EP300 genomic redistribution following E-cadherin inhibition in MCF7 cancer cells: evidence for early epigenetic reprogramming.

TLE1 promotes EMT in A549 lung cancer cells through suppression of E-cadherin

The Role of EMT in Pancreatic Cancer Progression

Background: We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with estrogen receptor negativity and tumors of African American women, both associated with aggressive tumors. BP1 is also expressed in metastatic tumors, shown by immunostaining of 46 samples of inflammatory breast cancer; all cases were BP1 positive. Nine cases had metastasized, and all nine metastatic lymph nodes were BP1 positive. BP1 positive cells were observed in lymph channels and blood vessels. Here, we demonstrated BP1 induces the epithelial to mesenchymal transition (EMT), resulting in increased migratory ability. EMT, a process by which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells become more invasive, migratory and can lead to metastasis. Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT, is activated by BP1. Materials and Methods: RNA levels of markers of EMT were verified by real-time PCR and their protein levels by Western blotting and, in some cases, by confocal microscopy. Chromatin immunoprecipitation (ChIP) was performed to verify the BP1 binding site on the Twist promoter. A scratch test was used to measure migratory ability. BP1 knockdown by siRNA transfection was also performed. Recombinant BP1 (rpBP1) was produced in E. coli. Results: Increased expression levels of Twist were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. We demonstrated that BP1 can upregulate Twist expression in two different breast cancer cell lines, MCF-7 and Hs578T cells, by binding to the Twist promoter. BP1 upregulates mesenchymal marker expression and down-regulates epithelial marker expression, consistent with EMT. BP1 also promotes breast cancer cell migratory ability, shown by the scratch test. In addition, cells grown in medium supplemented with rpBP1 showed increased Twist expression and migration. Experiments evaluating the effects of siBP1 on EMT are underway. Conclusions: BP1 stimulates Twist expression in MCF-7 and HS578T breast cancer cells, resulting in a more mesenchymal cell phenotype. We therefore hypothesize that BP1 induces EMT by upregulating Twist expression, and may result in metastasis. If BP1 is involved in EMT, it may be a good target for therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-15.

Abstract #6017 Background: Increasing evidence indicates the fundamental role of epithelial to mesenchymal transition (EMT) in breast cancer progression and this is viewed as an essential early step in tumor cell invasion and metastasis. One of the pivotal events of EMT is down-regulation of E-cadherin. Recent findings have demonstrated the importance of the miR-200 microRNA family in the regulation of E-cadherin and EMT. In view of these findings, we examined the relationship between expression of members of the miR-200 family and status of E-cadherin levels in various human breast cancer cell lines.
 Materials and Methods: Using quantitative real-time PCR analysis, we analyzed the expression of miR-200b, miR-200c, and miR-141 in 15 human breast cancer cell lines. Additionally, we measured the level of ZEB1, ZEB2, and E-cadherin proteins using Western blot analysis and E-cadherin promoter methylation by methylation-specific PCR.
 Results: We found a substantial down-regulation of miR-200b, miR-200c, and miR-141 in the breast cancer cell lines. The extent of down-regulation was correlated with metastatic potential of cells. The down-regulation of members of the miR-200 family was associated with up-regulation of ZEB1 and ZEB2, transcriptional repressors of E-cadherin, and a subsequent decrease in the E-cadherin levels. More importantly, inhibition of E-cadherin expression was not due to its promoter hypermethylation. Treatment of breast cancer cells with demethylating agent 5-aza-2'-deoxycytidine resulted in over-expression of miR-200b and miR-200c, increase of the level of E-cadherin, and diminished metastatic properties of cancer cells.Discussion: Previous studies have revealed the important role of promoter methylation-related inhibition of E-cadherin in EMT in human breast cancer. However, the transcriptional silencing of E-cadherin is not always related to promoter hypermethylation, which indicates the existence of other mechanisms in E-cadherin down-regulation. In the present study, we demonstrated the crucial role of members of the miR-200 family in up-regulation of ZEB1 and ZEB2 proteins leading to subsequent E-cadherin down-regulation in human breast cancer cell lines. The extent of down-regulation of the miR-200 family was dependent of cancer stage and was associated with metastatic potential of cancer cell lines. This suggests that expression of miR-200 may be a powerful diagnostic marker determining breast cancer stage and cancer invasive phenotype. More importantly, the results indicate the great potential for reversing the invasive phenotype of cancer cells by up-regulation of miR-200 microRNAs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6017.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse

BP1 Upregulates Twist, a Trigger of EMT and Cancer Metastasis Bin-Jin Hwang, Jinguen Rheey, and Patricia E. Berg George Washington University, Department of Biochemistry and Molecular Biology, Washington, D.C. Background. We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Increasing evidence suggests that homeobox genes are important in malignant transformation and tumorigenesis, and our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with breast cancer aggressiveness: 100% of estrogen receptor negative tumors and 89% of the tumors of African American women are BP1 positive. Now we have evidence that BP1 may regulate the epithelial to mesenchymal transition (EMT). EMT, a process in which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells to become more invasive and migratory, and can lead to metastasis. BP1 upregulates Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT. Our objective is to determine whether BP1 plays a role in induction of EMT. Materials and Methods. The expression levels of Twist, E-cadherin and vimentin were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. RNA levels were verified by real-time PCR and protein levels by Western blotting and, in some cases, by confocal microscopy. Summary. Twist mRNA expression on microarrays is higher in MCF-7 cells overexpressing BP1 compared with cells containing an empty vector. Real-time PCR confirmed that Twist mRNA is increased five-fold in cells with high levels of BP1, and Western blots demonstrated that Twist protein is also elevated in the same cells. We have discovered a potential DNA binding site for pBP1 in the Twist promoter, suggesting that BP1 may directly regulate Twist. This will be verified by ChIP. Expression of two genes regulated by Twist was also examined: Twist activates vimentin and represses E-cadherin. Cells overexpressing pBP1 also show increased vimentin and decreased E-cadherin. The effect of increased pBP1 on cell migration is under investigation. Conclusions: BP1 stimulates Twist expression in MCF-7 breast cancer cells, resulting in increased vimentin and decreased E-cadherin levels. We therefore hypothesize that BP1 is upstream of Twist and induces EMT. If BP1 is required for EMT, it may be a good target for therapy of metastatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2298.

BackgroundIron overload has been found in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and is thought to be involved in disease progression; however, the underlying mechanism is complex and not yet fully understood. We sought to assess the in vitro role of iron in the progression of fibrosis in lung epithelial cells, and examine the possible regulation of iron and IPF.MethodsErastin was used to establish a cell model of iron accumulation in mouse lung epithelial cell line 12 (MLE-12). A Cell Counting Kit-8 assay and annexin V staining were applied to measure cell viability and apoptosis, quantitative polymerase chain reaction (qPCR) and quantitative immunoblot analysis of the protein was conducted to analyze the expression of E-cadherin, N-cadherin, α-smooth muscle actin (α-SMA), Vimentin and β-Actin. The autophagy was visualized by microtubule-associated protein 1A/1B-light chain 3 (LC3) staining and western blot.ResultsThe results showed that cell proliferation was significantly inhibited and apoptotic and necrotic cells were significantly increased with 2 µM of erastin treatment. Western blotting showed that reactive oxygen species (ROS) production and the level of heme oxygenase-1 were increased in the cells. Epithelial-mesenchymal transition (EMT) represented by the suppression of E-cadherin and the upregulation of α-smooth muscle actin (α-SMA) and Vimentin was induced by erastin. Additionally, autophagy represented by activated LC3B and up-regulated Beclin-1 were also induced by erastin. To further ascertain the role of autophagy in erastin-induced EMT, chloroquine, which is an autophagy inhibitor, was employed, and was found to effectively reduce EMT in this process.ConclusionsThese results support the role of the enhanced accumulation of iron as a mechanism for increasing the vulnerability of lung epithelial cells to iron-driven oxidant injury that triggers further autophagy during EMT.

Objective To investigate into the biological effect of lipopolysaccharide(LPS) combined with transforming growth factor-β1(TGF-β1) on MCF-7 breast cancer cells and discuss the molecular mechanism underneath the phenomena. Methods The morphology of MCF-7 cells was observed under the inverted microscope.Rhodamine-phalloidine was used for staining the actin cytoskeleton.Real-time quantitative polymerase chain reaction(Real-time PCR) was used to detect genetic expression of E-cadherin, Vimentin,Snail-2, Twist and matrix metalloproteinase-9(MMP-9).Transwell was used to examine cell migration and invasion.Zymography experiment was used to test active protein MMP-9.Western blotting was used to detect signal pathways of nuclear factor-κB(NF-κB), extracellular signal-regulated kinase(ERK) and Smad. Results Almost all the cells in the group of LPS combined with TGF-β1(L+ T group)acquired fibroblastoid properties along with obvious cytoskeleton changes.L+ T group had E-cadherin down-regulated obviously while Snail-2, Twist and MMP-9 up-regulated sharply.The number of migrated cells in control group, LPS group, TGF-β1 group, L+ T group were 6. 8±4. 1, 10. 2±4. 3, 39. 5± 3. 8, 69. 7±4. 4 respectively.The number of invaded cells in control group, LPS group, TGF-β1 group, L+ T group were 4. 4±1. 1,6. 8±2. 4,32. 1±2. 3, 57. 9±4. 5 respectively.The active protein of MMP-9 was the most in L+ T group.L+ T group has much higher phosphorylated ERK and phosphorylated Smad-2 than TGF-β1 group's. Conclusion LPS cooperates with TGF-β1 to promote the epithelial to mesenchymal transition of MCF-7 breast cancer cells, and stimulation of LPS and TGF-β1 on MCF-7 make those cells have stronger capabilities of migration and invasion. Key words: Breast cancer; Transforming growth factor-β1; Lipopolysaccharide; Tumor metastasis; Epithelial-mesenchymal transition

BackgroundDuring cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance.MethodsThe expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay.ResultsInduction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2.ConclusionTaken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

IntroductionResistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.MethodsWe used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen.ResultsWe found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation.ConclusionsETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.

Epithelial-to-mesenchymal transition (EMT) or its reversal mesenchymal-to-epithelial transition (MET) are of critical importance throughout development and is implicated in wound healing, fibrotic disorders, and cancer metastasis. The ability of a cell to undergo EMT and MET, cellular plasticity, underlies tumor progression through multi-step metastasis and acquisition of chemotherapy resistance as alterations in the cellular and molecular level confer changes in motility, stemness properties, gene expression, and cellular dynamics. These changes facilitate invasion and dissemination of cancer cells. Epigenetic modifications leading to cancer cell plasticity are partially driven by histone modifying proteins including lysine (K)-specific demethylase 6A (KDM6A). KDM6A is a member of the COMPASS-like protein complex, and catalyzes the removal of methyl groups from H3K27me3, facilitating gene expression. KDM6A regulation of H3K27me3 has been associated with bivalent programs in gene expression, associates with oncogenic pathways, and contributes to low survival prognoses in breast cancer. There is critical need to elucidate the mechanisms of KDM6A suppression to understand its control on cellular plasticity. Previously we have shown that KDM6A is suppressed upon EMT. In this study, we sought to identify the regulatory mechanisms of KDM6A expression and how its activity is required to maintain epithelial cellular identity. To investigate the role of KDM6A in the context of EMT, KDM6A expression was compromised using CRISPR-Cas9 to determine the impact on H3K27me3, gene expression and cellular identity. Breast cancer cell lines were transduced with CRISPR-Cas9 and GFP via lentiviral transduction. KDM6A KO and control cell lines were generated via a CRISPR KO. We observed that the KDM6A suppression or inhibition profoundly impacts cellular identity, leading to gain of mesenchymal and stemness properties. Moreover, KDM6A was determined to be regulated through sub-cellular localization leading to partial sequestration of COMPASS-like protein complex outside the nucleus. These findings were validated via Western blot analysis of subcellular protein fractions of breast cancer cell lines and KDM6A KO cell lines and well as immunofluorescence and enzyme activity analysis. We conclude that KDM6A functions as a master regulator of epithelial cellular identity which is controlled by multi-factorial mechanisms including transcriptional suppression and altered protein localization. Citation Format: Charli Worth, Provas Das, Kelsey Johnson, Joseph Taube. KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B015.

Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression post-transcriptionally. Dysregulation of miRNA has been implicated in the development and progression of prostate cancer. Through next generation miRNA sequencing, we recently identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Of the five miRNAs, miR-301a had the strongest association with prostate cancer recurrence. Overexpression of miR-301a in prostate cancer cells, PC3, and LNCaP resulted in increased growth both in vitro and in xenografted tumors. We therefore sought to examine its role in prostate carcinogenesis in greater detail. We examined the effect of miR-301a expression on biochemical recurrence and metastasis among 585 men treated with radical prostatectomy for prostate cancer. We examined the mechanism of growth deregulation by miR-301a in prostate cancer cells using analysis of the miRome of prostate cancer cell lines, quantitative PCR, and Western blotting. High levels of miR-301a (above the median) were associated with an increased risk of biochemical recurrence (adjusted hazard ratio [aHR] 1.42, 95% confidence interval (CI) 1.06-1.90, P = 0.002) but not of metastasis (aHR 0.84, 95%CI 0.41-1.70, P = 0.6) after adjustment for known prognostic factors. RNA transcriptome sequencing analysis of miR-301a overexpressing prostate cancer cell lines identified the tumor suppressor p63 as a potential direct miR-301a target. Transcriptome sequencing, qPCR and Western blotting showed that miR-301a induced epithelial-mesenchymal transition (EMT) in prostate cancer cells through a pathway initiated by p63 inhibition. Luciferase assay verified p63 as a direct target of miR-301a. Loss of p63 resulted in miR-205 downregulation, releasing Zeb1 and Zeb2 from inhibition, culminating in Zeb1/Zeb2 suppression of E-cadherin. This pathway of growth alteration mediated by miR-301a upregulation was shown to be valid in prostate cancer cell lines and patient-derived tumors. These data indicate that miR-301a functions as an oncogene in prostate cancer by directly targeting the p63 tumor suppressor leading to loss of E-cadherin and EMT. Hence, miR-301a may serve as a novel biomarker in prostate cancer as well as a therapeutic target for prostate cancer management. Prostate 76:869-884, 2016. © 2016 Wiley Periodicals, Inc.

BackgroundEpithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice.MethodsEMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student’s t tests were performed to determine significance (p < 0.05).ResultsEMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of these cells were globally vimentin-positive rather than exhibiting regional EMT, and they expressed higher SNA1 than their in vitro counterparts. E-cadherin suppression caused a trend toward reduced lung metastasis, whereas E-cadherin overexpression resulted in the reverse trend, consistent with the increased proliferation rate and predominantly epithelial phenotype of MDA-MB-468 cells outside the primary xenograft. This was also originally observed in WT xenografts. Furthermore, we found that patients with breast cancer that expressed E-cadherin were more likely to have metastases.ConclusionsE-cadherin expression promotes growth of primary breast tumors and conceivably the formation of metastases, supporting a role for MET in metastasis. E-cadherin needs to be reevaluated as a tumor suppressor.