Hypoxia and aberrant activation of epidermal growth factor receptor (EGFR) are considered important features of various malignancies. However, whether hypoxia can directly trigger EGFR activation and its clinical implications remain unclear. In this study, we demonstrated that in oral cancer, a typical hypoxic tumor, hypoxia can induce chronic but constitutive phosphorylation of wild-type EGFR in the absence of ligands. Oral cancer cell lines exhibit different EGFR phosphorylation responses to hypoxia. In hypoxic HN4 and HN6 cells, ubiquitination-mediated endocytosis, lysosomal sorting, and degradation lead to low levels of EGFR phosphorylation. However, in CAL-27 and HN30 cells, a novel HIF-1α-induced long noncoding RNA (lncRNA), EUDAL, can compete with the E3 ligase/adaptor complex c-Cbl/Grb2 for binding to EGFR, stabilizing phosphorylated EGFR (pEGFR) and resulting in sustained activation of EGFR and its downstream STAT3/BNIP3 signaling. STAT3/BNIP3-mediated autophagy leads to antitumor drug resistance. A high EUDAL/EGFR/STAT3/autophagy pathway activation predicts poor response to chemotherapy in oral cancer patients. Collectively, hypoxia can induce noncanonical ligand-independent EGFR phosphorylation. High EUDAL expression facilitates sustained EGFR phosphorylation in hypoxic tumor cells and leads to autophagy-related drug resistance.
Humans ; ErbB Receptors/metabolism* ; Mouth Neoplasms/pathology* ; RNA, Long Noncoding/genetics* ; Drug Resistance, Neoplasm/genetics* ; Cell Line, Tumor ; Phosphorylation ; Signal Transduction ; STAT3 Transcription Factor/metabolism* ; Cell Hypoxia ; Autophagy ; Proto-Oncogene Proteins c-cbl/metabolism*

The present study aims to investigate the effect of Cbl-b, a member of E3 ubiquitin ligase family, on interleukin-1 (IL-1) pathway in synoviocytes. The protein expression levels of Cbl-b and IL-1-induced matrix metalloproteinase 13 (MMP-13) in synoviocytes were analyzed by Western blot. Collagen substrates were incubated with the conditioned medium collected from synoviocytes cultures and then subjected to SDS-PAGE for analysis of collagen degradation. The results showed that compared with wild-type cells, Cbl-b-deficient cells expressed more MMP-13 protein and had enhanced ability to degrade collagens under IL-1 stimulation. These data suggest that Cbl-b may negatively regulate IL-1-triggered degradation of collagen matrix in synoviocytes.
Collagen ; metabolism ; Epithelial Cells ; enzymology ; Humans ; Interleukin-1 ; metabolism ; Matrix Metalloproteinase 13 ; metabolism ; Proto-Oncogene Proteins c-cbl ; metabolism ; Signal Transduction

BACKGROUNDTransient sublethal ischemia is known as ischemic preconditioning, which enables cells and tissues to survive subsequent prolonged lethal ischemic injury. Ischemic preconditioning exerts neuroprotection through phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Cbl-b belongs to the Casitas B-lineage lymphoma (Cbl) family, and it can regulate the cell signal transduction.The roles of ubiquitin ligase Cbl-b and PI3K/Akt pathway and the relationship between them in oxygen-glucose deprivation preconditioning (OGDPC) in PC12 cells were investigated in the present study.

METHODSOxygen and glucose deprivation (OGD) model in PC12 cells was used in the present study. The 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nuclear staining with Hoechst 33258, and Western blotting were applied to explore the roles of Cbl-b and PI3K/Akt pathway and the relationship between them in OGDPC in PC12 cells.

RESULTSCell viability was significantly changed by OGD and OGDPC. OGD significantly decreased cell viability compared with the control group (P < 0.05), and preconditioning could rescue this damage was demonstrated by the increase of cell viability (P < 0.05). The expression of Cbl-b was significantly increased after OGD treatment. However, the activation of Akt and GSK3β was greatly inhibited. Preconditioning could inhibit the increase of Cbl-b caused by OGD and increase the activation of Akt and GSK3β. LY294002, a specific inhibitor of PI3K, could effectively inhibit the increase of Akt and GSK3β after preconditioning treatment. It partly inhibited the decrease of Cbl-b expression after preconditioning treatment.

CONCLUSIONUbiquitin ligase Cbl-b and PI3K/Akt pathway are differently involved in OGDPC in PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Cell Survival ; Glucose ; deficiency ; Ischemic Preconditioning ; Oxygen ; metabolism ; PC12 Cells ; Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-cbl ; genetics ; metabolism ; Rats ; Signal Transduction ; physiology

BACKGROUND AND OBJECTIVEc-Cbl and Cbl-b are two ubiquitous members of the Casitas B-lineage lymphoma (Cbl) family, which play important roles in the downregulation of epidermal growth factor receptor (EGFR) by acting as E3 ubiquitin ligases and multiadaptor proteins. This study investigated the expression of c-Cbl, Cbl-b, and EGFR in gastric carcinoma and its clinical significance.

METHODSThe expressions of c-Cbl, Cbl-b, and EGFR were detected by immunohistochemistry using tissue microarrays consisting of 124 specimens of gastric carcinoma and 16 specimens of normal gastric mucosa. The relationship between the expressions of c-Cbl, Cbl-b, and EGFR and clinicopathologic factors of gastric carcinoma were analyzed statistically.

RESULTSThe positive rates of c-Cbl, Cbl-b, and EGFR were higher in the gastric carcinoma group than in the normal group (71.0% vs. 18.0%, P<0.01; 82.3% vs. 25.0%, P<0.01; 56.5% vs. 12.5%, P<0.01, respectively). The expression of c-Cbl was positively correlated with depth of invasion (r=0.219, P=0.015), and TNM staging (r=0.266, P=0.003). The expression of Cbl-b was positively correlated with lymph node metastasis (r=0.190, P<0.034) and TNM staging (r=0.298, P<0.001). The expression of EGFR was positively correlated with depth of invasion (r=0.286, P<0.001) and TNM staging (r=0.362, P=0.000). The expression of both c-Cbl and Cbl-b was positively correlated with EGFR (r=0.241, P=0.007; r=0.183, P=0.042, respectively). Synchronous strong-positive expressions of c-Cbl, Cbl-b, and EGFR were observed in 27 specimens of gastric carcinoma, most of which were at advanced stage.

CONCLUSIONSOverexpressions of c-Cbl, Cbl-b, and EGFR are closely related to the invasion and progression of gastric carcinoma. c-Cbl and Cbl-b may serve as novel molecular markers for gastric carcinoma.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; metabolism ; Disease Progression ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Proto-Oncogene Proteins c-cbl ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Young Adult

The study was aimed to explore the mechanism of SYK and CBL family of ubiquitin ligases in Bufalin-induced HL-60 cells apoptosis. Cell viability was tested by trypan blue staining and apoptosis was detected by using flow cytometry. The expressions of CBL and CBL-b and the phosphorylation of SYK were detected by using immunoprecipitation and Western blot. The results showed that Bufalin inhibited HL-60 cell proliferation in time- and dose-dependent manners. IC(50) of suppressing cell viability at 24, 48 and 72 hours were about 26.3, 7.8 and 2.0 nmol/L respectively. The high dose of bufalin already induced apoptosis of HL-60 cells at 8 hours. SYK was quickly phosphorylated, and the expressions of CBL and CBL-b were down-regulated after treatment with Bufalin. It is concluded that SYK activation and CBL protein down-regulation may be involved in Bufalin-induced HL-60 cell apoptosis.
Apoptosis ; drug effects ; Bufanolides ; pharmacology ; Cell Proliferation ; drug effects ; Down-Regulation ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Proto-Oncogene Proteins c-cbl ; metabolism ; Signal Transduction ; Syk Kinase

After binding to its ligand, epidermal growth factor receptor (EGFR) dimerizes and is autophosphorylated. These events initiate the signal transduction process, which regulates a plethora of biologic activity. The duration and strength of these signals are controlled by many regulatory mechanisms, including downregulating activated EGFR primarily via endocytosis and ubiquitination-dependent lysomal degradation. The interaction between EGFR and the ubiquitin ligase Cbl/adaptor protein CIN85, as well as ESCRT complex recruitment play important roles in the process of downregulating EGFR. Tumorigenesis results when the de-sensitization process of EGFR is halted by its own mutation or a mutation that abrogates Cbl E3 ligase activity.
Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Down-Regulation ; Endocytosis ; Epidermal Growth Factor ; pharmacology ; Humans ; Mutation ; Proto-Oncogene Proteins c-cbl ; metabolism ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Signal Transduction ; Ubiquitin ; metabolism

In recent years, major progress has been obtained in studying the early events in TCR-mediated signaling. c-Cb1 has been found to be a negative regulatory factor of the tyrosine kinases in ZAP-70/SyK family. The studies on LAT, SLP-76, ItK and Vav have shown their roles in the signal transduction of Ras and phospholipase Cx1 to Ca2+. Micro-glycolipid raft also plays important role in T cell activation. This minireview shows a brief introduction to the process of TCR-mediated signaling.
Animals ; Humans ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-cbl ; Receptors, Antigen, T-Cell ; immunology ; physiology ; Signal Transduction ; immunology ; physiology ; Ubiquitin-Protein Ligases