Repression Of E-cadherin Research Articles

Hysteresis control of epithelial-mesenchymal transition dynamics conveys a distinct program with enhanced metastatic ability

Epithelial-mesenchymal transition (EMT) have been extensively characterized in development and cancer, and its dynamics have been modeled as a non-linear process. However, less is known about how such dynamics may affect its biological impact. Here, we use mathematical modeling and experimental analysis of the TGF-β-induced EMT to reveal a non-linear hysteretic response of E-cadherin repression tightly controlled by the strength of the miR-200s/ZEBs negative feedback loop. Hysteretic EMT conveys memory state, ensures rapid and robust cellular response and enables EMT to persist long after withdrawal of stimuli. Importantly, while both hysteretic and non-hysteretic EMT confer similar morphological changes and invasive potential of cancer cells, only hysteretic EMT enhances lung metastatic colonization efficiency. Cells that undergo hysteretic EMT differentially express subsets of stem cell and extracellular matrix related genes with significant clinical prognosis value. These findings illustrate distinct biological impact of EMT depending on the dynamics of the transition.

Foxf2 plays a dual role during transforming growth factor beta-induced epithelial to mesenchymal transition by promoting apoptosis yet enabling cell junction dissolution and migration

BackgroundThe most life-threatening step during malignant tumor progression is reached when cancer cells leave the primary tumor mass and seed metastasis in distant organs. To infiltrate the surrounding tissue and disseminate throughout the body, single motile tumor cells leave the tumor mass by breaking down cell-cell contacts in a process called epithelial to mesenchymal transition (EMT). An EMT is a complex molecular and cellular program enabling epithelial cells to abandon their differentiated phenotype, including cell-cell adhesion and cell polarity, and to acquire mesenchymal features and invasive properties.MethodsWe employed gene expression profiling and functional experiments to study transcriptional control of transforming growth factor (TGF)β-induced EMT in normal murine mammary gland epithelial (NMuMG) cells.ResultsWe identified that expression of the transcription factor forkhead box protein F2 (Foxf2) is upregulated during the EMT process. Although it is not required to gain mesenchymal markers, Foxf2 is essential for the disruption of cell junctions and the downregulation of epithelial markers in NMuMG cells treated with TGFβ. Foxf2 is critical for the downregulation of E-cadherin by promoting the expression of the transcriptional repressors of E-cadherin, Zeb1 and Zeb2, while repressing expression of the epithelial maintenance factor Id2 and miRNA 200 family members. Moreover, Foxf2 is required for TGFβ-mediated apoptosis during EMT by the transcriptional activation of the proapoptotic BH3-only protein Noxa and by the negative regulation of epidermal growth factor receptor (EGFR)-mediated survival signaling through direct repression of its ligands betacellulin and amphiregulin. The dual function of Foxf2 during EMT is underscored by the finding that high Foxf2 expression correlates with good prognosis in patients with early noninvasive stages of breast cancer, but with poor prognosis in advanced breast cancer.ConclusionsOur data identify the transcription factor Foxf2 as one of the important regulators of EMT, displaying a dual function in promoting tumor cell apoptosis as well as tumor cell migration.

PHF8 upregulation contributes to autophagic degradation of E-cadherin, epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma

BackgroundPlant homeodomain finger protein 8 (PHF8) serves an activator of epithelial-mesenchymal transition (EMT) and is implicated in various tumors. However, little is known about PHF8 roles in hepatocellular carcinoma (HCC) and regulating E-cadherin expression.MethodsPHF8 expression pattern was investigated by informatic analysis and verified by RT-qPCR and immunochemistry in HCC tissues and cell lines. CCK8, xenograft tumor model, transwell assay, and tandem mCherry-GFP-LC3 fusion protein assay were utilized to assess the effects of PHF8 on proliferation, metastasis and autophagy of HCC cells in vitro and in vivo. ChIP, immunoblot analysis, rescue experiments and inhibitor treatment were used to clarify the mechanism by which PHF8 facilitated EMT, metastasis and autophagy.ResultsPHF8 upregulation was quite prevalent in HCC tissues and closely correlated with worse overall survival and disease-relapse free survival. Furthermore, PHF8-knockdown dramatically suppressed cell growth, migration, invasion and autophagy, and the expression of SNAI1, VIM, N-cadherin and FIP200, and increased E-cadherin level, while PHF8-overexpression led to the opposite results. Additionally, FIP200 augmentation reversed the inhibited effects of PHF8-siliencing on tumor migration, invasion and autophagy. Mechanistically, PHF8 was involved in transcriptionally regulating the expression of SNAI1, VIM and FIP200, rather than N-cadherin and E-cadherin. Noticeably, E-cadherin degradation could be accelerated by PHF8-mediated FIP200-dependent autophagy, a crucial pathway complementary to transcriptional repression of E-cadherin by SNAI1 activation.ConclusionThese findings suggested that PHF8 played an oncogenic role in facilitating FIP200-dependent autophagic degradation of E-cadherin, EMT and metastasis in HCC. PHF8 might be a promising target for prevention, treatment and prognostic prediction of HCC.

Tetraspanin CD82 represses Sp1-mediated Snail expression and the resultant E-cadherin expression interrupts nuclear signaling of β-catenin by increasing its membrane localization

Tetraspanin membrane proteins form physical complexes with signaling molecules and have been suggested to influence the signaling events of associated molecules. Of the tetraspanin proteins, CD82 has been shown to promote homotypic cell-cell adhesion, which partially accounts for its role in suppressing cancer invasion and metastasis. We found here that CD82-induced cell-cell adhesion is attributed to increased E-cadherin expression through CD82-mediated downregulation of the E-cadherin repressor Snail. The Snail repression by CD82 resulted from the reduced binding of the Sp1 transcription factor to the Snail gene promoter. Notably, high CD82 expression did not allow the fibronectin matrix to induce Sp1 phosphorylation, implicating CD82 inhibition of the fibronectin-integrin signaling-dependent Sp1 activation. Meanwhile, E-cadherin upregulated by CD82 pulled β-catenin up to the membrane region, and consequently reduced the amount of cytoplasmic β-catenin that was able to move into to the nucleus. The Wnt signal-induced nuclear translocation of β-catenin was also inhibited by the CD82 function of upregulating E-cadherin. Overall, high CD82 expression was likely to suppress fibronectin adhesion-induced Sp1 activation signaling for Snail expression, resulting in continuous E-cadherin expression, which contributed not only to the maintenance of strong cell-cell adhesion but also to the blockage of nuclear β-catenin signaling.

Hypoxia promotes acquisition of aggressive phenotypes in human malignant mesothelioma

BackgroundHypoxia is a hallmark of the solid tumor microenvironment and is associated with poor outcomes in cancer patients. The present study was performed to investigate mechanisms underlying the hypoxia-induced phenotypic changes using human malignant mesothelioma (HMM) cells.MethodsHypoxic conditions were achieved by incubating HMM cells in the air chamber. The effect of hypoxia on phenotype changes in HMM cells was investigated by performing in vitro clonogenicity, drug resistance, migration, and invasion assays. Signaling pathways and molecules involved in the more aggressive behaviors of HMM cells under hypoxia were investigated. A two-tailed unpaired Student’s t-test or one-way ANOVA with Bonferroni post-test correction was used in this study.ResultsHypoxic conditions upregulated hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α in parallel with the upregulation of its target, Glut-1, in HMM cells. In vitro clonogenicity of HMM cells was significantly increased in hypoxic conditions, but the proliferation of cells at a high density in hypoxia was lower than that in normoxic conditions. The expression levels of HIF-2α and Oct4 were increased in hypoxic HMM cells. The percentage of cells with high CD44 expression was significantly higher in HMM cells cultured in hypoxia than those cultured in normoxia. Hypoxia significantly enhanced the resistance of HMM cells to cisplatin, which occurred through cytoprotection against cisplatin-induced apoptosis. While cisplatin treatment decreased the ratio of Bcl-2 to Bax in normoxic condition, hypoxia conversely increased the ratio in HMM cells treated with cisplatin. Hypoxia increased the mobility and invasiveness of HMM cells. Epithelial to mesenchymal transition was promoted, which was indicated by the repression of E-cadherin and the concomitant increase of vimentin in HMM cells.ConclusionsThe data illustrated that hypoxic conditions augmented the aggressive phenotypes of HMM cells at the biological and molecular levels. The present study provides valuable background information beginning to understand aggressiveness of HMM in tumor microenvironments, suggesting that a control measure for tumor hypoxia may be an effective therapeutic strategy to reduce the aggressiveness of cancer cells in HMM patients.

UBE2C Directly Targeted by mirR-548-5p Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein ZEB1/2 in NSCLC

Background: Recent evidence indicates that miR-548-5p dysregulation plays a vital role in tumor progression. Aberrant miR-5485p expression may be linked to tumorigenesis since miRNAs play important roles in various cellular processes and cancer metastasis. UBE2C is a type of ubiquitination enzyme catalyzing the degradation of proteins into smaller polypeptides and amino acids. UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis and transcriptional processes. However, the molecular mechanism of UBE2C as a growth factor regulating the tumorigenesis and metastasis of lung cancer is not clearly. EMT progression is mediated by the EMT-inducing transcriptional factors ZEB1/2, which is an important characteristic for cancer metastasis. What molecules regulate the expression of ZEB1/2 and then regulate the EMT in the lung cancer is also not clearly. We found that miR-548-5p, UBE2C, ZEB1/2 have the similar functions in tumor progression and metastasis. However, the relationship between miR-548-5p, UBE2C and ZBE1/2 in the tumorigenesis and development of lung cancer is still unknown. Methods: the mRNA expression of miR-548-5p, UBE2C was analyzed by RT-PCR assay. The protein level of UBE2C and ZEB1/2 was analyzed by western blot and Immunofluorescent staining. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter and gene transcription were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of UBE2C and 5'UTRZEB1/ 2. Results: In our study, we found that miR-548-5p was significantly downregulated in lung cancer tissue specimens. Our objective was to elucidate the functions, direct target genes, and molecular mechanisms of miR-548-5p in lung cancer tissue. We found that miR-548-5p downregulated the expression of its direct substrate, 4,5-ubiquitin-conjugating enzyme E2C (UBE2C). UBE2C is a proto-oncogene that is activated in lung cancer. However, its role in lung cancer metastasis is unclear. We found here that UBE2C expression was higher in non-small-cell lung cancer (NSCLC) than in the normal tissue adjacent to it and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by arresting the cell cycle and inhibiting apoptosis. We used reverse transcription (RT)-PCR assay to determine miR548-5p and UBE2C expression in lung cancer patients. Overexpression of miR-548-5p and UBE2C were established in lung cancer cells. Methyl thiazolyl tetrazolium (MTT)- and colony formation assays were performed to evaluate their effects on cell proliferation. We also performed cell migration- and invasion assays on lung cancer cells overexpressing miR548-5p and under expressing UBE2C. The results indicated that the aforementioned treatments significantly suppressed lung cancer cell proliferation, migration, and invasion. Moreover, luciferase reporter and Chromatin immunoprecipitation assay indicated that miR-548-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Using RT-PCR and western blot (WB), we also assessed epithelial-mesenchymal transition (EMT). We found that UBE2C knockdown downregulated the expression of the mesenchymal marker vimentin. In contrast, the epithelial marker E-cadherin was upregulated. Therefore, UBE2C promoted EMT. Bioinformatics assays coupled with WB and luciferase assays revealed that UBE2C directly binds to the 5′-untranslated region of the E-cadherin repressor, ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: the miR-548-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene promoting EMT in lung cancer cells by directly targeting the 5'-UTR of E-cadherin repressor ZEB1/2. The miR-548-5p, UBE2C, and ZEB1/2 constitute the miR548-5p-UBE2C-ZEB1/2 signal axis which enhances cancer cell invasiveness by directly interacting with the EMT maker protein. We believe that the miR-548-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target in lung cancer therapy Funding: This work was supported by the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067, ZR2017MH125 and ZR2015PH004), and Research Foundation of Binzhou Medical University (BY2015KYQD25 and BY2015KJ14). Declaration of Interest: The authors declare no competing financial interests. Ethical Approval: The experimental protocol was approved by the Research Ethics Committee of Binzhou Medical University, China (No. 2017-016-01 for human lung cancer specimen and No. 2017-009-09 for mouse experiments in vivo) and the written informed consent was obtained from all subjects. Informed consent was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th Edition of the AJCC Cancer Staging Manual: Stomach (2010)

SREBP1, targeted by miR-18a-5p, modulates epithelial-mesenchymal transition in breast cancer via forming a co-repressor complex with Snail and HDAC1/2

The progression of localized breast cancer to distant metastasis results in a poor prognosis and a high mortality rate. In this study, the contributions of miRNAs to tumor progression and the regulatory mechanisms leading to their expression alterations were investigated. Using highly lung-metastatic sub-lines from parental breast cancer cells, miRNA expression profiling revealed that the miR-17-92 cluster is significantly downregulated and the miR-18a-5p is the most evidently decreased. Ectopic expression and inhibition of miR-18a-5p demonstrated its capacity in suppressing migration and invasion of breast cancer cells. Further research identified sterol regulatory element binding transcription protein 1 (SREBP1), the master transcription factor that controls lipid metabolism, as a candidate target of miR-18a-5p. SREBP1 is overexpressed and strongly associated with worse clinical outcomes in breast cancer. Functionally SREBP1 promotes growth and metastasis of breast cancer both in vitro and in vivo. To unravel the underlying mechanism of SREBP1-mediated metastasis, mRNA profiling and subsequent gene set enrichment analyses (GSEA) were performed and SREBP1 was demonstrated to be significantly associated with epithelial-mesenchymal transition (EMT). Furthermore, SREBP1-mediated repression of E-cadherin was found to be deacetylation dependent and was augmented by recruiting Snail/HDAC1/2 repressor complex. In the light of these data, we propose that reduced expression of miR-18a-5p and concomitant overexpression of SREBP1 lead to induction of EMT states that in turn, promote breast cancer progression and metastasis. Taken together, our study reveals the crucial role of miR-18a-5p and SREBP1 in the EMT and metastasis, thus providing promising drug targets for tailored therapy in the advanced breast cancer setting.

Abstract 3960: The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes

Abstract Introduction: In cancer patients, the development of metastases is associated with a poor clinical prognosis. Metastatic dissemination requires cancer cells to detach from the primary tumor and invade adjacent or distant tissue. This process involves epithelial-to-mesenchymal transition (EMT), whereby tumor cells lose cell-cell adhesion mediated by E-cadherin repression. The CD44 family of transmembrane glycoproteins comprises alternatively spliced variants that are involved in many cellular processes. The isoform CD44v6 has been shown to play a major role in tumor growth and metastasis. CD44v6 acts as a co-receptor for the receptor tyrosine kinases (RTKs) c-MET, RON and VEGFR-2, and is preferentially expressed in a variety of epithelial cancers. We have previously shown that the co-receptor function of CD44v6 for c-MET is the driving force for metastasis. c-Met and RON facilitate metastasis through MAPK signaling and VEGFR-2 plays a role in angiogenesis. AMC303, an allosteric and selective peptide inhibitor of CD44v6, prevents phosphorylation of the CD44v6 dependent RTKs and respective cellular processes. Methods: The effect of AMC303 on cancer cell function in response to the growth factors HGF, MSP and VEGF was studied in various assays. Cell migration and invasion was analyzed in scratch assays and the Boyden Chamber. Morphological changes of EMT were monitored using microscopy in scattering assays in the presence of growth factors. EMT markers were analyzed by Western blot and immunohistochemistry. Tube formation was microscopically analyzed. Results: AMC303 dose dependently and at low nanomolar concentrations (IC50 10 - 20nM) prevents HGF- or VEGF-induced activation of c-MET/VEGFR-2 in various cancer cells of pancreatic, lung and colonic origin or endothelial cells, which cannot be overcome by excess HGF and VEGF. HGF- and MSP-dependent cell migration, invasion and wound closure in scratch assays was prevented by AMC303 using different cancer cell lines. AMC303 had a strong inhibitory effect on HGF- and MSP- induced cell scattering, a morphological feature of EMT. Consistent with this, AMC303 inhibited the downregulation of HGF induced EMT markers (E-cadherin). In primary endothelial cells, AMC303 blocked activation of VEGFR-2 and VEGF-A induced formation of a tubular network. Conclusions: Phosphorylation of CD44v6 dependent RTKs is inhibited in primary endothelial cells and different epithelial tumor cells by binding of AMC303 to the extracellular domaine of CD44v6. This novel mode of action results in strong anti-tumor and anti-metastatic effects of AMC303 involving inhibition of migration and invasion of tumor cells and prevention of EMT. AMC303 is currently tested in a First-in-Human Phase I/Ib study in cancer patients with solid epithelial tumors. Citation Format: Thorsten Läufer, Martin Augsten, Vanessa Al-Rawi, Birgit Hotz, Yvonne Heneka, Oliver Coutelle, Alexandra Matzke-Ogi. The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes [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 3960.

Abstract 5456: Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR

Abstract EMT is a vital process in development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens junction proteins E-cadherin, Claudin and ZO-1 on the membrane. Cells with EMT are elongated spindle like structures due to upregulation of mesenchymal markers Vimentin, N-cadherin and transcription repressor of E-cadherin, ZEB1. EMT may be responsible for Tyrosine kinase inhibitor (TKI) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. This acquired resistance to TKIs is due to a secondary T790M mutation in the kinase domain which could be responsible for inducing EMT. An EMT regulator, p120-catenin when it is no longer bound to membranous E-cadherin forms a complex with Kaiso factor suppressing its transcription repressor activity, promoting oncogenesis. PRMT1, another EMT inducer is also overexpressed in NSCLC. Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing a CSC marker ABCB1. Thus, we investigated EMT characteristics in wild type EGFR TKI resistant H358, H2170 cells and TKI resistant EGFR mutant H1975 cells (L858R and T790M mutation) and TKI sensitive EGFR mutant H3255 cells (L858R mutation). The modulation of EMT biomarkers was determined by immunoblotting and qPCR. Expression of ABCB1 and E-cadherin was measured using flow cytometry. Key EMT regulators such as PRMT1 and p120-catenin were upregulated 4.0 and 3.2-fold, respectively. Hence, knockdown of p120-catenin and PRMT1 were performed using siRNA or an inhibitor and examined by immunoblotting and MTT. EMT transcription factors such as Slug, Snail and Twist were also upregulated 3.18, 6.2 and 1.68-folds and E-cadherin, Claudin and ZO-1 were downregulated by 8.6, 11.6 and 15.2-folds in H1975 cells compared to H3255 cells. Also, N-cadherin, ZEB1 and Vimentin were upregulated by 4.9, 3.0 and 10.7-fold. Immunofluorescence studies with Vimentin showed that H1975 cells were elongated and have a mesenchymal phenotype compared to the H3255. 90% colocalization of p120-catenin and Kaiso factor was seen in H1975 cells whereas 10% colocalization was seen in H3255 cells. In resistant NSCLC cells with wild type and mutated EGFR, flow cytometry studies showed significant increase in expression of CSC marker ABCB1 whereas E-cadherin expression was decreased. Knockdown of PRMT1 by Furamidine, increased erlotinib efficacy by 27% in H1975 cells. Similarly, knockdown of p120-catenin by siRNA increased erlotinib efficacy by 40% in H1975 cells. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which methylate's Twist, upregulates transcription factors and p120-catenin which represses E-cadherin and Kaiso factor, activating EMT in H1975 cells. Citation Format: Sanjana Singh, Tsatsral Iderzorig, Pragya Kumar, Neelu Puri. Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR [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 5456.

Abstract 5003: RNase activity of MCPIP1 regulates key elements of mesenchymal phenotype of clear cell renal cell carcinoma cells

Abstract Introduction: Monocyte chemotactic protein 1-induced protein 1 (MCPIP1) regulates the mRNA of proinflammatory cytokines, such as IL-6, IL-1, IL-12 and IL-2 and influences NFκB and AP1 activity. MCPIP1 may also suppress miRNA biosynthesis. Last findings show that MCPIP1 affects viability and proliferation of tumor cells, degrades the mRNA of antiapoptotic gene transcripts in breast cancer cells and reduces tumor growth and metastatic disease of breast cancer. We have already demonstrated that MCPIP1 decreases during clear cell renal cell carcinoma (ccRCC) progression and is responsible for better tumor vascularity and the secretion of proangiogenic factors. Moreover, MCPIP1 downregulation promotes the metastatic spread of ccRCC cells. Aim: The main objective of our study was to determine the role of RNase activity of MCPIP1 in the regulation of key elements in the EMT signaling axis and the acquisition of mesenchymal phenotype of ccRCC. Materials and methods: Microarray analysis of tumor tissue was used to evaluate EMT signaling axis during ccRCC progression. For stable overexpression of MCPIP1, doxycycline-dependent TetON system was used: pLIX MCPIP1 for MCPIP1 overexpression, pLIX PURO as a control and mutant form pLIX D141N, with abolished MCPIP1 RNase activity. The level of genes and proteins were studied by real-time PCR, western blot, flow cytometry and immunoprecipitation. Chemotaxis, invasion and motility assays were performed to check migration and invasion. Foxn1nu/Foxn1nu and NOD-SCID mice were used in in vivo experiments. Results: Microarray analysis revealed that the expression of mesenchymal markers increases during ccRCC progression together with decreased level of MCPIP1 protein in tumor tissue. We demonstrate that endonuclease activity of MCPIP1 regulates the mRNA expression and protein level of key elements in the EMT signaling pathways e.g. Snai1 and Snai2, ß-catenin, vimentin, E-cadherin and N-cadherin. Mutated form of MCPIP1 lacking RNAse activity upregulates β-catenin and vimentin and suppressed E-cadherin. Moreover, our study revealed that MCPIP1 influences the cytosolic or nuclear location of the E-cadherin repressors, transcription factor Snai1 and Snai2 and ß-catenin. Furthermore, motile activity of ccRCC cells is strongly increased in ccRCC with abolished MCPIP1 RNase activity. We also show that initial overexpression of MCPIP1 causes no tumor growth whereas the lack of RNase activity of MCPP1 led to an increase of tumor growth. Conclusions: We showed that endonuclease activity of MCPIP1 directly enhanced the mesenchymal phenotype of ccRCC through regulation of cell motility, expression and position of key elements in the EMT signaling pathways and tumor initiation. Acknowledgement: The study was supported by research grants from the National Science Centre 2013/09/D/NZ/00249, 2015/19/B/NZ5/01405 and KNOW 35p/3/2015. Citation Format: Katarzyna Miekus, Paulina Marona, Judyta Górka, Jolanta Jura. RNase activity of MCPIP1 regulates key elements of mesenchymal phenotype of clear cell renal cell carcinoma cells [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 5003.

Juglone suppresses epithelial-mesenchymal transition in prostate cancer cells via the protein kinase B/glycogen synthase kinase-3β/Snail signaling pathway.

Epithelial-mesenchymal transition (EMT) serves an important role in the metastasis of prostate cancer. Juglone is a natural compound isolated from plants that is reported to possess potent cytotoxic properties. However, there are no studies on the anti-EMT effect of juglone in prostate cancer, or its potential underlying mechanisms of action. In the present study, the effect of juglone on the EMT of prostate cancer cells was investigated. Transwell assays were used to demonstrate that juglone inhibits the migration and invasion of the prostate cancer (PC) LNCaP and LNCaP-AI cell lines. Results from western blot analysis demonstrated that juglone increases the expression of the epithelial marker E-cadherin while decreasing the expression of mesenchymal markers (N-cadherin and Vimentin) in a dose-dependent manner. The data from the present study also revealed that juglone downregulates the expression of Snail, a repressor of E-cadherin and an inducer of EMT. Furthermore, juglone prevented inactivation of glycogen synthase kinase-3β (GSK-3β), an endogenous inhibitor of Snail in a dose-dependent manner. Lithium chloride (LiCl), a GSK-3β inhibitor, prevented juglone-mediated downregulation of Snail expression and upregulation of E-cadherin. In addition, phosphorylation and subsequent activation of protein kinase B (Akt), which is known to phosphorylate GSK-3β at serine 9 (Ser9), leading to its inhibition, were significantly decreased by juglone in LNCaP and LNCaP-AI cells. Inhibition of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway by LY294002 augmented juglone-mediated GSK-3β activity by inhibiting Ser9 phosphorylation. These findings indicated that juglone suppresses EMT via the Akt/GSK-3β/Snail pathway, consequently decreasing the invasiveness of PC cells.

DNA methyltransferase 3A isoform b contributes to repressing E-cadherin through cooperation of DNA methylation and H3K27/H3K9 methylation in EMT-related metastasis of gastric cancer

DNA methyltransferase 3A (DNMT3A) has been recognised as a key element of epigenetic regulation in normal development, and the aberrant regulation of DNMT3A is implicated in multiple types of cancers, especially haematological malignancies. However, its clinical significance and detailed functional role in solid tumours remain unknown, although abnormal expression has gained widespread attention in these cancers. Here, we show that DNMT3A isoform b (DNMT3Ab), a member of the DNMT3A isoform family, is critical for directing epithelial–mesenchymal transition (EMT)-associated metastasis in gastric cancer (GC). DNMT3Ab is positively linked to tumour-node-metastasis (TNM) stage, lymph node metastasis and poor prognosis in GC patients. Overexpression of DNMT3Ab promotes GC cell migration and invasion as well as EMT through repression of E-cadherin. Meanwhile, DNMT3Ab promotes lung metastasis of GC in vivo. Mechanistic studies indicate that DNMT3Ab mediates the epigenetic inaction of the E-cadherin gene via DNA hypermethylation and histone modifications of H3K9me2 and H3K27me3. Depletion of DNMT3Ab effectively restores the expression of E-cadherin and reverses TGF-β-induced EMT by reducing DNA methylation, H3K9me2 and H3K27me3 levels at the E-cadherin promoter. Importantly, DNMT3Ab cooperated with H3K9me2 and H3K27me3 contributes to the transcriptional regulation of E-cadherin in a Snail-dependent manner. Further, gene expression profiling analysis indicates that multiple metastasis-associated genes and oncogenic signalling pathways are regulated in response to DNMT3Ab overexpression. These results identify DNMT3Ab as a crucial regulator of metastasis-related genes in GC. Targeting the DNMT3Ab/Snail/E-cadherin axis may provide a promising therapeutic strategy in the treatment of metastatic GC with high DNMT3Ab expression.

Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer

Colorectal cancer (CRC) is the most common malignant disease worldwide due to its metastasis via the epithelial-mesenchymal transition (EMT) process. E-cadherin and Wnt signaling are emerging as potential targets for suppressing the EMT. In this context, Axin2 has been recognized as a negative regulator that inhibits glycogen synthase kinase 3β (GSK3β)-mediated degradation of Snail1, a transcriptional repressor of E-cadherin. However, Axin2 can also impede Wnt signaling via β-catenin degradation. Therefore, Axin2 may serve as either a promoter or suppressor of tumors, and the effects of its inhibition on the cell proliferation and metastasis of CRC require further elucidation. Here, esculetin (ES), a coumarin, was found to have the most potential effects on both β-catenin-responsive transcriptional and E-cadherin promoter activities. ES also showed anti-proliferative and anti-invasive activities in CRC cells. Mechanistically, Axin2 suppression by ES contributed to E-cadherin-mediated Wnt signaling inhibition. Moreover, the ability of ES to inhibit tumor growth and metastasis via Axin2 suppression was further supported in an HCT116-implanted orthotopic mouse model. Collectively, these findings suggest that targeting the Axin2/E-cadherin axis by ES may be an attractive therapeutic strategy for the treatment of metastatic CRC.

TRPS1 Suppresses Breast Cancer Epithelial-mesenchymal Transition Program as a Negative Regulator of SUZ12

Breast cancer (BC) is among the most common malignant diseases and metastasis is the handcuff of treatment. Cancer metastasis is a multistep process associated with the epithelial-mesenchymal transition (EMT) program. Several studies have demonstrated that transcriptional repressor GATA binding 1 (TRPS1) played important roles in development and progression of primary BC. In this study we sought to identify the mechanisms responsible for this function of TRPS1 in the continuum of the metastatic cascade. Here we described that TRPS1 was significantly associated with BC metastasis using public assessable datasets. Clinically, loss of TRPS1 expression in BC was related to higher histological grade. In vitro functional study and bioinformatics analysis revealed that TRPS1 inhibited cell migration and EMT in BC. Importantly, we identified SUZ12 as a novel target of TRPS1 related to EMT program. ChIP assay demonstrated TRPS1 directly inhibited SUZ12 transcription by binding to the SUZ12 promoter. Loss of TRPS1 resulted in increased SUZ12 binding and H3K27 tri-methylation at the CDH1 promoter and repression of E-cadherin. In all, our data indicated that TRPS1 maintained the expression of E-cadherin by inhibiting SUZ12, which might provide novel insight into how loss of TRPS1 contributed to BC progression.

MiR-448 inhibits the epithelial-mesenchymal transition in breast cancer cells by directly targeting the E-cadherin repressor ZEB1/2.

Recently, accumulating evidence provides that dysregulation of microRNAs (miRNAs) is considered to play vital roles in tumor progression. Based on microRNA arrays, we found that microRNA-448 (miR-448) was significantly downregulated in breast cancer tissue specimens. In our study, we were in an effort to clarify the function, the direct target gene, and the molecular mechanisms of miR-448 in breast cancer. By quantitative RT-PCR, we analyzed the expression of miR-448 in 16 patients with BC. Overexpression of miR-448 was established by transfecting miR-448-mimics into MDA-MB-231 and MCF-7 cells, methyl thiazolyl- tetrazolium and colony formation assays were performed to evaluate its effects on cell proliferation. We also performed cell migration and invasion assays in breast cells overexpressing miRNA-448. All the results indicated that overexpression of miR-448 in breast cancer cells markedly suppressed cell proliferation, migration, and invasion. Through the quantitative RT-PCR and Western Blots, we also evaluated epithelial-mesenchymal transition. We found that overexpression of miR-448 also downregulated the expression of vimentin, a well-known mesenchymal marker. Meanwhile, the epithelial marker E-cadherin was unregulated, suggesting that miR-448 inhibited epithelial-mesenchymal transition . Bioinformatics assay coupled with Western Blot and luciferase assays revealed that miR-448 directly binds to the 3'UTR of E-cadherin repressor ZEB1/2, resulting in suppression of epithelial-mesenchymal transition in breast cancer cells. Impact statement In our study, we revealed that miR-448 played a vital role in breast cancer development and we also uncovered the mechanisms of it. Following is the short description of the main findings: miR-448 is downregulated in BC. miR-448 regulates cell proliferation, migration, and invasion in BC. miR-448 specifically regulates ZEB1/2 through binding to the 3'UTR in BC cells. miR-448 inhibits cell migration, invasion, and EMT by targeting to the 3'UTR of ZEB1/2.

CD44 exerts a functional role during EMT induction in cisplatin-resistant head and neck cancer cells.

A number of studies report that epithelial to mesenchymal transition (EMT) supports the generation and maintenance of cancer stem cells (CSCs), which show tumor seeding ability and drug resistance; however, the molecular mechanisms underlying induction of EMT-associated tumor malignancy remain unclear. The present study reports that oral cancer cells switch from expressing the CD44 variant form (CD44v) to expressing the standard form (CD44s) during acquisition of cisplatin-resistance, which resulted in EMT induction. CD44s induced an EMT phenotype in cisplatin resistant cells by up-regulating ZEB1, a transcriptional repressor of E-cadherin. More importantly, CD44s up-regulated ZEB1 by suppressing microRNA-200c, which is a non-coding RNA that directly represses the ZEB1 gene. These results demonstrate the importance of the association between platinum resistance and CD44s during EMT induction in oral cancer cells.

High Glucose-Induced Reactive Oxygen Species Stimulates Human Mesenchymal Stem Cell Migration Through Snail and EZH2-Dependent E-Cadherin Repression

Background/Aims: Glucose plays an important role in stem cell fate determination and behaviors. However, it is still not known how glucose contributes to the precise molecular mechanisms responsible for stem cell migration. Thus, we investigate the effect of glucose on the regulation of the human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) migration, and analyze the mechanism accompanied by this effect. Methods: Western blot analysis, wound healing migration assays, immunoprecipitation, and chromatin immunoprecipitation assay were performed to investigate the effect of high glucose on hUCB-MSC migration. Additionally, hUCB-MSC transplantation was performed in the mouse excisional wound splinting model. Results: High concentration glucose (25 mM) elicits hUCB-MSC migration compared to normal glucose and high glucose-pretreated hUCB-MSC transplantation into the wound sites in mice also accelerates skin wound repair. We therefore elucidated the detailed mechanisms how high glucose induces hUCB-MSC migration. We showed that high glucose regulates E-cadherin repression through increased Snail and EZH2 expressions. And, we found high glucose-induced reactive oxygen species (ROS) promotes two signaling; JNK which regulates γ–secretase leading to the cleavage of Notch proteins and PI3K/Akt signaling which enhances GSK-3β phosphorylation. High glucose-mediated JNK/Notch pathway regulates the expression of EZH2, and PI3K/Akt/GSK-3β pathway stimulates Snail stabilization, respectively. High glucose enhances the formation of EZH2/Snail/HDAC1 complex in the nucleus, which in turn causes E-cadherin repression. Conclusion: This study reveals that high glucose-induced ROS stimulates the migration of hUCB-MSC through E-cadherin repression via Snail and EZH2 signaling pathways.

Hyperuricemia and Progression of Chronic Kidney Disease: Role of Phenotype Transition of Renal Tubular and Endothelial Cells.

Although the clinical implication of hyperuricemia in chronic kidney disease has been an issue of active debate, recent data suggested a causative role of uric acid (UA) in the development of renal disease. Afferent arteriopathy, an induction of oxidative stress and an activation of local inflammation, have been regarded as the mechanisms of UA-induced renal disease, which contribute to glomerular hypertrophy and interstitial fibrosis via endothelial dysfunction. However, there have been rare studies on the direct effect of UA on phenotype transition of renal cells such as epithelial-to-mesenchymal transition (EMT) or endothelial-to-mesenchymal transition (EndoMT). We have reported that UA-induced EMT of cultured renal tubular cells, which was blocked by the organic anion transport inhibitor, probenecid. UA increased the expression of snail and slug, the transcriptional repressors of E-cadherin, which resulted in a downregulation of E-cadherin production. UA also increased the degradation of E-cadherin via ubiquitination. UA also induced EndoMT with an increase in ROS generation and glycocalyx shedding of cultured vascular endothelial cells. Treatment with antioxidants ameliorated UA-induced EndoMT. In the kidney of hyperuricemic rats, there was an evidence of EMT before the development of significant tubulointerstitial fibrosis, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA) in renal tubular cells. Allopurinol significantly inhibited UA-induced EMT with an amelioration of renal fibrosis. In addition, endothelial staining in peritubular capillaries (PTC) was substantially decreased with de-novo expression of α-SMA in endothelial cells of PTC. Key Messages: UA per se induced a phenotypic transition of epithelial and endothelial cells via an induction of oxidative stress and glycocalyx shedding, which could be one of the mechanisms of UA-induced kidney disease.

PDE4a predicts poor prognosis and promotes metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.

Phosphodiesterases (PDEs) was found to be involved in a variety of cancer pathologies by modulating the degradation of levels of cAMP/cGMP. However, the prognostic significance and biological effect of PDE4a in hepatocellular carcinoma (HCC) have not been understood completely. In the present study, PDE4a expression was detected in a cohort of HCC and matched adjacent liver tissues (n = 210) by immunohistochemistry staining and Western immunoblotting assay, And in vitro experiments were conducted to determine the effect of PDE4a on metastatic capacity of HCC cells. The data here displayed that the majority of HCC patients had higher PDE4a expression in tumor tissues compared to matched adjacent liver tissues and enhanced PDE4a expression in tumor tissues was associated positively with HBV infection, liver cirrhosis, higher serum AFP level, advanced TNM stage, vascular embolus, intrahepatic metastases and portal vein tumor thrombus (PVTT). Survival analyses suggested that higher PDE4a was indicated the poor prognosis of HCCs after liver resection. Ectopic expression of PDE4a in Huh7 cells leaded to significant repression of E-cadherin and up-regulated the expression of N-cadherin and Vimentin, and facilitated migration and invasion abilities. Silencing PDE4a in MHCC97h cells acquired the opposite results. Taken together, PDE4a triggered EMT in HCC cells and acted as a predictive factor candidate and a potential therapeutic target for HCC.

Sirtuin 7 promotes colorectal carcinoma proliferation and invasion through the inhibition of E-cadherin.

Sirtuin 7 (Sirt7) is a member of the sirtuin protein family and is implicated in various carcinomas; however, the function of Sirt7 in colorectal carcinoma (CRC) remains unclear. The present study aimed to explore the biological function of Sirt7 in CRC tissues and cell lines, and to investigate the potential underlying mechanism by performing reverse transcription-quantitative polymerase chain reaction analyses, western blot analyses, luciferase reporter assays, cell proliferation and invasion assays. It was demonstrated that Sirt7 presented a higher expression in CRC tissues and cell lines compared with that in normal tissues and cells, and this higher expression was correlated with the tumor size, the tumor, node and metastasis stage and distant metastasis. Knockdown of Sirt7 repressed the proliferation ability of SW620 and HCT116 cells in vitro, while ectopic expression of Sirt7 increased the epithelial-mesenchymal transition and invasion in HT29 and SW480 cells. Notably, these functional effects of Sirt7 were exerted through the repression of E-cadherin. Thus, the data of the present study indicated a novel mechanistic role for Sirt7 as an oncogene in CRC malignancy, and Sirt7 may be a potential therapeutic target.