Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021)

The contribution of partial and full epithelial-to-mesenchymal transition to breast cancer progression

Breast cancer is a leading cause of mortality in women worldwide. The lethality associated with the disease is primarily a consequence of the systemic spread of cancer cells and the formation of metastasis, particularly in the lung, liver, brain and bones. Multiple cellular and molecular mechanisms may contribute to the complex process of metastasis formation. In order to devise clinically effective strategies to combat metastatic disease, these need to be better understood. Epithelial-to-mesenchymal transition (EMT) is a cellular program of trans- differentiation which is critical for embryonic development and wound healing but which may also contribute to the dissemination of cancer cells. During an EMT, epithelial cancer cells lose their cell-cell adhesion, dedifferentiate, and acquire a migratory and invasive phenotype. Experimental induction of an EMT is sufficient for metastasis formation. However, whether a spontaneous EMT is required for metastasis in an unperturbed system in vivo is a major focus of current research. Within recent years, it has become clear that EMT is not a binary switch but covers a spectrum of intermediate EMT hybrid states which differ in their functional characteristics and metastatic potential. However, due to the transient and reversible nature of the process, the extent to which cancer cells spontaneously undergo a partial or full EMT in vivo and the functional consequences regarding metastasis remain unknown. With my PhD work, I have aimed at assessing the contribution of partial and full EMT to breast cancer progression and metastasis. To this end, I have established two novel color-switching lineage tracing models in transgenic mice. Based on an irreversible switch from mCherry to GFP expression, this model allows to visualize and track cancer cells that have undergone a partial or full EMT, even if they re-differentiate by undergoing a mesenchymal-to-epithelial transition (MET). I show that cancer cells mostly transition between epithelial/mesenchymal hybrid states but rarely undergo a full EMT. Furthermore, cells which have undergone a partial EMT are highly enriched in lung metastases compared to primary tumors. In particular, metastasis with a mosaic composition of mCherry and GFP positive cells are observed, pointing towards a collective dissemination of cells that have undergone a partial EMT together with epithelial cancer cells. In contrast, cells that have undergone a full EMT retain a more quiescent mesenchymal phenotype and do not colonize the lung. In conclusion, these data suggest that although a full EMT may not be required, a partial EMT contributes to experimental breast cancer metastasis. In addition, I have further characterized the mammary gland-specific flippase driver-line which we have generated for the lineage tracing experiments. These mice may serve as a versatile tool for studying mammary gland biology and breast carcinogenesis. In summary, my PhD work provides novel insights into the dynamics of EMT and MET in vivo, as well as the contribution of partial and full EMT to breast cancer metastasis. Furthermore, our newly established mouse models offer novel opportunities to study the contribution of partial and full EMT towards distinct aspects of breast cancer progression in vivo

Breast cancer as an example of tumour heterogeneity and tumour cell plasticity during malignant progression

Heterogeneity within a tumour increases its ability to adapt to constantly changing constraints, but adversely affects a patient's prognosis, therapy response and clinical outcome. Intratumoural heterogeneity results from a combination of extrinsic factors from the tumour microenvironment and intrinsic parameters from the cancer cells themselves, including their genetic, epigenetic and transcriptomic traits, their ability to proliferate, migrate and invade, and their stemness and plasticity attributes. Cell plasticity constitutes the ability of cancer cells to rapidly reprogramme their gene expression repertoire, to change their behaviour and identities, and to adapt to microenvironmental cues. These features also directly contribute to tumour heterogeneity and are critical for malignant tumour progression. In this article, we use breast cancer as an example of the origins of tumour heterogeneity (in particular, the mutational spectrum and clonal evolution of progressing tumours) and of tumour cell plasticity (in particular, that shown by tumour cells undergoing epithelial-to-mesenchymal transition), as well as considering interclonal cooperativity and cell plasticity as sources of cancer cell heterogeneity. We review current knowledge on the functional contribution of cell plasticity and tumour heterogeneity to malignant tumour progression, metastasis formation and therapy resistance

A Transgenic MMTV-Flippase Mouse Line for Molecular Engineering in Mammary Gland and Breast Cancer Mouse Models.

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice

Hierarchy of TGFβ/SMAD, Hippo/YAP/TAZ, and Wnt/ β-catenin signaling in melanoma phenotype switching

In melanoma, a switch from a proliferative melanocytic to an invasive mesenchymal phenotype is based on dramatic transcriptional reprogramming which involves complex interactions between a variety of signaling pathways and their downstream transcriptional regulators. TGFβ/SMAD, Hippo/YAP/TAZ, and Wnt/ β-catenin signaling pathways are major inducers of transcriptional reprogramming and converge at several levels. Here, we report that TGFβ/SMAD, YAP/TAZ, and β-catenin are all required for a proliferative-to-invasive phenotype switch. Loss and gain of function experimentation, global gene expression analysis, and computational nested effects models revealed the hierarchy between these signaling pathways and identified shared target genes. SMAD-mediated transcription at the top of the hierarchy leads to the activation of YAP/TAZ and of β-catenin, with YAP/TAZ governing an essential subprogram of TGFβ-induced phenotype switching. Wnt/β-catenin signaling is situated further downstream and exerts a dual role: it promotes the proliferative, differentiated melanoma cell phenotype and it is essential but not sufficient for SMAD or YAP/TAZ-induced phenotype switching. The results identify epistatic interactions among the signaling pathways underlying melanoma phenotype switching and highlight the priorities in targets for melanoma therapy.ISSN:2575-107

Hierarchy of TGFβ/SMAD, Hippo/YAP/TAZ, and Wnt/β-catenin signaling in melanoma phenotype switching

In melanoma, a switch from a proliferative melanocytic to an invasive mesenchymal phenotype is based on dramatic transcriptional reprogramming which involves complex interactions between a variety of signaling pathways and their downstream transcriptional regulators. TGFβ/SMAD, Hippo/YAP/TAZ, and Wnt/β-catenin signaling pathways are major inducers of transcriptional reprogramming and converge at several levels. Here, we report that TGFβ/SMAD, YAP/TAZ, and β-catenin are all required for a proliferative-to-invasive phenotype switch. Loss and gain of function experimentation, global gene expression analysis, and computational nested effects models revealed the hierarchy between these signaling pathways and identified shared target genes. SMAD-mediated transcription at the top of the hierarchy leads to the activation of YAP/TAZ and of β-catenin, with YAP/TAZ governing an essential subprogram of TGFβ-induced phenotype switching. Wnt/β-catenin signaling is situated further downstream and exerts a dual role: it promotes the proliferative, differentiated melanoma cell phenotype and it is essential but not sufficient for SMAD or YAP/TAZ-induced phenotype switching. The results identify epistatic interactions among the signaling pathways underlying melanoma phenotype switching and highlight the priorities in targets for melanoma therapy

miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis

Epithelial tumour cells can gain invasive and metastatic capabilities by undergoing an epithelial-mesenchymal transition. Transcriptional regulators and post-transcriptional effectors like microRNAs orchestrate this process of high cellular plasticity and its malignant consequences. Here, using microRNA sequencing in a time-resolved manner and functional validation, we have identified microRNAs that are critical for the regulation of an epithelial-mesenchymal transition and of mesenchymal tumour cell migration. We report that miR-1199-5p is downregulated in its expression during an epithelial-mesenchymal transition, while its forced expression prevents an epithelial-mesenchymal transition, tumour cell migration and invasion in vitro, and lung metastasis in vivo. Mechanistically, miR-1199-5p acts in a reciprocal double-negative feedback loop with the epithelial-mesenchymal transition transcription factor Zeb1. This function resembles the activities of miR-200 family members, guardians of an epithelial cell phenotype. However, miR-1199-5p and miR-200 family members share only six target genes, indicating that, besides regulating Zeb1 expression, they exert distinct functions during an epithelial-mesenchymal transition

Parsing β-catenin’s cell adhesion and Wnt signaling functions in malignant mammary tumor progression

During malignant progression, epithelial cancer cells dissolve their cell–cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell–cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin’s transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)–PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin’s transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial–mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin’s transcriptional activities upon stimulation with Wnt3a or during TGF-β–induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin–dependent transcription in malignant tumor progression of breast cancer.Fil: Buechel, David. Universidad de Basilea; SuizaFil: Sugiyama, Nami. Universidad de Basilea; SuizaFil: Rubinstein, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; ArgentinaFil: Saxena, Meera. Universidad de Basilea; SuizaFil: Kalathur, Ravi K.R.. Universidad de Basilea; Suiza. Royal Children’s Hospital; AustraliaFil: Lüönd, Fabiana. Universidad de Basilea; SuizaFil: Vafaizadeh, Vida. Universidad de Basilea; SuizaFil: Valenta, Tomas. Universitat Zurich; SuizaFil: Hausmann, George. Universitat Zurich; SuizaFil: Cantù, Claudio. Linköping University; SueciaFil: Basler, Konrad. Universitat Zurich; SuizaFil: Christofori, Gerhard. Universidad de Basilea; Suiz

Distinct contributions of partial and full EMT to breast cancer malignancy

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance.ISSN:1534-5807ISSN:1878-155

Distinct contributions of partial and full EMT to breast cancer malignancy

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance