Epithelial-mesenchymal Cell Transformation Research Articles

A novel humanized Frizzled-7-targeting antibody enhances antitumor effects of Bevacizumab against triple-negative breast cancer via blocking Wnt/\u03b2-catenin signaling pathway

BackgroundAnti-angiogenic therapy has been widely applied to the clinical treatment of malignant tumors. However, the efficacy of such treatments has been called into question, especially in triple-negative breast cancer (TNBC). Bevacizumab, the first anti-angiogenic agent approved by FDA, actually increases invasive and metastatic properties of TNBC cells, resulting from the activation of Wnt/β-catenin signaling in response to hypoxia. As a critical receptor of Wnt/β-catenin signaling, Frizzled-7 (Fzd7) is aberrantly expressed in TNBC, indicating Fzd7 a potential target for developing drugs to be combined with anti-angiogenic agents.MethodsHybridoma technique and antibody humanization technique were utilized to generate a Fzd7-targeting antibody (SHH002-hu1). Biolayer interferometry (BLI) assay and near infrared (NIR) imaging were conducted to detect the affinity and targeting ability of SHH002-hu1. Next, whether SHH002-hu1 could suppress the invasion and migration of TNBC cells induced by Bevacizumab were validated, and the underlying molecular mechanisms were elucidated by luciferase reporter and western blot assays. The nude-mice transplanted TNBC models were established to assess the anti-TNBC activities of SHH002-hu1 when combined with Bevacizumab. Then, the effects on putative TNBC stem-like cells and Wnt/β-catenin signaling were evaluated by immunofluorescence (IF). Further, the tumor-initiating and self-renew capacity of TNBC cells were studied by secondary nude mouse xenograft model and sphere formation assay. In addition, the effects of SHH002-hu1 on the adaptation of TNBC cells to hypoxia were evaluated by the detection of vasculogenic mimicry (VM) and hypoxia-inducible factor-1α (HIF-1α) transcriptional activity.ResultsThe novel humanized antibody targeting Fzd7 (SHH002-hu1) exhibited extremely high affinity with Fzd7, and specifically targeted to Fzd7+ cells and tumor tissues. SHH002-hu1 repressed invasion, migration and epithelial-mesenchymal cell transformation (EMT) of TNBC cells induced by Bevacizumab through abating Wnt/β-catenin signaling. SHH002-hu1 significantly enhanced the capacity of Bevacizumab to inhibit the growth of TNBC via reducing the subpopulation of putative TNBC stem-like cells, further attenuating Bevacizumab-enhanced tumor-initiating and self-renew capacity of TNBC cells. Moreover, SHH002-hu1 effectively restrained the adaptation of TNBC cells to hypoxia via disrupting Wnt/β-catenin signaling.ConclusionSHH002-hu1 significantly enhances the anti-TNBC capacity of Bevacizumab, and shows the potential of preventing TNBC recurrence, suggesting SHH002-hu1 a good candidate for the synergistic therapy together with Bevacizumab.

TGF-β Signaling Promotes Tissue Formation during Cardiac Valve Regeneration in Adult Zebrafish

Cardiac valve disease can lead to severe cardiac dysfunction and is thus a frequent cause of morbidity and mortality. Its main treatment is valve replacement, which is currently greatly limited by the poor recellularization and tissue formation potential of the implanted valves. As we still lack suitable animal models to identify modulators of these processes, here we used adult zebrafish and found that, upon valve decellularization, they initiate a rapid regenerative program that leads to the formation of new functional valves. After injury, endothelial and kidney marrow-derived cells undergo cell cycle re-entry and differentiate into new extracellular matrix-secreting valve cells. The TGF-β signaling pathway promotes the regenerative process by enhancing progenitor cell proliferation as well as valve cell differentiation. These findings reveal a key role for TGF-β signaling in cardiac valve regeneration and establish the zebrafish as a model to identify and test factors promoting cardiac valve recellularization and growth.

Crosstalk of Oncogenic Signaling Pathways during Epithelial-Mesenchymal Transition.

Epithelial–mesenchymal transition (EMT) and cell transformation have been well-documented in multiple cancer cell models and are believed to be one of the earliest events in tumor progression. Genetic and epigenetic modifications shift cells toward either end of the EMT spectrum, and can be influenced by the microenvironment surrounding a tumor. EMT and mesenchymal–epithelial transition are critical to normal function and development and an intricate network of transcription factors and transcriptional regulators tightly regulates these processes. As evidenced in normal and transformed cell lines, many signaling pathways trigger EMT during development and differentiation. The signaling pathways include those triggered by different members of the transforming growth factor superfamily, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, hypoxia-inducible factor, Wnt, Notch, and many others. Functional redundancies allow cells to undergo EMT even if these key transcriptional regulators are lacking, but these same redundancies also make these pathways particularly susceptible to gain-of-function mutations or constitutive signal activation; the “forced” transition toward either a mesenchymal or epithelial phenotype.

TGFbeta Type III and TGFbeta Type II receptors have distinct activities during epithelial-mesenchymal cell transformation in the embryonic heart.

During the early stages of heart development, progenitors for the heart valves and septa come from endothelial cells via a developmental process known as "epithelial-mesenchymal cell transformation." This process is restricted to the atrioventricular (AV) canal and outflow tract portions of the embryonic heart. TGFbeta signal transduction pathways play critical roles during epithelial-mesenchymal cell transformation in heart development. Previously, we showed that both TGFbeta Type II (TbetaRII) and Type III (TbetaRIII) receptors are required to mediate epithelial mesenchymal cell transformation in chick heart. Further, distinct TGFbeta2 and TGFbeta3 activities correspond to separate components of the embryonic cell transformation process. Studies by others of TGFbeta-mediated inhibition of cell proliferation produced a model where TbetaRIII functions by facilitating TGFbeta2 binding to TbetaRII. In the present study, we provide evidence that TbetaRIII mediates distinct cellular responses from those of TbetaRII. Blocking antibody for TbetaRIII, but not antibody against TbetaRII, specifically inhibits the endothelial cell-cell separation step. Examination of developmental markers, perturbed by blocking TbetaRIII antibody, revealed a pattern of expression distinctively different from that of TbetaRII antibody treatment. These data show that a distinct TbetaRIII-mediated process is required for endothelial cell-cell separation during epithelial mesenchymal cell transformation. As TGFbeta2 mediates endothelial cell-cell separation, the data point to a specific association of TGFbeta2 and TbetaRIII in the cell separation step of epithelial mesenchymal cell transformation. We conclude that distinct TbetaRII and TbetaRIII signal transduction pathways mediate epithelial-mesenchymal cell transformation in the heart.

Slug is an Essential Target of TGFβ2 Signaling in the Developing Chicken Heart

An epithelial–mesenchymal cell transformation (EMT) occurs during the development of endocardial cushions in the atrioventricular (AV) canal of the heart. This is a complex developmental process regulated by multiple extracellular signals and signal transduction pathways. It was recently shown that the transcription factor Slug is expressed in the AV canal and is required for initial steps of EMT. Treatment of AV canal explants with either antisense oligodeoxynucleotides toward Slug or anti-TGFβ2 antibody inhibited initial steps of EMT. Others have identified roles for HGF and BMP during EMT in the heart. Both HGF and BMP are known to regulate Slug in other cell types. To determine whether TGFβ2 or other signaling factors regulate Slug expression during EMT in the heart, we cultured AV canal explants in the presence of anti-TGFβ2 antibody, anti-TGFβ3 antibody, pertussis toxin, retinoic acid, noggin, or anti-HGF antibody. Only treatment with anti-TGFβ2 antibody or retinoic acid inhibited Slug expression in AV canal explants. Consistent with these data, we found that retinoic acid disrupted initial steps of EMT, while antagonists of BMP and HGF signaling disrupted later steps of EMT. Transfection of AV canal explants with Slug rescued the inhibitory effect of anti-TGFβ2 antibody but not retinoic acid on EMT. Slug is thus an essential target of TGFβ2 signaling during EMT in the developing chicken heart.

Trichloroethylene inhibits development of embryonic heart valve precursors in vitro.

Previous epidemiological studies with humans and laboratory studies with chickens and rats linked trichloroethylene (TCE) exposure to cardiac defects. Although the cardiac defects in humans and laboratory animals produced by TCE are diverse, a majority of them involves valvular and septal structures. Progenitors of the valves and septa are formed by an epithelial-mesenchymal cell transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract areas of the heart. Based on these studies, we hypothesized that TCE might cause cardiac valve and septa defects by specifically perturbing epithelial-mesenchymal cell transformation. We tested this hypothesis using an in vitro chick-AV canal culture model. This study shows that TCE affected several elements of epithelial-mesenchymal cell transformation. In particular, TCE blocked the endothelial cell-cell separation process that is associated with endothelial activation. Moreover, TCE inhibited mesenchymal cell formation throughout the concentration range tested (50-250 ppm). In contrast, TCE had no effect on the cell migration rate of the fully formed mesenchymal cells. Finally, the expression of 3 proteins (selected as molecular markers of epithelial-mesenchymal cell transformation) was analyzed in untreated and TCE-treated cultures. TCE inhibited the expression of the transcription factor Mox-1 and extracellular matrix (ECM) protein fibrillin 2. In contrast, TCE had no effect on the expression of alpha-smooth muscle actin. These data suggest that TCE may cause cardiac valvular and septal malformations by inhibiting endothelial separation and early events of mesenchymal cell formation in the heart.

TGFβ2 and TGFβ3 Have Separate and Sequential Activities during Epithelial–Mesenchymal Cell Transformation in the Embryonic Heart

Heart valve formation is initiated by an epithelial–mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor β (TGFβ) mediates EMT in the AV canal, and TGFβ1 and 2 isoforms are expressed in the mouse heart while TGFβ 2 and 3 are expressed in the avian heart. Depletion of TGFβ3 in avian or TGFβ2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFβ isoforms participate in valve formation. In this study, we examined the localization and function of TGFβ2 and TGFβ3 in the chick heart during EMT. TGFβ2 was present in both endothelium and myocardium before and after EMT. TGFβ2 antibody inhibited endothelial cell–cell separation. In contrast, TGFβ3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFβ3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFβ2 and 3 increased fibrillin 2 expression. However, only TGFβ2 treatment increased cell surface β-1,4-galactosyltransferase expression. These data suggest that TGFβ2 and TGFβ3 are sequentially and separately involved in the process of EMT. TGFβ2 mediates initial endothelial cell–cell separation while TGFβ3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.

Epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor beta 3.

During early cardiac development, the progenitor cells of the heart valves and membranous septa undergo an epithelial-mesenchymal transformation. Previous studies have shown that this transformation depends on the activity of a transforming growth factor beta (TGF beta) molecule produced by the heart. In the present study, we have used modified antisense oligodeoxynucleotides generated to nonconserved regions of TGF beta 1, -2, -3, and -4 to examine the possible roles of these members in this transformation. A phosphoramidate-modified oligonucleotide complementary to TGF beta 3 mRNA was capable of inhibiting normal epithelial-mesenchymal transformation by 80%. Unmodified oligonucleotides to TGF beta 3, modified oligonucleotides to TGF beta 1, -2, and -4, and two modified control oligonucleotides were unable to inhibit the transformation. These data demonstrate that a specific member of the TGF beta family, TGF beta 3, is essential for the epithelial-mesenchymal cell transformation.

Signal transduction of a tissue interaction during embryonic heart development.

During early cardiac development, progenitors of the valves and septa of the heart are formed by an epithelial-mesenchymal cell transformation of endothelial cells of the atrioventricular (AV) canal. We have previously shown that this event is due to an interaction between the endothelium and products of the myocardium found within the extracellular matrix. The present study examines signal transduction mechanisms governing this differentiation of AV canal endothelium. Activators of protein kinase C (PKC), phorbol myristate acetate (PMA) and mezerein, both produced an incomplete phenotypic transformation of endothelial cells in an in vitro bioassay for transformation. On the other hand, inhibitors of PKC (H-7 and staurosporine) and tyrosine kinase (genistein) blocked cellular transformation in response to the native myocardium or a myocardially-conditioned medium. Intracellular free calcium concentration ([Ca2+]i) was measured in single endothelial cells by microscopic digital analysis of fura 2 fluorescence. Addition of a myocardial conditioned medium containing the transforming stimulus produced a specific increase in [Ca2+]i in "competent" AV canal, but not ventricular, endothelial cells. Epithelial-mesenchymal cell transformation was inhibited by pertussis toxin but not cholera toxin. These data lead to the hypothesis that signal transduction of this tissue interaction is mediated by a G protein and one or more kinase activities. In response to receptor activation, competent AV canal endothelial cells demonstrate an increase in [Ca2+]i. Together, the data provide direct evidence for a regional and temporal regulation of signal transduction processes which mediate a specific extracellular matrix-mediated tissue interaction in the embryo.

Epithelial-mesenchymal cell transformation in the embryonic heart can be mediated, in part, by transforming growth factor β

Progenitor cells of the valves and membranous septa of the vertebrate heart are formed by transformation of a specific population of endothelial cells into mesenchyme. Previous studies have shown that this epithelial-mesenchymal cell transformation is mediated by a signal produced by the myocardium of the atrioventricular (AV) canal and transferred across the extracellular matrix. Data are presented here that transforming growth factor β (TGFβ1 or TGFβ2), in combination with an explant of ventricular myocardium, will produce an epithelial-mesenchymal transformation by cultured AV canal endothelial cells in vitro. Alone, neither component is capable of producing this effect. The factor provided by the ventricular explant cannot be substituted by either epidermal growth factor or basic fibroblast growth factor. Further experiments show that an antibody that blocks TGFβ activity is effective in preventing the epithelial-mesenchymal cell transformation normally produced by AV canal myocardium. Control antibodies are without effect. By immunological criteria, a member of the TGFβ family of molecules can be demonstrated in the chicken embryo and heart at the time overt valvular formation begins. Together, these data show that TGFβ1 can produce mesenchymal cell formation in vitro and provide evidence that a member of the TGFβ family is present and plays a role in the process of epithelial-mesenchymal cell transformation in the embryonic heart.