Novel Regulator Of Epithelial-mesenchymal Transition Research Articles

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells.

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

EDIL3 promotes epithelial\u2013mesenchymal transition and paclitaxel resistance through its interaction with integrin \u03b1V\u03b23 in cancer cells

Epithelial–mesenchymal transition (EMT) has recently been associated with tumor progression, metastasis, and chemotherapy resistance in several tumor types. We performed a differential gene expression analysis comparing paclitaxel-resistant vs. paclitaxel-sensitive breast cancer cells that showed the upregulation of EDIL3 (EGF Like Repeats and Discoidin I Like Domains Protein 3). This gene codifies an extracellular matrix protein that has been identified as a novel regulator of EMT, so we studied its role in tumor progression and paclitaxel response. Our results demonstrated that EDIL3 expression levels were increased in paclitaxel-resistant breast and prostate cancer cells, and in subsets of high-grade breast and prostate tumors. Moreover, we observed that EDIL3 modulated the expression of EMT markers and this was impaired by cilengitide, which blocks the EDIL3–integrin αVβ3 interaction. EDIL3 knockdown reverted EMT and sensitized cells to paclitaxel. In contrast, EDIL3 overexpression or the culture of cells in the presence of EDIL3-enriched medium induced EMT and paclitaxel resistance. Adding cilengitide resensitized these cells to paclitaxel treatment. In summary, EDIL3 may contribute to EMT and paclitaxel resistance through autocrine or paracrine signaling in cancer cells. Blockade of EDIL3–integrin αVβ3 interaction by cilengitide restores sensitivity to paclitaxel and reverts EMT in paclitaxel-resistant cancer cells. Combinations of cilengitide and taxanes could be beneficial in the treatment of subsets of breast and prostate cancers.

Transcriptomic and computational analysis identified LPA metabolism, KLHL14 and KCNE3 as novel regulators of Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a complex biological program between physiology and pathology. Here, amniotic epithelial cells (AEC) were used as in vitro model of transiently inducible EMT in order to evaluate the transcriptional insights underlying this process. Therefore, RNA-seq was used to identify the differentially expressed genes and enrichment analyses were carried out to assess the intracellular pathways involved. As a result, molecules exclusively expressed in AEC that experienced EMT (GSTA1-1 and GSTM3) or when this process is inhibited (KLHL14 and KCNE3) were identified. Lastly, the network theory was used to obtain a computational model able to recognize putative controller genes involved in the induction and in the prevention of EMT. The results suggested an opposite role of lysophosphatidic acid (LPA) synthesis and degradation enzymes in the regulation of EMT process. In conclusion, these molecules may represent novel EMT regulators and also targets for developing new therapeutic strategies.

GCNT2 induces epithelial-mesenchymal transition and promotes migration and invasion in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in the world. The prognosis of patients with ESCC is dismal with a 5-year survival of about 15%. Thus, identification of novel diagnostic and prognostic biomarkers for ESCC patients is urgently needed. Here, we found that manipulation of I-branching N-acetylglucosaminyltransferase (GCNT2) expression had no effect on cell proliferation. Notably, overexpression of GCNT2 promoted the migration and invasion, and this effect was associated with increased expression of N-cadherin and vimentin and decreased expression of E-cadherin in KYSE30 and EC9706 cells. Knockdown of GCNT2 decreased the expression of N-cadherin and vimentin, increased the expression of E-cadherin, and inhibited the migration and invasion in KYSE150 and EC109 cells. The expression of GCNT2 was significantly higher in tumour tissues than in paratumour tissues through tissue microarray analysis. More importantly, overall survival was significantly lower in patients with high GCNT2 expression than those with low GCNT2 expression. Collectively, our findings establish GCNT2 as a novel regulator of epithelial-mesenchymal transition (EMT) and a candidate prognostic indicator of outcome in ESCC patients. SIGNIFICANCE OF THE STUDY: Our study suggested that GCNT2 was highly expressed in patients with ESCC and predicted adverse outcome. Overexpression of GCNT2 induces EMT and promotes migration and invasion in ESCC cells. Therefore, GCNT2 may act as a candidate prognostic indicator of outcome and a novel target in ESCC patients.

N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.

N-Glycans are involved in numerous biologic processes, such as cell adhesion, migration, and invasion. To distinguish the functions of complex high-mannose types of N-glycans, we used the clustered, regularly interspaced, short palindromic repeats/Cas9 system to establish N-acetylglucosaminyltransferase (GnT)-I-knockout (KO) cells. Loss of GnT-I greatly induced cell-cell adhesion and decreased cell migration. In addition, the expression levels of epithelial-mesenchymal transition (EMT) markers such as α-SMA, vimentin, and N-cadherin were suppressed, whereas the expression of claudin-1 was promoted, suggesting a mesenchymal-epithelial transition-like phenotype, an opposite process to the EMT, was occurred in the KO cells. The phosphorylation levels of Smad-2, epidermal growth factor receptor, and integrin-mediated focal adhesion kinase (FAK) were consistently suppressed. Furthermore, the restoration of GnT-I in the KO cells suppressed the cell-cell adhesion and augmented the expression of EMT markers as well as that of FAK activation. The expression levels of integrins were upregulated in the KO cells, although their functions were decreased, whereas their expression levels were downregulated in the rescued cells, which suggests a negative feedback loop between function and expression. Finally, we also found that the expression of GnT-I was important for cell survival, resistance to cancer drugs, and increased colony formation. The results of the present study demonstrate that GnT-I works as a switch to turn on/off EMT, which further supports the notion that on most surface receptors, the N-glycans differentially play essential roles in biologic functions.-Zhang, G., Isaji, T., Xu, Z., Lu, X., Fukuda, T., Gu, J. N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.

Sex-determining region Y-box protein 3 induces epithelial-mesenchymal transition in osteosarcoma cells via transcriptional activation of Snail1

BackgroundThe transcription factor sex-determining region Y-box protein 3 (SOX3) plays important roles in various types of cancer. However, its expression and function have not yet been elucidated in osteosarcoma (OS).MethodsThe expression levels of SOX3 in OS tissues and OS cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effects of SOX3 expression on OS cell biological traits were investigated by overexpressing and downregulating SOX3 protein. The expression of epithelial-mesenchymal transition (EMT) markers and transcription factors associated with EMT (EMT-TFs), were detected simultaneously. The mechanism underlying SOX3-mediated Snail1 expression was further investigated.ResultsSOX3 was upregulated in human OS tissues. SOX3 overexpression promoted the EMT, migration and invasion in OS cells. The downregulation of SOX3 resulted in opposing effects. Furthermore, SOX3 upregulation enhanced the expression of the transcriptional repressor Snail1 by binding to its promoter region. Additionally, a positive correlation among the expression of SOX3, Snail1, and E-cadherin was demonstrated in human OS tissues.ConclusionsSOX3 promotes migration, invasiveness, and EMT in OS cells via transcriptional activation of Snail1 expression, suggesting that SOX3 is a novel regulator of EMT in OS and may serve as a therapeutic target for the treatment of OS metastasis.

XRN2 promotes EMT and metastasis through regulating maturation of miR-10a.

MicroRNAs (miRNAs) have been proposed as critical regulatory molecules in the epithelial-mesenchymal transition (EMT) program. However, the roles of mature miRNA biogenesis during EMT process needs to be defined. Here we determined that increased expression of XRN2 induced EMT and promoted metastasis in vitro and in vivo. Furthermore, we uncovered that XRN2 functions as pro-metastatic gene, which accelerates miR-10a maturation by binding pre-miR-10a in a DICER-independent manner. These findings suggest that XRN2 is a novel regulator of EMT that contributes to the metastatic processes in lung cancer through a novel miRNA regulatory mechanism.

Abstract B22: Novel dual regulation of epithelial-mesenchymal transition and anoikis resistance prevents metastases of triple negative breast cancer

Abstract Triple negative breast cancers (TNBC) are associated with a higher rate of metastatic recurrence and poorer prognosis due to intrinsic biological aggressiveness and lack of targeted therapies. Novel strategies to prevent metastasis are needed to improve outcomes for patients diagnosed with TNBC. Epithelial-mesenchymal transition (EMT) and anoikis resistance are processes recognized as contributing to treatment resistance and enhanced metastatic potential. In a functional genomic screen, we identified several candidates as novel dual regulators of EMT and anoikis sensitivity of triple negative breast cancer cells, that can be pharmacologically targeted to prevent metastases. One highly validated candidate, PTK6/Brk, specifically enhances anoikis resistance and metastatic potential of triple negative breast cancer cells via E-cadherin regulation. Overexpression of PTK6 in non-transformed immortalized breast epithelial cells is sufficient to promote an EMT, whereas PTK6 inhibition (either PTK6 shRNA or kinase inhibitor treatment) enhances E-cadherin expression and suppresses migration and lung colonization of TNBC cells. PTK6-dependent E-cadherin regulation and anoikis resistance is specifically dependent on levels of SNAIL, a transcriptional repressor. SNAIL down-regulation by PTK6 inhibition is directly responsible for modulation of anoikis sensitivity, which is in turn causally linked to lung colonization potential. PTK6 inhibition promotes the degradation of SNAIL via a novel mechanism independent of GSK3β and Fbox proteins known to regulate Snail ubiquitination. Using an siRNA screening approach, we identified novel E3 ligase candidates responsible for SNAIL ubiquitination and degradation downstream of PTK6 inhibition. PTK6 is a representative novel regulator of EMT and anoikis resistance that can be targeted to prevent metastases of patient triple negative tumors. Citation Format: Koichi Ito, Sun Hee Park, Anupma Nayak, Hanna Y. Irie. Novel dual regulation of epithelial-mesenchymal transition and anoikis resistance prevents metastases of triple negative breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B22.

Abstract B34: CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression

Abstract The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by epithelial cancer cells. To identify novel regulators of EMT, we carried out cDNA screens that covered 500 human kinases. Subsequent characterization of candidate kinases led us to uncover cyclin-dependent kinase-like 2 (CDKL2) as a novel potent promoter for EMT and breast cancer progression. CDKL2-expressing human mammary gland epithelial cells displayed enhanced mesenchymal traits and stem cell-like phenotypes, which was acquired through activating a ZEB1/E-cadherin/beta-catenin positive feedback loop and regulating CD44 mRNA alternative splicing to promote conversion of CD24-high cells to CD44-high cells. Furthermore, CDKL2 enhanced primary tumor formation and metastasis in a breast cancer xenograft model. Notably, CDKL2 is expressed significantly higher in mesenchymal human breast cancer cell lines than in epithelial lines, and its over-expression/amplification in human breast cancers is associated with shorter disease-free survival. Taken together, our study uncovered a major role for CDKL2 in promoting EMT and breast cancer progression. Citation Format: Linna Li, Mohit Hulsurkar, Robert Amato, Jeffrey Chang, Guangwei Du, Wenliang Li. CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B34.

MicroRNA-181a regulates epithelial-mesenchymal transition by targeting PTEN in drug-resistant lung adenocarcinoma cells.

Chemoresistance is an inevitable occurrence in lung adenocarcinoma, which has been associated with decreased expression of the phosphatase and tensin homolog deleted on chromosome ten (PTEN). Therefore, it is important to identify novel molecular mechanisms to suppress chemoresistance in lung adenocarcinoma cells. Paclitaxel- and cisplatin-resistant A549 lung carcinoma cell derivatives were developed by long-term serial culture. The metastatic properties of the cells were assessed using wound-healing assays, migration assays, invasion assays, morphological examination, and western blot analysis/RT-PCR of genes associated with the epithelial-mesenchymal transition (EMT). To identify novel regulators of EMT in A549 cells, differentially expressed miRNAs in drug-resistant cells were identified by microarray analysis. The role of miR-181a was established by transfection with specific mimic and inhibitor followed by functional assays. Luciferase assays were performed to assess the ability of miR-181a to target the PTEN promoter, and regulation of PTEN expression by miR-181a was assessed by western blot analysis and RT-PCR. Paclitaxel- and cisplatin-resistant A549 cells acquired metastatic properties and EMT phenotype and had reduced PTEN expression as compared to sensitive cells. miR‑181a was identified as a differentially expressed miRNA in drug-resistant A549 cells, and miR-181a mimic and inhibitor were shown to affect migration, invasion, morphology and expression of EMT-associated genes. PTEN was identified as a direct target of miR-181a. Our findings demonstrate that miR-181a expression in lung adenocarcinoma is associated with EMT progression, potentially through targeting of PTEN. Regulation of miR-181a may provide a novel strategy for overcoming resistance to paclitaxel and cisplatin in lung adenocarcinoma.

Abstract 1433: CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression

Abstract The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by epithelial cancer cells. To identify novel regulators of EMT, we carried out cDNA screens that covered 500 human kinases. Subsequent characterization of candidate kinases led us to uncover cyclin-dependent kinase-like 2 (CDKL2) as a novel potent promoter for EMT and breast cancer progression. CDKL2-expressing human mammary gland epithelial cells displayed enhanced mesenchymal traits and stem cell-like phenotypes, which was acquired through activating a ZEB1/E-cadherin/beta-catenin positive feedback loop and regulating CD44 mRNA alternative splicing to promote conversion of CD24-high cells to CD44-high cells. Furthermore, CDKL2 enhanced primary tumor formation and metastasis in a breast cancer xenograft model. Notably, CDKL2 is expressed significantly higher in mesenchymal human breast cancer cell lines than in epithelial lines, and its over-expression/amplification in human breast cancers is associated with shorter disease-free survival. Taken together, our study uncovered a major role for CDKL2 in promoting EMT and breast cancer progression. Citation Format: Linna Li, Jeffrey Chang, Guangwei Du, Robert Amato, Wenliang Li. CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression. [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 1433. doi:10.1158/1538-7445.AM2015-1433

Abstract 1432: Role of ubiquitin-specific proteases 47 in epithelial-mesenchymal transition

Abstract Metastatic spread from primary tumor is the last phase of cancer development. Approximately 90% of deaths from cancer are caused by metastasis. During the transition from a benign tumor to a metastatic one, tumor cells lose cell-cell contacts and gain cell motility for invasion. This is called epithelial-mesenchymal transition (EMT). EMT is considered to be a critical contributor of cancer metastasis. Hypoxia, which plays a critical role in development and tumor progression, orchestrates EMT through the coordinated regulation of different EMT regulators. In this study, we found that a series of ubiquitin specific proteases (USP), particularly USP47, were induced in human colon cancer cells (DLD-1, HT-29 and SW-480) under the hypoxic condition based on cDNA microarray data. USPs are a subfamily of deubiquitinating enzymes (DUB) that primarily belong to the cysteine protease family and are known to remove the monoubiquitin and polyubiquitin chains from specific target proteins. Knockdown of USP47 substantially decreased expression of mesenchymal markers. Furthermore, hypoxia-inducible factor (HIF), especially HIF-2α, was found to regulate transcription of USP47. These results suggest USP47 as a novel regulator of EMT under hypoxic conditions. Citation Format: Bae-Jung Choi, Young-Joon Surh. Role of ubiquitin-specific proteases 47 in 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 1432. doi:10.1158/1538-7445.AM2015-1432

EDIL3 is a novel regulator of epithelial-mesenchymal transition controlling early recurrence of hepatocellular carcinoma

Patients with advanced hepatocellular carcinoma (HCC) continue to have a dismal prognosis. Early recurrence, metastases and angiogenesis are the major obstacles to improve the outcome of HCC. Epithelial-mesenchymal transition (EMT) is a key contributor to cancer metastasis and recurrence, which are the major obstacles to improve prognosis of HCC. Combining gene expression profiles of HCC samples with or without early recurrence and established cell lines with epithelial or mesenchymal phenotype, EDIL3 was identified as a novel regulator of EMT. The expression of EDIL3 was evaluated by quantitative PCR, Western blotting or immunohistochemistry. The effects of EDIL3 on the angiogenesis and metastasis of HCC cells were examined by wound healing, Matrigel invasion and tube formation assay in vitro and orthotopic xenograft mouse model of HCC in vivo. The signaling pathways of EDIL3 mediated were investigated through microarray and Western blotting analysis. EDIL3 was identified as a novel regulator of EMT, which contributes to angiogenesis, metastasis and recurrence of HCC. EDIL3 induces EMT and promotes HCC migration, invasion and angiogenesis in vitro. Mechanistically, overexpression of EDIL3, which was regulated by the downregulation of miR-137 in HCC, triggered the activation of ERK and TGF-β signaling through interactions with αvβ3 integrin. Blocking ERK and TGF-β signaling overcomes EDIL3 induced angiogenesis and invasion. Using the orthotopic xenograft mouse model of HCC, we demonstrated that EDIL3 enhanced the tumorigenic, metastatic and angiogenesis potential of HCC in vivo. EDIL3-mediated activation of TGF-β and ERK signaling could provide therapeutic implications for HCC.

PRMT1 Is a Novel Regulator of Epithelial-Mesenchymal-Transition in Non-small Cell Lung Cancer

Protein arginine methyl transferase 1 (PRMT1) was shown to be up-regulated in cancers and important for cancer cell proliferation. However, the role of PRMT1 in lung cancer progression and metastasis remains incompletely understood. In the present study, we show that PRMT1 is an important regulator of epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion, which are essential processes during cancer progression, and metastasis. Additionally, we have identified Twist1, a basic helix-loop-helix transcription factor and a well-known E-cadherin repressor, as a novel PRMT1 substrate. Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique "methyl arginine mark" for active E-cadherin repression. Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs. Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer.

SOX5 promotes epithelial-mesenchymal transition and cell invasion via regulation of Twist1 in hepatocellular carcinoma.

The transcription factor sex determining region Y-box protein 5 (SOX5) plays important roles in various types of cancers. However, the expression and function of SOX5 in hepatocellular carcinoma (HCC) have not been elucidated. Here, we found that SOX5 is significantly up-regulated in HCC tissues and cell lines. Gain- and loss-of-function studies demonstrated that SOX5 promoted HCC cell migration and invasion. In addition, we revealed that SOX5 is linked to epithelial-mesenchymal transition (EMT) by regulation of Twist1. Our results indicate for the first time that SOX5 is a novel regulator of EMT in HCC and may be a potential therapeutic target for HCC metastasis.

Kisspeptin-10 inhibits the migration of breast cancer cells by regulating epithelial-mesenchymal transition.

Breast cancer is the leading cause of cancer-related death in women. Kisspeptin-10 (KP-10) is a shorter fragment of KISS1. In the present study, we demonstrated the antitumor effects of KP-10 on human breast cancer cell lines, MDA-MB-231 and MDA-MB-157, both in vitro and in vivo. KP-10 was observed to induce apoptosis and inhibit the mobility of MDA-MB-231 and MDA-MB-157 cells. Correspondingly, KP-10 suppressed tumor growth in established xenograft tumor models and improved the survival rate of tumor-bearing mice. The formation of intratumoral microvessels was inhibited following treatment with KP-10. Finally, we confirmed that KP-10 inhibited cell mobility via epithelial-mesenchymal transition (EMT). Overall, the present study demonstrated that KP-10 suppressed breast cancer and human umbilical vein endothelial cell (HUVEC) growth both in vivo and in vitro. KP-10 is a novel regulator of EMT in breast cancer cells. However, additional studies are needed to confirm these results in other cell types.

Protocadherin 9 inhibits epithelial–mesenchymal transition and cell migration through activating GSK-3β in hepatocellular carcinoma

Protocadherin 9 (PCDH9) was found frequently lost in hepatocellular carcinoma (HCC). Here we investigated the role of PCDH9 in the development of HCC. We confirmed that PCDH9 was down-regulated in HCC tissues and cell lines compared with the adjacent non-tumor tissues. PCDH9 downregulation was significantly associated with malignant portal vein invasion of HCC patients. Gain- and loss-of-function studies revealed that downregulation of PCDH9 facilitated tumor cell migration and epithelial–mesenchymal transition (EMT). We identified PCDH9 as a novel regulator of EMT by increasing the activity of GSK-3β and inhibiting Snail1, indicating its potential therapeutic value for reducing metastasis of HCC.

Effects of microRNA-30a on migration, invasion and prognosis of hepatocellular carcinoma.

The role of microRNA-30a (miR-30a) deregulation in tumor progression and its downstream signaling pathways remain unknown. Here we confirmed significant downregulation of miR-30a in hepatocellular carcinoma (HCC) tissues and cell lines compared with non-tumor counterparts. MiR-30a downregulation was significantly associated with worse disease-free survival (DFS) of HCC patients. Gain- and loss-of-function studies revealed that downregulation of miR-30a facilitated tumor cell migration, invasion and epithelial-mesenchymal transition (EMT). We identified SNAI1 as a direct target of miR-30a and demonstrated miR-30a as a novel regulator of EMT by targeting SNAI1, indicating its potential therapeutic value for reducing invasion and metastasis of HCC.

Abstract A160: Characterization of novel regulators for epithelial-mesenchymal transition.

Abstract The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits of breast cancer cells. Recently, a few kinases, such as FYN and c-Met, were reported to participate in regulating EMT. However, the roles of the majority of >700 human kinases in EMT and breast cancer progression are still unknown. Here we carried out human kinase cDNA screens to identify novel regulators of EMT. Many unknown EMT regulators and a few known ones such as PI3K, PDGFRA, FYN and c-Met, were found to up-regulate mesenchymal marker vimentin. Some also increase expression of fibronectin and N-cadherin, as well as EMT transcriptional regulators, such as ZEB1/2, Snail and Slug. At the same time, the cells acquired mesenchymal phenotypes, such as increased migration/invasion activity, enhanced capability to survive and proliferate in growth factor deprivation condition or under drug treatments. Interestingly, FACS analysis also showed a substantial increase in CD44hiCD24low population in kinase-transducted mammary gland epithelial cells. The increase of CD44hi population could be attributed to increase of a CD44 mesenchymal variant - the standard form CD44s and decrease of the epithelial variant CD44 v8-10. And this alternation was associated with down-regulation of ESRP1 and ESRP2, two splicing factors that regulate CD44 splicing and EMT. Furthermore, the mammary gland epithelial cells undergoing EMT showed increase capability of forming mammosphere and differentiating into other lineages, such as osteoblast and adipocyte. Importantly, some of these novel EMT regulators are associated with TNBC, stemness and poor prognostics of human breast cancers. One novel regulator, a poorly studied serine/threonine protein kinase, was tested in vivo and found to be capable of generating primary tumor and metastasis at much higher efficiency. Further study of these novel EMT regulators promises to provide new mechanisms of breast cancer progression, and may also lead to development of new prognostic markers and therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A160. Citation Format: Linna Li, Emily Lu, Jeffrey Chang, Wenliang Li. Characterization of novel regulators for epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A160.

Role of fucosyltransferase IV in epithelial–mesenchymal transition in breast cancer cells

Epithelial–mesenchymal transition (EMT) is a crucial step in tumor progression and has an important role during cancer invasion and metastasis. Although fucosyltransferase IV (FUT4) has been implicated in the modulation of cell migration, invasion and cancer metastasis, its role during EMT is unclear. This study explores the molecular mechanisms of the involvement of FUT4 in EMT in breast cancer cells. Breast cancer cell lines display increased expression of FUT4, which is accompanied by enhanced appearance of the mesenchymal phenotype and which can be reversed by knockdown of endogenous FUT4. Moreover, FUT4 induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt, and inactivation of GSK3β and nuclear translocation of NF-κB, resulting in increased Snail and MMP-9 expression and greater cell motility. Taken together, these findings indicate that FUT4 has a role in EMT through activation of the PI3K/Akt and NF-κB signaling systems, which induce the key mediators Snail and MMP-9 and facilitate the acquisition of a mesenchymal phenotype. Our findings support the possibility that FUT4 is a novel regulator of EMT in breast cancer cells and a promising target for cancer therapy.