Oncogene database

1894

ECT2

epithelial cell transforming 2;ECT2;epithelial cell transforming 2

epithelial cell transforming sequence 2 oncogene|epithelial cell-transforming sequence 2 oncogene|protein ECT2

3q26.1-q26.2

protein-coding

Occupational exposure of humans to mixtures of insoluble and soluble nickel (Ni) compounds correlates with increased incidences of lung, sinus, and pharyngeal tumors. Specific insoluble Ni compounds are carcinogenic to animals by inhalation and induce morphological and neoplastic transformation of cultured rodent cells. Our objectives were to (1) understand mechanisms of nickel ion-induced cell transformation, hence carcinogenesis and (2) develop biomarkers of nickel ion exposure and nickel ion-induced cell transformation. We isolated mRNAs from green nickel oxide (NiO), crystalline nickel monosulfide (NiS), and 3-methylcholanthrene (MCA) transformed C3H/10T1/2 Cl 8 cell lines, and determined by mRNA differential display that nine mRNA fragments were differentially expressed between Ni transformed and non-transformed 10T1/2 cell lines. Fragment R2-5 was expressed at higher steady-state levels in the transformed cell lines. R2-5 had 100% sequence identity to part of the coding region of Ect2, a mouse proto-oncogene encoding a GDP-GTP exchange factor. The 3.9-kb Ect2 transcript was expressed at 1.6- to 3.6-fold higher steady-state levels in four Ni transformed, and in two MCA-transformed, cell lines. Ect2 protein was expressed at 3.0- to 4.5-fold higher steady-state levels in Ni-transformed and in MCA-transformed cell lines. The Ect2 gene was amplified by 3.5- to 10-fold in Ni transformed, and by 2.5- to 3-fold in MCA transformed cell lines. Binding of nickel ions to enzymes of DNA synthesis likely caused amplification of the Ect2 gene. Ect2 gene amplification and over-expression of Ect2 mRNA and protein can cause microtubule disassembly and cytokinesis, contributing to induction and maintenance of morphological, anchorage-independent, and neoplastic transformation of these cell lines. Over-expression of Ect2 protein is a useful biomarker to detect exposure to nickel compounds and nickel ion-induced morphological and neoplastic cell transformation.

As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB(+)- and (-)-matched cells using short hairpin RNA. In the RB(-) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(-) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(-) cells resulted in cytokinesis arrest, suggesting that RB(-) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.

The ECT2 (epithelial cell transforming sequence 2) proto-oncogene encodes a guanine nucleotide exchange factor for Rho GTPases, and regulates cytokinesis. ECT2 plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of glioma. In this study, we investigated relationships between ECT2 expression, tumor histology, and prognosis in glioma patients. ECT2 mRNA expression was examined using quantitative real-time PCR, while its protein expression was examined by immunohistochemistry of 54 glioma tissue samples. expressions of ECT2 mRNA and protein were markedly increased in high-grade gliomas compared to low-grade gliomas. Patients in whom expression of ECT2 mRNA and protein in tumor tissues was the lowest survived longer than patients who had higher expression. In vitro, ECT2 siRNA inhibited glioma cell proliferation and invasion. These data suggest that increased expression of ECT2 contribute to promotion of tumor invasiveness and progression, implying that evaluation of ECT2 expression is a prognostic marker for glioma patients.

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCiota oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCiota-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCiota directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCiota or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCiota regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCiota-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCiota-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCiota-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCiota-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

We have developed an efficient expression cloning system that allows rapid isolation of complementary DNAs able to induce the transformed phenotype. We searched for molecules expressed in epithelial cells and possessing transforming potential to fibroblasts, and cloned a cDNA for the normal receptor of a growth factor secreted by NIH/3T3 cells. Here we report a second novel transforming gene, ect2. The isolated cDNA is activated by amino-terminal truncation of the normal product. The Ect2 protein has sequence similarity within a central core of 255 amino acids with the products of the breakpoint cluster gene, bcr (ref. 5), the yeast cell cycle gene, CDC24 (ref. 6), and the dbl oncogene. Each of these genes encodes regulatory molecules or effectors for Rho-like small GTP-binding proteins. The baculovirus-expressed Ect2 protein could bind highly specifically to Rho and Rac proteins, whereas the dbl product showed broader binding specificity to Rho family proteins. Thus ect2 is a new member of an expanding family, whose products have transforming properties and interact with Rho-like proteins of the Ras superfamily.