Seika, P.; Puttapaka, S. N.; Hong, S. M.; Scott, A.; slosberg, J.; Bovo Minto, S.; Haigis, K. M.; Kulkarni, S.

Background and Aims: The tumor microenvironment in colorectal cancer (CRC) is richly innervated, yet the contribution of the enteric nervous system (ENS) to CRC biology remains poorly defined. ENS neurons express proenkephalin (PENK), which can be processed by proprotein convertase 1/3 (PCSK1) to generate Methionine-enkephalin (MENK), a bioactive peptide with growth-regulatory potential. We hypothesized that an ENS-derived PCSK1 MENK axis restrains CRC proliferation through opioid growth factor receptor (OGFr) signaling and is modulated by stress-associated glucocorticoid receptor (GR) signaling and GLP1 receptor (GLP1R) activity. Methods: Publicly available human CRC single-cell RNA-sequencing datasets were analyzed for OGFr expression. PCSK1 and MENK expression in murine ENS and tumor associated tissue was assessed by immunofluorescence. Functional studies were performed using murine CRC organoids, and primary murine ENS neurons in mono- and co-culture. CRC proliferation was quantified by EdU incorporation following treatment with recombinant MENK, recombinant PCSK1, OGFr synthetic ligand naloxone, or PCSK1 inhibitors. Effects of dexamethasone and liraglutide on PCSK1 expression in ENS-containing murine tissue were evaluated. Results: OGFr was enriched in CRC cells and positively associated with KRAS gene expression. A subset of adult murine colonic myenteric neurons expressed PCSK1 and MENK. MENK dose-dependently suppressed proliferation of CRC organoid cells. ENS neurons also suppressed CRC proliferation in a PCSK1 dependent manner. Dexamethasone reduced, whereas liraglutide increased, PCSK1 expression. Conclusions: These findings define a previously unrecognized ENS derived neuro oncologic pathway that is associated with reduced CRC cell proliferation and identify the GR/GLP1R PCSK1 MENK axis as a potentially actionable therapeutic node. Summary: This study identifies a neuronal PCSK1 MENK pathway in the ENS that directly suppresses colorectal cancer growth through local OGFr activation, revealing a previously unrecognized neuropeptidergic mechanism of tumor control within the intestinal microenvironment.