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1.
Methods Mol Biol ; 2374: 231-242, 2022.
Article in English | MEDLINE | ID: mdl-34562257

ABSTRACT

Preclinical mouse models of lung cancer have been vital experimental tools to elucidate cancer biology and test novel therapeutic regimens. Two main models are most commonly used-genetically engineered mouse models and xenograft transplantation models. The most common xenograft model employs subcutaneous transplantation of tumor cells. However, the subcutaneous space is a foreign environment to lung cancer cells and does not appropriately model the tumor-stromal interactions of endogenous lung cancers. Here, we present an orthotopic mouse model of lung cancer that utilizes direct injection of cancer cells into the lung parenchyma that allows many potential studies including interactions of lung fibroblast Hedgehog pathway activity and tumor epithelia. The protocol describes this procedure and its potential applications for lung cancer research.


Subject(s)
Lung Neoplasms , Animals , Cell Line, Tumor , Disease Models, Animal , Fibroblasts , Hedgehog Proteins , Lung , Mice , Neoplasm Transplantation , Transplantation, Heterologous
2.
Oncogene ; 39(16): 3258-3275, 2020 04.
Article in English | MEDLINE | ID: mdl-32108165

ABSTRACT

Activation of the Hedgehog (Hh) signaling pathway by mutations within its components drives the growth of several cancers. However, the role of Hh pathway activation in lung cancers has been controversial. Here, we demonstrate that the canonical Hh signaling pathway is activated in lung stroma by Hh ligands secreted from transformed lung epithelia. Genetic deletion of Shh, the primary Hh ligand expressed in the lung, in KrasG12D/+;Trp53fl/fl autochthonous murine lung adenocarcinoma had no effect on survival. Early abrogation of the pathway by an anti-SHH/IHH antibody 5E1 led to significantly worse survival with increased tumor and metastatic burden. Loss of IHH, another Hh ligand, by in vivo CRISPR led to more aggressive tumor growth suggesting that IHH, rather than SHH, activates the pathway in stroma to drive its tumor suppressive effects-a novel role for IHH in the lung. Tumors from mice treated with 5E1 had decreased blood vessel density and increased DNA damage suggestive of reactive oxygen species (ROS) activity. Treatment of KrasG12D/+;Trp53fl/fl mice with 5E1 and N-acetylcysteine, as a ROS scavenger, decreased tumor DNA damage, inhibited tumor growth and prolonged mouse survival. Thus, IHH induces stromal activation of the canonical Hh signaling pathway to suppress tumor growth and metastases, in part, by limiting ROS activity.


Subject(s)
Adenocarcinoma of Lung/drug therapy , Hedgehog Proteins/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Tumor Suppressor Protein p53/genetics , Acetylcysteine/pharmacology , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Animals , Antibodies, Anti-Idiotypic/pharmacology , Blood Vessels/drug effects , Cell Proliferation/drug effects , DNA Damage/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Ligands , Lung/metabolism , Lung/pathology , Mice , Mutation/genetics , Neoplasm Metastasis , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects
3.
Cancer Res ; 77(16): 4448-4459, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28652248

ABSTRACT

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K-mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448-59. ©2017 AACR.


Subject(s)
Carcinoma, Squamous Cell/therapy , Lung Neoplasms/therapy , Phosphoinositide-3 Kinase Inhibitors , Zinc Finger Protein GLI1/antagonists & inhibitors , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Drug Synergism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Transfection , Xenograft Model Antitumor Assays , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolism
4.
Mol Cancer Ther ; 15(5): 866-76, 2016 05.
Article in English | MEDLINE | ID: mdl-26823493

ABSTRACT

Deregulation of Hedgehog (Hh) pathway signaling has been associated with the pathogenesis of various malignancies, including basal cell carcinomas (BCC). Inhibitors of the Hh pathway currently available or under clinical investigation all bind and antagonize Smoothened (SMO), inducing a marked but transient clinical response. Tumor regrowth and therapy failure were attributed to mutations in the binding site of these small-molecule SMO antagonists. The antifungal itraconazole was demonstrated to be a potent SMO antagonist with a distinct mechanism of action from that of current SMO inhibitors. However, itraconazole represents a suboptimal therapeutic option due to its numerous drug-drug interactions. Here, we show that posaconazole, a second-generation triazole antifungal with minimal drug-drug interactions and a favorable side-effect profile, is also a potent inhibitor of the Hh pathway that functions at the level of SMO. We demonstrate that posaconazole inhibits the Hh pathway by a mechanism distinct from that of cyclopamine and other cyclopamine-competitive SMO antagonists but, similar to itraconazole, has robust activity against drug-resistant SMO mutants and inhibits the growth of Hh-dependent BCC in vivo Our results suggest that posaconazole, alone or in combination with other Hh pathway antagonists, may be readily tested in clinical studies for the treatment of Hh-dependent cancers. Mol Cancer Ther; 15(5); 866-76. ©2016 AACR.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Basal Cell/metabolism , Hedgehog Proteins/metabolism , Signal Transduction/drug effects , Skin Neoplasms/metabolism , Triazoles/pharmacology , Animals , Antifungal Agents/pharmacology , Carcinoma, Basal Cell/drug therapy , Carcinoma, Basal Cell/pathology , Cell Line, Tumor , Disease Models, Animal , Gene Knockout Techniques , Humans , Mice , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Smoothened Receptor/genetics , Smoothened Receptor/metabolism , Xenograft Model Antitumor Assays
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