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A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

ABSTRACT: Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.

SUBMITTER: Schonhuber N

PROVIDER: S-EPMC4270133 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

Schönhuber Nina N Seidler Barbara B Schuck Kathleen K Veltkamp Christian C Schachtler Christina C Zukowska Magdalena M Eser Stefan S Feyerabend Thorsten B TB Paul Mariel C MC Eser Philipp P Klein Sabine S Lowy Andrew M AM Banerjee Ruby R Yang Fangtang F Lee Chang-Lung CL Moding Everett J EJ Kirsch David G DG Scheideler Angelika A Alessi Dario R DR Varela Ignacio I Bradley Allan A Kind Alexander A Schnieke Angelika E AE Rodewald Hans-Reimer HR Rad Roland R Schmid Roland M RM Schneider Günter G Saur Dieter D

Nature medicine 20141019 11

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carci ...[more]

PMID: 25326799

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Project description:Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection.

Project description:Cell-specific expression of genes offers the possibility to use their promoters to drive expression of Cre-recombinase, thereby allowing for detailed expression analysis using reporter gene systems, cell lineage tracing, conditional gene deletion, and cell ablation. In this context, current data suggest that the integrin α11 subunit has the potential to serve as a fibroblast biomarker in tissue regeneration and pathology, in particular in wound healing and in tissue- and tumor fibrosis. The mesenchyme-restricted expression pattern of integrin α11 thus prompted us to generate a novel ITGA11-driver Cre mouse strain using a ϕC31 integrase-mediated knock-in approach. In this transgenic mouse, the Cre recombinase is driven by regulatory promoter elements within the 3 kb segment of the human ITGA11 gene. β-Galactosidase staining of embryonic tissues obtained from a transgenic ITGA11-Cre mouse line crossed with Rosa 26R reporter mice (ITGA11-Cre;R26R) revealed ITGA11-driven Cre expression and activity in mesenchymal cells in a variety of mesenchymal tissues in a pattern reminiscent of endogenous α11 protein expression in mouse embryos. Interestingly, X-gal staining of mouse embryonic fibroblasts (MEFs) isolated from the ITGA11-Cre;R26R mice indicated heterogeneity in the MEF population. ITGA11-driven Cre activity was shown in approximately 60% of the MEFs, suggesting that the expression of integrin α11 could be exploited for isolation of different fibroblast populations. ITGA11-driven Cre expression was found to be low in adult mouse tissues but was induced in granulation tissue of excisional wounds and in fibrotic hearts following aortic banding. We predict that the ITGA11-Cre transgenic mouse strain described in this report will be a useful tool in matrix research for the deletion of genes in subsets of fibroblasts in the developing mouse and for determining the function of subsets of pro-fibrotic fibroblasts in tissue fibrosis and in different subsets of cancer-associated fibroblasts in the tumor microenvironment.

Dataset Information

A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

Publications

A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

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