ABSTRACT
Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.
Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , MicroRNAs/genetics , 3' Untranslated Regions/genetics , Animals , Asian People/genetics , Carcinogens/pharmacology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Cell Division , Dioxins/pharmacology , Gene Expression Regulation, Neoplastic , Genes, Reporter , Humans , Liver Neoplasms/pathology , Liver Neoplasms/therapy , Mice , MicroRNAs/drug effects , Models, Animal , Models, Genetic , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Transcription, Genetic/drug effects , Up-Regulation , White People/geneticsABSTRACT
H-Ras is a binary switch that is activated by multiple co-factors and triggers several key cellular pathways one of which is MAPK. The specificity and magnitude of downstream activation is achieved by the spatio-temporal organization of the active H-Ras in the plasma membrane. Upon activation, the GTP bound H-Ras binds to Galectin-1 (Gal-1) and becomes transiently immobilized in short-lived nanoclusters on the plasma membrane from which the signal is propagated to Raf. In the current study we show that stabilizing the H-Ras-Gal-1 interaction, using bimolecular fluorescence complementation (BiFC), leads to prolonged immobilization of H-Ras.GTP in the plasma membrane which was measured by fluorescence recovery after photobleaching (FRAP), and increased signal out-put to the MAPK module. EM measurements of Raf recruitment to the H-Ras.GTP nanoclusters demonstrated that the enhanced signaling observed in the BiFC stabilized H-Ras.GTP nanocluster was attributed to increased H-Ras immobilization rather than to an increase in Raf recruitment. Taken together these data demonstrate that the magnitude of the signal output from a GTP-bound H-Ras nanocluster is proportional to its stability.
Subject(s)
MAP Kinase Signaling System , Nanostructures/chemistry , ras Proteins/chemistry , ras Proteins/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cell Survival , Cricetinae , Fluorescence Recovery After Photobleaching , Galectin 1/chemistry , Galectin 1/metabolism , Guanosine Triphosphate/metabolism , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Protein Stability , Time Factors , raf Kinases/chemistry , raf Kinases/metabolismABSTRACT
The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters. Gal-1 is therefore an integral structural component of the H-Ras-signaling nanocluster. Increasing Gal-1 levels increases the stability of H-Ras nanoclusters, leading to enhanced effector recruitment and signal output. Elements in the H-Ras C-terminal hypervariable region and an activated G-domain are required for H-Ras-Gal-1 interaction. Palmitoylation is not required for H-Ras-Gal-1 complex formation, but is required to anchor H-Ras-Gal-1 complexes to the plasma membrane. Our data suggest a mechanism for H-Ras nanoclustering that involves a dual role for Gal-1 as a critical scaffolding protein and a molecular chaperone that contributes to H-Ras trafficking by returning depalmitoylated H-Ras to the Golgi complex for repalmitoylation.
