Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

Epithelial–mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin–vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.     

Detecting and targeting mesenchymal-like subpopulations within squamous cell carcinomas

Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44⁺CD24⁻ stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.Key words: EMT, squamous cell carcinoma, head and neck cancer, esophageal cancer, chemotherapy resistance, salinomycin, tumor heterogeneity     

Triclosan Potentiates Epithelial-To-Mesenchymal Transition in Anoikis-Resistant Human Lung Cancer Cells

Alteration of cancer cell toward mesenchymal phenotype has been shown to potentiate tumor aggressiveness by increasing cancer cell metastasis. Herein, we report the effect of triclosan, a widely used antibacterial agent found in many daily products, in enhancing the epithelial-to-mesenchymal transition (EMT) in aggressive anoikis resistant human H460 lung cancer cells. EMT has been long known to increase abilities of the cells to increase migration, invasion, and survival in circulating system. The present study reveals that treatment of the cancer cells with triclosan at the physiologically related concentrations significantly increased the colony number of the cancer cells assessed by tumor formation assay. Also, the mesenchymal-like morphology and decrease in cell-to-cell adhesion were observed in triclosan-treated cells. Importantly, western blot analysis revealed that triclosan-treated cells exhibited decreased E-cadherin, while the levels of EMT markers, namely N-cadherin, vimentin, snail and slug were found to be significantly up-regulated. Furthermore, EMT induced by triclosan treatment was accompanied by the activation of focal adhesion kinase/ATP dependent tyrosine kinase (FAK/Akt) and Ras-related C3 botulinum toxin substrate 1 (Rac1), which enhanced the ability of the cells to migrate and invade. In conclusion, we demonstrated for the first time that triclosan may potentiate cancer cells survival in detached condition and motility via the process of EMT. As mentioned capabilities are required for success in metastasis, the present study provides the novel toxicological information and encourages the awareness of triclosan use in cancer patients.     

SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM⁻) breast cancer cells to an epithelial EpCAM^+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM⁻ breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.     

Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells

The role of estrogen in the motility and invasion of breast cancer cells is controversial. Although estrogen receptor (ER)-positive breast tumors are considered less aggressive and more differentiated they still undergo metastasis. In many types of epithelial cancers, the ability to undergo metastasis has been associated with a loss of epithelial features and acquisition of mesenchymal properties leading to migration of individual cells, a process known as epithelial-to-mesenchymal transition (EMT). In this report, we show that a subset of ER-positive breast cancer cells can acquire mesenchymal-like features and motility in a reversible manner. In MCF-7 breast cancer cells estrogen-promoted acquisition of mesenchymal-like features while antiestrogens, such as tamoxifen, prevented this transition. Moreover, pharmacological inhibition of Src family kinases decreased the ability of estrogen to promote epithelial-to-mesenchymal-like transition. In addition to mesenchymal-like motility, a subset of estrogen-treated cells also moved as cell clusters (collective motility). While membrane localization of E-cadherin/beta-catenin was decreased in fibroblast-like cells, enhanced levels of E-cadherin/beta-catenin were detected in motile cell clusters. Thus, during tumor progression, estrogen may foster motility and invasion of ER-positive breast cancer by promoting simultaneously reversible EMT-like changes and collective motility. These studies suggest that antiestrogen therapy and Src family kinase inhibitors may decrease development of metastases in ER-positive breast cancer by blocking estrogen-dependent migration of human breast cancer cells.     

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial-mesenchymal transition

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through EGFR mutations, c-MET amplification, or epithelial-mesenchymal transition (EMT). It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here we examined whether gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 (TM4SF5), which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinib-resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell-cell contacts, and gefitinib-resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinib-resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib-sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. Together, these results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells.     

EMT impairs breast carcinoma cell susceptibility to CTL-mediated lysis through autophagy induction

Epithelial to mesenchymal transition (EMT) has become one of the most exciting fields in cancer biology. While its role in cancer cell invasion, metastasis and drug resistance is well established, the molecular basis of EMT-induced immune escape remains unknown. We recently reported that EMT coordinately regulates target cell recognition and sensitivity to specific lysis. In addition to the well-characterized role for EMT in tumor phenotypic change including a tumor-initiating cell phenotype, we provided evidence indicating that EMT-induced tumor cell resistance to cytotoxic T-lymphocytes (CTLs) also correlates with autophagy induction. Silencing of BECN1 in target cells that have gone through the EMT restored CTL susceptibility to CTL-induced lysis. Although EMT may represent a critical target for the development of novel immunotherapy approaches, a more detailed understanding of the inter-relationship between EMT and autophagy and their reciprocal regulation will be a key determinant in the rational approach to future tumor immunotherapy design.     

Activation of Notch-1 enhances epithelial-mesenchymal transition in gefitinib-acquired resistant lung cancer cells

Despite an initial response to EGFR tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer, most patients eventually become resistant and result in treatment failure. Recent studies have shown that epithelial to mesenchymal transition (EMT) is associated with drug resistance and cancer cell metastasis. Strong multiple gene signature data indicate that EMT acts as a determinant of insensitivity to EGFR-TKI. However, the exact mechanism for the acquisition of the EMT phenotype in EGFR-TKI resistant lung cancer cells remains unclear. In the present study, we showed that the expression of Notch-1 was highly upregulated in gefitinib-resistant PC9/AB2 lung cancer cells. Notch-1 receptor intracellular domain (N1IC), the activated form of the Notch-1 receptor, promoted the EMT phenotype in PC9 cells. Silencing of Notch-1 using siRNA reversed the EMT phenotype and restored sensitivity to gefitinib in PC9/AB2 cells. Moreover, Notch-1 reduction was also involved in inhibition of anoikis as well as colony-formation activity of PC9/AB2 cells. Taken together, these results provide strong molecular evidence that gefitinib-acquired resistance in lung cancer cells undergoing EMT occurs through activation of Notch-1 signaling. Thus, inhibition of Notch-1 can be a novel strategy for the reversal of the EMT phenotype thereby potentially increasing therapeutic drug sensitivity to lung cancer cells.     

Intratumoral heterogeneity: Clonal cooperation in epithelial-to-mesenchymal transition and metastasis

Although phenotypic intratumoral heterogeneity was first described many decades ago, the advent of next-generation sequencing has provided conclusive evidence that in addition to phenotypic diversity, significant genotypic diversity exists within tumors. Tumor heterogeneity likely arises both from clonal expansions, as well as from differentiation hierarchies existent in the tumor, such as that established by cancer stem cells (CSCs) and non-CSCs. These differentiation hierarchies may arise due to genetic mutations, epigenetic alterations, or microenvironmental influences. An additional differentiation hierarchy within epithelial tumors may arise when only a few tumor cells trans-differentiate into mesenchymal-like cells, a process known as epithelial-to-mesenchymal transition (EMT). Again, this process can be influenced by both genetic and non-genetic factors. In this review we discuss the evidence for clonal interaction and cooperation for tumor maintenance and progression, particularly with respect to EMT, and further address the far-reaching effects that tumor heterogeneity may have on cancer therapy.     

SET-mediated NDRG1 inhibition is involved in acquisition of epithelial-to-mesenchymal transition phenotype and cisplatin resistance in human lung cancer cell

Development of resistance to therapy continues to be a serious clinical problem in lung cancer management. Cancer cells undergoing epithelial-to-mesenchymal transition (EMT) have been shown to play roles in resistance to chemotherapy. Here, we utilized a proteomics-based method and identified a significant downregulation of the metastasis suppressor NDRG1 in drug resistant lung cancer cells. We showed that downregulation of DNRG1 constitutes a mechanism for acquisition of EMT phenotype and endows lung cancer cells with an increased resistance to cisplatin. We also identified a signal cascade, namely, SET---| PP2A---| c-myc---| NDRG1, in which upregulation of SET is critical for inhibition of NDRG1. We also found that blockade of SET (or reactivation of PP2A) by FTY720 reverted EMT, restored drug sensitivity, and inhibited invasiveness and growth of lung tumor xenografts. Together, our results indicated a functional link between SET-mediated NDRG1 regulation and acquisition of EMT phenotype and drug resistance, and provided an evidence that blockade of SET-driven EMT can overcome drug resistance and inhibit tumor progression.     

YAP参与调控细胞上皮-间充质转化的研究进展

上皮-间充质转化(epithelial-mesenchymal transition,EMT)是上皮来源细胞在各种理化因素作用下经历表型转化获得间充质样细胞表型的过程.研究表明,有多种信号分子参与EMT的发生,并在胚胎发育、器官损伤修复和肿瘤的发生发展过程中起着关键作用.Yes相关蛋白(yes-associated protein,YAP)作为Hippo信号通路的下游效应分子,被广泛报道参与EMT的进程,调控多种基因的表达,起到调节细胞增殖、凋亡、器官发育和修复等作用.最新研究表明,YAP活性的变化直接介导肿瘤细胞的迁移和侵袭等能力的变化,而这些变化都伴随着EMT的发生.因此,YAP蛋白跟EMT的发生密切相关.本文就近年来关于YAP调控组织发育、器官纤维化及在肿瘤发生发展中的作用,以及相关分子机制的研究进行综述,并将阐明其与EMT之间的相互关系,以期为EMT的研究提供新的视角,进而为相关疾病的治疗提供新的分子靶点和诊断治疗策略.     

Hypoxia,partial EMT and collective migration: Emerging culprits in metastasis

Epithelial-mesenchymal transition (EMT) is a cellular biological process involved in migration of primary cancer cells to secondary sites facilitating metastasis. Besides, EMT also confers properties such as stemness, drug resistance and immune evasion which can aid a successful colonization at the distant site. EMT is not a binary process; recent evidence suggests that cells in partial EMT or hybrid E/M phenotype(s) can have enhanced stemness and drug resistance as compared to those undergoing a complete EMT. Moreover, partial EMT enables collective migration of cells as clusters of circulating tumor cells or emboli, further endorsing that cells in hybrid E/M phenotypes may be the ‘fittest’ for metastasis. Here, we review mechanisms and implications of hybrid E/M phenotypes, including their reported association with hypoxia. Hypoxia-driven activation of HIF-1α can drive EMT. In addition, cyclic hypoxia, as compared to acute or chronic hypoxia, shows the highest levels of active HIF-1α and can augment cancer aggressiveness to a greater extent, including enriching for a partial EMT phenotype. We also discuss how metastasis is influenced by hypoxia, partial EMT and collective cell migration, and call for a better understanding of interconnections among these mechanisms. We discuss the known regulators of hypoxia, hybrid EMT and collective cell migration and highlight the gaps which needs to be filled for connecting these three axes which will increase our understanding of dynamics of metastasis and help control it more effectively.     

Multiple roles for basement membrane proteins in cancer progression and EMT

Metastasis or the progression of malignancy poses a major challenge in cancer therapy and is the principal reason for increased mortality. The epithelial-mesenchymal transition (EMT) of the basement membrane (BM) allows cells of epithelial phenotype to transform into a mesenchymal-like (quasi-mesenchymal) phenotype and metastasize via the lymphovascular system through a metastatic cascade by intravasation and extravasation. This helps in the progression of carcinoma from the primary site to distant organs. Collagen, laminin, and integrin are the prime components of BM and help in tumor cell metastasis, which makes them ideal cancer drug targets. Further, recent studies have shown that collagen, laminin, and integrin can be used as a biomarker for metastatic cells. In this review, we have summarized the current knowledge of such therapeutics, which are either currently in preclinical or clinical stages and could be promising cancer therapeutics.Data availabilityNot applicable     

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.     

HOX cluster-embedded lncRNAs and epithelial-mesenchymal transition in cancer: Molecular mechanisms and therapeutic opportunities

Although there has been substantial improvement in the treatment modalities, cancer remains the major cause of fatality worldwide. Metastasis, recurrence, and resistance to oncological therapies are the leading causes of cancer mortality. Epithelial-mesenchymal transition (EMT) is a complex biological process that allows cancer cells to undergo morphological transformation into a mesenchymal phenotype to acquire invasive potential. It encompasses reversible and dynamic ontogenesis by neoplastic cells during metastatic dissemination. Hence, understanding the molecular landscape of EMT is imperative to identify a reliable clinical biomarker to combat metastatic spread. Accumulating evidence reveals the role of HOX (homeobox) cluster-embedded long non-coding RNAs (lncRNAs) in EMT and cancer metastasis. They play a crucial role in the induction of EMT, modulating diverse biological targets. The present review emphasizes the involvement of HOX cluster-embedded lncRNAs in EMT as a molecular sponge, chromatin remodeler, signaling regulator, and immune system modulator. Furthermore, the molecular mechanisms behind therapy resistance and the potential use of novel drugs targeting HOX cluster-embedded lncRNAs in the clinical management of distant metastasis will be discussed.     

CCN5/WISP-2: A micromanager of breast cancer progression

The gain of plasticity by a subset of cancer cells is a unique but common sequence of cancer progression from epithelial phenotype to mesenchymal phenotype (EMT) that is followed by migration, invasion and metastasis to a distant organ, and drug resistance. Despite multiple studies, it is still unclear how cancer cells regulate plasticity. Recent studies from our laboratory and others’ proposed that CCN5/WISP-2, which is found intracellularly (in the nucleus and cytoplasm) and extracellularly, plays a negative regulator of plasticity. It prevents the EMT process in breast cancer cells as well as pancreatic cancer cells. Multiple genetic insults, including the gain of p53 mutations that accumulate over the time, may perturb CCN5 expression in non-invasive breast cancer cells, which ultimately helps cells to gain invasive phenotypes. Moreover, emerging evidence indicates that several oncogenic lesions such as miR-10b upregulation and activation of TGF-β-signaling can accumulate during CCN5 crisis in breast cancer cells. Collectively, these studies indicate that loss of CCN5 activity may promote breast cancer progression; application of CCN5 protein may represent a novel therapeutic intervention in breast cancer and possibly pancreatic cancer.     

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor,saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.