Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.tumor stroma | cancer therapy | Sonic hedgehog | hedgehog agonist | cerulein P ancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer-related death in the United States and is the most lethal of common human malignancies, with a 5-y survival rate of ∼7% (1, 2). The most effective chemotherapy regimens for metastatic or locally advanced inoperable disease are largely palliative and are capable of extending overall survival by only several months (3, 4). Even localized disease, treatable with surgery followed by adjuvant chemotherapy, has a dismal 5-y survival rate of 24% (1). Among gastrointestinal malignancies, PDA is unique in that it is predominantly driven by oncogenic Kras activity. In addition, PDA pathogenesis is marked by a striking desmoplastic reaction to invading tumor cells. This desmoplasia includes a dense extracellular matrix with abundant stromal fibroblasts and influences the cellular biology of the tumor as well as its response to chemotherapeutic agents.Hedgehog (Hh) signaling has been thought to play a role in PDA desmoplasia and tumor progression but is notable during embryonic development of the pancreas for its absence in the region of embryonic endoderm from which the pancreas forms (5-7). This absence of activity is required for normal specification of early pancreatic progenitor fate, and pharmacologic or antibody treatments that inhibit Hh ...
Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
A robust genome editing pipeline is critical to the vitality of a modern genetic laboratory. Previous studies have shown that Cas9 ribonucleoprotein (RNP)-based editing can be highly effective in Caenorhabditis elegans, particularly...
Mutations in Isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1–5. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert alpha-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation6–10. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models (GEMMs) expressing mutant IDH in the adult liver show aberrant response to hepatic injury, characterized by HNF4α silencing, impaired hepatocyte differentiation and markedly elevated levels of cell proliferation. Moreover, mutant IDH and activated Kras, genetic alterations that co-exist in a subset of human IHCCs4,5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel GEMM of IDH-driven malignancy.
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