Interplay Between Types of Cadherin in Breast Cancer
Interplay Between Types of Cadherin in Breast Cancer
Introduction: Deregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)-β signaling. Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer.
Methods: The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis.
Results: VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of β-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression was forced.
Conclusions: Our results highlight the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents the re-expression of E-cadherin and localization of β-catenin at the plasma membrane of mesenchymal mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.
Cadherins are a family of transmembrane proteins that, together with their associated intracellular catenins, have important functions in cell-cell adhesion. Different cell types express different members of the cadherin family. Epithelial (E)-cadherin is a key component of adherens junctions in epithelial cells and functions as a suppressor of tumor growth and invasion. Perturbation of its function leads to an invasive phenotype in many tumors. Neural (N)-cadherin is expressed in neural tissues and fibroblasts, where it mediates a less stable and more dynamic form of cell-cell adhesion. Vascular endothelial (VE)-cadherin is the primary component of endothelial cell adherens junctions and has an important function in regulating vascular permeability and angiogenesis. Because of the important role played by cadherins in cell recognition, adhesion, and signaling, modulation of their function and expression has significant implications for the progression of tumors. For instance, a switch from E-cadherin to N-cadherin expression contributes to increased tumor cell migration, invasion and metastasis. Aberrant expression of VE-cadherin was first detected in aggressive melanoma cells and in some cases of sarcoma. A recent study from our group has revealed that VE-cadherin is expressed aberrantly in a subset of tumor cells in human breast cancer. In a mouse mammary carcinoma model, VE-cadherin expression was induced in cancer cells that had undergone epithelial-mesenchymal transition (EMT). Functional experiments showed that VE-cadherin promotes malignant tumor cell proliferation and invasion by enhancing the protumorigenic transforming growth factor-beta (TGF-β) pathway. However, the functional interaction between VE-cadherin and N-cadherin during tumor progression is poorly characterized to date.
EMT was first described by Elizabeth Hay in the 1980s as a central process in early embryonic morphogenesis. The initial step of EMT includes the loss of epithelial markers such as E-cadherin via its transcriptional repression and the gain of mesenchymal markers such as vimentin. As a consequence, the cadherin-binding partner β-catenin can dissociate from the E-cadherin complex at the plasma membrane and translocate to the nucleus where it participates in EMT signaling and activates genes involved in tumor progression. Epithelial cells then lose their typical baso-apical polarization as cell-cell junctions disassemble. Additionally, the cytoskeleton undergoes dynamic cortical actin remodeling and gains the front-rear polarization that facilitates cell movement. Finally, cell-matrix adhesion changes as proteolytic enzymes such as matrix metalloproteases are activated. The transition from an epithelial to mesenchymal phenotype is reversible; for example, several rounds of EMT and mesenchymal-epithelial transition (MET) occur during development as cells differentiate and the complex three dimensional structure of internal organs forms. There is increasing evidence that EMT also facilitates the dissemination of tumor cells to form distant metastasis. Various publications have described a switch between the epithelial and mesenchymal phenotypes through EMT and MET in models of colorectal, bladder, ovarian and breast cancer. These findings indicate that the phenotypic conversion of tumor cells in the metastatic cascade is multifaceted, with EMT being critical for the initial transformation from benign to invasive carcinoma and the spreading of tumor cells, but MET occurring at the site of metastatic colonization.
The mouse mammary carcinoma model that we have previously used to study the expression of cadherins utilizes tumor cell lines that represent different stages of tumor progression: Ep5 cells are tumorigenic mammary epithelial cells transformed by the v-Ha-Ras oncogene, whereas Ep5ExTu cells, isolated from Ep5 cell tumors grown in mice, have undergone EMT in vivo and present a mesenchymal, invasive and angiogenic phenotype. We observed that VE-cadherin expression is induced in these murine breast cancer cells during (TGF-β-mediated) EMT. On the other hand, E-cadherin expression was downregulated, and N-cadherin levels remained unchanged. Silencing VE-cadherin expression inhibited tumor cell proliferation and invasion in vitro, and experimental tumor growth in mice. However, the role of N-cadherin and its potential interaction with VE-cadherin in this model is unclear. Here, we investigate the influence of N-cadherin on EMT and tumor progression in Ep5ExTu cells. Silencing N-cadherin significantly decreased VE-cadherin expression and stimulated Ep5ExTu cells to re-express E-cadherin at the cell surface. This promoted localization of β-catenin at the plasma membrane and induced the cells to undergo MET. Efficient silencing of N-cadherin expression in Ep5ExTu cells consistently inhibited tumor growth, and complete tumor regression was even seen in some cases. Taken together, these results reveal a novel interplay between classical cadherins in breast cancer progression.
Abstract and Introduction
Abstract
Introduction: Deregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)-β signaling. Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer.
Methods: The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis.
Results: VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of β-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression was forced.
Conclusions: Our results highlight the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents the re-expression of E-cadherin and localization of β-catenin at the plasma membrane of mesenchymal mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.
Introduction
Cadherins are a family of transmembrane proteins that, together with their associated intracellular catenins, have important functions in cell-cell adhesion. Different cell types express different members of the cadherin family. Epithelial (E)-cadherin is a key component of adherens junctions in epithelial cells and functions as a suppressor of tumor growth and invasion. Perturbation of its function leads to an invasive phenotype in many tumors. Neural (N)-cadherin is expressed in neural tissues and fibroblasts, where it mediates a less stable and more dynamic form of cell-cell adhesion. Vascular endothelial (VE)-cadherin is the primary component of endothelial cell adherens junctions and has an important function in regulating vascular permeability and angiogenesis. Because of the important role played by cadherins in cell recognition, adhesion, and signaling, modulation of their function and expression has significant implications for the progression of tumors. For instance, a switch from E-cadherin to N-cadherin expression contributes to increased tumor cell migration, invasion and metastasis. Aberrant expression of VE-cadherin was first detected in aggressive melanoma cells and in some cases of sarcoma. A recent study from our group has revealed that VE-cadherin is expressed aberrantly in a subset of tumor cells in human breast cancer. In a mouse mammary carcinoma model, VE-cadherin expression was induced in cancer cells that had undergone epithelial-mesenchymal transition (EMT). Functional experiments showed that VE-cadherin promotes malignant tumor cell proliferation and invasion by enhancing the protumorigenic transforming growth factor-beta (TGF-β) pathway. However, the functional interaction between VE-cadherin and N-cadherin during tumor progression is poorly characterized to date.
EMT was first described by Elizabeth Hay in the 1980s as a central process in early embryonic morphogenesis. The initial step of EMT includes the loss of epithelial markers such as E-cadherin via its transcriptional repression and the gain of mesenchymal markers such as vimentin. As a consequence, the cadherin-binding partner β-catenin can dissociate from the E-cadherin complex at the plasma membrane and translocate to the nucleus where it participates in EMT signaling and activates genes involved in tumor progression. Epithelial cells then lose their typical baso-apical polarization as cell-cell junctions disassemble. Additionally, the cytoskeleton undergoes dynamic cortical actin remodeling and gains the front-rear polarization that facilitates cell movement. Finally, cell-matrix adhesion changes as proteolytic enzymes such as matrix metalloproteases are activated. The transition from an epithelial to mesenchymal phenotype is reversible; for example, several rounds of EMT and mesenchymal-epithelial transition (MET) occur during development as cells differentiate and the complex three dimensional structure of internal organs forms. There is increasing evidence that EMT also facilitates the dissemination of tumor cells to form distant metastasis. Various publications have described a switch between the epithelial and mesenchymal phenotypes through EMT and MET in models of colorectal, bladder, ovarian and breast cancer. These findings indicate that the phenotypic conversion of tumor cells in the metastatic cascade is multifaceted, with EMT being critical for the initial transformation from benign to invasive carcinoma and the spreading of tumor cells, but MET occurring at the site of metastatic colonization.
The mouse mammary carcinoma model that we have previously used to study the expression of cadherins utilizes tumor cell lines that represent different stages of tumor progression: Ep5 cells are tumorigenic mammary epithelial cells transformed by the v-Ha-Ras oncogene, whereas Ep5ExTu cells, isolated from Ep5 cell tumors grown in mice, have undergone EMT in vivo and present a mesenchymal, invasive and angiogenic phenotype. We observed that VE-cadherin expression is induced in these murine breast cancer cells during (TGF-β-mediated) EMT. On the other hand, E-cadherin expression was downregulated, and N-cadherin levels remained unchanged. Silencing VE-cadherin expression inhibited tumor cell proliferation and invasion in vitro, and experimental tumor growth in mice. However, the role of N-cadherin and its potential interaction with VE-cadherin in this model is unclear. Here, we investigate the influence of N-cadherin on EMT and tumor progression in Ep5ExTu cells. Silencing N-cadherin significantly decreased VE-cadherin expression and stimulated Ep5ExTu cells to re-express E-cadherin at the cell surface. This promoted localization of β-catenin at the plasma membrane and induced the cells to undergo MET. Efficient silencing of N-cadherin expression in Ep5ExTu cells consistently inhibited tumor growth, and complete tumor regression was even seen in some cases. Taken together, these results reveal a novel interplay between classical cadherins in breast cancer progression.
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