ABSTRACT
The non-receptor tyrosine kinase Src is a major player in multiple physiological responses including growth, survival and differentiation. Overexpression and/or oncogenic mutation in the Src gene have been documented in human tumors. The v-Src protein is an oncogenic mutant of Src, which promotes cell survival, migration, invasion and division. GRIM-19 is an antioncogene isolated using a genome-wide knockdown screen. Genes associated with Retinoid-IFN-induced Mortality (GRIM)-19 binds to transcription factor STAT3 and ablates its pro-oncogenic effects while v-Src activates STAT3 to promote its oncogenic effects. However, we found that GRIM-19 inhibits the pro-oncogenic effects of v-Src independently of STAT3. Here, we report the identification of functionally inactivating GRIM-19 mutations in a set of head and neck cancer patients. While wild-type GRIM-19 strongly ablated v-Src-induced cell migration, cytoskeletal remodeling and tumor metastasis, the tumor-derived mutants (L(71)P, L(91)P and A(95)T) did not. These mutants were also incapable of inhibiting the drug resistance of v-Src-transformed cells. v-Src downregulated the expression of Pag1, a lipid raft-associated inhibitor of Src, which was restored by wild-type GRIM-19. The tumor-derived mutant GRIM-19 proteins failed to upregulate Pag1. These studies show a novel mechanism that deregulates Src activity in cancer cells.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Transformation, Neoplastic/pathology , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Mutation/genetics , NADH, NADPH Oxidoreductases/genetics , Oncogene Protein pp60(v-src)/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Movement , Cell Proliferation , Fluorescent Antibody Technique , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunoenzyme Techniques , Mice , Mice, Nude , Oligonucleotide Array Sequence Analysis , Oncogene Protein pp60(v-src)/genetics , Phosphorylation , RNA, Messenger/genetics , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.
Subject(s)
Cell Movement , Cytoskeleton/chemistry , NADH, NADPH Oxidoreductases/physiology , Oncogene Protein pp60(v-src)/antagonists & inhibitors , Animals , Cell Transformation, Neoplastic , Cortactin/metabolism , Mice , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , Phosphorylation , Structure-Activity RelationshipABSTRACT
We have isolated a novel interferon (IFN)-retinoid regulated cell death regulatory protein genes associated with retinoid-IFN-induced mortality (GRIM)-19 earlier. To understand its mechanism of action, we have employed a yeast-two-hybrid screen and identified serine protease HtrA2 as its binding partner. GRIM-19 physically interacts with HtrA2 and augments cell death in an IFN/all-trans retinoic acid (RA)-dependent manner. In the presence of GRIM-19, the HtrA2-driven destruction of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP) is augmented. These interactions were disrupted by an human herpes virus-8 (HHV-8)-coded oncoprotein, vIRF1, and conferred resistance to IFN/RA-induced cell death. These data show a critical role of HtrA2 in a cytokine-induced cell death response for the first time and its inhibition by a viral protein.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/physiology , Mitochondrial Proteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , Serine Endopeptidases/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cell Survival/physiology , Drug Resistance , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , High-Temperature Requirement A Serine Peptidase 2 , Humans , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Interferon-beta/pharmacology , Mitochondrial Proteins/genetics , NADH, NADPH Oxidoreductases/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine Endopeptidases/genetics , Transfection , Tretinoin/pharmacology , Two-Hybrid System Techniques , Viral Proteins/genetics , Viral Proteins/metabolism , X-Linked Inhibitor of Apoptosis Protein/genetics , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
BACKGROUND: Investigations have been conducted on the potential of pyruvate, a normal tissue metabolite, in the prevention of cataract formation. METHODS: Cataract was induced by maintaining young rats on a diet containing 30% galactose. The progress of cataract was monitored by visual inspection and ophthalmoscopic and slit-lamp examinations. The protective effect of pyruvate was assessed by incorporating it in the galactose diet and drinking water. The progress of cataract was substantially thwarted by this dietary regimen. RESULTS: Substantial morphogenetic changes in the lenses of the galactosaemic animals, which by themselves can offer obstruction to light penetration through the lens and scattering, were significantly attenuated. These changes were ascertained by histological detection of errors in cellular differentiation and their migration in unwanted areas. CONCLUSIONS: As per previous studies, the pyruvate effect is attributed to its direct effect on the biochemistry of lens related to the inhibition of oxidative stress, as well as to its effect on tissue physiology related to the lifelong process of organogenesis, characteristic of this tissue.
