GNAS R201C enhances peritoneal carcinomatosis in K RAS G12D -driven gastrointestinal cancer models

Background KRAS G12D and GNAS R201C co-mutations are common in low-grade appendiceal mucinous neoplasms and promote peritoneal metastasis. We examined the role of GNAS R201C in tumor progression using syngeneic mouse models. MethodsWe generated cdx2-Cre;LSL-Kras G12D ;Rosa26R-LSL-rtTA;TetO-Gnas R201C (cdx2-Kras/Gnas) mice, where KRAS G12D is constitutively active and GNAS R201C is doxycycline (dox)-inducible in the intestine. Protein levels were compared in RPPA between dox- and dox+ appendiceal organoids (cdx2-Kras/Gnas) generated from cdx2-Kras/Gnas mice. Pancreatic IPMN cells (p48-Kras/Gnas) were tested in a syngeneic peritoneal carcinomatosis model. Tumor burden, signaling, and survival were assessed. ResultsIn cdx2-Kras/Gnas mice, phospho-p44/42 MAPK Erk1/2 was robustly increased in cecum and colon without histologic abnormalities after 3-9 months of dox administration. In cdx2- Kras/Gnas organoids, dox induced GNAS R201C expression (>3000-fold) and increased phosphorylated PKA substrates, accelerating organoid growth (doubling time: 78.5 h in dox+ vs. 121 h in dox−). In RPPA, proteins related to 1) survival and anabolic signaling (AKT2 pS474), 2) ER stress response and amino acid biosynthesis (PERK, Calnexin, and ASNS), and 3) glycolytic and lipid metabolism (Hexokinase II, ACSL, and UGT1A) were increased in dox+ vs. dox− organoids. To test the role of Gnas R201C in peritoneal carcinomatosis, cdx2-Kras/Gnas and p48-Kras/Gnas cells were IP implanted in C57BL/6 mice. No peritoneal tumor formation was detected in mice after cdx2-Kras/Gnas cell IP implantation, whereas p48-Kras/Gnas cells formed pancreatic tumor, and extensive peritoneal and visceral metastases including liver and mesenteric lymph nodes in dox+ mice, reducing median survival to 11.5 days (log-rank p = 0.0368). Dox− mice implanted with p48-Kras/Gnas cells showed minimal disease. CD3 + immune cells were locally enriched in dox+ pancreatic tumors, whereas minimal infiltration was observed in dox− tumors. Conclusion GNAS R201C cooperates with KRAS G12D to promote peritoneal metastasis and alter the tumor microenvironment, suggesting GNAS signaling as a therapeutic target.