ABSTRACT
Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFß1 mRNA, and addition of exogenous TGFß1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFßR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFß signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.
Subject(s)
Lung Neoplasms/genetics , Protein Kinase C-alpha/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Signal Transduction/genetics , Transforming Growth Factor beta/genetics , p38 Mitogen-Activated Protein Kinases/genetics , Animals , Bronchioles/metabolism , Bronchioles/pathology , Cells, Cultured , Disease Models, Animal , Enzyme Activation , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Protein Kinase C-alpha/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathology , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/metabolism , Stem Cells/pathology , Transforming Growth Factor beta/metabolism , WT1 Proteins/genetics , WT1 Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVES: The hepatocyte growth factor receptor (Met) is frequently overexpressed in Head and Neck Squamous Cell Carcinoma (HNSCC), correlating positively with high-grade tumors and shortened patient survival. As such, Met may represent an important therapeutic target. The purpose of this study was to explore the role of Met signaling for HNSCC growth and locoregional dissemination. MATERIALS AND METHODS: Using a lentiviral system for RNA interference, we knocked down Met in established HNSCC cell lines that express high levels of the endogenous receptor. The effect of Met silencing on in vitro proliferation, cell survival and migration was examined using western analysis, immunohistochemistry and live cell imaging. In vivo tumor growth, dissemination and mouse survival was assessed using an orthotopic tongue mouse model for HNSCC. RESULTS: We show that Met knockdown (1) impaired activation of downstream MAPK signaling; (2) reduced cell viability and anchorage independent growth; (3) abrogated HGF-induced cell motility on laminin; (4) reduced in vivo tumor growth by increased cell apoptosis; (5) caused reduced incidence of tumor dissemination to regional lymph nodes and (6) increased the survival of nude mice with orthotopic xenografts. CONCLUSION: Met signaling is important for HNSCC growth and locoregional dissemination in vivo and that targeting Met may be an important strategy for therapy.
Subject(s)
Proto-Oncogene Proteins c-met/metabolism , Tongue Neoplasms/metabolism , Animals , Apoptosis/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Indoles/pharmacology , Lymphatic Metastasis , Mice , Mice, Nude , Neoplasms, Experimental , Piperazines/pharmacology , Proto-Oncogene Proteins c-met/drug effects , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Sulfonamides/pharmacologyABSTRACT
The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor beta1, but lower concentrations of tumour necrosis factor alpha, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.