PO.MCB03.01 · 分子与细胞生物学
GNAS R201C在KRAS G12D驱动的胃肠道癌症模型中增强腹膜癌病
GNAS R201C enhances peritoneal carcinomatosis in K RAS G12D -driven gastrointestinal cancer models
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：KRAS G12D和GNAS R201C共突变常见于低级别阑尾黏液性肿瘤，并促进腹膜转移。我们使用同基因小鼠模型研究了GNAS R201C在肿瘤进展中的作用。
方法：我们构建了cdx2-Cre;LSL-Kras G12D;Rosa26R-LSL-rtTA;TetO-Gnas R201C (cdx2-Kras/Gnas)小鼠，其中KRAS G12D组成型激活，而GNAS R201C在肠道中可由多西环素(dox)诱导。通过RPPA比较由cdx2-Kras/Gnas小鼠产生的dox-与dox+阑尾类器官(cdx2-Kras/Gnas)中的蛋白水平。在同基因腹膜癌病模型中测试了胰腺IPMN细胞(p48-Kras/Gnas)。评估了肿瘤负荷、信号传导和生存情况。
结果：在cdx2-Kras/Gnas小鼠中，给予dox 3-9个月后，磷酸化p44/42 MAPK Erk1/2在盲肠和结肠中显著增加，但无组织学异常。在cdx2-Kras/Gnas类器官中，dox诱导GNAS R201C表达(>3000倍)并增加磷酸化PKA底物，加速类器官生长(倍增时间：dox+组78.5 h对比dox−组121 h)。在RPPA中，与dox−类器官相比，dox+类器官中与以下相关的蛋白增加：1)生存和合成代谢信号(AKT2 pS474)，2)ER应激反应和氨基酸生物合成(PERK、Calnexin和ASNS)，3)糖酵解和脂质代谢(Hexokinase II、ACSL和UGT1A)。为测试Gnas R201C在腹膜癌病中的作用，将cdx2-Kras/Gnas和p48-Kras/Gnas细胞腹腔内植入C57BL/6小鼠。cdx2-Kras/Gnas细胞腹腔内植入后小鼠未检测到腹膜肿瘤形成，而p48-Kras/Gnas细胞形成胰腺肿瘤，并在dox+小鼠中形成广泛的腹膜和内脏转移，包括肝脏和肠系膜淋巴结，使中位生存期降至11.5天(log-rank p = 0.0368)。植入p48-Kras/Gnas细胞的dox−小鼠仅表现出极轻微的疾病。CD3+免疫细胞在dox+胰腺肿瘤中局部富集，而在dox−肿瘤中观察到极少的浸润。
结论：GNAS R201C与KRAS G12D协同促进腹膜转移并改变肿瘤微环境，提示GNAS信号传导可作为治疗靶点。
查看英文原文 English abstract
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.
利益披露 Disclosure
I. Ito, None..
S. Chowdhury, None..
J. P. Shen, None.