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.
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•Lineage tracing of partial and full EMT cells in breast cancer metastasis•Partial EMT cells cycle between hybrid EMT and epithelial stages•Partial, but not full, EMT cells are required for metastasis formation•Both partial and full EMT cells contribute to chemoresistance
Lüönd et al. establish genetic lineage tracing systems to monitor mammary tumor cells undergoing early partial and late full epithelial-mesenchymal transition (EMT). They demonstrate that partial EMT cells, but not full EMT cells, are required for lung metastasis, while both contribute to the development of chemoresistance.
An epithelial-mesenchymal transition (EMT) represents a basic morphogenetic process of high cell plasticity underlying embryogenesis, wound healing, cancer metastasis and drug resistance. It involves ...a profound transcriptional and epigenetic reprogramming of cells. A critical role of epigenetic modifiers and their specific chromatin modifications has been demonstrated during EMT. However, it has remained elusive whether epigenetic control differs between the dynamic cell state transitions of reversible EMT and the fixed differentiation status of irreversible EMT. We have employed varying EMT models of murine breast cancer cells to identify the key players establishing epithelial-mesenchymal cell plasticity during reversible and irreversible EMT. We demonstrate that the Mbd3/NuRD complex and the activities of histone deacetylases (HDACs), and Tet2 hydroxylase play a critical role in keeping cancer cells in a highly metastatic mesenchymal state. Combinatorial interference with their functions leads to mesenchymal-epithelial transition (MET) and efficiently represses metastasis formation by invasive murine and human breast cancer cells in vivo.
Changes in EphA2 signaling can affect cancer cell-cell communication and motility through effects on actomyosin contractility. However, the underlying cell-surface interactions and molecular ...mechanisms of how EphA2 mediates these effects have remained unclear. We demonstrate here that EphA2 and membrane-anchored membrane type-1 matrix metalloproteinase (MT1-MMP) were selectively up-regulated and coexpressed in invasive breast carcinoma cells, where, upon physical interaction in same cell-surface complexes, MT1-MMP cleaved EphA2 at its Fibronectin type-III domain 1. This cleavage, coupled with EphA2-dependent Src activation, triggered intracellular EphA2 translocation, as well as an increase in RhoA activity and cell junction disassembly, which suggests an overall repulsive effect between cells. Consistent with this, cleavage-prone EphA2-D359I mutant shifted breast carcinoma cell invasion from collective to rounded single-cell invasion within collagen and in vivo. Up-regulated MT1-MMP also codistributed with intracellular EphA2 in invasive cells within human breast carcinomas. These results reveal a new proteolytic regulatory mechanism of cell-cell signaling in cancer invasion.
Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo ...cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP-negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain-containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion.
Pygopus 2 (Pygo2) is a coactivator of Wnt/β-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me
) and participate in chromatin reading and writing. It remains unknown ...whether the Pygo2-H3K4me
association has a functional relevance in breast cancer progression
. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me
was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/β-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFβ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction potentiated Wnt/β-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and β-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me
interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes β-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.
Kaposi sarcoma (KS), an angioproliferative disease associated with Kaposi sarcoma herpesvirus (KSHV) infection, harbors a diversity of cell types ranging from endothelial to mesenchymal cells of ...unclear origin. We developed a three-dimensional cell model for KSHV infection and used it to demonstrate that KSHV induces transcriptional reprogramming of lymphatic endothelial cells to mesenchymal cells via endothelial-to-mesenchymal transition (EndMT). KSHV-induced EndMT was initiated by the viral proteins vFLIP and vGPCR through Notch pathway activation, leading to gain of membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive properties and concomitant changes in viral gene expression. Mesenchymal markers and MT1-MMP were found codistributed with a KSHV marker in the same cells from primary KS biopsies. Our data explain the heterogeneity of cell types within KS lesions and suggest that KSHV-induced EndMT may contribute to KS development by giving rise to infected, invasive cells while providing the virus a permissive cellular microenvironment for efficient spread.
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► KSHV induces mesenchymal reprogramming of lymphatic endothelial cells (LECs) in 3D ► KSHV-activated Notch-MT1-MMP axis regulates LEC reprogramming ► KSHV-reprogrammed LECs are invasive ► 3D microenvironment of infected LECs leads to viral gene expression changes
Poly(l-lactic acid-co-glycolic acid)/hydroxyapatite (PLGA/HAp) composites were fabricated by the in situ polymerization of l-lactide and glycolide in porous HAp disks, using lipase MM, derived from ...Mucor miehei, as a catalyst. Various PLGA/HAp composites were obtained by changing the feed ratio of l-lactide and glycolide. The fourier transform infrared spectroscopy, scanning electron microscopy and porosity measurements showed that the porous HAp was completely filled with PLGA after polymerization at 100 °C for 9 days. Lactyl unit fractions (FL ) of obtained PLGA calculated from the 1 H nuclear magnetic resonance were consistent with the feed fraction of l-lactide (fL ). The PGA/HAp, PLGA20/HAp, PLGA50/HAp, PLGA80/HAp and PLLA/HAp composites showed maximum bending strengths of 91.1 MPa, 78.8 MPa, 73.4 MPa, 54.3 MPa and 67.0 MPa, respectively. These values were 4.7-2.8 times greater than that of the untreated porous HAp disks and were suitable for artificial bone materials. The cell adhesion and proliferation properties of these materials with osteoblast-like MC3T3-E1 cells suggest that these PLGA/HAp composites have suitably bioactive surfaces. The PLGA/HAp composites showed higher alkaline phosphatase activity after cultivation of rat bone marrow stromal cells.
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).
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•A dual recombinase lineage tracing system for cancer cells undergoing EMT•Enables live imaging of cancer cells undergoing early or late EMT•Supports flow-cytometry-mediated isolation of cancer cells in various stages of EMT•Enables single-cell transcriptomic studies of cancer cells undergoing EMT
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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.
Tumor cells use membrane type 1 matrix metalloproteinase (MT1-MMP) for invasion and metastasis. However, the signaling mechanisms that underlie MT1-MMP regulation in cancer have remained unclear. ...Using a systematic gain-of-function kinome screen for MT1-MMP activity, we have here identified kinases that significantly enhance MT1-MMP activity in tumor cells. In particular, we discovered an MT1-MMP/FGF receptor-4 (FGFR4) membrane complex that either stimulates or suppresses MT1-MMP and FGFR4 activities, depending on a tumor progression-associated polymorphism in FGFR4. The FGFR4-R388 allele, linked to poor cancer prognosis, increased collagen invasion by decreasing lysosomal MT1-MMP degradation. FGFR4-R388 induced MT1-MMP phosphorylation and endosomal stabilization, and surprisingly, the increased MT1-MMP in return enhanced FGFR4-R388 autophosphorylation. A phosphorylation-defective MT1-MMP was stabilized on the cell surface, where it induced simultaneous FGFR4-R388 internalization and dissociation of cell–cell junctions. In contrast, the alternative FGFR4-G388 variant down-regulated MT1-MMP, and the overexpression of MT1-MMP and particularly its phosphorylation-defective mutant vice versa induced FGFR4-G388 degradation. These results provide a mechanistic basis for FGFR4-R388 function in cancer invasion.
Aberrant expression and polymorphism of fibroblast growth factor receptor 4 (FGFR4) has been linked to tumor progression and anticancer drug resistance. We describe here a novel mechanism of tumor ...progression by matrix degradation involving epithelial-to-mesenchymal transition in response to membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk variant. Both FGFR4 and MT1-MMP were upregulated in tissue biopsies from several human cancer types including breast adenocarcinomas, where they were partially coexpressed at the tumor/stroma border and tumor invasion front. The strongest overall coexpression was found in prostate carcinoma. Studies with cultured prostate carcinoma cell lines showed that the FGFR4-R388 variant, which has previously been associated with poor cancer prognosis, increased MT1-MMP-dependent collagen invasion. In this experimental model, knockdown of FGFR4-R388 or MT1-MMP by RNA interference blocked tumor cell invasion and growth in collagen. This was coupled with impaired phosphorylation of FGFR substrate 2 and Src, upregulation of E-cadherin, and suppression of cadherin-11 and N-cadherin. These in vitro results were substantiated by reduced MT1-MMP content and in vivo growth of prostate carcinoma cells after the FGFR4-R388 gene silencing. In contrast, knockdown of the alternative FGFR4-G388 allele enhanced MT1-MMP and invasive tumor cell growth in vivo and within three-dimensional collagen. These results will help to explain the reported association of the FGFR4-R388 variant with the progression and poor prognosis of certain types of tumors.
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