Epithelial Cell Transforming Sequence 2 Research Articles

GINS2 regulates temozolomide chemosensitivity via the EGR1/ECT2 axis in gliomas

Temozolomide (TMZ), a DNA alkylating agent, has become the primary treatment for glioma, the most common malignancy of the central nervous system. Although TMZ-containing regimens produce significant clinical response rates, some patients inevitably suffer from inferior treatment outcomes or disease relapse, likely because of poor chemosensitivity of glioma cells due to a robust DNA damage response (DDR). GINS2, a subunit of DNA helicase, contributes to maintaining genomic stability and is highly expressed in various cancers, promoting their development. Here, we report that GINS2 was upregulated in TMZ-treated glioma cells and co-localized with γH2AX, indicating its participation in TMZ-induced DDR. Furthermore, GINS2 regulated the malignant phenotype and TMZ sensitivity of glioma cells, mostly by promoting DNA damage repair by affecting the mRNA stability of early growth response factor 1 (EGR1), which in turn regulates the transcription of epithelial cell-transforming sequence 2 (ECT2). We constructed a GINS2–EGR1–ECT2 prognostic model, which accurately predicted patient survival. Further, we screened Palbociclib/BIX-02189 which dampens GINS2 expression and synergistically inhibits glioma cell proliferation with TMZ. These findings delineate a novel mechanism by which GINS2 regulates the TMZ sensitivity of glioma cells and propose a promising combination therapy to treat glioma.

Long noncoding RNA MAPKAPK5-AS1 promotes metastasis through regulation miR-376b-5p/ECT2 axis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common diseases threatening human health worldwide. However, the molecular mechanisms of HCC are still unclear. Here, we identified a differentially expressed lncRNA called MAPKAPK5-AS1(abbreviation: MK5-AS1) and elucidated its role and molecular mechanism in the development of HCC. Real-time PCR (RT-PCR) was used to verify the expression of MK5-AS1 in hepatocarcinoma cell lines and tumor tissues of HCC patients. The biological functions of MK5-AS1 in HCC cells was assessed both in vitro and in vivo assays. The Lncbase, miRDB and TargetScan databases were used to predict the lncRNA-miRNA-mRNA interactions. RNA immunoprecipitation (RIP) and double luciferase reporter gene assays further verified the interactions. MK5-AS1 expression was significantly upregulated in HCC tissues and cell lines. Furthermore, high MK5-AS1 expression was positively associated with tumor progression and poor prognosis. In vitro and in vivo experiments confirmed that overexpressed MK5-AS1 promoted migration and invasion of HCC cells. Bioinformatics analysis based on Lncbase, miRDB and TargetScan databases showed MK5-AS1 competitively bound to miR-376b-5p that prevented epithelial cell transforming sequence 2 (ECT2) from miRNA-mediated degradation, thus facilitated HCC metastasis. Our results established a tumor promotive role of MK5-AS1 in HCC pathogenesis.

CircSETD3 mediates acquired resistance to gefitinib in non-small lung cancer cells by FXR1/ECT2 pathway.

Gefitinib is the first-line treatment for non-small cell lung cancer (NSCLC) harboring EGFR sensitive mutation. However, acquired resistance significantly limits its therapeutic efficacy. CircSETD3 has been reported to promote gefitinib resistance in NSCLC cells, however, its underlying mechanisms have not been fully clarified. The expression of circSETD3 were detected in NSCLC patients who received gefitinib as first-line treatment, including 20 gefitinib-sensitive patients and 20 acquired gefitinib-resistant patients. Cell viability were examined by CCK8 assay. The mRNA and protein levels were detected by qRT-PCR and western blot. Using RNA pull-down assay followed by mass spectrometry to identified proteins that interact with circSETD3. The interaction between circSETD3 and fragile X-related protein-1 (FXR1) were further validated by RNA immunoprecipitation (RIP) and pull-down analysis. Fuorescence in situ hybridization (FISH) and immunofluorescence (IF) assays was used for the identification of sub-location of circSETD3 and FXR1 in cells. The effect of circSETD3 overexpression and knockdown on NSCLC tumor growth to gefitinib sensitivity was detected using the mouse xenograft model. CircSETD3 was significantly upregulated in gefitinib-resistant NSCLC cells, and decreased the gefitinib sensitivity in vitro and in vivo. Mechanically, circSETD3 facilitated FXR1 binding to its downstream mRNA target, epithelial cell-transforming sequence 2 (ECT2), promoting ECT2 mRNA decay, which further inhibited cellular apoptosis. CircSETD3/FXR1/ECT2 axis plays a critical role in the acquired resistance to gefitinib in NSCLC. Our results highlight the potential of circSETD3 as a biomarker and therapeutic target for NSCLC patients with acquired gefitinib resistance.

ECT2 promotes malignant phenotypes through the activation of the AKT/mTOR pathway and cisplatin resistance in cervical cancer.

Epithelial cell transforming sequence 2 (ECT2) is expressed at high levels in various malignancies and contributes to malignant phenotypes in cancers. However, ECT2 is still not fully understood regarding its function and carcinogenic mechanism in cervical cancer. This research indicated that ECT2 expression was elevated in cervical cancer based on bioinformatics analysis and clinical specimens. Experiments in vitro and in vivo confirmed that ECT2 knockdown could suppress the proliferation and metastasis of cervical carcinoma cells. In addition, we found that silencing ECT2 could enhance the sensitivity to cisplatin and promote cell apoptosis. Mechanistically, we observed that ECT2 knockdown could inhibit the AKT/mTOR pathway and activate apoptosis, while ECT2 overexpression induced the opposite effect. The relationship between ECT2 and AKT was further confirmed by immunoprecipitation and rescue experiments. We found that the ECT2 and AKT could interact to form a complex, and knockdown AKT could offset all of the effects induced by ECT2. Our study emphasized the key point of ECT2 in the reversal of cisplatin resistance, and ECT2 could become a potential therapeutic target in cervical cancer.

Screening of four key genes in esophageal carcinoma based on TCGA and GEO data and verification of anti-proliferative effect of LAPTM4B knockdown in esophageal carcinoma cells invitro

Esophageal carcinoma (ESCA) is one of the most prevalent and aggressive malignancies of the gastrointestinal tract and constitutes sixth primary cause of cancer-related death worldwide. It is urgently needed to identify effective therapeutic targets. Differentially expressed genes (DEGs) involved in ESCA were identified via bioinformatics analysis. Four DEGs were selected for further analysis using Gene Expression Profiling Interactive Analysis, Human Protein Atlas, UALCAN web portal, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. 5-ethynyl-2′-deoxyuridine incorporation and cell counting kit-8 assays were used to evaluate cell proliferation. Western blot analysis was used to detect the protein levels of lysosomal-associated transmembrane protein 4B (LAPTM4B), Notch1, hairy and enhancer of split 1 (Hes1), and hairy and enhancer of split-related with YRPW motif 1 (Hey1). Results showed that LAPTM4B, Bcl-2 homology domain 3 (BH3)-interacting domain death agonist (BID), epithelial cell transforming sequence 2 (ECT2), and aurora kinase A (AURKA) were upregulated in several types of tumors including ESCA and correlated with tumor stage and tumor histology based on bioinformatics analysis. KEGG pathway analysis suggested that LAPTM4B-associated genes were significantly enriched in Notch pathway. Meanwhile, BID-, ECT2-, and AURKA-correlated genes were particularly enriched in p53 signaling pathway. Additionally, we found that LAPTM4B silencing inhibited cell proliferation and Notch pathway in ESCA cells. Notch1 overexpression abrogated LAPTM4B knockdown-induced proliferation reduction in ESCA cells. In conclusion, LAPTM4B silencing inhibited proliferation in ESCA cells by inactivating the Notch pathway.

LncRNA FGD5-AS1 enhances the proliferation and stemness of hepatocellular carcinoma cells through targeting miR-223 and regulating the expression of ECT2 and FAT1.

Hepatocellular carcinoma (HCC) is common and causes many deaths worldwide. The aim of this study is to explore the mechanism by which long non-coding RNA FGD5-AS1 regulates HCC cell proliferation and stemness. Tumor and normal adjacent tissues were harvested from HCC patients. Real-time quantitative reverse transcription-PCR was applied to examine the expression of FGD5-AS1, miR-223, Epithelial cell transforming sequence 2 (ECT2) and FAT1. The protein levels of ECT2, FAT1, proliferating cell nuclear antigen (PCNA), OCT4, CD133 and CD90 were analyzed by western blot. The localization of FGD5-AS1 was examined by Fluorescence in situ hybridization. Cell proliferation was analyzed with CCK-8 and colony formation assays. Spheroid formation was used for analyzing cell stemness. Gene interaction was examined by RNA immunoprecipitation and luciferase activity assays. A subcutaneous xenograft mouse model was established to analyze HCC growth and stemness in vivo. Immunohistochemistry staining was used to analyze the expression PCNA and OCT4 in subcutaneous tumors. FGD5-AS1 was upregulated in HCC and its high expression indicated poor prognosis of patients. High expression of FGD5-AS1 enhanced HCC cell proliferation and stemness. Knockdown of FGD5-AS1 restrained tumor growth and stemness in mice. FGD5-AS1 directly sponged miR-223 and promoted the expression of ECT2 and FAT1 in HCC. Both knockdown of miR-223 and overexpression of ECT2 and FAT1 reversed FGD5-AS1 silencing-mediated suppression of HCC cell proliferation and stemness. FGD5-AS1 directly sponged miR-223 and promoted the expression of ECT2 and FAT1 in HCC, thus enhancing HCC cell proliferation and stemness. Our study identifies potential prognostic biomarkers and therapeutic targets for HCC.

High expression of ECT2 and E2F1 is associated with worse clinical manifestations and prognosis in patients with oral squamous cell carcinoma

To detect the expression, prognostic value, and possible effects of epithelial cell transforming sequence 2 (ECT2) and E2F1 in patients with oral squamous cell carcinoma (OSCC). Seventy subjects hospitalized for OSCC surgical treatment in the Fourth Hospital of Hebei Medical University were selected for this study. Expression levels of ECT2 and E2F1 were detected by qRT-PCR, Western blot, and immunohistochemistry. The Kaplan-Meier method and Cox risk proportional regression analysis were used to analyze the relationship between different expression levels of ECT2 or E2F1 and the survival of patients with OSCC in 3 years. Relative expression levels of E2F1 mRNA (1.31 ± 0.15) and ECT2 mRNA (3.95 ± 0.72) in OSCC tissues, compared to adjacent normal tissues (0.87 ± 0.11, 1.03 ± 0.23, all p < 0.05). ECT2 was highly expressed in 42 (60.00%) OSCC samples and E2F1 was highly expressed in 45 (64.29%) samples. The expression of ECT2 and E2F1 was related to clinical stage, lymphatic metastasis, tumor differentiation grade, and tumor diameter in OSCC patients. The higher the expression of ECT2 and E2F1, the lower the 3-year survival rate of patients. ECT2 high expression (HR=2.407, p < 0.001), E2F1 high expression of E2F1 (HR = 2.159, p = 0.013), Clinical stages (III+IV) (HR = 1.362, p = 0.012), medium and low differentiation (HR = 1.522, p = 0.015), lymphatic metastasis (HR = 1.951, p < 0.001), and tumor diameter (≥3 cm) (HR = 1.824, p = 0.002) could be independent factors for 3-year survival of patients with OSCC. The expression of ECT2 and E2F1 in OSCC was significantly up-regulated, which was closely related to clinical stage, lymph node metastasis, tumor size, and 3-year survival of OSCC patients.

Upregulation of ECT2 Predicts Adverse Clinical Outcomes and Increases 5-Fluorouracil Resistance in Gastric Cancer Patients.

Background The abnormal expression and prognosis prediction of epithelial cell transforming sequence 2 (ECT2) in gastric cancer (GC) has been reported. However, the effect of ECT2 on 5-fluorouracil (5-Fu) resistance in GC is unclear. This research aims to solve the abovementioned problems. Methods Gene expression was detected by RT-qPCR and Western blot analysis. Cell viability was evaluated by the colony formation assay, MTT assay, and flow cytometric analysis. Transwell and wound healing assays were used to detect cell metastasis. Results Upregulation of ECT2 was found in stomach adenocarcinoma (STAD) and GC tissues. In addition, high ECT2 expression can predict adverse clinical outcomes in GC patients. More importantly, ECT2 knockdown weakened the resistance of 5-FU in GC cells. ECT2 silencing reduced the cell migratory and invasive abilities of GC cells treated with 5-FU. We also found that downregulation of ECT2 increased 5-FU sensitivity in GC cells by downregulating P-gp, MRP1, and Bcl-2. Conclusion Upregulation of ECT2 can predict adverse clinical outcomes and increase 5-FU resistance in GC patients.

MiR-30a-5p Regulates Viability, Migration, and Invasion of Lung Adenocarcinoma Cells via Targeting ECT2.

Objective The abnormal expression of epithelial cell transforming sequence 2 (ECT2) is often considered the driving factor for the growth and invasion of tumors. This study was performed to investigate the regulatory effect of miR-30a-5p and ECT2 on lung adenocarcinoma (LUAD), which provides a basis for the effective clinical treatment of LUAD. Methods The mature miRNAs, expression data of mRNAs, and clinical data of LUAD were downloaded from The Cancer Genome Atlas (TCGA). The expression levels of ECT2 mRNA and miR-30a-5p in cancer cell lines were detected by qRT-PCR. Western blot was performed to test the expression of ECT2 protein. The targeting relationship between miR-30a-5p and ECT2 was verified by dual-luciferase assay. The CCK-8 method and Transwell assay were conducted to test the viability, migratory, and invasive abilities of cells. Results ECT2 expression was upregulated in LUAD and was significantly correlated with the LUAD clinical stage and pathologic T stage, and the expression of its upstream regulatory gene miR-30a-5p was downregulated. miR-30a-5p targeted ECT2 in LUAD. Downregulation of ECT2 could inhibit the viability, migration, and invasion of LUAD cells, which could be reversed by simultaneously suppressing the expression of miR-30a-5p. Conclusion Our results suggested that miR-30a-5p repressed the malignant progression of LUAD via downregulating ECT2. miR-30a-5p and ECT2 may be effective targets for LUAD patients.

ECT2 promotes lung adenocarcinoma progression through extracellular matrix dynamics and focal adhesion signaling.

Lung adenocarcinoma (LAC) is the most prevalent form of lung cancer. Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor that has been implicated in oncogenic and malignant phenotypes of LAC. Here, we identified an oncogenic role of ECT2 in the extracellular matrix (ECM) dynamics of LAC cells. We showed that suppression of ECT2 decreased adhesion and spreading of LAC cells on ECM components. Morphologically, ECT2‐depleted cells exhibited a rounded shape and cytoskeletal changes. Examination of transcriptional changes by RNA sequencing revealed a total of 1569 and 828 genes whose expressions were altered (absolute fold change and a difference of >2 fold) in response to suppression of ECT2 in two LAC cells (Calu‐3 and NCI‐H2342), respectively, along with 298 genes that were common to both cell lines. Functional enrichment analysis of common genes demonstrated a significant enrichment of focal adhesions. In accord with this observation, we found that ECT2 suppression decreased the expression level of proteins involved in focal adhesion signaling including focal adhesion kinase (FAK), Crk, integrin β1, paxillin, and p130Cas. FAK knockdown leads to impaired cell proliferation, adhesion, and spreading of LAC cells. Moreover, in LAC cells, ECT2 binds to and stabilizes FAK and is associated with the formation of the focal adhesions. Our findings provide new insights into the underlying role of ECT2 in cell‐ECM dynamics during LAC progression and suggest that ECT2 could be a promising therapeutic avenue for lung cancer.

Effect of Ect2 Expression on the Growth of Triple-Negative Breast Cancer Cells with Paclitaxel Intervention.

ObjectTo identify the expression levels of ECT2 (epithelial cell transforming sequence 2) in triple-negative breast cancer (TNBC) before and after administration of paclitaxel (PTX) and explore the interaction between ECT2 and PTX in breast cancer treatment.MethodsLentiviral (LV) packaging ECT2 overexpression and interference plasmids were constructed for in vitro assays. The effects of ECT2 expression on the TNBC cell line (HCC1806), particularly its roles in the proliferation, invasion, migration and apoptosis and cell cycle, were evaluated using the CCK-8 and other methods before and after PTX treatment. In nude mouse xenograft settings were performed to detect cell apoptosis and Ki-67 expression levels by TUNEL and immunohistochemical staining, respectively.ResultsIn the vitro assays, before and after the PTX treatment, comparison of the LV-ECT2 and sh-ECT2 groups and the remaining three groups (control, LV-NC, sh-NC) showed statistically significant differences in terms of cell proliferation, invasion and migration and apoptosis and changes in the cell cycle. In the vivo assays, the control, LV-ECT2 and sh-ECT2 groups markedly outweighed the corresponding PTX-treated groups. The LV-ECT2, PTX, sh-ECT2 and sh-ECT2-PTX were all significantly different from the control group in terms of body weight and tumour size changes. Cell apoptosis occurred in the PTX, sh-ECT2 and sh-ECT2-PTX groups. About the Ki-67 proliferation index, the PTX, LV-ECT2-PTX, sh-ECT2 and sh-ECT2-PTX groups were significantly different from the control group.ConclusionECT2, which is a major driving factor in the growth of breast cancer cells, plays an important role in regulating TNBC growth. PTX therapy had significantly improved efficacy after silencing ECT2. This finding indicates that the inhibition of ECT2 expression may facilitate the treatment of breast cancer as a new regimen and provide a theoretical basis for the development of new targeted drugs as a replacement for PTX in breast cancer treatment.

Upregulated microRNA-194 impairs stemness of cholangiocarcinoma cells through the Rho pathway via inhibition of ECT2.

Cholangiocarcinoma (CCA) is devastating for its delayed presence,difficulty in diagnosis, and high mortality. Other studies have supported the important role of microRNAs (miRNAs) in the pathogenesis of CCA, and the role of miR-194 was investigated in several human cancers, though, the molecular mechanism of miR-194 in CCA stem cells remains largely unknown. We aimed to identify the functional significance of miR-194 in CCA. The microarray-based analysis was applied to detect the epithelial cell transforming sequence 2 (ECT2) expression and predict the miRNA-regulated ECT2, followed by the identification of relationship between ECT2 and obtained miRNA by dual-luciferase reporter gene assay. The effects of depletion or ectopic expression of miR-194 on Rho pathway and the biological characteristics of CCA were assessed by reverse transcription quantitative polymerase chain reaction, immunoblotting, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, scratch test, Transwell, and flow cytometry. Lastly, tumor growth was assessed by xenograft tumor in nude mice. ECT2 was highly expressed while miR-194 was poorly expressed in CCA stem cells, and the targeting relation between ECT2 and miR-194 was proved. More important, the elevated expression of miR-194 or ECT2 silencing inhibited the Rho pathway, and further promoted the apoptosis and suppressed the stem cell proliferation, migration, and invasion of CCA in vitro. miR-194 inhibited the tumor growth in vivo. In a word, miR-194 inhibits ECT2 and blocks the activation of Rho signaling pathway, thus promoting apoptosis, inhibiting proliferation and migration of CCA stem cells, and suppressing tumor growth. The mechanism can be regarded as a target for treating CCA.

The Oncogene ECT2 Contributes to a Hyperplastic, Proliferative Lung Epithelial Cell Phenotype in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) and lung cancer are progressive lung diseases with a poor prognosis. IPF is a risk factor for the development of lung cancer, and the incidence of lung cancer is increased in patients with IPF. The disease pathogenesis of IPF and lung cancer involves common genetic alterations, dysregulated pathways, and the emergence of hyperplastic and metaplastic epithelial cells. Here, we aimed to identify novel, common mediators that might contribute to epithelial cell reprogramming in IPF. Gene set enrichment analysis of publicly available non-small cell lung cancer and IPF datasets revealed a common pattern of misregulated genes linked to cell proliferation and transformation. The oncogene ECT2 (epithelial cell transforming sequence 2), a guanine nucleotide exchange factor for Rho GTPases, was highly enriched in both IPF and non-small cell lung cancer compared with nondiseased controls. Increased expression of ECT2 was verified by qPCR and Western blotting in bleomycin-induced lung fibrosis and human IPF tissue. Immunohistochemistry demonstrated strong expression of ECT2 staining in hyperplastic alveolar epithelial type II (ATII) cells in IPF, as well as its colocalization with proliferating cell nuclear antigen, a well-known proliferation marker. Increased ECT2 expression coincided with enhanced proliferation of primary mouse ATII cells as analyzed by flow cytometry. ECT2 knockdown in ATII cells resulted in decreased proliferation and collagen I expression in vitro. These data suggest that the oncogene ECT2 contributes to epithelial cell reprogramming in IPF, and further emphasize the hyperplastic, proliferative ATII cell as a potential target in patients with IPF and lung cancer.

Antimetastatic effects of calycosin on osteosarcoma and the underlying mechanism.

Osteosarcoma (OS) refers to a malignant tumor with potential invasiveness and metastasis; however, the current chemotherapy of OS is lacking. Thus, the alternative drug for treating OS is urgent to explore. Calycosin (CC) is evidenced in our previous study to play the anti-OS benefits for suppressing cancer cell proliferation. Consequently, further investigation of CC-medicated anti-invasive and metastatic effects against OS is needed. In the current study, the clinical samples of OS patients were collected for biological and staining assays, such as enzyme-linked immunosorbent assay and polymerase chain reaction. Meanwhile, the cell line and tumor-bearing nude mice were employed in assessing antimetastatic effects of CC against OS through biochemical tests and immunoassays. As a result, the OS patients exhibited upregulated neoplastic expressions of matrix metalloproteinase 2 (MMP2) and proliferating cell nuclear antigen (PCNA), cellular mRNAs and proteins of inhibitor of nuclear factor kappa-B alpha (IκBα), and epithelial cell transforming sequence 2 (ECT2). In cell-line study, CC-treated human OS cells exhibited induced cell apoptosis, reduced cell proliferation, and cellular MMP2 and PCNA concentration, inhibited cell migration, lowered expressions of IκBα ECT2 mRNAs, and proteins. In tumor-bearing nude mice study, CC-treated mice resulted in the dose-dependent reductions of tumor weights and intracellular MMP2 contents. As shown in further assays, neoplastic expressions of interleukin 6 protein, IκBα, ECT2 mRNAs, and proteins were downregulated dose-dependently in CC-treated tumor-bearing mice. In conclusion, these investigative findings suggest that CC may play the potential anti-invasive benefits against OS through suppressing metastasis-associated IκBα/ECT2 molecular pathway.

Epithelial cell transforming sequence 2 expression is associated with the progression of laryngeal squamous cell carcinoma.

Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor and its expression is associated with the development of malignant tumor types. However, to the best of our knowledge, there is no information on the role of ECT2 in the development and progression of laryngeal squamous cell carcinoma (LSCC). The present study aimed at investigating the expression pattern and potential role of ECT2 in the development and progression of LSCC. The expression of ECT2 in 81 pairs of LSCC and adjacent non-tumor tissues was characterized by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. ECT2 expression was upregulated in LSCC tissues and associated significantly with poor differentiation, higher stages, lymph node metastasis and poor survival in the sample population. The relative expression levels of ECT2 mRNA transcripts were correlated with the intensity of anti-ECT2 staining in 25 ECT2+LSCC specimens selected randomly. These results indicated that ECT2 expression was crucial for the progression of LSCC and may serve as a biomarker for the diagnosis and prognosis of LSCC.

Cytoplasmic expression of epithelial cell transforming sequence 2 in lung adenocarcinoma and its implications for malignant progression

Epithelial cell transforming sequence 2 (ECT2), a guanine nucleotide exchange factor, is predominantly localized in the nucleus of non-transformed cells and functions to regulate cytokinesis. ECT2 is also localized in the cytoplasm of cancer cells. Aberrant cytoplasmic expression of ECT2 is thought to drive tumor growth and invasion. In this study, we investigated the cytoplasmic expression of ECT2 and its prognostic and biological significance in lung adenocarcinoma. Western blotting of cellular fractions from the nucleus and cytoplasm was performed to determine the subcellular localization of ECT2 in lung adenocarcinoma cell lines. The cytoplasmic expression of ECT2 in 167 lung adenocarcinomas was evaluated by immunohistochemistry and its clinical significance was examined using Kaplan–Meier curves and Cox regression analysis. Scraping cytology specimens of 13 fresh lung adenocarcinomas were used to assess the subcellular localization of ECT2 and its phosphorylation at Thr790 (P-ECT2(T790)). We found that ECT2 was localized in both the nucleus and cytoplasm of lung adenocarcinoma cell lines and tumor tissues. Cytoplasmic expression of ECT2 was detected by immunohistochemistry in 83 (50%) of the lung adenocarcinomas, and was found to increase during cancer progression. It was expressed in 30 (29%) small adenocarcinomas ( ≤ 2 cm in diameter) and 53 (82%) advanced adenocarcinomas ( > 2 cm in diameter). Cytoplasmic positivity for ECT2 was associated with a poor outcome in terms of both disease-free and overall survival (both P < 0.001), and was an independent prognostic factor for overall survival (P = 0.025). Immunocytochemical staining for P-ECT2(T790) demonstrated cytoplasmic and membrane positivity in Calu-3 cells and scraping cytology specimens. Positive P-ECT2(T790) staining was correlated with cytoplasmic ECT2 expression in 6 of 13 scraped cytology specimens tested. In conclusion, our findings indicate that cytoplasmic ECT2 expression could promote the malignant progression of lung adenocarcinoma and may represent a potent therapeutic target for patients with lung adenocarcinoma.

Overexpression of ECT2 is a strong poor prognostic factor in ER(+) breast cancer.

Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor encoded by the ECT2 gene, which is located on the 3q26.31 chromosomal region and is directly associated with the occurrence of cancers. The aim of the present study was to examine the expression and prognostic importance of ECT2 in various breast cancer subtypes using the online tools, Gene Expression Profiling Interactive Analysis, Kaplan-Meier-plotter and bc-GenExMiner. ECT2 mRNA expression was significantly different in oestrogen receptor ER(+) breast cancer; overexpression of ECT2 was associated with poor prognosis in ER+ breast cancer. The mRNA expression levels of ECT2 were increased in basal-like breast cancer and triple negative breast cancer, but were not significant for prognostic prediction. We identified ECT2-correlated genes and their corresponding Gene Ontology (GO) enrichment terms. The results revealed that GO: 0005524 (protein binding) had the greatest number of correlated genes and also contained ECT2. This suggested that overexpression of ECT2 may be a significant prognostic factor for poor outcome in ER+ breast cancer; however, the precise role of ECT2 in breast cancer requires further investigation.

Quantitative phosphoproteomic analysis identifies novel functional pathways of tumor suppressor DLC1 in estrogen receptor positive breast cancer.

Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative Reverse Transcription PCR in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. The Cancer Genome Atlas (TCGA) data showed that down regulation of DLC1 is not due to methylation or mutations. To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we stably overexpressed DLC1-full-length (DLC1-FL) in T-47D breast cancer cells; this inhibited cell colony formation significantly in vitro compared to its control counterpart. Label-free global proteomic and TiO2 phosphopeptide enrichment assays (ProteomeXchange identifier PXD008220) showed that 205 and 122 phosphopeptides were unique to DLC1-FL cells and T-47D-control cells, respectively, whereas 6,726 were quantified by phosphoproteomics analysis in both conditions. The top three significant clusters of differentially phosphopeptides identified by DAVID pathway analysis represent cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Phosphoproteomics analysis documented an inverse relation between DLC1 expression and several phosphopeptides including epithelial cell transforming sequence 2 (ECT2). Decreased phosphorylation of ECT2 at the residue T359, critical for its active conformational change, was validated by western blot. In addition, the ECT2 T359-containing phosphopeptide was detected in both basal and luminal patient-derived breast cancers breast cancer phosphoproteomics data on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay portal. Together, for the first time, this implicates ECT2 phosphorylation in breast cancer, which has been proposed as a therapeutic target in lung cancer. In conclusion, this data suggests that low expression of DLC1 is associated with poor prognosis. Targeting ECT2 phosphopeptides could provide a promising mechanism for controlling poor prognosis seen in DLC1low ER+ breast cancer.

Progression and prognostic value of ECT2 in non-small-cell lung cancer and its correlation with PCNA.

PurposeEpithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor, which is involved in cell division regulation and cell cycle modulation. Recent evidence indicates that ECT2 is overexpressed in many human cancers. However, the exact prognostic value of ECT2 in lung cancer has not been elucidated.Patients and methodsIn the current study, we performed correlation and prognosis analyses using public databases and conducted immunohistochemical staining in tissue microarrays, using samples from 204 lung cancer patients with survival data.ResultsWe found that the expression of ECT2 was markedly increased in lung cancer tissues compared with normal tissues. Moreover, we demonstrated that the expression of ECT2 was related to tumor cell differentiation degree, TNM stage, lymph node metastasis, and prognosis in non-small-cell lung cancer (NSCLC). A correlation analysis indicated that ECT2 levels were significantly correlated with proliferating cell nuclear antigen (PCNA) levels in NSCLC. Furthermore, Kaplan–Meier analyses revealed that high ECT2 expression was associated with unfavorable overall survival (OS) and progression-free survival (PFS) in NSCLC patients.ConclusionTaken together, these results indicate that the overexpression of ECT2 contributes to tumor invasion and progression, suggesting that ECT2 is a potential prognostic marker for NSCLC patients.

Abstract 2695: Quantitative phosphoproteome analysis identifies novel functional pathways of tumor suppressor DLC1 in estrogen receptor-positive breast cancer

Abstract Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative polymerase chain reaction (qPCR) in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Mutation and methylation status of DLC1 were assessed in these datasets including The Cancer Genome Atlas (TCGA). To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we developed a knock-in model of DLC1 in T47D breast cancer cells. Label-free global proteomic and TiO2 phosphopeptide enrichment assays were performed and validated using Western blotting. Data are available via ProteomeXchange with identifier PXD008220. Here, we report that low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. Analysis of the TCGA data showed that down regulation of DLC1 is not due to methylation or mutations. Stable knock-in of DLC1-full-length inhibits cell colony formation significantly in vitro compared to its control counterpart. 6726 phosphopeptides were quantified by phosphoproteomics analysis in both conditons, whereas 205 and 122 were unique to DLC1 knock-in and T47D-control cells, respectively. Pathway analysis using DAVID showed the top three significant clusters of differentially identified phosphopeptides involving cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Decreased phosphorylation of epithelial cell transforming sequence 2 (ECT2) at the residue T359, critical for its active conformational change, was validated. In conclusion, this data suggests that high expression of DLC1 reduces cell growth and is associated with favorable prognosis. Our results document an inverse relation between ECT2 phosphorylation and DLC1 expression, promising a novel strategy for treating ER+ breast cancer. Citation Format: Yesim Gökmen-Polar, Jason D. True, Edyta Vieth, Yuan Gu, Xiaoping Gu, Guihong D. Qi, Amber L. Mosley, Sunil Badve. Quantitative phosphoproteome analysis identifies novel functional pathways of tumor suppressor DLC1 in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2695.