EMT States Research Articles

A 3D in vitro model to explore the inter-conversion between epithelial and mesenchymal states during EMT and its reversion.

Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Intermediate states, arising from inter-conversion between epithelial (E) and mesenchymal (M) states, are characterized by phenotypic heterogeneity combining E and M features and increased plasticity. Hybrid EMT states are highly relevant in metastatic contexts, but have been largely neglected, partially due to the lack of physiologically-relevant 3D platforms to study them. Here we propose a new in vitro model, combining mammary E cells with a bioengineered 3D matrix, to explore phenotypic and functional properties of cells in transition between E and M states. Optimized alginate-based 3D matrices provided adequate 3D microenvironments, where normal epithelial morphogenesis was recapitulated, with formation of acini-like structures, similar to those found in native mammary tissue. TGFβ1-driven EMT in 3D could be successfully promoted, generating M-like cells. TGFβ1 removal resulted in phenotypic switching to an intermediate state (RE cells), a hybrid cell population expressing both E and M markers at gene/protein levels. RE cells exhibited increased proliferative/clonogenic activity, as compared to M cells, being able to form large colonies containing cells with front-back polarity, suggesting a more aggressive phenotype. Our 3D model provides a powerful tool to investigate the role of the microenvironment on metastable EMT stages.

Abstract 2311: Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways

Abstract Cancer stem cells (CSCs) drive tumor growth as well as mediating metastasis and treatment resistance. In breast cancer (BC), CSCs exist in alternative mesenchymal (EMT) and epithelial (MET)-like states characterized by the expression of CD44+CD24- and aldehyde dehydrogenase (ALDH) respectively. BCSCs display phenotypic plasticity allowing them to transition between EMT and MET states in a process regulated by the tumor microenvironment. The plasticity of BCSCs suggests that it may be necessary to simultaneously target alternative BCSC states to achieve maximal eradication of these cell populations. In order to develop strategies to target these BCSC states, we measured the cellular bioenergetics of EMT and MET BCSCs compared to non-stem bulk tumor cells as well as the sensitivity of distinct BCSC states to inhibitors of glucose and hydroperoxide metabolism. Using a Seahorse XCF instrument, we found that both MET and EMT BCSCs display higher glycolytic potential than bulk tumor cells. Increased glycolysis correlated with elevated expression of hexokinase 2, a rate limiting glycolytic enzyme in cancer. Interestingly, the glycolytic inhibitor, 2-deoxyglucose (2DG), specifically inhibited EMT BCSCs in a dose dependent fashion; however, MET BCSCs were completely refractory to this treatment. Proteomic and RNA-seq analyses revealed that two important arms of hydroperoxide metabolism, the thioredoxin- and glutathione-mediated antioxidant pathways, were robustly up-regulated in MET BCSCs, suggesting that these cells are rendered resistant to glycolysis inhibition via an enhanced anti-oxidant defense. Pharmacologic inhibition of this antioxidant defense by Auranofin (an inhibitor of thioredoxin reductase) is sufficient to deplete MET BCSCs in SUM149 BC cells which were rescued by NAC, a ROS scavenger, or catalase, a specific enzyme capable of degrading intracellular H2O2. Finally, administration of 2DG together with Auranofin and BSO (an inhibitor of glutathione metabolism) synergistically suppressed tumor growth in a patient derived xenograft (PDX) model by suppressing both EMT and MET BCSCs. This study suggests that utilizing metabolic inhibition to simultaneously target EMT and MET BCSCs is a viable therapeutic strategy with important clinical implications. Citation Format: Ming Luo, April Davis, Sean McDermott, Evelyn Jiagge, Michael Brooks, Elizabeth Gheordunescu, Tahra Luther, Shawn G. Clouthier, Sarah Conley, Douglas R. Spitz, Max S. Wicha. Targeting EMT and MET breast cancer stem cell states through simultaneous inhibition of glycolytic and antioxidant pathways. [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 2311. doi:10.1158/1538-7445.AM2015-2311

Abstract SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance

Abstract SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance

Abstract 2220: Annexin A3 regulates MET-like aldehyde dehydrogenase positive breast cancer stem cells

Abstract Background: Many cancers, including breast cancer, are hierarchically organized and contain a subpopulation of self-renewing cells that display stem cell properties. Breast cancer stem cells (BCSCs) drive tumor growth and metastasis and contribute to therapeutic resistance. Recent studies suggest that BCSCs exist in alternative mesenchymal-like (EMT) and epithelial-like (MET) states which are characterized by expression of the BCSC markers CD44+/CD24- and aldehyde dehydrogenase (ALDH), respectively. BCSCs maintain the plasticity to transition between EMT and MET-like states in a process regulated by the tumor microenvironment. However, the pathways involved in regulation of these stem cell states remain undefined. Approach and Results: In order to elucidate the mechanisms which regulate EMT and MET BCSCs, we performed gene expression and proteomic analysis of BCSC populations enriched for EMT (CD44+/CD24-) or MET, ALDH (Aldefluor-positive). Among the genes enriched in the MET BCSC populations were Annexin A3 (ANXA3). Validation by qPCR and western blotting confirmed increased ANXA3 mRNA and protein expression in the ALDH+ fractions of MCF7, SUM 159, T47D and SUM 149 breast cancer cells, as well as normal mammary epithelial cells. Immunohistochemical analysis revealed that ANXA3 expression was significantly higher in breast cancers compared to normal breast tissue with over 75% of primary human tumors demonstrating significant ANXA3 expression. To determine the functional role of ANXA3 in regulating BCSCs we established breast cancer cell lines with inducible knockdown of ANXA3 utilizing a doxycycline (DOX) inducible shRNA. ANXA3 knockdown reduced cell proliferation and decreased colony formation in soft agar. In addition, ANXA3 knockdown significantly decreased the proportion of ALDH+ BCSCs. In contrast, ANXA3 knockdown increased the relative proportion of EMT-like (i.e. CD44+/CD24-) cells as well as expression of EMT regulatory genes including Twist1 and Snail1. In addition, ANXA3 knocked down significantly decreased tumorsphere formation in vitro and tumor initiating capacity of breast cancer cells in NOD/SCID mice. Conclusions: These results suggest that ANXA3 may play a significant role in regulating the self-renewal of the MET-like ALDH+ BCSC. Studies are underway to determine the mechanisms of this regulation and to assess the potential of ANXA3 as a cancer stem cell therapeutic target. Citation Format: Yadwinder S. Deol, Sean P. McDermott, David M. Lubman, Jenny C. Chang, Song Nie, Tahra K. Luther, Yang Cong, Ebrahim Azizi, Justin Colacino, Shawn G. Clouthier, Max Wicha. Annexin A3 regulates MET-like aldehyde dehydrogenase positive breast cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2015-2220

P-Cadherin Linking Breast Cancer Stem Cells and Invasion: A Promising Marker to Identify an "Intermediate/Metastable" EMT State.

Epithelial–mesenchymal transition (also known as EMT) is a fundamental mechanism occurring during embryonic development and tissue differentiation, being also crucial for cancer progression. Actually, the EMT program contributes to the dissemination of cancer cells from solid tumors and to the formation of micro-metastasis that subsequently develop into clinically detectable metastases. Besides being a process that is defined by the progressive loss of epithelial cell characteristics and the acquisition of mesenchymal features, EMT has also been implicated in therapy resistance, immune escape, and maintenance of cancer stem cell properties, such as self-renewal capacity. However, the majority of the studies usually neglect the progressive alterations occurring during intermediate EMT states, which imply a range of phenotypic cellular heterogeneity that can potentially generate more metastable and plastic tumor cells. In fact, few studies have tried to identify these transitory states, partly due to the current lack of a detailed understanding of EMT, as well as of reliable readouts for its progression. Herein, a brief review of evidences is presented, showing that P-cadherin expression, which has been already identified as a breast cancer stem cell marker and invasive promoter, is probably able to identify an intermediate EMT state associated with a metastable phenotype. This hypothesis is based on our own work, as well as on the results described by others, which suggest the use of P-cadherin as a promising EMT marker, clearly functioning as an important clinical prognostic factor and putative therapeutic target in breast carcinogenesis.

Biological and clinical significance of cancer stem cell plasticity.

In the past decade, the traditional view of cancers as a homogeneous collection of malignant cells is being replaced by a model of ever increasing complexity suggesting that cancers are complex tissues composed of multiple cell types. This complex model of tumorigenesis has been well supported by a growing body of evidence indicating that most cancers including those derived from blood and solid tissues display a hierarchical organization of tumor cells with phenotypic and functional heterogeneity and at the apex of this hierarchy are cells capable of self-renewal. These “tumor imitating cells” or “cancer stem cells” drive tumorigenesis and contribute to metastasis, treatment resistance and tumor relapse. Although tumor stem cells themselves may display both genetic and phenotypic heterogeneity, recent studies have demonstrated that cancer stem cells maintain plasticity to transition between mesenchymal-like (EMT) and epithelial-like (MET) states, which may be regulated by the tumor microenvironment. These stem cell state transitions may play a fundamental role in tumor progression and treatment resistance. In this review, we discuss the emerging knowledge regarding the plasticity of cancer stem cells with an emphasis on the signaling pathways and noncoding RNAs including microRNAs (miRNA) and long non-coding RNAs (lncRNAs) in regulation of this plasticity during tumor growth and metastasis. Lastly, we point out the importance of targeting both the EMT and MET states of CSCs in order to eliminate these lethal seeds of cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-014-0032-3) contains supplementary material, which is available to authorized users.

Abstract BS01-2: Moving breast cancer stem cell therapies to the clinic

Abstract The realization that many cancers, including breast cancer, are driven by cells which display stem cell properties has significant clinical implications. Furthermore, the demonstrated role of these cells in mediating tumor metastasis and treatment resistance suggests the need to develop strategies to specifically target CSC populations. Cancer stem cell self-renewal and survival pathways represent potential therapeutic targets. These self-renewal pathways are regulated by an interacting network of cell intrinsic pathways, as well as extrinsic factors from the tumor microenvironment. These mircroenvironmental factors include cytokines such as IL-6, IL-8 and TGFb. CSCs maintain the plasticity to transition between epithelial-like MET and mesenchymal-like EMT states, a process regulated by the tumor microenvironment through microRNA circuits. We have demonstrated that previously identified cancer stem cell markers are cancer stem cell state specific. CD44+/CD24- CSCs represent mesenchymal-like stem cells capable of tissue invasion which are largely quiescent. In contrast, Aldehyde dehydrogenase expression identifies a more epithelial-like cancer stem cell state associated with self-renewal. Reversible EMT/MET transitions play a crucial role in mediating tumor metastasis. Preclinical breast cancer models predict that the greatest efficacy of CSC targeting therapeutics will occur when they are used in the adjuvant setting, a concept supported by preclinical models and clinical trials. Tumor regression may reflect effects on bulk cell populations explaining the lack of correlation between tumor shrinkage and patient survival. In contrast, recurrence following adjuvant therapy may be mediated by CSCs, which possesses sufficient self-renewal to form clinically significant metastasies. The important role of HER2 signaling in regulating breast cancer stem cell self-renewal may account for the remarkable clinical efficacy of targeting HER2 in the adjuvant setting. Furthermore, the clinical benefit of such therapies in classically defined HER2-negative breast cancers may be due to selective expression of HER2 in CSCs in the absence of HER2 gene amplification. The clinical benefit of adjuvant trastuzumab in women whose breast cancers are currently classified as HER2-negative is currently being assessed in the randomized national clinical trial B47. These studies may demonstrate the need for reevaluating currently used clinical endpoints and clinical trial designs. Promising new technologies including the isolation and molecular characterization of circulating cancer stem cells may provide the opportunity for real time assessment of the efficacy of CSC targeting agents. A number of agents regulating BCSCs have entered early phase clinical trials which will determine whether effective targeting of CSCs improves patient outcome. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr BS01-2.

Abstract 5131: Modeling of EMT/MET transitions in breast cancer stem cells.

Abstract Background: Recent studies suggest that breast cancer stem-like cells (BCSCs) exist in two freely interconvertible states: an EMT- and an MET-like state, and that conversion between these two states is regulated by both intracellular and microenvironmental signaling (Liu et al. 2012, Korkaya et al. 2012). Using a mathematical model, we quantify rates of transition between the EMT and MET-like states. We further explore BCSC behavior in response to alterations in microenvironmental signaling and examine key regulators of BCSC population dynamics. Methods: We employ a continuous-time Markov chain model to quantify rates of transition between the EMT and MET-like state for BCSCs. Our data-based modeling approach employs parameter estimates derived from mouse xenograft experiments from the Wicha laboratory (Liu et al. 2012) to estimate the equilibrium EMT- and MET-like state distributions. We expand our model to simulate BCSC population counts and EMT and MET state transitions in response to alterations in the complex regulatory network involving IL-6, HER2, miRNA, NF-κB, and TGF-β signaling. Results: We estimate the rate of BCSC transition from an EMT- to MET-like state to be 0.08 per cell per day, and the rate of transition from an MET- to EMT-like state to be 0.013 per cell per day. Simulation studies examining the effects of abrogating microenvironmental signals individually and in combinations revealed that slowing both self-renewal rate and the rate of transition from an MET- to EMT-like state is essential to eradicating the total BCSC population. The fastest decline in the BCSC population was observed when IL-6 is inhibited and the activation of NF-κB via HER2 signaling and IL-6 feedback was blocked. Conclusions: Rates of transition between EMT- and MET-like states are estimated to occur frequently and these events are much more likely than dedifferentiation. Predictions from our mathematical model match experimental data (Korkaya et al. 2012) showing the efficacy of combined HER2 and IL-6 blockade in reducing the BCSC population. Citation Format: Mary E. Sehl, Miki Shimada, Kenneth Lange, Max S. Wicha. Modeling of EMT/MET transitions in breast cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5131. doi:10.1158/1538-7445.AM2013-5131

Abstract 3385: A systems view of epithelial – mesenchymal transition signaling states

Abstract Epithelial – mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and ‘epigentically-fixed’ EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell-cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49fhi/EpCAM-/lo and CD44hi/CD24lo respectively. 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 3385. doi:10.1158/1538-7445.AM2011-3385