Inhibition Of EMT Research Articles

Abstract PR10: Silencing of semaphorin 7a attenuates breast tumor progression

Abstract Determining what drives conversion of non-lethal to lethal breast cancers is of utmost importance for understanding and blocking tumor metastasis. We have identified semaphorin 7a (Sem7a) as a potent driver of human breast cancer progression, suggesting it may represent a novel target to inhibit metastasis. Sem7a is a GPI membrane anchored protein that promotes attachment and spreading in multiple cell types1,2; previous reports have identified a role for Sem7a in mouse mammary tumorigenesis, including promotion of tumor growth and EMT, as well as induction of angiogenic cytokine production by macrophages and metastatic outgrowth in the lung3-5. However, Sem7a has not been investigated in human breast tumors, so we utilized publicly available microarray datasets and found that SEMA7A gene expression is increased in ~45% of all breast cancers. Further, we found SEMA7A upregulation was associated with poor prognosis, which is consistent with a role in metastasis. Specifically, breast cancer patients with high expression of SEMA7A are at risk for early recurrence and have decreased overall and distant metastasis-free survival rates. We used in vitro and in vivo approaches to assess the role of SEM7a in human breast tumor progression. In vivo, we show that shRNA mediated silencing of Sem7a significantly slows tumor growth, delays tumor progression from in situ to locally invasive disease, and decreases tumor associated lymphangiogenesis. Furthermore, having previously shown that collagen I and COX-2 drive tumor progression in our breast cancer models6,7, we identified a relationship between COX-2 and Sem7a expression at the mRNA and protein levels. Finally, using three- dimensional spheroid and tube formation assays, we show that semaphorin 7a promotes tumor cell invasion on collagen, and lymphangiogenesis, via activation of the β1-integrin receptor. Our results indicate that Sem7a may be novel target for blocking multiple aspects of breast tumor progression that lead to metastasis.

HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer.

AIMTo investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells.METHODSImmunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments.RESULTSIn GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility.CONCLUSIONHER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC.

Abstract LB-B21: BI-69A11 inhibits Epithelial to Mesenchymal Transition exploiting β-catenin pathway in colon carcinoma

Abstract EMT is a potential biological phenomena required for metastatic dissemination. Although targeting EMT for therapeutic implication has been gaining interest to the scientific community, only few molecules have succeeded to employ their anti-metastatic effect so far. BI-69A11 has been earlier proved for its anti-tumor activity in prostate and colon carcinoma. This study aims to elucidate the biological consequence of BI-69A11 on EMT in colon carcinoma in vitro and in vivo. Our findings showed that BI-69A11 increased epithelial signature marker E-cadherin and reciprocally decreased the mesenchymal markers snail, slug and vimentin. Moreover, cellular migration, invasion and adhesion were potentially hampered by BI-69A11 through β-catenin dependent pathway. The underlying molecular mechanism behind BI-69A11 induced inhibition of EMT includes suppression of nuclear transport of β-catenin, diminished phosphorylation of β-catenin accompanied with enhanced E-cadherin- β-catenin complex formation at the membrane. Moreover, BI-69A11 controlled the transcriptional activity and reversed nuclear translocation by decrease in the phosphorylation at ser552 and ser45/Thr41 residue. The growing interaction between E-cadherin and β-catenin complex also promoted the E-cadherin transcription leading to the reversal of EMT. BI-69A11 caused increasing accumulation of vinculin in the membrane that in turn strengthened the focal adhesion junction leading to the inhibition of metastasis. Overall, these evidences present a thoughtful insight towards underlying molecular mechanism of BI-69A11 induced reversal of EMT in colorecteal carcinoma in vitro and in vivo. Citation Format: Ipsita Pal, Mahitosh Mandal. BI-69A11 inhibits Epithelial to Mesenchymal Transition exploiting β-catenin pathway in colon carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B21.

Fucoidan Elevates MicroRNA-29b to Regulate DNMT3B-MTSS1 Axis and Inhibit EMT in Human Hepatocellular Carcinoma Cells.

Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC). Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3′-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1), a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-β receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin) and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9), by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-β signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.

Abstract 4650: Novel resveratrol-analog HPIMBD inhibits breast cancer cell metastasis by reversal of epithelial-mesenchymal transition

Abstract Epithelial-mesenchymal transition (EMT) is a biological process that mediates a phenotypical change in the polarized epithelial cells to mesenchymal cells. The process of EMT increases a cell's migratory and invasive properties. It is well documented that the process of EMT plays a very critical role in cancer metastasis. The objective of this study was to characterize the role of a novel Resveratrol analog 4-(E)-{4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD) on EMT in human breast cancer cells. Human breast cancer cells MDA-MB-231 were treated with Resveratrol (Res) and HPIMBD for up to 48 hours. Protein expression levels of E-cadherin, matrix metalloproteases (MMPs), snail, slug, zeb1/2 and nuclear migration of β-catenin were quantified after treatment with Res or HPIMBD and compared with that of the vehicle-treated control cells. mRNA expression levels of β-catenin and wnt-7b were also quantified. Migration MDA-MB-231 cells was determined after treatment with Res or HPIMBD by wound healing assay. Treatment with HPIMBD significantly up-regulated E-cadherin protein expression levels. The up-regulation of E-cadherin by HPIMBD was associated with suppression of snail, slug and Zeb1 protein levels. HPIMBD significantly down-regulated mRNA expression levels of β-catenin and wnt-7b. Furthermore, HPIMBD restricted nuclear migration of β-catenin. Treatment of breast cancer cells with HPIMBD resulted in down-regulation of MMP3 and MMP9 but not MMP2 protein expression levels. Treatment with HPIMBD also prevented breast cancer cell migration. Our studies demonstrtae that HPIMBD decreases the metastatic properties of breast cancer cells by inhibition of EMT and down-regulation of wnt/β catenin pathway. These findings suggest that HPIMBD may have a potential in inhibtion of EMT and may be explored as as a potential therapy to prevent breast cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Anwesha Chatterjee, Amruta Ronghe, Subhash Padhye, Hari K. Bhat. Novel resveratrol-analog HPIMBD inhibits breast cancer cell metastasis by reversal of epithelial-mesenchymal transition. [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 4650. doi:10.1158/1538-7445.AM2015-4650

Abstract 2677: Decrease in phospho-S6 expression under MEK inhibitor (MEKi) treatment as a potential predictive biomarker of response to MEKi alone or in combination with PI3K-mTOR pathway inhibitor in pancreatic adenocarcinoma in vitro and ex vivo models

Abstract Context: Activating KRAS mutations are frequent (>90%) in PDAC and drive downstream deregulation of both MAPK and PI3K-mTOR pathways. MEK inhibitors (MEKi) are under clinical evaluation in PDAC, in combination with other agents including PI3K-mTOR inhibitors. RAS and BRAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition under treatment have been suggested as predictive markers for MEKi, but remain unvalidated in PDAC. We explored the cellular and molecular effects of MEKi GSK1120212 (GSK212) alone or in combination with PI3K-mTOR inhibitors, in PDAC cell lines and ex vivo culture system. Material and methods: GSK212 is an allosteric non-ATP competitive MEKi. Everolimus (Ev) is a mTORC1 inhibitor and BKM120 a pan-class PI3K inhibitor. Effects on proliferation were evaluated by MTT assay. Combinations were analyzed by the Chou-Talalay method. Protein expression was assessed by Western blot. Ex vivo drug evaluation used selected concentrations of drugs on cultures of fresh tissue slices from patient surgical specimens. Apoptosis and proliferation were evaluated by caspase 3 and MIB-1 (Ki67) immunostaining, respectively. Correlations between protein expressions were explored using linear regression. Results: MIAPaCa-2 and PANC-1 are two mesenchymal KRAS mutated/BRAF wild-type PDAC cell lines with very different response to GSK212, MIAPaCa-2 being sensitive (72h-IC50 = 0.009μM) and PANC-1 resistant (72h-IC50 = 33μM). MIAPaCa-2 was more sensitive than PANC-1 to Ev (IC50 = 23.3μM vs 47.0μM) and BKM120 (IC50 = 4.19μM vs 31.6μM). Combination of GSK212 and Ev or BKM120 for 72h resulted in synergistic effects in MIAPaCa-2 sensitive cell line but not in PANC-1. GSK212 (0.1μM) treatment resulted in pERK extinction in both cell lines but in decreased pS6 expression in MIAPaCa-2 sensitive cell line only. Combination therapy resulted in extinction of pERK and pS6 expression in both cell lines. Apoptosis induction in MIAPaCa-2 was confirmed by PARP cleavage from 48h. Two tumor specimens were cultured ex vivo: (a) GSK212 (0.1 μM) treatment for 48h induced apoptosis (caspase 3 expression) concomitantly with a decrease in pS6 expression; (b) Ev (1μM) or BKM120 (0.1μM) exerted antiproliferative effects but did not induce apoptosis; (c) combinations resulted in higher caspase 3 expression associated with a higher decrease in pS6 expression compared to MEKi alone. Linear regression showed significant correlation between caspase 3 and pS6 (R2 = 0.5798, p = 0.0282, and R2 = 0.9423, p = 0.013, for tumor 1 and 2, respectively). Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with “classical” biomarkers of response to MEKi. Decrease in pS6 expression under MEKi treatment may be an early monitoring biomarker of response to MEKi and MEK/mTOR pathway inhibitor combination. Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Jérôme Cros, Pierre Bourgoin, Philippe Bourget, Maria Serova, Armand De Gramont, Sandrine Faivre, Eric Raymond, Philippe Ruszniewski, Pascal Hammel. Decrease in phospho-S6 expression under MEK inhibitor (MEKi) treatment as a potential predictive biomarker of response to MEKi alone or in combination with PI3K-mTOR pathway inhibitor in pancreatic adenocarcinoma in vitro and ex vivo models. [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 2677. doi:10.1158/1538-7445.AM2015-2677

Correlation of decreased pS6 expression under MEK inhibitor (MEKi) with response to MEKi alone or combined with PI3K-mTOR inhibitor (PI3Ki-mTORi) in pancreatic adenocarcinoma (PAC) in vitro and ex vivo models.

e15262 Background: MEKi are under clinical evaluation in combination with other agents including PI3Ki-mTORi. RAS/RAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition during treatment have...

MicroRNA-340 Inhibits Esophageal Cancer Cell Growth and Invasion by Targeting Phosphoserine Aminotransferase 1

Background/Aims: Emerging evidence indicates that microRNA (miR)-340 is downregulated in various human cancers, suggesting that it acts as a tumor suppressor. The aim of the present study was to evaluate the expression and role of miR-340 in human esophageal squamous cell carcinoma (ESCC). Methods: The expression of miR-340 was examined in 64 paired ESCC and adjacent non-tumor tissues by quantitative real time PCR. The effects of miR-340 on ESCC cell proliferation and metastasis were examined by MTT and Matrigel invasion assays. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. Targets of miR-340 were identified by bioinformatics and verified by luciferase reporter assays, quantitative real-time PCR, and western blotting. Results: MiR-340 was significantly downregulated in ESCC tumor tissues compared to adjacent non-tumor tissues and in ESCC cell lines compared to esophageal endothelial cells. Overexpression of miR-340 inhibited ESCC cell growth, colony formation, and invasion, and tumor growth in a xenograft mouse model. PSAT1 was identified as a direct target of miR-340 and its ectopic expression partially reversed the miR-340 mediated inhibition of viability, invasion and EMT in ESCC cells. The expression of miR-340 was negatively correlated with that of PSAT1 in human ESCC samples. Conclusion: MiR-340 functions as a tumor suppressor by modulating the expression of PSAT1 and may contribute to the progression and invasiveness of ESCC.

In vitro inhibitory effects of terpenoids from Chloranthus multistachys on epithelial–mesenchymal transition via down-regulation of Runx2 activation in human breast cancer

From Chloranthus multistachys, three terpenoids – lupeol (1), henrilabdane B (2), and istanbulin A (3) were isolated. Structures of compounds were established by NMR and MS. We reported here that ISTA (3) suppressed cell invasion, but lupeol (1) and henrilabdane B (2) did not. Furthermore, ISTA significantly inhibited the ability of adhesion and migration in vitro. Next, mechanisms of ISTA-induced inhibitory effects on in vitro metastasis were investigated. Sequential treatment data revealed that ISTA dramatically inhibited EGF-induced EMT. Western blot indicated that ISTA also significantly suppressed expression of E-cadherin, vimentin, and slug. In addition, ISTA inhibited Runx2 activation and phosph-Runx2 expression. Collectively, ISTA exhibited significant inhibitory effects on in vitro metastatic potential via inducing EMT inhibition, which may be associated with inhibition of transcriptional activity of Runx2.

Cisplatin sensitivity is enhanced in non-small cell lung cancer cells by regulating epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2.

BackgroundEpithelial-mesenchymal transition (EMT) has been believed to be related with chemotherapy resistance in non-small cell lung cancer (NSCLC). Recent studies have suggested eIF5A-2 may function as a proliferation-related oncogene in tumorigenic processes.MethodsWe used cell viability assays, western blotting, immunofluorescence, transwell-matrigel invasion assay, wound-healing assay combined with GC7 (a novel eIF5A-2 inhibitor) treatment or siRNA interference to investigate the role of eIF5A-2 playing in NSCLC chemotherapy.ResultsWe found low concentrations of GC7 have little effect on NSCLC viability, but could enhance cisplatin cytotoxicity in NSCLC cells. GC7 also could reverse mesenchymal phenotype in NCI-H1299 and prevented A549 cells undergoing EMT after TGF-β1 inducement. eIF5A-2 knockdown resulted in EMT inhibition.ConclusionOur data indicated GC7 enhances cisplatin cytotoxicity and prevents the EMT in NSCLC cells by inhibiting eIF5A-2.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2466-14-174) contains supplementary material, which is available to authorized users.

Network modeling of TGFβ signaling in hepatocellular carcinoma epithelial-to-mesenchymal transition reveals joint sonic hedgehog and Wnt pathway activation.

Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue, and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is TGFβ, which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We have constructed an EMT network of 70 nodes and 135 edges by integrating the signaling pathways involved in developmental EMT and known dysregulations in invasive HCC. We then used discrete dynamic modeling to understand the dynamics of the EMT network driven by TGFβ. Our network model recapitulates known dysregulations during the induction of EMT and predicts the activation of the Wnt and Sonic hedgehog (SHH) signaling pathways during this process. We show, across multiple murine (P2E and P2M) and human HCC cell lines (Huh7, PLC/PRF/5, HLE, and HLF), that the TGFβ signaling axis is a conserved driver of mesenchymal phenotype HCC and confirm that Wnt and SHH signaling are induced in these cell lines. Furthermore, we identify by network analysis eight regulatory feedback motifs that stabilize the EMT process and show that these motifs involve cross-talk among multiple major pathways. Our model will be useful in identifying potential therapeutic targets for the suppression of EMT, invasion, and metastasis in HCC.

Mammary Morphogenesis and Regeneration Require the Inhibition of EMT at Terminal End Buds by Ovol2 Transcriptional Repressor

Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition [EMT]). However, how epithelial plasticity is kept in check in epithelial cells during tissue development and regeneration remains to be fully understood. Here we show that restricting the EMT of mammary epithelial cells by transcription factor Ovol2 is required for proper morphogenesis and regeneration. Deletion of Ovol2 blocks mammary ductal morphogenesis, depletes stem and progenitor cell reservoirs, and leads epithelial cells to undergo EMT in vivo to become nonepithelial cell types. Ovol2 directly represses myriad EMT inducers, and its absence switches response to TGF-β from growth arrest to EMT. Furthermore, forced expression of the repressor isoform of Ovol2 is able to reprogram metastatic breast cancer cells from a mesenchymal to an epithelial state. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity in development and cancer.

Novel PAK4 inhibitors for pancreatic cancer therapy.

233 Background: Pancreatic cancer (PC) is a deadly disease in urgent need of novel molecularly targeted drugs. Gene copy number amplification studies in PC patient cohorts has shown amplification of the p21-activated kinase (PAK) family member PAK4. PAK4 acts as a key effector of the Rho family GTPases downstream of Ras signaling. Moreover, PAK4 protein is over-expressed in PC cell lines but not in normal human pancreatic ductal epithelial (HPDE) cells. Most importantly, RNA interference of PAK4 has been shown to suppress PC cell proliferation. These studies clearly make PAK4 an attractive therapeutic target especially because direct targeting of Kras has been a failure. Methods: We have identified a new class of PAK4 allosteric modulators that show anti-proliferative activity against several PC cell lines (IC50s <250nM) while sparing normal HPDE (IC50s5 fold higher). Results: Cell growth inhibition is concurrent with apoptosis induction and suppression of colony formation in the PC cell lines (and not in HPDE cells). Our small molecule PAK4 allosteric modulator, KPT-7189, suppresses PAK4 protein expression and caused reversal of anti-apoptotic signaling. PAK4 RNA interference enhances KPT-7189 activity, and co-immunoprecipitation experiments showed disruption of PAK4 binding partners. KPT-7189 also inhibited spheroid forming ability of highly resistant PC cells carrying markers of cancer stem cells (CSCs;triple positive for CD33+CD44+EpCAM+) consistent with epithelial-to-mesenchymal (EMT) phenotype. Molecular analyses of KPT-7189 treated CD33+CD44+EpCAM+ spheroids showed suppression of EMT and CSC markers with re-expression of epithelial phenotype markers. Another potent PAK allosteric modulator in the same series, KPT-7651 showing good oral bioavailability (%F = 95%) was well tolerated by mice with a maximum tolerated dose of 60 mg/kg following oral administration. Pre-clinical animal efficacy trial in sub-cutaneous, orthotopic and LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mice model is currently under investigation. Conclusions: This is the first proof of concept study demonstrating the development of a PAK4-targeted drug for the treatment of PC, and thus further pre-clinical and clinical investigations are warranted.

Research advance on mechanism and application of HATs and HDACs in epithelial-mesenchymal transition of lung cancer

肺癌是危害我国人民健康与生命的重大疾病之一, 肺癌的复发和死亡多源于肿瘤转移。上皮细胞的间质化过程(epithelial-mesenchymal transition, EMT)是肺癌转移中的一个关键步骤, 此过程涉及E-cadherin表达下调, 并受到EMT转录因子调控。组蛋白乙酰转移酶(histone acetyltransferases, HATs)和组蛋白去乙酰化酶(histone deacetyltransferases, HDACs)是催化组蛋白乙酰化和去乙酰化的蛋白家族, 不仅在肿瘤进程中发挥重要功能, 近年来发现它们同样参与肺癌EMT过程。HATs与HDACs和某些EMT转录因子有相互作用。而且, 这些EMT转录因子的功能受乙酰化调控, 并影响肺癌EMT进程。本文将分别介绍HATs和HDACs参与肺癌EMT的作用机理, 从分子机制方面对它们之间的相互作用进行探讨, 并讨论HDAC抑制剂在抑制EMT和肺癌治疗方面的潜在应用价值, 以期为相关基础研究和临床实践提供借鉴。

Abstract C71: DEAR1 orchestrates selective control over the epithelial-mesenchymal transition axis of TGFβ's function

Abstract Little is known about the cellular mechanisms that govern the context dependent roles of the critical cytokine TGFβ, which functions in breast cancer as either a tumor suppressor to restrict proliferation in early stage disease or a master driver of epithelial-mesenchymal transition during invasion and metastasis. We previously reported the discovery of the TRIM protein DEAR1(annotated as TRIM62) as a gene that is mutated and homozygously deleted in early onset breast cancers and functions as a dominant regulator of acinar morphogenesis in three dimensional (3D) culture, suggesting that DEAR1 is a novel tumor suppressor that regulates both polarity and tissue architecture. Herein we demonstrate that loss of function of DEAR1 in immortal mammary epithelial cells (HMECs) resulted in a failure of acinar morphogenesis in the presence of TGFβ and was required for TGFβ-induced cell migration, invasion and anoikis resistance, all of which key indicators of TGFB-driven EMT. However, DEAR1 loss did not affect TGFβ-induced proliferation suppression, indicative that DEAR1 exerts a selective negative control over TGFβ-mediated EMT oncogenic functions while preserving TGFβ's growth suppressive functions. These results indicate that the mechanisms governing the dual functions of TGFβ are separable and that it is possible to selectively block the deleterious arm of TGFβ's function. In addition, DEAR1 suppressed TGFβ-SMAD3 signaling by binding to and ubiquitinating SMAD3, the major effector of TGFβ-meditated EMT. Furthermore, treatment of DEAR1 knockdown HMECs with a SMAD3 inhibitor or SMAD3 shRNA rescued the DEAR1 loss of function phenotype as evidence by the downregulation of expression of EMT markers and inhibition of migration induced by TGFβ in strong support that DEAR1 specifically blocks TGFβ-induced EMT through the regulation of SMAD3 protein levels and suggestive that results could have clinical significance for stratifying breast cancers for targeted therapies aimed at the oncogenic arm of this pathway. These data demonstrate a significant role for DEAR1 in the control of TGFβ-induced EMT as well as present a novel paradigm for regulation of one of the most important pathways aberrant in human breast cancer. Citation Format: Nanyue Chen, Ret., Seetharaman Balasenthil, Yu Cao, Ann M. Killary. DEAR1 orchestrates selective control over the epithelial-mesenchymal transition axis of TGFβ's function. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C71.

Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/β-Catenin Signaling and Apoptosis Induction

Honokiol, an active compound of Magnolia officinalis, exerted many anticancer effects on various types of cancer cells. We explored its effects on the elimination of cancer stem-like side population (SP) cells in human oral squamous cell carcinoma SAS cells. The sorted SP cells possessed much higher expression of stemness genes, such as ABCG2, ABCC5, EpCAM, OCT-4, CD133, CD44, and β-catenin, and more clonogenicity as compared with the Non-SP cells. After 48 h of treatment, honokiol dose dependently reduced the proportion of SP from 2.53% to 0.09%. Apoptosis of honokiol-treated SP cells was evidenced by increased annexin V staining and cleaved caspase-3 as well as decreased Survivin and Bcl-2. Mechanistically, honokiol inhibited the CD44 and Wnt/β-catenin signaling of SP cells. The Wnt signaling transducers such as β-catenin and TCF-4 were decreased in honokiol-treated SP cells, while the β-catenin degradation promoting kinase GSK-3α/β was increased. Consistently, the protein levels of β-catenin downstream targets such as c-Myc and Cyclin D1 were also downregulated. Furthermore, the β-catenin-related EMT markers such as Slug and Snail were markedly suppressed by honokiol. Our findings indicate honokiol may be able to eliminate oral cancer stem cells through apoptosis induction, suppression of Wnt/β-catenin signaling, and inhibition of EMT.

Dual Inhibiting Senescence and Epithelial-to-Mesenchymal Transition by Erythropoietin Preserve Tubular Epithelial Cell Regeneration and Ameliorate Renal Fibrosis in Unilateral Ureteral Obstruction

This study aims to investigate the renoprotective effect of recombinant human erythropoietin (rhEPO) treatment could preserve tubular epithelial cell regeneration and ameliorate renal fibrosis by dual inhibition of stress-induced senescence and EMT in unilateral ureteric obstruction (UUO) mouse model. UUO or sham-operated mice were randomly assigned to receive rhEPO or vehicle treatment and were sacrificed on days 3, 7, and 14. Kidney specimens were fixed for histopathological and immunohistochemical study. The expression of S100A4, TGF-β1, BMP-7, Smad2/3, Smad1/5/8, and p16INK4a was determined by western blot and real-time RT-PCR. Vehicle treated UUO mice had increased tubular atrophy and interstitial fibrosis within 3 to 14 days. An increase in TGF-β1, Smad2/3, S100A4, and p16INK4a expression and a decrease in BMP-7 and Smad1/5/8 expression were observed in the obstructed kidneys. p16INK4a was positively correlated with TGF-β1/Smad2/3 and negatively correlated with BMP-7/Smad1/5/8 in UUO mice. rhEPO treatment significantly suppressed the upregulation of TGF-β, Smad2/3, S100A4, and p16INK4a and preserved the downregulation of BMP-7 and Smad1/5/8, resulting in markedly reduced TA/IF compared to the vehicle treated mice. The renoprotective effects of rhEPO could ameliorate renal TA/IF by modulating senescence and EMT which could be a part of therapeutic option in patients with chronic kidney disease.

Knockdown of Musashi-1 Results in Tumor Growth Arrest Through Inhibition of c-MYC, Notch-1 and EMT by Let-7a, Mir-144 and Mir-200a MicroRNAs Dependent Mechanisms Respectively

Knockdown of Musashi-1 Results in Tumor Growth Arrest Through Inhibition of c-MYC, Notch-1 and EMT by Let-7a, Mir-144 and Mir-200a MicroRNAs Dependent Mechanisms Respectively

Abstract 1157: Identification of miRNAs that target the Epithelial-Mesenchymal Transition regulator, Zeb2, using transcriptome PCR arrays

Abstract Epithelial-mesenchymal transition (EMT) is the process of molecular reprogramming from polarized immotile epithelial cells to motile mesenchymal cells and is activated during cancer cell invasion of tissues and metastasis. Zeb2 is a transcription factor that has been implicated in EMT, in part, through decreasing levels of E-cadherin. Loss of E-cadherin leads to cell motility which is required for tumor invasion and metastasis. The miRNA family of miR-200 has been shown to inhibit EMT through direct targeting of Zeb2. Currently, only members of the miR-200 family (miR-200bc/429, miR-200a/141) have been identified to target and decrease the expression of Zeb2. Therefore, to identify additional potential miRNAs that target Zeb2, a reverse genetics approach was used. The SureFind Transcriptome PCR Array is an array of 90 cDNA preparations obtained from HeLa cells that were transfected with various miRNA mimics to simulate overexpression of the mature miRNAs. Using a cancer-focused array, qPCR was performed to quantify the expression of Zeb2 wherein changes in mRNA expression identified specific miRNAs that alter Zeb2 expression. In addition to confirming miR-200c (miR-200ab/141/429 were not on the array) as a regulator of Zeb2, five new miRNAs, not previously predicted to target Zeb2 using bioinformatics or reported in the literature, were identified as targeting Zeb2 mRNA. Four of the miRNAs, Let-7g, miR-122, miR-142-5p, and miR-29b, caused at least a two-fold decrease in Zeb2 expression compared to the negative control, which was from cells transfected with a negative mimic control. In addition one miRNA, miR-34c-5p caused a 2.4 fold increase in Zeb2 expression, identifying it as a potential positive regulator of Zeb2 expression. Next to determine if the identified miRNAs inhibit translation of Zeb2, a reporter consisting of firefly luciferase coupled to the 3’UTR of Zeb2 was used to measure the activity of these miRNAs toward the 3’UTR of Zeb2. Let-7g, miR-142-5p, miR-200c, and miR-29b inhibited luciferase activity by at least 1.5 fold compared to negative mimic control. Interestingly, miR-34c-5p and miR-122 exhibited no statistical difference in luciferase activity compared to the negative control. An explanation for this is that miR-34c-5p and miR-122 do not target the 3’UTR of Zeb2 or that they require contextual cues from full-length RNA for their activity. In conclusion, five novel miRNA/Zeb2 interactions were identified in addition to the previously reported miR-200 family. To date only miR-200 has been implicated in EMT though it is possible that let-7g, miR-122, miR-142-5p and miR-29b maybe involved in inhibition of EMT through downregulation of Zeb2 expression. Additionally, these experiments demonstrated that the SureFIND Transcriptome PCR Arrays identify novel miRNA/gene interactions. This application is for research use only, not diagnostic/clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1157. doi:10.1158/1538-7445.AM2011-1157

MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.MethodsTotal RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.ResultsBased on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.ConclusionsThese results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.