to TrkA receptors encouraged the intracellular Warburg effect in PC and DRG cells. TrkA blocking‐up could restrain NGF induced PC cell migration and neural invasion. Mechanistically, NGF could upregulate TDE‐miR‐21‐5p levels, and DRG cells took up TDE to activate the Warburg effect and stimulate nociceptor gene expression. miR‐21‐5p inhibitor could abolish the facilitative effect of NGF on PNI in MiaPaCa‐2 cells. In vivo tumorigenesis experiments, Tanezumab markedly alleviated nerve invasion of PC cells as well as relieved nociceptive conduction in animal models. Conclusions. These findings displayed that NGF/TrkA encouraged the neuroinvasive potential of PC cells by activating the Warburg effect in DRG cells through upregulation of TDE‐miR‐21‐5p expression.

Recently, evidence has shown that GOT1 expression is upregulated in pancreatic cancer tissues and promotes cancer development, but the specific mechanism remains unclear. We found that GOT1 expression was upregulated in pancreatic cancer cell-derived exosomes. When PANC-1 cells were incubated with exosomes alone or transfected together with si-GOT1, we found that exosomes enhanced cell proliferation, invasion and migration, promoted ferroptosis, and si-GOT1 reversed the effects of exosomes. The results of online bioinformatics database analysis indicated that CCR2 was a potential binding protein of GOT1 and is highly expressed in pancreatic cancer tissues. PANC-1 cells were transfected with pcDNA-CCR2 or si-CCR2, and it was found that pcDNA-CCR2 enhanced cell proliferation, invasion and migration, promoted ferroptosis, and si-CCR2 had an opposite effect. Next, exosome-treated cells were transfected with si-GOT1 alone or together with pcDNA-CCR2, and we found that exosomes promoted CCR2 expression, promoted cell proliferation and invasion, and inhibited ferroptosis, the transfection of si-GOT1 abolished the effect of exosomes, and the transfection of pcDNA-CCR2 again reversed the effect of si-GOT1. Furthermore, when exosome-treated cells were transfected with si-GOT1 alone or co-incubated with Nrf2 activator NK-252, we found that si-GOT1 reversed the promoting effect of exosomes on Nrf2 and HO-1 expression, as well as its inhibitory effect on ferroptosis, but this effect was abrogated by NK-252. In vivo studies showed that knockdown of GOT1 expression inhibited tumor formation compared with tumor tissues formed upon exosome induction, which was mediated by promoting ferroptosis via suppressing the protein expression of GOT1, CCR2, Nrf2 and HO-1 in tumor tissues.