ZEB1 Levels Research Articles

Vitamin D Regulates CXCL12/CXCR4 and Epithelial-to-Mesenchymal Transition in a Model of Breast Cancer Metastasis to Lung.

Vitamin D deficiency is associated with poor cancer outcome in humans, and administration of vitamin D or its analogs decreases tumor progression and metastasis in animal models. Using the mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) model of mammary cancer, we previously demonstrated a significant acceleration of carcinogenesis in animals on a low vitamin D diet and a reduction in spontaneous lung metastases when mice received vitamin D through perfusion. We investigate here the action mechanism for vitamin D in lung metastasis in the same non-immunodeficient model and demonstrate that it involves the control of epithelial to mesenchymal transition as well as interactions between chemokine C-X-C motif chemokine 12 (CXCL12) and its receptor C-X-C chemokine receptor type 4 (CXCR4). In vitro, 10-9M vitamin D treatment modifies the phenotype of MMTV-PyMT primary mammary tumor cells and significantly decreases their invasiveness and mammosphere formation capacity by 40% and 50%, respectively. Vitamin D treatment also inhibits phospho-signal transducer and activator of transcription 3 (p-STAT3), zinc finger E-box-binding homeobox 1 (Zeb1), and vimentin by 52%, 75%, and 77%, respectively, and increases E-cadherin by 87%. In vivo, dietary vitamin D deficiency maintains high levels of Zeb1 and p-STAT3 in cells from primary mammary tumors and increases CXCL12 expression in lung stroma by 64%. In lung metastases, vitamin D deficiency increases CXCL12/CXCR4 co-localization by a factor of 2.5. These findings indicate an involvement of vitamin D in mammary cancer "seed" (primary tumor cell) and "soil" (metastatic site) and link vitamin D deficiency to epithelial-to-mesenchymal transition (EMT), CXCL12/CXCR4 signaling, and accelerated metastasis, suggesting vitamin D repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing spread to distant organs.

Small molecule inhibitor of nicotinamide N-methyltransferase shows anti-proliferative activity in HeLa cells

The anti-proliferative effects of 5-methylquinolinium (5MQ) of nicotinamide N-methyltransferase (NNMT) have not been previously investigated on a cervical cancer cell line. NNMT is a metabolic enzyme that is correlated with tumour progression and metastasis. 5MQ is a small molecule inhibitor of NNMT. 0.1–500 μM of 5MQ was tested on the HeLa epithelial cervical cancer cell line. Cell viability was assessed with the MTT test. TWIST, ZEB1, SERPIN1, SIRT1, CD16, mRNA and various protein expression levels were analysed with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively. 5MQ significantly inhibited HeLa cell proliferation in a concentration and time-dependent manner. Increased cell shrinkage, loss of cellular adhesions and apoptotic bodies were observed in HeLa cells after 5MQ treatment. Following treatment with 5MQ, ZEB1, SIRT1, CD16 mRNA levels were increased while TWIST and SERPIN1 mRNA levels were reduced. Expressions of oncogenic proteins phospho-Akt and SIRT1 were decreased. 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. IMPACT STATEMENT What is already known on this subject? NNMT is a cytosolic enzyme involved in tumour progression, metastasis and treatment resistance. It was overexpressed in many human malignancies. 5-amino-1-methylquinolinium (5MQ) is a novel small molecule inhibitor of NNMT that has shown promising results in the treatment of obesity and in senescent muscle regeneration. 5MQ has not been tested on the HeLa cervical cancer cell line, previously. What do the results of this study add? In this study, 5MQ was tested on the HeLa cervical cancer cell line for the first time and the molecular changes associated with 5MQ treatment were analysed. 5MQ demonstrated significant anti-proliferative activity on HeLa cells, which displayed morphological signs of apoptosis. Treatment of HeLa cells with 5MQ led to an increase in ZEB1, SIRT1 mRNA while TWIST mRNA was decreased. Phospho-Akt and Sirtuin1 protein expressions were decreased. What are the implications of these findings for clinical practice and/or further research? 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. 5MQ treatment was associated with a decrease in the expression of phospho-Akt and Sirtuin1 proteins, both of which have been reported to maintain tumour progression. 5MQ can further be investigated and modified for anti-cancer therapy.

Circ-LRP6 mediates epithelial-mesenchymal transition and autophagy in oral squamous cell carcinomas.

Tumor metastasis seriously affects the therapeutic effect and prognosis of cancer patients. Here, we studied the role of has_circ_0000378 (circ-LRP6) in oral squamous cell carcinoma (OSCC) metastasis to explore new ideas and schemes for clinical treatment. The expressions of circ-LRP6 in OSCC and normal tissues from matched controls were measured by real-time PCR (RT-PCR). Levels of epithelial-mesenchymal transition (EMT) transcription factors, P62 and LC3B, were determined by Western blot analysis and immunofluorescence (IF) assay. Furthermore, we evaluated the effects of circ-LRP6 downregulation on migration, invasion, and autophagy using CCK8, transwell assays, transmission electron microscopy (TEM), and immunofluorescence (IF) assay. The expression of circ-LRP6 in OSCC tissues was high. Downregulation of circ-LRP6 reduced the EMT process of SCC-15 cells, as evidenced by increased E-cadherin and decreased vimentin and Zeb1 levels. Downregulation of circ-LRP6 also decreased autophagy as shown by increased levels of P62 and decreased LC3B in SCC-15 cells. Autophagy revulsant rapamycin (RAPA) rescued the inhibitory effect of circ-LRP6 on LC3B, vimentin, and Zeb1. circ-LRP6 promoted EMT and autophagy of OSCC and increased autophagy could rescue EMT in OSCC cells inhibited by circ-LRP6 siRNA.

RHBDD1 silencing inhibited cell growth and invasion of non-small cell lung cancer by mediating ZEB1/PI3K/AKT signaling pathway.

Rhomboid domain containing 1 (RHBDD1) gene, which was reported to be upregulated in human several cancer, was associated with carcinogenesis. However, the potential biological function of RHBDD1 in non-small cell lung cancer (NSCLC) carcinogenesis remains still not known. In this study, we aimed to investigate the role of RHBDD1 and its underlying molecular mechanism in NSCLC. The gene RHBDD1 expression was detected in NSCLC tissues and matched nontumor adjacent tissues. In vitro experiments, NSCLC cell lines (A549, H1650, H358 and H1299) were performed to investigate the biological function of RHBDD1 and its molecular mechanism. Our findings showed that the mRNA and protein expression levels of RHBDD1 were notably increased in human NSCLC tissues and cell lines, especially in A549 and H1650 cells. Moreover, silencing of RHBDD1 by RNAi notably inhibited NSCLC cell proliferation and increased cell apoptosis. Caspase-3/7 activity was remarkably increased in cells treated with RHBDD1 siRNA. RHBDD1 silencing notably reduced the number of invading cells. Furthermore, our findings showed that silencing of RHBDD1 notably inhibited the mRNA and protein expression levels of ZEB1 in A549 and H1650 cells. The phosphorylation of PI3K and AKT was also remarkably decreased by RHBDD1 silencing. ZEB1/AKT overexpression reversed the effect of RHBDD1 silencing on NSCLC cell growth and invasion. Taken together, our findings indicated that RHBDD1 silencing inhibited cell growth and invasion of non-small cell lung cancer by mediating ZEB1/PI3K/AKT signaling pathway, implying that RHBDD1 was possibly a potential diagnostic and therapeutic target for NSCLC treatment.

Rhynchophylline improves trophocyte mobility potential by upregulating ZEB1 level via the inhibition of miR-141-3p level.

Preeclampsia (PE) is characterized by the impaired invasive ability of trophocytes, which can be modulated by microRNAs (miRs). In the current study, the effects of rhynchophylline (Rhy) on the viability and invasive ability of trophocytes were explored by focusing on miR-141-3p/ZEB1 axis. The level of miR-141-3p was modulated in human trophocytes and the changes in cell viability, apoptosis, invasive ability, and ZEB1 level were detected. Then the trophocytes with miR-141-3p overexpression were treated with Rhy and the effects on trophocyte phenotypes were assessed. The induced miR-141-3p level suppressed cell viability, induced apoptosis, and inhibited invasion and ZEB1 level in trophocytes. The treatment of Rhy restored the viability and invasive ability of trophocytes under the overexpression of miR-141-3p, indicating the protective effects of Rhy on trophocytes. The findings in the current study highlighted the protective effects of Rhy on trophocytes during PE progression, which was associated with the inhibition of miR-141-3p.

CircRNA DNA methyltransferase 1 silence inhibits breast cancer development by regulating micoRNA-485-3p/zinc finger E-box binding homeobox 1 axis.

Circular RNAs (circRNAs) are associated with tumorigenesis of breast cancer. Nevertheless, how and whether circRNA DNA methyltransferase 1 (circ-DNMT1) controls breast cancer development remains poorly understood. The paired tumor and paracancer tissues (n = 41) were obtained from breast cancer patients. Circ-DNMT1, microRNA (miR)-485-3p, and zinc finger E-box binding homeobox 1 (ZEB1) abundances were measured by quantitative reverse transcription polymerase chain reaction and western blot. Cell colony formation, migration, invasion, and apoptosis were analyzed by colony formation analysis, transwell analysis, and flow cytometry. Target relationship was evaluated via dual-luciferase reporter analysis, RNA immunoprecipitation, and pull-down. The in vivo experiments were conducted using a xenograft model. Circ-DNMT1 and ZEB1 levels were upregulated in breast cancer, and miR-485-3p was downregulated. Circ-DNMT1 knockdown restrained cell colony formation, migration, and invasion and increased apoptosis. MiR-485-3p was negatively regulated by circ-DNMT1, and miR-485-3p knockdown mitigated the effect of circ-DNMT1 silence on breast cancer development. ZEB1 was targeted via miR-485-3p, miR-485-3p overexpression repressed cell colony formation, migration, and invasion and triggered apoptosis by decreasing ZEB1. Circ-DNMT1 silence reduced ZEB1 expression via regulating miR-485-3p. Circ-DNMT1 knockdown reduced xenograft tumor growth. Circ-DNMT1 knockdown constrains breast cancer development via modulating miR-485-3p/ZEB1 axis.

Dihydromyricetin Inhibits Tumor Growth and Epithelial-Mesenchymal Transition through regulating miR-455-3p in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) leads to poor prognosis due to high aggressiveness and common chemoresistance. Dihydromyricetin (DMY), the main bioactive compound isolated from Ampelopsis grossedentata, exhibits broad anti-tumor effects. This study aimed to investigate the inhibitory effect of DMY on CCA tumor growth and epithelial-mesenchymal transition (EMT) and its underlying mechanism in CCA. DMY treatment significantly inhibited cell proliferation and EMT in CCA cell lines. The expression of ZEB1 and vimentin were down-regulated, while the level of E-cadherin was increased after DMY treatment. By analyzing the TCGA dataset, we found that miR-455 expression was significantly downregulated, while the level of ZEB1 was up-regulated in human CCA tumor tissues compared to normal samples. Mechanistic studies showed that ZEB1 was a direct target of miR-455-3p in CCA. Moreover, DMY treatment potently increased miR-455-3p expression and inhibited ZEB1 expression. Inhibition of miR-455-3p expression abolished DMY's inhibitory effects on tumor growth and EMT in both CCA cells and cell-engrafted nude mice. Finally, DMY significantly suppressed the expressions of p-PI3K and p-AKT, while silencing miR-455-3p remarkably abrogated the inhibitory effect. In conclusion, DMY suppresses tumor growth and EMT through regulating miR-455-3p in human cholangiocarcinoma, suggesting a potential option for CCA treatment.

Resveratrol enhances the sensitivity of FL118 in triple-negative breast cancer cell lines via suppressing epithelial to mesenchymal transition.

We aimed to investigate whether resveratrol (RSV) could sensitize human triple-negative breast cancer (TNBC) cells to FL118-induced cell death, epithelial to mesenchymal transition (EMT), invasion, and migration. The effects of sequential administration of RSV and FL118 on MDA-MB-436 and MDA-MB-468 cells were evaluated in terms of cell viability, cytotoxicity, apoptosis, cell cycle distribution, active caspase-3/7 levels, migration and invasion. Furthermore, mRNA and protein levels of EMT associated genes and proteins were also evaluated. Sequential administration of RSV and FL118 inhibited the cell viability in both TNBC cell lines. Meanwhile sequential administration of RSV and FL118 also dramatically reduced the migratory and invasive capabilities, it also reversed the EMT process in both TNBC cells. Moreover, sequential administration of RSV and FL118 led to a significant increase of apoptotic cells, as well as active Caspase-3/7 levels. Sequential administration of RSV and FL118 caused TNBC cells accumulating in the G1 phase, and markedly suppressed the mRNA and protein levels of N-cadherin, β-catenin, Vimentin, Snail, and Slug, and also significantly downregulated mRNA levels of Fibronectin, Twist1, Twist2, Zeb1, and Zeb2 genes, while enhanced the mRNA and protein levels of E-cadherin genes. RSV sensitized TNBC cells to FL118 via facilitating apoptosis, migration, invasion, and EMT and enhancing intracellular entrapment of FL118. Thus, our results suggest that since RSV enhanced the in vitro anticancer activity of FL118 in BC, it may be a potential therapeutic agent in advanced BC.

Involvement of Wnt/β-catenin pathway in the inhibition of invasion and epithelial-mesenchymal transition in ovarian cancer cells

Purpose: To investigate the effects of zerumbone on cell invasion, epithelial-mesenchymal transition (EMT) and the potential signaling pathway involved in ovarian cancer cells.Methods: Caov-3 cell proliferation was assessed using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay. Wound healing assay was used to determine Caov-3 cell migration while cell invasion was evaluated using Transwell assay. Protein expression was determinedby western blot.Results: Cell viability was reduced by 5, 10, 20, and 50 μM zerumbone (p < 0.05) in a concentrationdependent manner while cell migration and invasion were inhibited by 10 and 20 μM zerumbone (p < 0.05). Protein expression levels of E-cadherin and cytoplasm β-catenin were upregulated by zerumbone (p < 0.05) in a concentration-dependent manner. On the other hand, protein expression levels of Ncadherin, vimentin, ZEB1, nuclear β-catenin, and c-Myc were suppressed by zerumbone (p < 0.05) also in a concentration-dependent manner.Conclusion: The results demonstrate that zerumbone inhibits cell proliferation, migration and invasion, but represses the EMT process via inactivation of Wnt/β-catenin signaling pathway.
 Keywords: Zerumbone, Ovarian cancer, Wnt/β-catenin pathway, Epithelial-mesenchymal transition

Comparison of Snail1, ZEB1, E-Cadherin Expression Levels in HPV-Induced Cervical Cancer.

Background:Molecular profiling techniques are the rapid detection of biomarkers in the human papillomavirus (HPV) infected cells. We aimed to measure the expression level of three cell factors including Snail1, ZEB-1, and E-cadherin in cervical cancer (CC), precancerous and healthy samples, simultaneously, to find potential biomarkers.Methods:The expression level of the mentioned cell factors were investigated in 72 CC patients, precancerous patients, and healthy controls by using Real-Time PCR.Results:The results demonstrated a significant reduction in the expression level of E-cadherin in cancer and precancerous cases than that in healthy cases; whereas the expression level of ZEB-1 and Snail1 were upregulated in cancer and precancerous samples. The receiver operating characteristic (ROC) analyses shows the highest AUC value emerged for Snail1: 1(95% CI: 1-1) in comparing CC and healthy groups with a sensitivity of 100.0 % and specificity of 100.0%.Conclusion:The molecular biomarker Snail1 may be helpful to early diagnosis and prognosis of CC in the HPV-infected human populations. Considering the increased expression level of Snail1 in cancer and precancerous tissue compared to healthy tissue as well as the area under the ROC curve, Snail1 can be used for early detection of CC.

Combined inhibition of CD73 and ZEB1 by Arg-Gly-Asp (RGD)-targeted nanoparticles inhibits tumor growth

Abnormal expression of several macromolecules within tumor milieu helps the development of neoplasia and immune suppression in various cancers. ZEB-1 and CD73 are important factors in tumor progression, which their overexpression in tumor site enhances several cancer hallmarks, including proliferation, angiogenesis, metastasis, migration, and invasion. In this study, we decided to inhibit the expression of these factors in the tumor site by using RGD-conjugated chitosan lactate (RGD-CL) nanoparticles (NPs) encapsulating CD73/ZEB-1 siRNA molecules, in vitro and in vivo. The NPs were about 127 nm in size, non-toxic, and RGD conjugation to NPs could efficiently increase cell transfection through interaction with αvβ3 integrins expressed on cancer cells and tumoral endothelial cells.Moreover, RGD-conjugated CL NPs containing siRNAs could significantly reduce the ZEB-1 and CD73 expression levels in cancer cells. Following transfection, cancer cells showed a significant reduction in migration and proliferation. Furthermore, the administration of these NPs into 4T1 and CT26 tumor-bearing mice resulted in tumor suppression and prolonged survival. These findings indicate the importance of targeting the CD73/ZEB1 axis in cancer cells, which could encourage their use in cancer patients in the near future.

Abstract PR03: Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype

Abstract The purpose of this study was to determine the role of BAP1 levels in cutaneous melanoma (CM). BAP1 is a tumor suppressor in which loss of heterozygosity (LOH) from mutation and copy number alteration is well described in germline and somatic cancers. Although BAP1 genomic alterations in CM are extremely rare (2% of 665 samples from 5 datasets), marked variability in BAP1 expression is observed in CM. We show that low nuclear BAP1 levels portend a significantly worse clinical outcome in stage III CM (n=37, log rank p ≤0.01 for both overall survival and progression-free survival). Gene Set Enrichment Analysis (GSEA) revealed low BAP1 expression to be most highly ranked with an increased epithelial–mesenchymal transition (EMT) gene expression profile in CM tumors (n=379, FDR q = 1.34E-26) and cell lines (n=53, FDR q = 2.86E-116). We identify the expression of ZEB1, a master regulator of EMT, to be significantly associated with low BAP1 expression in CM tumors and cell lines (p= 1.5E-04 and 3.3E-05, respectively). Analysis of the BAP1 promoter indicates three canonical ZEB1 binding sites. Functional experiments show ZEB1 to bind to the BAP1 promoter, and luciferase activity assays indicate that ZEB1 acts as a transcriptional suppressor of BAP1 expression with differential utilization of the promoter binding sites. Targeted reduction of endogenous ZEB1 caused increased BAP1 levels, while targeted reduction of BAP1 did not modulate ZEB1 levels, consistent with ZEB1 having a suppressive effect on BAP1. Phenotypically, targeted reduction of BAP1 in CM cells resulted in a switch from a more differentiated, melanocytic state, to a less differentiated, more migratory and invasive phenotype. Extinguishing melanocyte-specific BAP1 in mice with a BRAF V600E mutant genetic background resulted in the emergence of primary melanoma tumors, with a marked EMT gene expression profile, and resultant metastases. Given the phenotypic changes associated with BAP1 levels in our mouse and human studies, we then tested the effect of modulating BAP1 on BRAF targeted therapy. Exogenous expression of BAP1 sensitized BRAF inhibitor (vemurafenib)-resistant melanoma cells, while the targeted reduction of BAP1 desensitized BRAF inhibitor-sensitive melanoma cells. BRAF mutant/BAP1 loss mice failed to exhibit a marked response to vemurafenib treatment compared to control mice. These data implicate regulation of BAP1 to be a major mechanism that characterizes a highly malignant and treatment-resistant subset of tumors. Our study indicates that nongenomic reduction in BAP1 through ZEB1 transcriptional modulation may be a key factor in aggressive CM. This abstract is also being presented as Poster A30. Citation Format: Haifeng Zhu, Junna Oba, Xiaoxing Yu, Caitlin A. Creasy, Marie-Andrée Forget, Fernando Carapeto, Cara L. Haymaker, Chang-Jiun Wu, Tatiana V. Karpinets, Wei-Lien Wang, Michael T. Tetzlaff, Alexander J. Lazar, Gordon B. Mills, Amanda R. Moore, Yu Chen, Jianhua Zhang, Jeffrey E. Gershenwald, Jennifer A. Wargo, Chantale Bernatchez, Patrick Hwu, P. Andrew Futreal, Scott E. Woodman. Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR03.

Epigenetic reprogramming of epithelial-mesenchymal transition promotes ferroptosis of head and neck cancer

Ferroptosis is a newly defined form of cell death induced by iron-dependent accumulation of lethal lipid peroxidation. Ferroptosis represent a therapeutic strategy to suppress therapy-resistant cancer cells with more property of epithelial-mesenchymal transition (EMT). However, epigenetic reprogramming of EMT has been rarely studied in the context of ferroptosis susceptibility. Therefore, we examined the therapeutic potentiality of EMT epigenetic reprogramming in promoting ferroptosis in head and neck cancer (HNC) cells. The effects of ferroptosis inducers and EMT inhibition or induction were tested in HNC cell lines and mouse tumor xenograft models. These effects were analyzed concerning cell viability and death, lipid reactive oxygen species and iron production, labile iron pool, glutathione contents, NAD/NADH levels, and mRNA/protein expression. Cell density and the expression levels of E-cadherin, vimentin, and ZEB1 were associated with the different susceptibility to ferroptosis inducers. CDH1 silencing or ZEB1 overexpression increased the susceptibility to ferroptosis, whereas CDH overexpression or ZEB1 silencing decreased the susceptibility, in vitro and in vivo. Histone deacetylase SIRT1 gene silencing or pharmacological inhibition by EX-527 suppressed EMT and consequently decreased ferroptosis, whereas SIRT inducers, resveratrol and SRT1720, increased ferroptosis. MiR-200 family inhibitors induced EMT and increased ferroptosis susceptibility. In HNC cells with low expression of E-cadherin, the treatment of 5-azacitidine diminished the hypermethylation of CDH1, resulting in increased E-cadherin expression and decreased ferroptosis susceptibility. Our data suggest that epigenetic reprogramming of EMT contributes to promoting ferroptosis in HNC cells.

Polysaccharide isolated from persimmon leaves (Diospyros kaki Thunb.) suppresses TGF-β1-induced epithelial-to-mesenchymal transition in A549 cells

In the present study, to verify the effect of polysaccharides derived from persimmon leaves (PLE) at epithelial-to-mesenchymal transition (EMT), A549 cells were treated with TGF-β1 alone or co-treated with TGF-β1 and PLE (50 and 75 μg/mL). PLE-treated cells showed higher expression of E-cadherin and lower expression of N-cadherin and vimentin compared to TGF-β1-treated cells by inhibiting the levels of transcription factors, including Snail, Slug, and ZEB1, all associated with EMT. PLE also significantly decreased migration, invasion, and anoikis resistance through TGF-β1 mediated EMT suppression, whereby PLE inhibited the levels of MMP-2 and MMP-9 while cleaving PARP. These inhibitory effects of PLE against EMT, migration, invasion, and anoikis resistance were determined by activating the canonical SMAD2/3 and non-canonical ERK/p38 signaling pathways. Therefore, these results suggest that PLE could be used as a potential chemical therapeutic agent for early metastasis of lung cancer in vitro.

Abstract 6061: NR2F1 limits dissemination of early cancer mammary epithelial cells

Abstract Cancer cells have been found to disseminate during very early and sometimes asymptomatic stages of tumor progression. At secondary organs, early disseminated cancer cells (eDCCs) can enter a non-proliferative state for prolonged periods of time. Intriguingly, eDCCs also carry metastasis- initiating capacity. However, the mechanisms by which these early cancer cells complete all steps of metastasis including metastasis-initiating capacity are unknown. Here we report that the orphan nuclear receptor and lineage commitment regulator NR2F1 serves as an early barrier to early dissemination propelled by HER2 signaling. NR2F1 loss of function in HER2 early cancer cells induced cell motility, loss of E-cadherin junctions, disorganized laminin-V deposition, decreased β-catenin membrane localization and increased micro-invading cells with CK14+/CK18 phenotype. Importantly, upon NR2F1 depletion in HER2 early cancer acini, TWIST, ZEB1 and PRRX1 levels were upregulated. Our findings reveal a previously unrecognized function of NR2F1 as a suppressor of dissemination during early stages of breast tumor progression. Thus, therapies that restore and activate NR2F1 function might limit early dissemination and metastatic outgrowth. Citation Format: Maria Soledad Sosa, Nupura Kale, Nitisha Shrivastava, Carolina Rodriguez-Tirado, Maria Carlini, Jiayi Ji, Javier Bravo-Cordero. NR2F1 limits dissemination of early cancer mammary epithelial cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6061.

Long-Noncoding RNA PCAT6 Aggravates Osteosarcoma Tumourigenesis via the MiR-143-3p/ZEB1 Axis.

IntroductionThe long-noncoding RNA PCAT6 plays an important regulatory role in the development of several cancers. However, the expression pattern and underlying mechanisms of PCAT6 in osteosarcoma (OS) are yet unknown.MethodsWe used real-time PCR to measure PCAT6 expression in 106 tumor pairs and corresponding non-tumor tissues from OS patients. Statistical analyses were applied to evaluate the prognostic value and associations of PCAT6 expression with clinical parameters. Furthermore, the PCAT6 was silenced with siRNA in OS cells. Moreover, phenotype of PCAT6 silenced OS cells was measured using colony formation, CCK-8, cell migration and invasion assay. Finally, the molecular mechanism of PCAT6/miR-143-3p/ZEB1 axis in OS progression was explored.ResultsThe expression level of PCAT6 in OS tissues was significantly elevated as compared with that in the adjacent normal bone tissues and that high PCAT6 expression closely correlated with the malignant phenotype and poor survival among patients with OS. Multivariate analyses revealed PCAT6 overexpression as an independent prognostic factor for the poor outcome of patients with OS. Functional assay results demonstrated that the knockdown of PCAT6 expression notably suppressed the proliferation, migration, and invasion of OS cells. An elevated PCAT6 level aggravated the malignant phenotype of OS cells via ZEB1 expression upregulation. Mechanistic studies revealed that PCAT6 could sponge endogenous miR-143-3p and inhibit its activity, resulting in an increase in ZEB1 level. Finally, we demonstrated that the tumour-promoting role of PCAT6 in OS was dependent on the regulation of the miR-143-3p/ZEB1 axis.ConclusionThese findings highlight the potential role of PCAT6, which could serve as a valuable prognostic indicator for patients with OS.

PHRF1 promotes migration and invasion by modulating ZEB1 expression.

PHRF1 (PHD and RING finger domain-containing protein 1) suppresses acute promyelocytic leukemia (APL) by promoting TGIF (TG-interacting factor) ubiquitination, while the PML-RARα protein interferes with PHRF1-mediated TGIF breakdown to facilitate APL. Beyond its role in APL tumorigenesis, PHRF1 contributes to non-homologous end-joining by linking H3K36 methylation and Nbs1 upon DNA damage insults. However, little is known regarding its function in tumor invasion. Here we highlight the unreported details of PHRF1 in the invasion of lung cancer cells by modulating the transcriptional level of ZEB1, a prominent regulator involved in epithelial-mesenchymal transition. PHRF1 associated with the phosphorylated C-terminal repeat domain of Rpb1, the large subunit of RNA polymerase II, through its C-terminal Set2 Rpb1 Interacting (SRI) domain. Chromatin immunoprecipitation revealed that PHRF1 bound to the proximal region adjacent to the transcription start site of ZEB1. SRI-deleted PHRF1 neither associated with Rpb1 nor increased ZEB1's expression. Collectively, PHRF1 might take the stage at migration and invasion by modulating the expression of ZEB1.

MicroRNA-708 prevents ethanol-induced hepatic lipid accumulation and inflammatory reaction via direct targeting ZEB1

Alcoholic liver disease (ALD) was a global liver disease which divided into liver inflammation, fatty liver, alcoholic hepatitis or cirrhosis. Abnormal expression levels of some microRNAs (miRNA) family members often lead to ALD and other liver diseases. MicroRNA-708 (miR-708) was known to suppress the proliferation and metastasis of hepatocellular carcinoma (HCC), but its role in the progression of ALD was not clear. In this study, the expression level of miR-708 was down-regulated in ethanol-induced L0-2 cells. ZEB1 could decrease the PPAR-α expression while increase the SREBP-1 expression. Meanwhile, the expression levels of TNF-α and IL-6 were up-regulated by ZEB1. Of note, ZEB1 aggravated the apoptotic rate of L0-2 cells induced by ethanol via inhibiting p-AKT and p-mTOR of AKT/mTOR signaling pathway. What's more, it was demonstrated that miR-708 family members particularly target ZEB1 3′-UTR regions and can down-regulate the expression level of ZEB1 in L0-2 cells. Sum up, these results indicated that miR-708 might inhibit the liver inflammation and lipid accumulation by targeting ZEB1 via regulating AKT/mTOR signaling pathway.

Long noncoding RNA HOTAIR promotes breast cancer development by targeting ZEB1 via sponging miR-601

BackgroundBreast cancer (BC) is a common malignancy worldwide. It has been reported that long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is abnormally expressed in BC. However, the role of HOTAIR in the malignancy of BC is worth further discussion. This study aims to clarify the function and molecular mechanism of HOTAIR in BC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of HOTAIR, microRNA (miR)-601 and zinc finger E-box binding homeobox 1 (ZEB1). Cell counting kit-8 (CCK-8) and transwell assay were used to detect the proliferation, migration and invasion of cells. Further, the protein levels of AKT, phosphorylated-AKT (p-AKT), ZEB1 and Ki-67 were confirmed by western blot (WB) assay. Moreover, dual-luciferase reporter assay was applied to examine the targeting relationship between HOTAIR and miR-601 or miR-601 and ZEB1. In addition, animal experiments were conducted to verify the effect of HOTAIR on BC tumor growth in vivo.ResultsHOTAIR was upregulated in BC tissues and cells, and its knockdown suppressed the proliferation, migration, invasion and the activity of AKT signaling pathway of BC cells. HOTAIR could serve as a sponge of miR-601. Further experiments revealed that miR-601 inhibitor could reverse the inhibition effect of HOTAIR silencing on the progression of BC. Meanwhile, ZEB1 was a target of miR-601, and its overexpression could invert the suppression effect of miR-601 overexpression on the progression of BC. Additionally, ZEB1 expression was regulated by HOTAIR and miR-601. Furthermore, interference of HOTAIR could attenuate BC tumor growth in vivo.ConclusionIn short, this study demonstrated that HOTAIR promoted the proliferation, migration, invasion of BC through regulating the miR-601/ZEB1 axis, which provided a theoretical basis for the research on lncRNA-directed therapeutics in BC.

Hypoxia improved vasculogenesis in distraction osteogenesis through Mesenchymal-Epithelial transition (MET), Wnt/β-catenin signaling pathway, and autophagy

IntroductionThe osteogenesis rate of distraction osteogenesis is 4–6 times faster than that of infants, far beyond fracture healing. However, the osteogenesis mechanism of DO is complicated and inconclusive owing to two significant elements: mechanical tension which is well explored and trauma caused by bone fracture. Vasculogenesis and EPCs are critical for successful bone regeneration during DO. Thus, this study aimed to explore the effects of hypoxia caused by trauma or CoCl2 on the vasculogenesis of DO and EPCs. Material and methodsMandibular DO and BF models were generated using 6 beagle dogs with a distraction rate of 1 mm per day for 7 days or acute lengthening for 7 mm. The vasculogenesis in DO-gap or BF-gap were assessed via histological analyses, qRT-PCR and immunofluorescence staining. Dog bone marrow EPCs were isolated and cultured with or without 0.1 mM CoCl2. The effect of hypoxia caused by CoCl2 were subsequently valuated via in vitro assays including Cell Counting Kit-8, transwell assay, qRT-PCR, western blot, and immunofluorescence staining. ResultsHistological analyses, qRT-PCR and immunofluorescence staining revealed that vasculogenesis markedly accelerated in DO-gap compared with BF-gap, and the DO-gap displayed more positive to CD133, CD34, HIF-1α, E-cadherin, beclin1, β-catenin, VEGF, bFGF, and less positive to ZEB1 than BF-gap. In addition, in vitro analyses revealed CoCl2 treatment enhanced EPCs proliferation and migration, and the levels of HIF-1α, E-cadherin, β-catenin, beclin1, VEGF, bFGF of EPCs were increased, but the level of ZEB1 was decreased. ConclusionOur studies showed that hypoxia promoted vasculogenesis in DO and EPCs, and the mechanism may involve autophagy, Wnt/β-catenin signaling pathway, and Mesenchymal-Epithelial transition (MET).