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Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.

Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson SA - J. Cell Biol. (2000)

Bottom Line: To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis.These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin.The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA. rhazan@smtplink.mssm.edu

ABSTRACT
E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

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Histopathology of metastatic lesions in nude mice. 5-μm sections of the pancreas of mice injected with N-cad-17 cells were stained with either H&E (A) or cytokeratin antibodies, followed by DAB detection (B). Cytokeratin immunoreactivity of the N-cad-17 primary tumor (C) was compared with the pancreatic lesion produced by this tumor in B. Sections from the salivary gland of mice injected with N-cad-5 cells were stained with anticytokeratin antibodies, followed by DAB detection (D) and sections from the omentum (E) and primary tumor of N-cad-5–injected mice (F) were stained with FITC-conjugated cytokeratin antibodies. N-cad-8 metastatic lesions were detected in lung sections by H&E (G) and cytokeratin/DAB detection (H). N-cad-15 cells were found in the lumbar spinal muscle (I) using double fluorescent staining with antiactin, followed by secondary antibodies coupled to rhodamine (red) and FITC-conjugated anticytokeratin antibodies (green). Bar: (A) 100 μm; (G and H) 50 μm; (B, D, and E) 33 μm; (C, F, and I) 20 μm.
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Figure 5: Histopathology of metastatic lesions in nude mice. 5-μm sections of the pancreas of mice injected with N-cad-17 cells were stained with either H&E (A) or cytokeratin antibodies, followed by DAB detection (B). Cytokeratin immunoreactivity of the N-cad-17 primary tumor (C) was compared with the pancreatic lesion produced by this tumor in B. Sections from the salivary gland of mice injected with N-cad-5 cells were stained with anticytokeratin antibodies, followed by DAB detection (D) and sections from the omentum (E) and primary tumor of N-cad-5–injected mice (F) were stained with FITC-conjugated cytokeratin antibodies. N-cad-8 metastatic lesions were detected in lung sections by H&E (G) and cytokeratin/DAB detection (H). N-cad-15 cells were found in the lumbar spinal muscle (I) using double fluorescent staining with antiactin, followed by secondary antibodies coupled to rhodamine (red) and FITC-conjugated anticytokeratin antibodies (green). Bar: (A) 100 μm; (G and H) 50 μm; (B, D, and E) 33 μm; (C, F, and I) 20 μm.

Mentions: Of a total of 20 mice injected with N-cadherin–expressing cell lines, 13 mice (65%) had primary tumors. Similarly, 10 out of 12 mice (83%) injected with control MCF-7 cells developed primary tumors. N-cadherin–expressing tumors grew slower on average, reaching a weight of ∼30% that of control MCF-7 or Neo-5 tumors (Table ). This is consistent with the slower growth rate of the N-cadherin–transfected cells in vitro (data not shown). Most (77%) of the 13 mice with N-cadherin–expressing primary tumors developed metastases in multiple sites, whereas none of the control mice bearing larger tumors had detectable metastases (Table ). Staining with H&E strongly suggested the presence of metastatic cells in the different organs (for examples see Fig. 5, A–G). The human epithelial origin of these cells was confirmed by staining with an anticytokeratin antibody either directly coupled to FITC (Fig. 5E, Fig. F, and Fig. I) or followed by secondary HRP detection (Fig. 5, B–D and H). This antibody reacted only with human and not with mouse cytokeratin. The sections of the primary tumors stained equally well, regardless of the method of detection, (peroxidase or FITC) (Fig. 5C and Fig. F). Fig. 5 shows a representative panel of sections from different organs in which metastases were found. In most organs (pancreas, salivary gland, omentum, and muscle, Fig. 5B, Fig. D, Fig. E, and Fig. I, respectively), large areas of metastatic growth were found, but micrometastases shown in a lung section (Fig. 5G and Fig. H) were also present. In a comparable analysis of tissue sections from mice injected with control MCF-7 or Neo-5 cells, no cytokeratin-reactive cells were ever found in the pancreas, lymph nodes, salivary gland, omentum, liver, lung, and skeletal muscle (Table ), or in other tissues (data not shown). As indicated in Table , whereas the metastases produced by other clones were more random, the two clones with the highest level of N-cadherin expression produced liver metastases in almost all injected mice.


Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.

Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson SA - J. Cell Biol. (2000)

Histopathology of metastatic lesions in nude mice. 5-μm sections of the pancreas of mice injected with N-cad-17 cells were stained with either H&E (A) or cytokeratin antibodies, followed by DAB detection (B). Cytokeratin immunoreactivity of the N-cad-17 primary tumor (C) was compared with the pancreatic lesion produced by this tumor in B. Sections from the salivary gland of mice injected with N-cad-5 cells were stained with anticytokeratin antibodies, followed by DAB detection (D) and sections from the omentum (E) and primary tumor of N-cad-5–injected mice (F) were stained with FITC-conjugated cytokeratin antibodies. N-cad-8 metastatic lesions were detected in lung sections by H&E (G) and cytokeratin/DAB detection (H). N-cad-15 cells were found in the lumbar spinal muscle (I) using double fluorescent staining with antiactin, followed by secondary antibodies coupled to rhodamine (red) and FITC-conjugated anticytokeratin antibodies (green). Bar: (A) 100 μm; (G and H) 50 μm; (B, D, and E) 33 μm; (C, F, and I) 20 μm.
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Figure 5: Histopathology of metastatic lesions in nude mice. 5-μm sections of the pancreas of mice injected with N-cad-17 cells were stained with either H&E (A) or cytokeratin antibodies, followed by DAB detection (B). Cytokeratin immunoreactivity of the N-cad-17 primary tumor (C) was compared with the pancreatic lesion produced by this tumor in B. Sections from the salivary gland of mice injected with N-cad-5 cells were stained with anticytokeratin antibodies, followed by DAB detection (D) and sections from the omentum (E) and primary tumor of N-cad-5–injected mice (F) were stained with FITC-conjugated cytokeratin antibodies. N-cad-8 metastatic lesions were detected in lung sections by H&E (G) and cytokeratin/DAB detection (H). N-cad-15 cells were found in the lumbar spinal muscle (I) using double fluorescent staining with antiactin, followed by secondary antibodies coupled to rhodamine (red) and FITC-conjugated anticytokeratin antibodies (green). Bar: (A) 100 μm; (G and H) 50 μm; (B, D, and E) 33 μm; (C, F, and I) 20 μm.
Mentions: Of a total of 20 mice injected with N-cadherin–expressing cell lines, 13 mice (65%) had primary tumors. Similarly, 10 out of 12 mice (83%) injected with control MCF-7 cells developed primary tumors. N-cadherin–expressing tumors grew slower on average, reaching a weight of ∼30% that of control MCF-7 or Neo-5 tumors (Table ). This is consistent with the slower growth rate of the N-cadherin–transfected cells in vitro (data not shown). Most (77%) of the 13 mice with N-cadherin–expressing primary tumors developed metastases in multiple sites, whereas none of the control mice bearing larger tumors had detectable metastases (Table ). Staining with H&E strongly suggested the presence of metastatic cells in the different organs (for examples see Fig. 5, A–G). The human epithelial origin of these cells was confirmed by staining with an anticytokeratin antibody either directly coupled to FITC (Fig. 5E, Fig. F, and Fig. I) or followed by secondary HRP detection (Fig. 5, B–D and H). This antibody reacted only with human and not with mouse cytokeratin. The sections of the primary tumors stained equally well, regardless of the method of detection, (peroxidase or FITC) (Fig. 5C and Fig. F). Fig. 5 shows a representative panel of sections from different organs in which metastases were found. In most organs (pancreas, salivary gland, omentum, and muscle, Fig. 5B, Fig. D, Fig. E, and Fig. I, respectively), large areas of metastatic growth were found, but micrometastases shown in a lung section (Fig. 5G and Fig. H) were also present. In a comparable analysis of tissue sections from mice injected with control MCF-7 or Neo-5 cells, no cytokeratin-reactive cells were ever found in the pancreas, lymph nodes, salivary gland, omentum, liver, lung, and skeletal muscle (Table ), or in other tissues (data not shown). As indicated in Table , whereas the metastases produced by other clones were more random, the two clones with the highest level of N-cadherin expression produced liver metastases in almost all injected mice.

Bottom Line: To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis.These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin.The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA. rhazan@smtplink.mssm.edu

ABSTRACT
E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

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Related in: MedlinePlus