RESUMEN
The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.
Asunto(s)
Carcinoma Ductal Pancreático , Metaplasia/genética , Metaplasia/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proliferación Celular/genética , Células Cultivadas , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Fibroblastos/citología , Fibroblastos/patología , Eliminación de Gen , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Páncreas/patología , Transducción de Señal/genética , Factor de Crecimiento Transformador alfa/metabolismo , Células Tumorales Cultivadas , Proteína Gli2 con Dedos de ZincRESUMEN
The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment.
Asunto(s)
Transformación Celular Neoplásica/patología , ADN Nucleotidiltransferasas/metabolismo , Modelos Animales de Enfermedad , Proteínas de Homeodominio/fisiología , Neoplasias Pancreáticas/patología , Transactivadores/fisiología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , ADN Nucleotidiltransferasas/genética , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Microambiente Tumoral , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development.