Epithelial-to-mesenchymal Transition States Research Articles

Tunable Hybrid Hydrogels of Alginate and Cell-Derived dECM to Study the Impact of Matrix Alterations on Epithelial-to-Mesenchymal Transition.

Epithelial-to-mesenchymal transition (EMT) is crucial for tumor progression, being linked to alterations in the extracellular matrix (ECM). Understanding the ECM's role in EMT can uncover new therapeutic targets, yet replicating these interactions in vitro remains challenging. It is shown that hybrid hydrogels of alginate (ALG) and cell-derived decellularized ECM (dECM), with independently tunable composition and stiffness, are useful 3D-models to explore the impact of the breast tumor matrix on EMT. Soft RGD-ALG hydrogels (200Pa), used as neutral bulk material, supported mammary epithelial cells morphogenesis without spontaneous EMT, allowing to define the gene, protein, and biochemical profiles of cells at different TGFβ1-induced EMT states. To mimic the breast tumor composition, dECM from TGFβ1-activated fibroblasts (adECM) are generated, which shows upregulation of tumor-associated proteins compared to ndECM from normal fibroblasts. Using hybrid adECM-ALG hydrogels, it is shown that the presence of adECM induces partial EMT in normal epithelial cells, and amplifes TGF-β1 effects compared to ALG and ndECM-ALG. Increasing the hydrogel stiffness to tumor-like levels (2.5kPa) have a synergistic effect, promoting a more evident EMT. These findings shed light on the complex interplay between matrix composition and stiffness in EMT, underscoring the utility of dECM-ALG hydrogels as a valuable in vitro platform for cancer research.

Reconstruction of single-cell lineage trajectories and identification of diversity in fates during the epithelial-to-mesenchymal transition

Exploring the complexity of the epithelial-to-mesenchymal transition (EMT) unveils a diversity of potential cell fates; however, the exact timing and mechanisms by which early cell states diverge into distinct EMT trajectories remain unclear. Studying these EMT trajectories through single-cell RNA sequencing is challenging due to the necessity of sacrificing cells for each measurement. In this study, we employed optimal-transport analysis to reconstruct the past trajectories of different cell fates during TGF-beta-induced EMT in the MCF10A cell line. Our analysis revealed three distinct trajectories leading to low EMT, partial EMT, and high EMT states. Cells along the partial EMT trajectory showed substantial variations in the EMT signature and exhibited pronounced stemness. Throughout this EMT trajectory, we observed a consistent downregulation of the EED and EZH2 genes. This finding was validated by recent inhibitor screens of EMT regulators and CRISPR screen studies. Moreover, we applied our analysis of early-phase differential gene expression to gene sets associated with stemness and proliferation, pinpointing ITGB4, LAMA3, and LAMB3 as genes differentially expressed in the initial stages of the partial versus high EMT trajectories. We also found that CENPF, CKS1B, and MKI67 showed significant upregulation in the high EMT trajectory. While the first group of genes aligns with findings from previous studies, our work uniquely pinpoints the precise timing of these upregulations. Finally, the identification of the latter group of genes sheds light on potential cell cycle targets for modulating EMT trajectories.

Complex and variable regulation of ΔNp63 and TAp63 by TGFβ has implications for the dynamics of squamous cell epithelial to mesenchymal transition

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Functional requirement of alternative splicing in epithelial-mesenchymal transition of pancreatic circulating tumor

Circulating tumor cells (CTCs) that undergo epithelial-to-mesenchymal transition (EMT) can provide valuable information regarding metastasis and potential therapies. However, the current studies on the EMT overlook the alternative splicing. Here, we used the single-cell full-length transcriptome data and mRNA-sequencing of CTCs to identify stage-specific alternative splicing of partial EMT and mesenchymal states during pancreatic cancer metastasis. We classified definitive tumor and normal epithelial cells via genetic aberrations and demonstrated dynamic changes in the epithelial-mesenchymal continuum in both epithelial cancer cells and CTCs. We provided the landscape of alternative splicing in CTCs at different stages of EMT, uncovering cell-type-specific splicing patterns and splicing events in cell-surface proteins suitable for therapies. We showed that MBNL1 governs cell fate through alternative splicing independently of changes in gene expression and affects the splicing pattern during EMT. We found a high frequency of events that contained multiple premature termination codons and were enriched with C and G nucleotides in close proximity, which influence the likelihood of stop codon readthrough and expand the range of potential therapeutic targets. Our study provides insights into the EMT transcriptome's dynamic changes and identifies potential diagnostic and therapeutic targets in pancreatic cancer.

Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFβ1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

A Cell-Fate Reprogramming Strategy Reverses Epithelial-to-Mesenchymal Transition of Lung Cancer Cells While Avoiding Hybrid States.

Network modeling unravels the highly complex and plastic process regulating epithelial and mesenchymal states in cancer cells and discovers therapeutic interventions for reversing epithelial-to-mesenchymal transition and enhancing chemosensitivity.

SNAIL driven by a feed forward loop motif promotes TGFβ induced epithelial to mesenchymal transition

Epithelial to Mesenchymal Transition (EMT) plays an important role in tissue regeneration, embryonic development, and cancer metastasis. Several signaling pathways are known to regulate EMT, among which the modulation of TGFβ (Transforming Growth Factor-β) induced EMT is crucial in several cancer types. Several mathematical models were built to explore the role of core regulatory circuit of ZEB/miR-200, SNAIL/miR-34 double negative feedback loops in modulating TGFβ induced EMT. Different emergent behavior including tristability, irreversible switching, existence of hybrid EMT states were inferred though these models. Some studies have explored the role of TGFβ receptor activation, SMADs nucleocytoplasmic shuttling and complex formation. Recent experiments have revealed that MDM2 along with SMAD complex regulates SNAIL expression driven EMT. Encouraged by this, in the present study we developed a mathematical model for p53/MDM2 dependent TGFβ induced EMT regulation. Inclusion of p53 brings in an additional mechanistic perspective in exploring the EM transition. The network formulated comprises a C1FFL moderating SNAIL expression involving MDM2 and SMAD complex, which functions as a noise filter and persistent detector. The C1FFL was also observed to operate as a coincidence detector driving the SNAIL dependent downstream signaling into phenotypic switching decision. Systems modelling and analysis of the devised network, displayed interesting dynamic behavior, systems response to various inputs stimulus, providing a better understanding of p53/MDM2 dependent TGF-β induced Epithelial to Mesenchymal Transition.

Abstract 962: A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance

Abstract Introduction: Epithelial to mesenchymal transition (EMT) is essential for cancer dissemination but its role in overt metastasis establishment remains an open issue. Multiple EMT states and great phenotypic plasticity have also been described, indicating the need for in-depth characterization of biological properties associated with different EMT phenotypes. The so-called hybrid phenotype, characterized by presence of both mesenchymal and epithelial markers, might in fact identify cancer cells with enhanced metastatic propensity. Materials and Methods: To assess the metastatic potential of different EMT phenotypes, we exploited two lung cancer cell lines with different properties (A549/epithelial and LT73/hybrid) and generated their respective sublines through four sequential rounds of in vitro migration assay (named M4). TGFβ treatment was also used to induce EMT. With a previously validated score based on the ratio between expression of epithelial (CDH1) or mesenchymal genes (SNAI2), cells were classified as: epithelial (A549)>hybrid (A549-M4, LT73)>mesenchymal (LT73-M4, A549-TGFβ). The disseminating/metastatic properties of the different lines were tested in vivo by subcutaneous and tail-vein injections in SCID mice. Interaction with NK cells (NKs), known to specifically target EMT cancer cells, was also assessed. Results and Discussion: Compared to parental cells, M4 cells showed up-regulation of EMT-associated genes, expansion of the subset of CD133+ lung cancer stem cells (CSC) and displayed in vivo higher ability to disseminate from primary tumors to murine lungs (2-fold change, p=0.004). However, hybrid cells (LT73 and A549-M4) showed an increased ability to generate overt lung metastases in experimental metastasis assays compared to either epithelial (A549) or mesenchymal cells (LT73-M4 and A549-TGFβ). The higher metastatic ability of hybrid cells was correlated with CSCs expansion and reduced NKs infiltration to the metastatic lungs. Expression of IL-8, a cytokine involved in NKs recruitment, was decreased in hybrid cells and they were less prone to chemoattract NKs in vitro compared to both epithelial and mesenchymal cells. Finally, in vivo prevention of NKs recruitment to the lungs using a neutralizing antibody, massively increased metastasis formation and expansion of CSCs in mice injected i.v. with mesenchymal A549-TGFβ (p=0.0006), but had limited effects in groups injected with both epithelial A549 and hybrid A549-M4. Conclusions: Our data indicate that the hybrid phenotype correlates with the highest metastatic potential related to efficient cancer cell dissemination, increased induction of stemness features and ability to avoid NKs recruitment. Conversely, fully mesenchymal cells show limited metastatic potential, possibly due the control exerted by NKs on their CSC subsets. Citation Format: Giulia Bertolini, Monica Parodi, Massimo Milione, Fabio Murianni, Giovanni Centonze, Giulia Taiè, Gabriella Sozzi, Massimo Vitale, Luca Roz. A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 962.

Abstract 414: Comprehensive molecular characterization of mechanisms involved in primary resistance to EGFR tyrosine kinase inhibitors

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) activating mutations have significantly improved patient´s treatment with these alterations. However, 15-20% of EGFR-mutated patients do not respond to the therapy, even harboring the same mutations than responders. Little is known about the mechanisms involved in this primary resistance. This work aims to characterize molecular aberrations in clones with intrinsic resistance to first and third generation EGFR-TKIs generated from non-small cell lung cancer (NSCLC) cell lines. Three Erlotinib-sensitive (HCC827, PC9 and H4006) and one Osimertinib-sensitive (H1975) NSCLC cell lines were used to generate primary resistance models through a pulsatile method in which cells were treated with a high concentration of the TKI during a short period of time. Survivor colonies were individually expanded in the presence of the inhibitor until resistant cell lines were established. Exome, transcriptome and methylome were then analyzed in selected clones. Erlotinib- and Osimertinib-resistant clones exhibited cross-resistance to other EGFR-targeted therapies, such as Afatinib and Cetuximab, but not to the chemotherapeutics Carboplatin or Cisplatin. Among derived clones, we found several genetic alterations in genes associated with TKI acquired resistance, including NTRK1, ARID2, ERBB3, RET, and other genes. MET amplification was exclusively associated with resistant HCC827-derived clones. We also identified non-genetic alterations that include increased expression and over-activation of well-known TKI resistance-associated proteins, such as AXL, FGFR1, or AKT, as well as induction of epithelial-to-mesenchymal transition (EMT) and stemness-related genes. Transcriptome and methylome analyses revealed EMT plasticity among resistant clones that could reflect different EMT states conferring resistance in pre-treated tumors. Molecular inhibition of MET, a recognized resistance mechanism, sensitizes MET-amplified resistant clones to Erlotinib. Combination treatments or genetic inhibitions will be further performed to validate our findings. Taken together, our results suggest that multiple genetic and non-genetic alterations already present before TKI treatment could account for primary resistance. We will employ integrative analysis of our multi-omic data to identify novel targets that allow to overcome EGFR-TKI resistance. Further clinical validation will help to prospectively select the best treatment option for EGFR-mutated patients for the sake of precision medicine. Citation Format: Juan Manuel Coya, Eva Álvarez, David Gómez-Sánchez, Aranzazu Rosado, Irene Ferrer, Luis Paz-Ares. Comprehensive molecular characterization of mechanisms involved in primary resistance to EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 414.

Cooperative interaction between ER\u03b1 and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

The epithelial to mesenchymal transition (EMT) has been proposed to contribute to the metastatic spread of breast cancer cells. EMT-promoting transcription factors determine a continuum of different EMT states. In contrast, estrogen receptor α (ERα) helps to maintain the epithelial phenotype of breast cancer cells and its expression is crucial for effective endocrine therapies. Determining whether and how EMT-associated transcription factors such as ZEB1 modulate ERα signaling during early stages of EMT could promote the discovery of therapeutic approaches to suppress metastasis. Here we show that, shortly after induction of EMT and while cells are still epithelial, ZEB1 modulates ERα-mediated transcription induced by estrogen or cAMP signaling in breast cancer cells. Based on these findings and our ex vivo and xenograft results, we suggest that the functional interaction between ZEB1 and ERα may alter the tissue tropism of metastatic breast cancer cells towards bone.

Distinct cytosine modification profiles define epithelial-to-mesenchymal cell-state transitions.

Background:Epithelial-to-mesenchymal transition (EMT) is an early step in the invasion-metastasis cascade, involving progression through intermediate cell states. Due to challenges with isolating intermediate cell states, genome-wide cytosine modifications that define transition are not completely understood.Methods:The authors measured multiple DNA cytosine modification marks and chromatin accessibility across clonal populations residing in specific EMT states.Results:Clones exhibiting more intermediate EMT phenotypes demonstrated increased 5-hydroxymethylcytosine and decreased 5-methylcytosine. Open chromatin regions containing increased 5-hydroxymethylcytosine CpG loci were enriched in EMT transcription factor motifs and were associated with Rho GTPases.Conclusion:The results indicate the importance of both distinct and shared epigenetic profiles associated with EMT processes that may be targeted to prevent EMT progression.

Quantifying the Epithelial-to-Mesenchymal Transition (EMT) from Bench to Bedside

Simple SummaryCell developmental programs used in wound healing and development such as the epithelial-to-mesenchymal transition (EMT) are frequently coopted by solid tumors to increase motility, plasticity, and invasive characteristics which promote metastasis. Identifying and quantifying the presence and extent of these programs can help to aid in patient prognosis and dictate therapeutic decision making. Here, we review the methods and findings to detect and quantify these cellular transitions in both laboratory and clinical settings.The epithelial-to-mesenchymal transition (EMT) and its reversal, the mesenchymal-to-epithelial transition (MET) are critical components of the metastatic cascade in breast cancer and many other solid tumor types. Recent work has uncovered the presence of a variety of states encompassed within the EMT spectrum, each of which may play unique roles or work collectively to impact tumor progression. However, defining EMT status is not routinely carried out to determine patient prognosis or dictate therapeutic decision-making in the clinic. Identifying and quantifying the presence of various EMT states within a tumor is a critical first step to scoring patient tumors to aid in determining prognosis. Here, we review the major strides taken towards translating our understanding of EMT biology from bench to bedside. We review previously used approaches including basic immunofluorescence staining, flow cytometry, single-cell sequencing, and multiplexed tumor mapping. Future studies will benefit from the consideration of multiple methods and combinations of markers in designing a diagnostic tool for detecting and measuring EMT in patient tumors.

Abstract P4-07-19: Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state

Abstract Background: Triple Negative Breast Cancer (TNBC) is an aggressive and heterogeneous subtype characterized by ER/PR/HER2 negative status. Much of the disease potential and aggressive nature of this subtype derives from inter- and intra-tumoral heterogeneity, which makes developing targeted therapies challenging. A key contributor to both heterogeneity in TNBC and later stage chemo-resistance and metastasis is the Epithelial-to-Mesenchymal transition (EMT). This developmental program is frequently exploited in the context of cancer to increase migratory abilities, invasiveness, metastatic potential, and resistance to chemotherapy. Indeed, EMT has been demonstrated and linked to poor prognosis and decreased survival in many solid cancer types. Cells have been found to reside in multiple stable intermediate states along the EMT spectrum, which confer increased aggressive, metastatic, and chemoresistance attributes to a heterogeneous tumor through increased stem-like characteristics. Identifying and targeting this disease-potentiating population in patient tumors is a major hurdle in overcoming metastatic disease. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in diagnostic pathology or cancer therapy have been minimal. One reason for this gap stems from our inability to accurately detect and quantify epithelial-mesenchymal heterogeneity in primary tumor specimens. Secondly, the significance of recently identified intermediate or partial EMT states to predicting tumor prognosis and therapy response are unclear. Approach & Results: To study the role of various states within the EMT spectrum and their regulatory networks, the heterogeneous breast cancer cell line, SUM149PT, was used to derive six single cell clones encompassing the spectrum of EMT states, from epithelial to mesenchymal. Interrogation of this model system in vivo has revealed increased tumor growth and metastatic potential in the intermediate EMT states when compared to the extreme epithelial and mesenchymal states. To further elucidate EMT states in vivo, we employ a 6-marker multi-round immunofluorescence-based staining approach to identify cells that reside in various states along the EMT spectrum. We subsequently used an entropy-based approach and nearest-neighbor analysis on these tumors with the purpose of scoring heterogeneity and overall EMT state. Notably, this analysis segregated stromal infiltrates and their contributions to aggregate EMT scoring, which has been a major hurdle in using EMT as a scoring metric in patient samples. Overall, SUM149 clone-derived tumors held true to the relative EMT states of the starting cell populations; intermediate-derived tumors displayed high heterogeneity while epithelial and mesenchymal clone-derived tumors had lower levels of heterogeneity, despite retaining different EMT scores. Decoupling of heterogeneity and EMT state in this way provides two metrics to assess potential metastatic ability of a tumor. This staining method and analysis has been successfully applied in a preliminary set of patient tumors, showing promise for these two factors, E-M Heterogeneity and EMT score, as a tumor prognostic indicator to inform therapeutic decision-making. Conclusions: EMT tumor states and EMT-derived intra-tumoral heterogeneity play an important role in tumor metastasis and disease progression. Here, we have validated a multiplexed staining approach to quantify these metrics within a tumor, while segregating out stromal infiltrating cells. In the future, this staining and quantification shows promise as a means of predicting patient prognosis and informing potential treatment options based on targeting EMT states Citation Format: Meredith S Brown, Behnaz Abdollahi, Nevena Ognjenovic, Kristen E Muller, Saeed Hassanpour, Diwakar R Pattabiraman. Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-19.

EBV latent membrane proteins promote hybrid epithelial-mesenchymal and extreme mesenchymal states of nasopharyngeal carcinoma cells for tumorigenicity.

EBV-encoded LMPs are consistently detected in nasopharyngeal carcinoma (NPC). Recent evidence suggests potential roles of LMP1 and LMP2A in Epithelial-to-mesenchymal transition (EMT) process in NPC. EMT engages in the generation and maintenance of cancer stem cells (CSCs) and confers on cancer cells increased tumor-initiating and metastatic potential, and higher resistance to anticancer therapies. However, how LMP1 and LMP2A regulate the EMT process to generate cells with different EMT states and its implications for tumor progression remain unclear. Here we report that LMP1 and LMP2A promote EMT that drives NPC cells from the epithelial-like state (E) (CD104+, CD44low) to epithelial-mesenchymal hybrid (E/M) state (CD104+, CD44high). Furthermore, LMP2A possesses an additional function in stabilizing LMP1 and increasing the level of LMP1 in NPC cells. The elevated LMP1 further forces the EMT to generate extreme-mesenchymal (xM) state cells (CD104-, CD44high). To define the tumorigenic features of cancer stem cells at different states in the EMT spectrum, E, E/M and xM subpopulations were isolated and tested for tumorigenic capability in a tumor xenograft animal model. We found that the cells with E/M phenotypes possess the highest tumor initiating capacity. However, the xM subpopulation exhibits increased vasculogenic mimicry, a hallmark of metastatic cancers. Taken together, coordinated action of LMP1 and LMP2A generates an array of intermediate subpopulations in the EMT spectrum that are responsible for distinct tumorigenic features of NPC such as tumor-initiation, vasculogenesis, and metastasis.

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for invivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

Calcium signaling induces a partial EMT.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

Analysis of Cellular EMT States Using Molecular Biology and High Resolution FISH Labeling.

Metastasis results from the ability of cancer cells to grow and to spread beyond the primary tumor to distant organs. Epithelial-to-Mesenchymal Transition (EMT), a fundamental developmental process, is reactivated in cancer cells, and causes epithelial properties to evolve into mesenchymal and invasive ones. EMT changes cellular characteristics between two distinct states, yet, the process is not binary but rather reflects a broad spectrum of partial EMT states in which cells exhibit various degrees of intermediate epithelial and mesenchymal phenotypes. EMT is a complex multistep process that involves cellular reprogramming through numerous signaling pathways, alterations in gene expression, and changes in chromatin morphology. Therefore, expression of key proteins, including cadherins, occludin, or vimentin must be precisely regulated. A comprehensive understanding of how changes in nuclear organization, at the level of single genes clusters, correlates with these processes during formation of metastatic cells is still missing and yet may help personalized prognosis and treatment in the clinic. Here, we describe methods to correlate physiological and molecular states of cells undergoing an EMT process with chromatin rearrangements observed via FISH labeling of specific domains.

ΔNp63-Regulated Epithelial-to-Mesenchymal Transition State Heterogeneity Confers a Leader-Follower Relationship That Drives Collective Invasion.

Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader-follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205-dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells. SIGNIFICANCE: These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.

A plausible accelerating function of intermediate states in cancer metastasis.

Epithelial-to-mesenchymal transition (EMT) is a fundamental cellular process and plays an essential role in development, tissue regeneration, and cancer metastasis. Interestingly, EMT is not a binary process but instead proceeds with multiple partial intermediate states. However, the functions of these intermediate states are not fully understood. Here, we focus on a general question about how the number of partial EMT states affects cell transformation. First, by fitting a hidden Markov model of EMT with experimental data, we propose a statistical mechanism for EMT in which many unobservable microstates may exist within one of the observable macrostates. Furthermore, we find that increasing the number of intermediate states can accelerate the EMT process and that adding parallel paths or transition layers may accelerate the process even further. Last, a stabilized intermediate state traps cells in one partial EMT state. This work advances our understanding of the dynamics and functions of EMT plasticity during cancer metastasis.

Control of Invasion by Epithelial-to-Mesenchymal Transition Programs during Metastasis.

Epithelial-to-mesenchymal transition (EMT) programs contribute to the acquisition of invasive properties that are essential for metastasis. It is well established that EMT programs alter cell state and promote invasive behavior. This review discusses how rather than following one specific program, EMT states are diverse in their regulation and invasive properties. Analysis across a spectrum of models using a combination of approaches has revealed how unique features of distinct EMT programs dictate whether tumor cells invade as single cells or collectively as cohesive groups of cells. It has also been shown that the mode of collective invasion is determined by the nature of the EMT, with cells in a trailblazer-type EMT state being capable of initiating collective invasion, whereas cells that have undergone an opportunist-type EMT are dependent on extrinsic factors to invade. In addition to altering cell intrinsic properties, EMT programs can influence invasion through non-cell autonomous mechanisms. Analysis of tumor subpopulations has demonstrated how EMT-induced cells can drive the invasion of sibling epithelial populations through paracrine signaling and remodeling of the microenvironment. Importantly, the variation in invasive properties controlled by EMT programs influences the kinetics and location of metastasis.