Ect2 Protein Research Articles

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

The YTH Domain Protein ECT2 Is an m6A Reader Required for Normal Trichome Branching in Arabidopsis.

Methylations at position N6 of internal adenosines (m6As) are the most abundant and widespread mRNA modifications. These modifications play crucial roles in reproduction, growth, and development by controlling gene expression patterns at the posttranscriptional level. Their function is decoded by readers that share the YTH domain, which forms a hydrophobic pocket that directly accommodates the m6A residues. While the physiological and molecular functions of YTH readers have been extensively studied in animals, little is known about plant readers, even though m6As are crucial for plant survival and development. Viridiplantae contains high numbers of YTH domain proteins. Here, we performed comprehensive evolutionary analysis of YTH domain proteins and demonstrated that they are highly likely to be actual readers with redundant as well as specific functions. We also show that the ECT2 protein from Arabidopsis thaliana binds to m6A-containing RNAs in vivo and that this property relies on the m6A binding pocket carried by its YTH domain. ECT2 is cytoplasmic and relocates to stress granules upon heat exposure, suggesting that it controls mRNA fate in the cytosol. Finally, we demonstrate that ECT2 acts to decode the m6A signal in the trichome and is required for their normal branching through controlling their ploidy levels.

Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles.

Our recent studies revealed a dose-dependent proinflammatory response to copper oxide nanoparticles (CuO NPs) in rats following short-term inhalation exposure for five consecutive days. Here transcriptomics approaches were applied using the same model to assess global gene expression in lung tissues obtained 1 day post-exposure and after a recovery period of 22 days from rats exposed to clean air or 6 hour equivalent doses of 3.3 mg m-3 (low dose) and 13.2 mg m-3 (high dose). Microarray analyses yielded about 1000 differentially expressed genes in the high-dose group and 200 in low-dose compared to the clean air control group, and less than 20 after the recovery period. Pathway analysis indicated cell proliferation/survival and inflammation as the main processes triggered by exposure to CuO NPs. We did not find significant perturbations of pathways related to oxidative stress. Upregulation of epithelial cell transforming protein 2 (Ect2), a known oncogene, was noted and ECT2 protein was upregulated in the lungs of exposed animals. Proliferation of alveolar epithelial cells was demonstrated based on Ki67 expression. The gene encoding monocyte chemoattractant protein 1 (or CCL2) was also upregulated and this was confirmed by immunohistochemistry. However, no aberrant DNA methylation of inflammation-associated genes was observed. In conclusion, we have found that inhalation of CuO NPs in rats causes upregulation of the oncoprotein ECT2 and the chemokine CCL2 and other proinflammatory markers as well as proliferation in bronchoalveolar epithelium after a short-term inhalation exposure. Thus, pathways known to be associated with neoplastic processes and inflammation were affected in this model.

P2.02-073 Cytoplasmic Mislocalization of ECT2 Protein Is Associated with Poor Prognosis in Lung Adenocarcinoma

P2.02-073 Cytoplasmic Mislocalization of ECT2 Protein Is Associated with Poor Prognosis in Lung Adenocarcinoma

Overexpression of epithelial cell transforming 2 protein in colorectal carcinoma predicts a poor prognosis.

Epithelial cell transforming 2 (Ect2) protein is a member of the human diffuse B-cell lymphoma family of guanine nucleotide exchange factors, which activate the Ras homolog gene family of small GTPases; however, the clinical implications of Ect2 in colorectal carcinoma (CRC) are unclear. The present study aimed to determine the relationship between Ect2 expression and prognosis in patients with CRC. Western blot analysis and immunohistochemistry assays were used to determine the expression of Ect2 in CRC and paired non-cancerous tissues from 66 patients. The correlation between Ect2 expression and clinicopathological parameters was assessed using χ2 tests. Patient survival was determined using the Kaplan-Meier method and log-rank test. Cox regression was used for multivariate analysis of prognostic factors. Results demonstrated that Ect2 protein was highly expressed in human CRC samples [29/45 (64.45%)] and significantly correlated with a poor prognosis (P<0.05). Compared with normal tissues, CRC tissues demonstrated higher expression levels of Ect2 mRNA [44/66 (66.67%)]. In addition, highly-expressed Ect2 was significantly associated with recurrence (P=0.023) and invasion (P=0.008) of CRC. High Ect2 expression levels in patients were associated with poorer overall survival (OS) and disease-free survival (DFS) compared with lower expression levels of Ect2. Based on multivariate analysis, Ect2 overexpression was significantly correlated with OS and DFS (P=0.015 and 0.020, respectively). In conclusion, Ect2 overexpression is an independent and important prognostic factor for OS and DFS in patients with CRC.

Elevated levels of epithelial cell transforming sequence 2 predicts poor prognosis for prostate cancer.

Epithelial cell transforming sequence 2 (Ect2) was originally reported as an oncogene that is involved in several types of human cancers. However, little is known about its expression and function in prostate cancer. Immunohistochemical staining for Ect2 was performed on a human tissue microarray. The staining intensity was analyzed in association with clinical pathological parameters such as Gleason score, pathological grade, clinical stage, tumor invasion, lymph node and distant metastasis. Furthermore, we repeated such analysis and investigated the prognostic value of Ect2 using the TCGA (The Cancer Genome Atlas) Dataset. Our immunohistochemical results showed that the expression levels of Ect2 protein were enhanced in human prostate cancer tissues. There existed positive correlations between the expression levels of Ect2 and several clinicopathological parameters, including advanced clinical stage, enhanced tumor invasion and lymph node metastasis. Similarly, we found that the expression levels of Ect2 were positively related to Gleason score, tumor invasion, lymph node metastasis and high distant metastasis in the TCGA Dataset. Kaplan-Meier analysis revealed that lower levels of Ect2 mRNA predicted higher overall survivals and biochemical recurrence (BCR)-free survivals in all patients or non-metastatic patients. Multivariate analysis by Cox regression showed that the expression of Ect2 could be an independent prognostic marker of poor BCR-free survivals. Therefore, levels of Ect2 may serve as a novel marker for the diagnosis or prognosis of prostate cancer.

Annexin V-induced rat Leydig cell proliferation involves Ect2 via RhoA/ROCK signaling pathway.

This study investigated the effect of annexin V on the proliferation of primary rat Leydig cells and the potential mechanism. Our results showed that annexin V promoted rat Leydig cell proliferation and cell cycle progression in a dose- and time-dependent manner. Increased level of annexin V also enhanced Ect2 protein expression. However, siRNA knockdown of Ect2 attenuated annexin V-induced proliferation of rat Leydig cells. Taken together, these data suggest that increased level of annexin V induced rat Leydig cell proliferation and cell cycle progression via Ect2. Since RhoA activity was increased following Ect2 activation, we further investigated whether Ect2 was involved in annexin V-induced proliferation via the RhoA/ROCK pathway, and the results showed that annexin V increased RhoA activity too, and this effect was abolished by the knockdown of Ect2. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated annexin V-induced proliferation and cell cycle progression. We thus conclude that Ect2 is involved in annexin V-induced rat Leydig cell proliferation through the RhoA/ROCK pathway.

Abstract 1853: Identification and characterization of genes in regions of recurrent genomic gain as putative therapeutic targets in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. PDAC presents with the worst prognosis of all solid tumors and the 5-year survival rate for patients with advanced PDAC is only 2%. Current chemotherapies fail to attenuate the aggressiveness of this disease and as such, novel therapeutic strategies are needed. The purpose of our study is to identify putative therapeutic targets in PDAC and characterize the role of a target gene in tumor progression. Towards this aim, we sought to identify genes which are recurrently gained or amplified in pancreatic tumors. If increased copy of specific genes confers neoplastic properties, selective targeting of such genes may have therapeutic implications. We obtained publically available copy number alteration data on 60 PDAC genomes characterized in four independent PDAC studies. Using bioinformatics and computational approaches, we identified 20 genomic loci that are gained in at least one sample in three of the four PDAC datasets. Our integrated analysis of the genes mapping to these 20 loci results in a catalogue of 710 protein-coding genes and 46 miRNA genes, which are candidate targets for further analysis. In order to delineate appropriate biological models for functional validation of these genes in PDAC, we obtained SNP array-based copy number data using the Illumina OmniExpress platform and gene expression analysis from Illumina HT-12 BeadChip arrays from 30 human PDAC cell lines. We identified genes from our candidate gene list in which copy number alteration and gene expression are correlated as computed by a Spearman rank correlation coefficient, α. This gene set was enriched for genes with high correlation between copy number and expression in comparison to simulated gene sets (p = 0.007). Our data suggest that Epithelial cell-transforming sequence 2 oncogene (ECT2) on 3q26.3 is a strong candidate for functional validation. This gene encodes a Rho-specific guanine exchange factor involved in various cellular processes including regulation of G1-to-S phase transition in cell-cycle progression. To validate our in silico findings, we have designed experiments to investigate the role of ECT2 amplification in PDAC, using the 30 human PDAC cell lines we have genetically analyzed. This will be accomplished through differential inhibition of the ECT2 protein, using RNAi and small molcules, in cell lines in which ECT2 is amplified, compared to appropriate control lines. In conclusion, we have identified a set of candidate target genes in PDAC and are currently validating the role of one of these targets, ECT2, in PDAC tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1853. doi:1538-7445.AM2012-1853

Targeting of the RhoGEF Ect2 to the Equatorial Membrane Controls Cleavage Furrow Formation during Cytokinesis

In animal cells, formation of the cytokinetic furrow requires activation of the GTPase RhoA by the guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cortex during anaphase is not understood. Here, we show that Ect2 concentrates at the equatorial membrane during cytokinesis in live cells. Ect2 membrane association requires a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids. Both guanine nucleotide exchange function and membrane targeting of Ect2 are essential for RhoA activation and cleavage furrow formation in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2's spindle midzone anchor complex, and is temporally coordinated with chromosome segregation through the activation state of CDK1. We propose that targeting of Ect2 to the equatorial membrane represents a key step in the delivery of the cytokinetic signal to the cortex.

Amplification of the Ect2 proto-oncogene and over-expression of Ect2 mRNA and protein in nickel compound and methylcholanthrene-transformed 10T1/2 mouse fibroblast cell lines

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.

The Tandem BRCT Domains of Ect2 Are Required for Both Negative and Positive Regulation of Ect2 in Cytokinesis

Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, we address the role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, we show that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, we provide evidence that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of our study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis.

Chromosomal Localization of the Human ECT2 Proto-oncogene to 3q26.1 → q26.2 by Somatic Cell Analysis and Fluorescence in Situ Hybridization

The mouse ect2 oncogene was identified in a search of mitogenic signal transducers using a novel expression cloning strategy. In this system, the introduction of an appropriate expression-cDNA library prepared from the mRNA of one cell type into another can result in the morphological transformation of the recipient cells. The predicted Ect2 protein has sequence similarity within a central core of 255 amino acids to the products of the breakpoint-cluster gene, bcr, the yeast cell cycle gene, CDC24, the dbl and vav oncogenes, and the RasGrf gene. Baculovirus-expressed Ect2 protein shows specific binding to Rho and Rac proteins, indicating that Ect2 is a new member of transforming proteins that can interact with Rho-like proteins of the Ras superfamily. The ect2 foci exhibit an unusual stellate morphology and are capable of forming tumors when subcutaneously inoculated in nude mice. These results indicate that the ect2 gene acts as an oncogene. To investigate the involvement of the ect2 in human tumors, the authors isolated the human homolog, ECT2, of mouse ect2 and determined its chromosomal localization. 13 refs., 1 fig.

Oncogene ect2 is related to regulators of small GTP-binding proteins

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.