Restoration Of E-cadherin Expression Research Articles

ClC-5 alleviates renal fibrosis in unilateral ureteral obstruction mice.

Renal fibrosis is the major feature of end-stage renal disease with high mortality. Chloride (Cl-) moving along Cl- channels has been suggested to play to an important role in renal function. This study aims to investigate the role of ClC-5 in renal fibrosis in unilateral ureteral occlusion (UUO) mice. C57BL/6 mice received UUO surgery followed by delivery of adeno-associated virus encoding ClC-5 cDNA (AAVClC-5). Western blotting, real-time PCR and histological analysis were used to investigate the effects of ClC-5 on renal fibrosis and underlying mechanisms. The expression of ClC-5 was significantly decreased in renal cortex of UUO mice and transforming growth factor-β1 (TGF-β1)-stimulated HK2 cells. Overexpression of ClC-5 in vivo markedly ameliorated UUO-induced renal injury and fibrosis. The increased expressions of plasminogen activator inhibitor type 1, connective tissue growth factor, collagen III and collagen IV were also inhibited by ClC-5 upregulation. Moreover, UUO-induced immune cell infiltration and inflammatory cytokines release were attenuated in mice infected with AAVClC-5. In addition, the in vivo and in vitro results showed that ClC-5 overexpression prevented epithelial-to-mesenchymal transition (EMT), concomitantly with a restoration of E-cadherin expression and a decrease of vimentin, α-SMA and S100A4 expressions. Furthermore, ClC-5 overexpression inhibited UUO- or TGF-β1-induced increase in nuclear factor kappa B (NF-κB) acetylation and matrix metalloproteinases-9 (MMP-9) expression. However, downregulation of ClC-5 in HK2 cells further potentiated TGF-β1-induced EMT and increase in NF-κB acetylation and MMP-9 expression. ClC-5 upregulation ameliorates renal fibrosis via inhibiting NF-κB/MMP-9 pathway signaling activation, suggesting that ClC-5 may be a novel therapeutic target for treating renal fibrosis and chronic kidney disease.

3-deazaneplanocin A protects against cisplatin-induced renal tubular cell apoptosis and acute kidney injury by restoration of E-cadherin expression

3-deazaneplanocin A (3-DZNeP) has been used as an inhibitor of enhancer of zeste homolog 2 (EZH2). Here, we explore the role and underlying mechanisms action of 3-DZNeP in abrogating cisplatin nephrotoxicity. Exposure of cultured mouse renal proximal tubular epithelial cells (mTECs) to cisplatin resulted in dose and time-dependent cleavage of caspase-3, decrease of cell viability, and increase of histone H3 lysine 27 trimethylation (H3K27me3), whereas expression levels of EZH2, a major methyltransferase of H3K27me3, were not affected. Treatment with 3-DZNeP significantly inhibited cisplatin-induced activation of caspase-3, apoptosis, loss of cell viability but did not alter levels of EZH2 and H3K27me3 in cultured mTECs. 3-DZNeP treatment did not affect activation of extracellular signal-regulated kinase (ERK) 1/2, p38 or c-Jun N-terminal kinases (JNK) 1/2, which contribute to renal epithelial cell death, but caused dose-dependent restoration of E-cadherin in mTECs exposed to cisplatin. Silencing of E-cadherin expression by siRNA abolished the cytoprotective effects of 3-DZNeP. In contrast, 3-DZNeP treatment potentiated the cytotoxic effect of cisplatin in H1299, a non-small cell lung cancer cell line that expresses lower E-cadherin levels. Finally, administration of 3-DZNeP attenuated renal dysfunction, morphological damage, and renal tubular cell death, which was accompanied by E-cadherin preservation, in a mouse model of cisplatin nephrotoxicity. Overall, these data indicate that 3-DZNeP suppresses cisplatin-induced tubular epithelial cell apoptosis and acute kidney injury via an E-cadherin-dependent mechanism, and suggest that combined application of 3-DZNeP with cisplatin would be a novel chemotherapeutic strategy that enhances the anti-tumor effect of cisplatin and reduces its nephrotoxicity.

Lack of microRNA-155 ameliorates renal fibrosis by targeting PDE3A/TGF-β1/Smad signaling in mice with obstructive nephropathy.

Although microRNA-155 (miR-155) is implicated in the pathogenesis of several fibrotic diseases, information regarding its functional role in renal fibrosis is limited. The current study aims to investigate the effects of miR-155 on renal fibrosis in unilateral ureteral occlusion (UUO) mice. MiR-155 level was significantly increased in renal tissues of UUO mice and TGF-β1-treated HK2 cells. Masson's trichrome staining showed that delivery of adeno-associated virus encoding miR-155 inhibitor led to a decrease in renal fibrosis induced by UUO. The increased expression of plasminogen activator inhibitor type 1, collagen III and collagen IV was also inhibited after miR-155 inhibition. In addition, miR-155 knockdown also prevented TGF-β1-induced epithelial-mesenchymal transition, concomitantly with a restoration of E-cadherin expression and a decrease of vimentin expression. Computational analysis revealed that miR-155 directly targets at 3'UTR of PDE3A. Overexpression of miR-155 suppressed the luciferase activity and protein expression of PDE3A, whereas inhibition of miR-155 increased PDE3A luciferase activity and expression. Furthermore, miR-155 inhibited TGF-β1-induced the increase of TGF-β1 expression and Smad-2/3 phosphorylation in HK2 cells. In contrast, knockdown of PDE3A reversed the effect of miR-155 inhibition on TGF-β1 expression. This study demonstrates that knockdown of miR-155 attenuates renal fibrosis via inhibiting TGF-β1/Smad signaling activation by targeting the upstream molecule PDE3A. This study suggests that miR-155 inhibition may be a novel therapeutic approach for preventing fibrotic kidney diseases.

Epidermal growth factor receptor targeting alters gene expression and restores the adhesion function of cancerous cells as measured by single cell force spectroscopy.

Loss of cell-cell adhesion function is a common characteristic of many human epithelial carcinomas that is frequently due to loss of E-cadherin expression. In cancer progression, loss of E-cadherin is associated with invasion and metastasis potential, hence restoration of its function may contribute to the metastasis inhibition. This study examined effect of Epidermal Growth Factor Receptor (EGFR/Her1) blockade on the E-cadherin expression, cellular adherence, and cell elasticity in two human epithelial cancer cell lines, MCF7 and A431. EGFR blocking agents as antibodies or small molecules target EGFR directly. Furthermore, due to intracellular signaling pathways they influence cell behavior and activities. The idea here is to investigate the effect of reduced activity of this signaling pathway using anti-EGFR Antibody (Cetuximab) and tyrosine kinase inhibitor (Lapatinib) on cell-cell adhesion and cell mechanical properties. Real-Time PCR analysis demonstrated that treatment of cells with considered drugs increased the expression of E-cadherin gene among samples. The atomic force microscopy-based single cell force spectroscopy technique was used to measure adhesive force of cancerous cells. Results indicated that inhibition of EGFR activity elevated cell-cell adhesion force, accompanied by stiffening of the cell bodies. In summary, Cetuximab and Lapatinib have been found to mediate cell-cell adhesion by restoration of E-cadherin expression and function. Our data suggest possible therapeutic potential for inhibition of metastasis via the blockade of EGFR signaling.

Abstract 1445: Overexpression of angiotensin II type 1 receptor induces epithelial-mesenchymal transition and promotes tumorigenesis of human breast cancer cells

Abstract Propose: Accumulating evidence suggests that angiotensin II type 1 receptor (AGTR1) is involved in tumorigenesis and metastasis in many types of cancer, however little is known about its role in breast cancer. Our objective of the present study is to investigate the effect of AGTR1 overexpression on cell proliferation, epithelial-mesenchymal transition (EMT), migration, invasion, angiogenesis and tumorigenesis of human breast cancer. Methods: To determine the role of AGTR1, MCF7 cells were stably transfected with an expression construct carrying the GFP cDNA for AGTR1. MTS assay, Matrigel invasion assay, wound healing assay, Western blotting and immunofluorescence were used. For in vivo study, parental MCF7 or AGTR1-MCF7 cells were subcutaneously injected into the left and right flanks of nude mice. Tumor growth and angiogenesis were determined. Results and conclusion: AGTR1-MCF7 cells, expressing high level of AGTR1 exhibited significant increases in cell proliferation, migration and invasion capacities compared with parental MCF7 cells. Furthermore, AGTR1-MCF7 cells displayed the mesenchymal phenotype, as evidenced by elongated spindle morphology, decreased E-cadherin level, increased N-cadherin and snail, and nuclear accumulation of smad3 and smad4. Interestingly, blockade of smad4 using small interfering RNA strongly induced mesenchymal to epithelial transition (MET) and restoration of E-cadherin expression in AGTR1-MCF7 cells, suggesting that AGTR1-induced EMT is ultimately mediated by smad4 activation. Finally, we showed that AGTR1 overexpression enhanced tumor volume (p<0.001, n = 10) and weight (p<0.01) as well as increased angiogenesis in human breast cancer xenograft in vivo. In summary, we demonstrate that AGTR1 overexpression promotes cell proliferation, EMT, angiogenesis and tumorigenic properties of breast cancer cells. Our findings suggest that AGTR1 could be a useful target of therapy in breast cancer. Citation Format: Eunhye Oh, Ji Young Kim, Youngkwan Cho, Nahyun Lee, Hyunsook An, Jun Suk Kim, Jae Hong Seo. Overexpression of angiotensin II type 1 receptor induces epithelial-mesenchymal transition and promotes tumorigenesis of human breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1445. doi:10.1158/1538-7445.AM2015-1445

MiR-520d-5p directly targets TWIST1 and downregulates the metastamiR miR-10b.

MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.

The Ethanolic Extract of Bark from Salix Aegyptiaca L. Inhibits the Metastatic Potential and Epithelial to Mesenchymal Transition of Colon Cancer Cell Lines

Willow bark extracts have been used for centuries as a natural pain killer. Recently their potential as anticancer agents has been reported. We have shown the high antioxidant activity, phenolic and flavonoid content in the ethanolic extract of bark (EEB) from Salix aegyptiaca, a species endogenous to the Middle East. We have also reported that incubation with EEB resulted in a reduction in cell proliferation through the induction of apoptosis and cell cycle arrest via the inhibition of phosphatidyl inositol 3-kinase/Protein kinase B and mitogen activated protein kinases signaling pathways as strongly as commercial inhibitors. The current study demonstrates the robust inhibition of anchorage-independent growth, motility, migration, and adhesion of colon cancer cell lines HCT-116 and HT-29 by EEB. These in vitro functional changes were accompanied by a restoration of E-cadherin expression, a reduction in EGFR, SNAI1, SNAI2, and Twist1 and the matrix metalloproteases MMP9 and MMP2. Many of these proteins are involved in the process of epithelial to mesenchymal transition, which is considered as a critical step in the progression of noninvasive tumor cells into malignant, metastatic carcinomas. We therefore propose that EEB from Salix aegyptiaca is a potent nutraceutical causing cancer chemoprevention by inhibiting epithelial to mesenchymal transition and can be consumed for its health promoting effects.

Restoration of E-cadherin expression by selective Cox-2 inhibition and the clinical relevance of the epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma.

BackgroundThe epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin has been thought to promote metastasis. Cyclooxygenase-2 (Cox-2) is presumed to contribute to cancer progression through its multifaceted function, and recently its inverse relationship with E-cadherin was suggested. The aim of the present study was to investigate whether selective Cox-2 inhibitors restore the expression of E-cadherin in head and neck squamous cell carcinoma (HNSCC) cells, and to examine the possible correlations of the expression levels of EMT-related molecules with clinicopathological factors in HNSCC.MethodsWe used quantitative real-time PCR to examine the effects of three selective Cox-2 inhibitors, i.e., celecoxib, NS-398, and SC-791 on the gene expressions of E-cadherin (CDH-1) and its transcriptional repressors (SIP1, Snail, Twist) in the human HNSCC cell lines HSC-2 and HSC-4. To evaluate the changes in E-cadherin expression on the cell surface, we used a flowcytometer and immunofluorescent staining in addition to Western blotting. We evaluated and statistically analyzed the clinicopathological factors and mRNA expressions of Cox-2, CDH-1 and its repressors in surgical specimens of 40 patients with tongue squamous cell carcinoma (TSCC).ResultsThe selective Cox-2 inhibitors upregulated the E-cadherin expression on the cell surface of the HNSCC cells through the downregulation of its transcriptional repressors. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. A univariate analysis showed that higher Cox-2 mRNA expression (p = 0.037), lower CDH-1 mRNA expression (p = 0.020), and advanced T-classification (p = 0.036) were significantly correlated with lymph node metastasis in TSCC. A multivariate logistic regression revealed that lower CDH-1 mRNA expression was the independent risk factor affecting lymph node metastasis (p = 0.041).ConclusionsThese findings suggest that the appropriately selective administration of certain Cox-2 inhibitors may have an anti-metastatic effect through suppression of the EMT by restoring E-cadherin expression. In addition, the downregulation of CDH-1 resulting from the EMT may be closely involved in lymph node metastasis in TSCC.

Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

BackgroundThe success of using glycolytic inhibitors for cancer treatment relies on better understanding the roles of each frequently deregulated glycolytic genes in cancer. This report analyzed the involvement of a key glycolytic enzyme, alpha-enolase (ENO1), in tumor progression and prognosis of human glioma.MethodsENO1 expression levels were examined in glioma tissues and normal brain (NB) tissues. The molecular mechanisms of ENO1 expression and its effects on cell growth, migration and invasion were also explored by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, Transwell chamber assay, Boyden chamber assay, Western blot and in vivo tumorigenesis in nude mice.ResultsENO1 mRNA and protein levels were upregulated in glioma tissues compared to NB. In addition, increased ENO1 was associated disease progression in glioma samples. Knocking down ENO1 expression not only significantly decreased cell proliferation, but also markedly inhibited cell migration and invasion as well as in vivo tumorigenesis. Mechanistic analyses revealed that Cyclin D1, Cyclin E1, pRb, and NF-κB were downregulated after stable ENO1 knockdown in glioma U251 and U87 cells. Conversely, knockdown of ENO1 resulted in restoration of E-cadherin expression and suppression of mesenchymal cell markers, such as Vimentin, Snail, N-Cadherin, β-Catenin and Slug. Furthermore, ENO1 suppression inactivated PI3K/Akt pathway regulating the cell growth and epithelial-mesenchymal transition (EMT) progression.ConclusionOverexpression of ENO1 is associated with glioma progression. Knockdown of ENO1 expression led to suppressed cell growth, migration and invasion progression by inactivating the PI3K/Akt pathway in glioma cells.

MicroRNA-30a-3p inhibits tumor proliferation, invasiveness and metastasis and is downregulated in hepatocellular carcinoma

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that regulate physiological and pathological processes by suppressing target gene expression. Altered expression of miR-30a-3p has been demonstrated in several cancers. However, little about how miR-30a-3p functions in these cancers has been reported, and the role of miR-30a-3p in hepatocellular carcinoma (HCC) is unknown. The purpose of this study was to identify the role and underlying molecular mechanism of action of miR-30a-3p in HCC. MethodsA total of 110 HCC patients, primarily treated by surgical removal of tumors, were involved in the study. HCC cell line Bel-7402 was selected to characterize the function of miR-30a-3p in vitro. ResultsOur results showed that in 83.6% of the 110 HCC patients, expression of miR-30a-3p was significantly downregulated (P < 0.0001) in tumors compared to adjacent normal tissues. In a clinicopathological correlation analysis, downregulation of miR-30a-3p correlated with a significantly higher incidence of portal vein tumor thrombus (PVTT, P = 0.009). Moreover, miR-30a-3p markedly inhibited the invasion and migration of Bel-7402 HCC cells in vitro. Furthermore, miR-30a-3p overexpression had an inhibitory effect on cell proliferation, induced apoptosis and increased arrest of cells in the S phase. We further demonstrated that miR-30a-3p regulates HCC cell function by a mechanism involving reduced vimentin and MMP3 expression and restoration of E-cadherin expression. Conclusionsour data suggest that miR-30a-3p is downregulated in HCC and acts as a tumor suppressor in vitro. Regulation of vimentin, E-cadherin and MMP3 by miR-30a-3p suggests a useful therapeutic strategy for tumors with reduced miR-30a-3p expression.

Effects of the Chinese herbal extract Songyou Yin on the residual hepatocellular carcinoma after chemotherapy in nude mice

To investigate the effects of a Chinese herbal extract Songyou Yin on residual hepatocellular carcinoma after chemotherapy in nude mice and the relevant mechanisms. Orthotopic nude mouse models bearing residual hepatocellular carcinoma after chemotherapy was established using human liver carcinoma MHCC97L cells. Three different doses of Songyon Yin (2.1 g/kg, 4.2 g/kg and 8.4 g/kg) were administered to the mice in the trial groups by intragastric gavage, respectively. The mice in the control group were administered physiological saline. The tumor growth, metastasis and survival in the mice of each group were recorded. The corresponding mechanisms were studied. The pulmonary metastasis rates of the control group and 2.1g/kg, 4.2g/kg, 8.4g/kg Songyou Yin treatment group were 86.7%, 73.3%, 40.0%, and 20.0%, respectively, and the survivals of these groups were 53.83 ± 4.71, 56.50 ± 6.09, 66.67 ± 5.61, 81.17 ± 7.36 days, respectively. Compared with the mice in the control group, mice in the 4.2 g/kg, 8.4 g/kg Songyou Yin treatment groups had a lower pulmonary metastasis rate (P = 0.021 and P = 0.001, respectively) and longer survival (P = 0.002 and P = 0.001, respectively). A restoration of E-cadherin expression and a concomitant reduction of N-cadherin expression were detected in the tumors of the 4.2 g/kg and 8.4 g/kg Songyou Yin treatment groups. Songyou Yin effectively inhibits the invasion and metastasis of the residual hepatocellular carcinoma after chemotherapy in nude mice through attenuating the epithelia-mesenchymal transition and prolongs the survival. Songyon Yin may have potential to promote the efficacy of chemotherapy in hepatocellular carcinoma.

Abstract PR04: The Six2 homeoprotein mediates breast cancer metastasis via repressing E-cadherin

Abstract Homeobox genes are master regulators of embryogenesis and it is widely known that misexpression of these transcription factors can lead to tumorigenesis/ metastasis. The Six1 homeoprotein has been implicated in tumor onset and metastasis of many different cancers, including breast cancer. However, little is known about whether related homeoproteins in the Six family can affect tumor onset or progression. Recent studies have shown that while Six1 knockdown dramatically decreases metastasis, knockdown cells that are capable of metastasizing have upregulated a related family member, Six2. Interestingly, Six2 regulates stem/ progenitor cell populations and maintains a mesenchymal progenitor phenotype in the developing kidney; however, the role of Six2 in breast cancer progression has not been explored. Here, we demonstrate for the first time that up-regulation of Six2 in breast cancer cells can promote metastasis in a mechanism that differs from its related family member, Six1. Knockdown (KD) of Six2 in 66cl4 mouse mammary carcinoma cells significantly decreases lung metastasis in an immune-competent orthotopic mouse model, as compared to control KD. In contrast to what is seen with Six1 KD in the same orthotopic mouse model, no significant inhibition of primary tumor size was observed with Six2 knockdown, suggesting that unlike Six1, the functions of Six2 may be confined to later stages of tumor progression. Overexpression of Six2 in the weakly metastatic 4TO7 mouse mammary carcinoma cell line led to changes in cell morphology, increased anchorage independent growth and significantly enhanced lung metastasis in an immune competent mouse model. Microarray analysis was performed to determine the mechanism by which Six2 mediates metastasis and the epithelial marker E-Cadherin, whose loss has been implicated in the metastatic potential of many cancers, was dramatically downregulated in the 4TO7-Six2 expressing cells. Importantly, restoration of E-cadherin expression reduced lung metastasis induced by Six2 and increased survival of the animals. We further determine that repression of E-cadherin by Six2 is mediated via multiple mechanisms. SIP1/Zeb2 (zinc finger E box binding homeobox2), an epithelial to mesenchymal transition (EMT) inducer, is increased in cells overexpressing Six2, and Zeb2 KD in these cells reverts E-cadherin expression back to the level as that observed in the control cells and importantly leads to a reduction in metastasis in vivo. In addition, we find that Six2 may also repress E-cadherin though an epigenetic mechanism, since repression of E-cadherin downstream of Six2 can be reversed by inhibiting DNA methylation. Finally, analysis of Six2 expression from public microarray datasets indicates that Six2 is increased in breast cancers compared to normal breast tissue and that high expression of SIX2 correlates with poor prognosis (distant metastasis free survival, overall survival and relapse free survival) in 1881 human breast tumors examined using the GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database. Moreover, a reverse correlation of E-cadherin and Six2 in breast cancer datasets suggest that Six2 promotes loss of E-cadherin in human breast cancer. Together, these data demonstrate that Six2 is a specific regulator of the metastatic process, through regulating E-cadherin expression. This abstract is also presented as Poster B094. Citation Format: Chu-An Wang, Paul Jedlicka, Aik-Choon Tan, Catherine Pham, Vadym Zaberezhnvv, Heide Ford. The Six2 homeoprotein mediates breast cancer metastasis via repressing E-cadherin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR04.

Abstract 290: EMT and cancer stem cells: role in radiation resistance of breast cancer.

Abstract Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. During EMT, epithelial cells lose epithelial characteristics, such as E-cadherin expression and gain mesenchymal properties, such as vimentin expression. EMT cells have stem cells properties and possess the characteristics of cell motility, invasiveness and chemotherapy resistance and these cancer stem cells may be responsible for mediating tumor metastasis and resistance to cancer treatments. However, very little is known about the role of EMT and cancer stem cells in modulating radiation response of human breast cancer cells. We compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) were more sensitive to increasing doses of radiation and had high expression of E-cadherin. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1-expression vector and isolated stable clones. These clones were selected and tested for radiosensitivity. MDA-MB-231 and Hs578t cells transfected with a control vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized the cells compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. Our preliminary data demonstrates that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which are relatively radiosensitive. Thus, there was a general correlation between EMT, based on loss of E-cadherin expression, and radioresistance. Overall, our results suggest that E-cadherin interacts with radiation and enhances the radioresponse of human breast cancer cells. Since it has been demonstrated that the process of EMT contributes to drug resistance and results in cells with CSC-like characteristics, we compared ALDH1 expression in ER positive (MCF-7) and ER negative (MDA-MB-231 and Hs578t) cell lines. Our data shows that ALDH1 expression correlates with the ER status of the breast cancer cells, with a higher number of ALDH1+ cells in ER negative compared to ER positive cell lines. Our preliminary investigation leads us to believe that potentially resistant breast cancer stem cell populations appear to be overrepresented in ER negative breast cancer cell lines. Our observation concurs with clinical data that ER negative cancers are resistant to radiation therapy compared to ER positive breast cancers. Citation Format: Anupama Munshi, Meghna Mehta, Yuhui Yuan, Huifeng Liu, Raymond E. Meyn, Rajagopal Ramesh. EMT and cancer stem cells: role in radiation resistance of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 290. doi:10.1158/1538-7445.AM2013-290

C‐Myc suppressed E‐cadherin through miR‐9 at the post‐transcriptional level

c-Myc oncoprotein is overexpressed in most human cancers and regulates different genes and pathways in different cell types. E-cadherin expression is repressed by MYC through a post-transcriptional mechanism, but the exact mechanism remains elusive. Since E-cadherin is a direct target of miR-9 and miR-9 can be activated by MYC and MYCN, this suggests that c-Myc negatively modulates E-cadherin through a microRNA pathway. We have established a c-Myc-inducible expression system in which the protein level and transcriptional activity of c-Myc is significantly upregulated upon doxycycline induction. Overexpressed c-Myc led to an EMT-like conversion in the T-REx-293 cells and resulted in a significant decrease in E-cadherin and an increase in Vimentin. Stem-loop RT-PCR showed elevated expression of miR-9 when c-Myc was induced to be overexpressed. Regarding the relationship of c-Myc, miR-9 and E-cadherin, the expression of miR-9 was curtailed by using antagomir-9 in induced overexpressing c-Myc. Restoration of E-cadherin expression became much stronger in the presence of c-Myc. Thus c-Myc represses E-cadherin at the post-transcriptional level through miR-9.

Smoking Induces Epithelial-to-Mesenchymal Transition in Non–Small Cell Lung Cancer through HDAC-Mediated Downregulation of E-Cadherin

Epidemiological studies have shown that most cases of lung cancers (85%-90%) are directly attributable to tobacco smoking. Although association between cigarette smoking and lung cancer is well documented, surprisingly little is known about the molecular mechanisms of how smoking is involved in epithelial-to-mesenchymal transition (EMT) through epigenetic changes. Here, we show that lung cancer patients with a smoking history have low E-cadherin levels and loss of E-cadherin is a poor prognostic factor in smokers. Moreover, the downregulation of E-cadherin correlates with the number of pack years. In an attempt to determine the role of long-term cigarette smoking on EMT, we observed that treatment of lung cell lines with cigarette smoke condensate (CSC) induces EMT through downregulation of epithelial markers, including E-cadherin and upregulation of mesenchymal markers. CSC decreases E-cadherin expression at the transcriptional level through upregulation of LEF1 and Slug, and knockdown of these two proteins increases E-cadherin expression. Importantly, chromatin immunoprecipitation assays suggest that LEF-1 and Slug binding to E-cadherin promoter is important for CSC-mediated downregulation of E-cadherin. The histone deacetylase (HDAC) inhibitor MS-275 reverses CSC-induced EMT, migration, and invasion through the restoration of E-cadherin expression. These results suggest that recruitment of HDACs by transcriptional repressors LEF-1 and Slug is responsible for E-cadherin suppression and EMT in cigarette smokers and provide a potential drug target toward the treatment of lung cancer.

Restoration of E-cadherin expression in pancreatic ductal adenocarcinoma treated with microRNA-101

To investigate the possibility of inhibiting the progression of pancreatic ductal adenocarcinoma (PDAC) by facilitating the expression of E-cadherin through the enforced expression of microRNA-101 (miR-101). In situ hybridization was conducted with archival tissue using a double digoxigenin-labeled probe. Chromatin immunoprecipitation (ChIP) assay was conducted with EZ-Magna ChIPTM A. Gene profile analysis, Western blot, and immunoprecipitation assays were performed using standard protocols. We found that decreased miR-101 expression observed in archival patient tissues was significantly associated with poor prognosis indicated by low-intensity staining in high-grade tumors. ChIP assays using anti-enhancer of zeste homolog 2 (EZH2) antibodies indicated not only the interaction of EZH2 to the CDH1 (E-cadherin) promoter, but also that this interaction was significantly diminished in cells transfected with pre-miR-101. We observed a global downregulation of trimethylated lysine 27 of H3 histone (H3K27me3) along with upregulation of the enzymes histone deacetylase -1 and -2 with the re-expression of miR-101. Further, we observed lesser levels of transcriptional factors that inhibit the CDH1 promoter with pre-miR-101 treatment. Western blot analysis confirmed the enhanced E-cadherin expression. PANC-1 cells transduced with pre-miR-101 displayed markedly attenuated growth in SCID mice. These results suggest the potential therapeutic use of miR-101-enforced expression for inhibition of PDAC.

P1-02-08: Modulation of EMT by Targeting E-Cadherin Restores Radiation Sensitivity in Human Breast Cancer Cells.

Abstract E-cadherin is the major adhesion protein associated with epithelial cells and loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Findings from our laboratory, suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. We observed that many ER-a negative lines, were also E-cadherin negative, and the ER-a positive lines were positive for E-cadherin expression. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) and had high expression of E-cadherin, were also more sensitive to increasing doses of radiation. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1 -expression vector and isolated stable clones, which were then tested for radiosensitivity. Cells transfected with the backbone vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized both, MDA-MB-231 and Hs578t cells, compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. These observations suggest that ER-a signaling might regulate E-cadherin and implied that this regulation might influence EMT and radioresistance. Overall, our results suggest that the EMT status of breast cancer cells governs their response to radiation. A detailed investigation of the mechanism is ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-08.

WT1 and Pax2 Re-Expression Is Required for Epithelial-Mesenchymal Transition in 5/6 Nephrectomized Rats and Cultured Kidney Tubular Epithelial Cells

Mature tubular epithelial cells in the adult kidney can undergo epithelial-mesenchymal transition (EMT), a phenotypic change that is linked to the pathogenesis of renal interstitial fibrosis. EMT may be considered the reverse of mesenchymal-epithelial transition, which occurs during normal kidney development. The Wilms’ tumor suppressor gene WT1 and the paired box 2 gene Pax2 are needed to induce mesenchymal-epithelial transition and play key roles in the progression of nephrogenesis. However, until now, WT1 and Pax2 have not been tested for their direct involvement in the process of renal tubular EMT. In this study, we explored the potential roles of WT1 and Pax2 in EMT that is induced in the remnant kidney of rats following 5/6 nephrectomy. We also examined WT1 and Pax2 in cultured renal tubular epithelial (NRK52E) cells treated with interleukin-1α and investigated the effects of blocking EMT using RNA interference. We showed that WT1 and Pax2 were re-expressed in the EMT models, and these were accompanied by decreased expression of E-cadherin and increased expression of vimentin, Snail and α-smooth muscle actin. Silencing WT1 and Pax2 by RNA interference blocked the interleukin-1α-induced EMT in the NRK52E cells, as reflected in the suppression of α-SMA and Snail expression, the restoration of E-cadherin expression and normal cell morphology. Our experiments suggested that the re-expression of WT1 and Pax2 in the tubular epithelial cells plays important roles in the promotion of EMT, and there may be therapeutic value in silencing Pax2 and WT1 to prevent or reverse renal fibrosis.

Methotrexate induces apoptosis through p53/p21-dependent pathway and increases E-cadherin expression through downregulation of HDAC/EZH2

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used as an anticancer drug in different kinds of human cancers. Here we investigated the anti-tumor mechanism of MTX against non-small cell lung cancer (NSCLC) A549 cells. MTX not only inhibited in vitro cell growth via induction of apoptosis, but also inhibited tumor formation in animal xenograft model. RNase protection assay (RPA) and RT-PCR demonstrated its induction of p53 target genes including DR5, p21, Puma and Noxa. Moreover, MTX promoted p53 phosphorylation at Ser15 and acetylaion at Lys373/382, which increase its stability and expression. The apoptosis and inhibition of cell viability induced by MTX were dependent on p53 and, partially, on p21. In addition, MTX also increased E-cadherin expression through inhibition of histone deacetylase (HDAC) activity and downregulation of polycomb group protein enhancer of zeste homologue 2 (EZH2). Therefore, the anticancer mechanism of MTX acts through initiation of p53-dependent apoptosis and restoration of E-cadherin expression by downregulation of HDAC/EZH2.

Abstract 3379: Role of E-cadherin in radiation resistance of human breast cancer cells

Abstract E-cadherin is the major adhesion protein associated with epithelial cells loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Our preliminary data suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) were more sensitive to increasing doses of radiation and had high expression of E-cadherin. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 cells with a CDH1-expression vector and isolated stable clones. These clones were selected and tested for radiosensitivity. MDA-MB-231 cells transfected with a control vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized the cells compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. Thus, there was a general correlation between EMT, based on loss of E-cadherin expression, and radioresistance. Overall, our results suggest that E-cadherin interacts with radiation and enhances the radioresponse of human breast cancer cells. A detailed investigation of the mechanism is ongoing. 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 3379.