Epithelial–Mesenchymal Transition in Cancer: Insights Into Therapeutic Targets and Clinical Implications

The epithelial–mesenchymal transition (EMT) is a process in which epithelial cells dedifferentiate to become mesenchymal cells [1–3]. The process requires them to lose their cell polarity, to relinquish their cell–cell adhesion, and to gain migratory and invasive properties. The mesenchymal cells are multipotent stromal cells (mesenchymal stem cells) that can differentiate into various cell types. EMT is essential for the developing embryo to enable mesoderm and neural tube formation. EMT is also necessary for wound healing and other physiological processes. On the other hand, EMT can also be an initiator for metastasis, cancer progression, organ fibrosis, and other pathological conditions. These are disorderly processes. The induction of EMT is complex (Fig. 1) [4]. A fundamental event appears to be the loss of E-cadherin. Transcription factors that can repress E-cadherin directly or indirectly are important to this process. SNAI1/Snail 1, SNAI2/Snail 2 (also known as Slug), ZEB1, ZEB2, E47, and Kruppel-like factor-8 (KLF8) can repress E-cadherin transcription by binding to its promoter. Indirect repressors, such as Twist, Goosecoid, TCF4 (sometimes referred to as immunoglobulin transcription factor 2), homeobox protein SIX1, and FOXC2 (fork-head box protein C2) are able to repress E-cadherin indirectly [5]. Other junction proteins, such as claudins and desmosomes, are also suppressed, facilitating EMT. Various transcription factors such as grainyhead-like protein 2 homologue (GRHL2) and ETS-related transcription factors ELF3 and ELF5 are downregulated during EMT [6, 7]. The induction of EMT is dependent on numerous signaling pathways including TGF-beta, FGF, EGF, HGF, Wnt/beta-catenin, Notch, and hypoxia-induced mechanisms. Ras-MAPK signaling activates Snail and Slug during EMT induction. Slug is involved in disrupting desmosomes, inducing cell spreading, and separating the cell–cell borders [8]. However, EMT involves a second phase that cannot be triggered by Slug. This phase includes the induction of cell motility, repression of the cytokeratin expression, and activation of vimentin expression [9]. Phosphatidylinositol 3′ kinase (PI3K)/AKT-axis activation is emerging as pivotal to EMT. Similarly, hedgehog, nuclear factorkappaB (NF-κB), and activating transcription factor-2 have been implicated [10]. Of course, the EMT processes are specific, so that while Wnt signaling pathways regulate EMT in embryonic development, cardiac valve formation, and cancer, other pathways are important towound healing and fibrosis [11]. EMT is central to two recent J Mol Med contributions. Kimura et al. report on the attenuation of EMT in retinal pigment epithelial cells and subretinal fibrosis by a retinoic-acid receptor-gamma (RAR-γ) agonist [12]. The retinal pigment epithelium belongs the neural retina and is indispensable for vision. In humans, proliferation and transformation (EMT switching) of retinal pigment epithelial cells after injury causes retinal disorders and loss of vision [13]. On the other hand, EMT could develop into a therapeutic target [14]. Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD), a devastating and common disease in older people. The authors began with retinal epithelial cells in culture. When subjected to TGF-β2, the cells expressed mesenchymal markers including α-smooth muscle actin and fibronectin. The cells also released interleukin-6. Paxillin was phosphorylated; MAPK constituents such as ERK, p38, and JNK were activated, as were Smad2 and AKT. A RAR-γ agonist, R667, attenuated the EMT responses. Furthermore, the cells treated * Friedrich C. Luft luft@charite.de

The epithelial to mesenchymal transition (EMT) is a highly conserved morphogenesis program that is essential for re-shaping and mobilizing epithelial cells during gastrulation, neural crest development and tissue regeneration. In cancer cells, EMT induction promotes acquisition of invasive cellular morphologies, stem cell-like properties and pro-survival mechanisms, which contribute to disease progression, therapy resistance and decreased overall survival. The identification of compounds that block or reverse EMT therefore represents an important therapeutic strategy to prevent cancer invasion and eradicate disseminated tumor cells. Unfortunately, current EMT inhibitors have shown limited clinical benefit, in part due to an incomplete understanding of the molecular mechanisms controlling EMT in development and cancer as well as a lack of screening platforms that recapitulate the complex physiological environment of EMT in the living animal. To overcome these obstacles, we have established a zebrafish Snail1-GFP lineage reporter strain to label dorsal neural tube progenitor cells before they undergo EMT to become neural crest, which allows us to visualize epithelial morphogenesis independent of later cell migration events. Thousands of Snail1-GFP embryos can be easily generated to perform whole animal-based screens with small molecule libraries to identify compounds that inhibit EMT in vivo. Our initial screening results using previously characterized EMT inhibitors showed that many compounds inhibited neural crest migration after EMT had occurred, but only one compound, an AXL receptor tyrosine kinase inhibitor called TP-0903, inhibited both epithelial morphogenesis and neural crest migration. TP-0903 significantly decreased twist1a expression, a canonical EMT transcription factor and blocked down-regulation of epithelial Cadherins. RNA-Seq analysis and chemical rescue experiments revealed that TP-0903 acts by inducing retinoic acid (RA) biosynthesis and triggering a RA-mediated transcriptional program. TP-0903 treatment of a number of human cell lines and mouse xenograph tumors showed that TP-0903 also inhibits EMT programs and survival in human cancer cells. These studies demonstrate the value and feasibility of using zebrafish neural crest development to identify effective EMT compounds in vivo. As such, we have identified TP-0903 as a new potential therapeutic for inhibiting EMT in cancer, and our findings support the hypothesis that RA-induced inhibition of EMT contributes to its current success in treating minimal residual disease in humans. Citation Format: Laura Jimenez, Clifford Whatcott, Jindong Wang, Steven Warner, Monique Morrison, David Bearss, Rodney A. Stewart. Identifying epithelial morphogenesis inhibitors in neural crest development and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA29.

Epithelial to mesenchymal transition (EMT) is a biological process that allows well-differentiated, polarized epithelial cells to undergo a conversion to motile, unpolarized mesenchymal cells. EMT plays crucial roles during implantation, embryogenesis, and organ development (Type 1 EMT), is associated with tissue regeneration and organ fibrosis (Type 2 EMT), and involved in cancer invasion, metastasis, and drug resistance (Type 3 EMT). Since aggressiveness and drug resistance are hallmarks of ductal pancreatic cancer, significant effort has been undertaken in recent years to elucidate molecular EMT mechanisms in this dismal malignancy. This represents a formidable challenge for several reasons: EMT is a dynamic process, both with regard to spatial and temporal heterogeneity. Moreover, EMT is induced and regulated by a complex network of traditional signaling pathways and new players like microRNAs. Interestingly, similar molecular characteristics link EMT-type cells also to the concept of cancer stem cells. This review tries to integrate the current knowledge regarding EMT and pancreatic cancer; furthermore to outline not only the perspective on novel EMT-associated therapeutic targets, but also on overcoming drug resistance by interfering with EMT.

Cancer is one of the leading causes of death. A primary tumor forms when cells start to proliferate in an uncontrolled way and stop reacting to restraining signals. Tumors that reach a critical volume induce angiogenesis, a vascular remodeling process that provides nutrients and oxygen to the degenerated cell mass. Upon further tumor progression to a malignant cancer, cells acquire the ability to invade the surrounding tissue. In order to do so, formerly epithelial tumor cells undergo an epithelial to mesenchymal transition (EMT). This process of cell-cell detachment, breaching through the basement membrane and gaining migratory capabilities is the first step of the metastatic cascade. Metastasis is a process that allows tumor cells to leave the primary lesion and disseminate via the vascular system to secondary sites. Metastases, as the fatal feature of cancer, lead in most of the cases to patients’ death. Furthermore, metastasis, as the end stage of malignant disease, until now is incurable. This is because metastases are spread systemically throughout the body. Moreover, they even can establish from a disseminated tumor cell months after the primary tumor has already been surgically removed. Another barely controllable feature of cancer is tumor relapse. Even after a thorough surgery with the aim to completely remove the primary tumor and eventual draining lymph nodes combined with chemotherapy, patients relapse. Resistance to chemotherapy and establishment of metastases has both been accounted for to the abundance of cancer stem cells (CSC) within the tumor mass. These CSCs are endowed with the ability to evade chemotherapeutic drugs, they are mesenchymal in nature, migratory and invasive, and, most importantly, they are able to establish cancer and metastasis de novo. The work of my thesis has been dedicated to investigate the process of metastasis and the function of cancer initiation. Tumor initiation has recently been associated with EMT. To understand the functional circumstances how EMT cells gain the ability to form tumors, I used cellular murine breast cancer models. These model systems allowed me to study cells’ behavior before and after EMT in vitro and in vivo. In vitro experiments validated the observation of others that EMT cells indeed resemble cancer stem cells by being able to form hollow spheres and being susceptible to the CSC-specific drug salinomycin. In vivo studies revealed that EMT cells initiate tumors with a much earlier onset and with a higher efficiency when limited amounts of cells were injected orthotopically into mice compared to their epithelial counterpart. Moreover, EMT cell-generated cancers are highly vascularized already in the early phase of tumor establishment. Knockdown studies of the main pro-angiogenic factor VEGF-A revealed that it is required for early tumor onset. Thus, tumor angiogenesis is not only an effect of early and fast EMT-tumor progression. Further supporting this notion are limiting dilution experiments, which suggest that tumor initiation in EMT cells is a multifactorial event. This concept was validated by the observation that 10 EMT cells could initiate a tumor, whereas VEGF‑A knockdown cells could not. Hence, EMT-induced tumor initiation is achieved by the ability of promoting angiogenesis. EMT can be induced by the cytokine TGFβ. Normally, cells that experience TGFβ signaling become quiescent or die due to induction of apoptosis. Cancer cells can overcome these effects and react to TGFβ by undergoing EMT. As part of my thesis, I could show that the transcription factor Dlx2 is an important switch that allows cells to react to TGFβ by undergoing EMT without facing apoptosis induction. Dlx2 exerts its anti-apoptotic, pro-survival function by directly reducing TGFβRI expression and inducing the expression of the epidermal growth factor receptor ligand betacellulin. Another feature of EMT is the gain of cell motility. Cell migration is extremely important for cancer cells in order to leave their primary site and to disseminate. I have found ephrinB2 expression upregulated during EMT. EphrinB2 is a member of the Eph-ephrin signaling network that is known to be crucial for cell-cell communication. Thereby, cells that do not belong to the same entity repulse each other, restricting intermingling of tissue. Furthermore, ephrinB2 is required for neuronal axon guidance and angiogenesis. In my thesis, I showed that EMT cells need ephrinB2 to efficiently migrate. As an explanation for this phenotype I described that a knockdown for ephrinB2 led to an over-stabilization of focal adhesions. Cells normally use focal adhesions to hold on to an extracellular matrix (ECM) surface. Over-stabilization of focal adhesions attaches cells too firmly to the ECM and, hence, cells cannot retract their rear-end anymore which decreases cell motility. In summary, I succeeded in gathering further insights into tumor initiation, EMT, and with this, the metastatic process.

[Background] EMT (epithelial-to-mesenchymal transition) was originally proposed as a process of organogenesis. In recent years, the association between EMT and cancer invasion and metastasis has been advocated and actively investigated in various cancers. In addition, chemoresistance and cancer stemness could be involved in EMT, and the elucidation of this association might contribute to improved outcomes of hepatocellular carcinoma (HCC). We utilized surgical specimens of HCC from our department to examine the clinical implications of EMT. [Methods] One hundred-one patients with hepatocellular carcinoma, who underwent resection in our department between 1994 and 2003 were analyzed. The mRNA expression of E-cadherin and Vimentin were measured by quantitative real-time PCR and EMT status of each patient was determined as follows: Vimentin/E-cadherin < 2 = Epithelial (E), Vimentin/E-cadherin ≤ 2 = Mesenchymal (M). Moreover, transcription factors which are involved in EMT (Twist, Snail, Slug, Zeb-1, and Zeb-2) and also IL-6 and its receptor IL-6R were measured. The correlation between these values and clinicopathological factors and prognosis were analyzed statistically. [Results] 1. AFP values were significantly higher in the epithelial group than in the mesenchymal group (P = 0.029) and PIVKA-II values also tended to be higher in the epithelial group as compared to the mesenchymal group. There was no difference in overall survival, but a significant difference was found in disease-free survival (P = 0.042), which showed that patients with a mesenchymal tumor were more prone to have an earlier recurrence than those with an epithelial tumor. 2. All transcription factors (Twist, Snail, Slug, Zeb-1, and Zeb-2) were more highly expressed in mesenchymal than in epithelial tumors, and in particular, Twist and Zeb-2 were significantly overexpressed (P = 0.0002, P = 0.0017, respectively). Overall survival and disease-free survival of Twist and Zeb-2 expression tumors were examined, but there was no difference between these groups. 3. IL-6 expression was also significantly higher in mesenchymal than in epithelial tumors, but there was no difference in IL-6R expression. [Discussion] Analysis of EMT status in HCC has been performed in the past, but the majority of these studies were done in vitro. Our study using resected surgical specimens suggest that EMT could be involved in cancer invasion and metastasis at the clinical level. In particular, Twist and Zeb-2 might be important for inducing EMT in HCC and patients with mesenchymal tumors are more prone to have early recurrence or metastases after resection. In addition, IL-6 might be an important factor for HCC EMT and could be a potential therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3402. doi:1538-7445.AM2012-3402

Epithelial–Mesenchymal Transition in Tumor Metastasis: A Method to the Madness

Nitric oxide (NO) has been shown to mediate contrasting effects on tumorigenesis i.e. at low levels NO is pro-tumorigenic and at high levels NO is anti-tumorigenic. Recent studies revealed that treatment of tumor cell lines with NO donors at high levels significantly inhibited cell growth and survival pathways such as the constitutively activated NF-κB. Tumor cells with activated NF-κB were reported to have the capacity to initiate the metastatic cascade via induction of epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by decreased expression of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin, fibronectin). In addition, the tumor cells exhibit invasive and migratory properties. Based on these above findings, we hypothesized that treatment of metastatic prostate carcinoma cell lines, PC-3 and DU-145 with DETANONOate (500-1000μM) may reverse the EMT phenotype. This hypothesis was tested and treatment of the cells with DETANONOate resulted in inhibition of the EMT phenotype through inhibition of NF-κB activity. Inhibition of NF-κB was shown to be due, in part, to S-nitrosylation of p50. In addition to the inhibition of EMT phenotype, treatment with DETANONOate also inhibited invasive properties. The dual roles of DETANONOate-mediated inhibition of NF-κB and EMT were corroborated by the use of the NF-κB inhibitor DHMEQ. We examined the underlying mechanism by which NF-κB-induced inhibition by DETANONOate resulted in the inhibition of EMT. The EMT-induced transcription factor Snail was highly expressed in PC-3 and DU-145 cells. Treatment with DETANONOate inhibited Snail expression concomitant with inhibition of EMT. The direct role of Snail inhibition by DETANONOate and the inhibition of EMT was corroborated in cells transfected with Snail siRNA and such treatment reversed the EMT phenotype, mimicking DETANONOate. Inhibition of the repressor activity of Snail by DETANONOate resulted in the upregulation of the Raf-1 kinase inhibitory protein (RKIP). The role of RKIP induction in the reversal of EMT was corroborated in cells overexpressing RKIP. The in vitro findings with DETANONOate-induced inhibition of EMT were validated in mice bearing PC-3 xenografts. Treatment with DETANONOate and analysis by IHC of tumor biopsies revealed that the EMT phenotype was reversed. There was an inverse correlation between Snail inhibition and RKIP induction. The present findings demonstrate, for the first time, that NO donors (high concentrations) inhibit the EMT phenotype and suggest their potential therapeutic applications in vivo in patients. These studies also suggest that the NF-κB/Snail/RKIP circuitry is dysregulated in tumor cells and is responsible, in part, for the EMT phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1466.

BackgroundEMT is the process whereby epithelial cells acquire mesenchymal characteristics. This process is highly regulated and required in development and tissue regeneration. However, in cancer the process becomes dysregulated leading...

Metastasis is responsible for most of the deaths related to cancer. Therefore, the most effective methods to decrease cancer morbidity and mortality consist of early detection, prevention, and treatment of metastasis. Epithelial-to-mesenchymal transition (EMT) is one of the processes involved in cancer progression and metastasis. EMT induces cellular and microenvironmental changes that result in loss of the epithelial phenotype and acquisition of mesenchymal properties, thus promoting cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including EGF and TGF-beta. Overexpression of transcription factors such as SNAIL also induce EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was treated with the growth factor EGF (10 ng/mL) to identify proteomic alterations and specific mechanisms involved in the induction of EMT and metastasis. EGF treatment led to EMT, which was validated by molecular, morphologic, and functional experiments. Subcellular proteome enrichment followed by target proteomics was performed to identify changes in protein expression between the subcellular compartments cytoplasm, nucleus, and membrane. Thirty micrograms of each subcellular compartment of cells induced (or not) to EMT were processed to obtain tryptic peptide digest for subsequent MRM analysis. Proteotypic peptides representing relevant proteins for EMT and cancer progression, including proteins identified in our previous studies, were selected to compose panels for MRM analysis. EGF treatment activated the ERK/MAPK signaling pathway, which is involved in cancer progression by deregulating cell growth. Changes in expression of important proteins related to the EMT process were observed after EGF treatment, such as CDH1, CDH2, VIME, SOD2, and TGFB1. Subcellular compartment analysis allowed the identification of the main location of the proteins selected as well as differential regulation between subcellular compartments and potential mechanisms of subcellular translocation resulting from the EGF treatment and EMT induction, such as the regulation of PRDX3. Changes in the cellular protein localization may lead to alterations in the protein function and may thus be correlated with tumor progression. Other proteins related to cancer progression, including cytoskeleton rearrangement and cell cycle control, were also regulated after EGF treatment in specific compartments, such as CDK1, ARPC2, CKAP5, CNN3, and ARHGDIB. Besides that, we could identify the sumoylation as a possible post-translational modification regulated during EMT, which was verified by the expression of SUMO1/2 proteins, mainly in the cytoplasmic compartment. This detailed analysis allowed a better comprehension of the mechanisms of the EMT induction by EGF in a breast cancer cell line and can help to identify the biologic process and proteins relevant to cancer progression and that can potentially serve as targets for metastasis inhibition and/or diagnosis. (Financial support: FAPESP 2015/08693-0, 2011-0947-1, 2016/03809-3; CISBi NAP/USP grant 12.1.17598.1.3 and CTC-CEPID 2013/08135-2.) Citation Format: Camila S. Palma, Mariana L. Grassi, Carolina H. Thomé, Aline Poersch, Guilherme P. Lanfredi, Vitor M. Faça. Proteomic analysis of epithelial-to-mesenchymal transition induced by EGF in breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A192.

Inflammation plays a pivotal role in the progression of solid malignant tumor with studies showing that the presence of specific inflammatory-immune cells, such as macrophages, at the tumor site leads to poor prognosis. M1 polarized macrophages secrete pro-inflammatory chemokines and cytokines which induce an epithelial to mesenchymal transition (EMT) in tumor cells; a process where epithelial cells acquire mesenchymal traits, thus facilitating tissue infiltration and consequent metastasis. EMT as a marker of cancer progression is understudied in thyroid cancer. In our present study, we evaluated the crosstalk between the macrophages and the epithelial cells in the thyroid tumor microenvironment with the use of an in vitro model system consisting of an immortalized thyroid cell line (Nthy-ori 3-1), three papillary cancer cell lines (BCPAP, 8505C and TPC-1) and one follicular cancer cell line (CGTHW-1). We observed the halt in proliferation of the thyroid cancer cells as the epithelial cells differentiate into mesenchymal cells under the influence of activated macrophage conditioned media. We also observed a statistically significant (p<0.05) upregulation of migration and invasion of thyroid tumor cells in presence of macrophage conditioned media. Using nuclear and cytoplasmic extracts from the thyroid cancer cell lines, we observed that EMT markers including E-cadherin, β-catenin, vimentin, NFk-B, snail, slug and twist are modulated in response to activated macrophage conditioned media. Downregulation of E-cadherin, required for maintenance of stable junction and β-catenin essential for cell-cell adhesion was noticed in response to macrophage conditioned media. Transcription factors like NFk-B, snail and slug were translocated to the nucleus, which is indicative of the initiation of EMT. Our data provides evidence that macrophage secreted proteins play a crucial role in epithelial-mesenchymal transition in thyroid tumor advancement and metastasis. We have successfully identified some significant markers including transcription factors such as NFk-B, slug, twist and snail that play a critical role in the induction of epithelial to mesenchymal transition in thyroid cancer. These findings will allow for the possible development of targeted therapy designed at inhibiting EMT and subsequent suppression of thyroid cancer metastasis and dissemination. Citation Format: Neha Y. Tuli, Jonathan Cabin, Robert Suriano, Robert Bednarczyk, Elyse Hanly, Jan Geliebter, Edward Shin, Raj K. Tiwari. Tumor microenvironment based modulation of thyroid cancer phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1107. doi:10.1158/1538-7445.AM2014-1107

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

STAT3-driven EMT in cancer metastasis and chemoresistance: A review.

The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-β, BMP, Wnt-β-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-β1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-β-induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.

BP1 Upregulates Twist, a Trigger of EMT and Cancer Metastasis Bin-Jin Hwang, Jinguen Rheey, and Patricia E. Berg George Washington University, Department of Biochemistry and Molecular Biology, Washington, D.C. Background. We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Increasing evidence suggests that homeobox genes are important in malignant transformation and tumorigenesis, and our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with breast cancer aggressiveness: 100% of estrogen receptor negative tumors and 89% of the tumors of African American women are BP1 positive. Now we have evidence that BP1 may regulate the epithelial to mesenchymal transition (EMT). EMT, a process in which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells to become more invasive and migratory, and can lead to metastasis. BP1 upregulates Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT. Our objective is to determine whether BP1 plays a role in induction of EMT. Materials and Methods. The expression levels of Twist, E-cadherin and vimentin were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. RNA levels were verified by real-time PCR and protein levels by Western blotting and, in some cases, by confocal microscopy. Summary. Twist mRNA expression on microarrays is higher in MCF-7 cells overexpressing BP1 compared with cells containing an empty vector. Real-time PCR confirmed that Twist mRNA is increased five-fold in cells with high levels of BP1, and Western blots demonstrated that Twist protein is also elevated in the same cells. We have discovered a potential DNA binding site for pBP1 in the Twist promoter, suggesting that BP1 may directly regulate Twist. This will be verified by ChIP. Expression of two genes regulated by Twist was also examined: Twist activates vimentin and represses E-cadherin. Cells overexpressing pBP1 also show increased vimentin and decreased E-cadherin. The effect of increased pBP1 on cell migration is under investigation. Conclusions: BP1 stimulates Twist expression in MCF-7 breast cancer cells, resulting in increased vimentin and decreased E-cadherin levels. We therefore hypothesize that BP1 is upstream of Twist and induces EMT. If BP1 is required for EMT, it may be a good target for therapy of metastatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2298.

Simple SummaryEpithelial-to-mesenchymal transition (EMT) is a cellular program that enables epithelial cells to transition toward a mesenchymal phenotype with augmented cellular motility. Although EMT is a fundamental, non-pathological process in embryonic development and tissue repair, it also confers biological aggressiveness to cancer cells, including invasive behavior, tumor- and metastasis-initiating cancer stem cell activity, and greater resistance to all the cancer treatment modalities. Whereas alterations in the metabolic microenvironment are known to induce EMT, it is also true that the EMT process involves a very marked metabolic remodeling. However, whether there is a causal or merely an ancillary relationship between metabolic rewiring and the EMT phenomenon has not yet been definitively clarified. Here, we combined several technology platforms to assess whether the accompanying changes in the metabolic profile and mitochondria functioning that take place during the EMT process are independent or not of the non-tumorigenic versus tumorigenic nature of epithelial cells suffering a mesenchymal conversion. Understanding the metabolic basis of the non-tumorigenic and tumorigenic EMT provides fundamental insights into the causation and progression of cancer and may, in the long run, lead to new therapeutic strategies.Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-RasV12-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial–cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. “Upper” and “lower” glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl–carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.