Cyclooxygenase-2 in Regulating Vascular Channel Formation
Cyclooxygenase-2 in Regulating Vascular Channel Formation
Introduction: Cyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor.
Methods: To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2.
Results: We found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E2. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation.
Conclusion: Antivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.
Tumor growth and metastasis are thought to be angiogenesis-related processes. However, it has recently been reported that an angiogenesis-independent pathway, in which tumors can feed themselves without the use of classical blood vessels, exists in very aggressive tumors of the lung and breast, as well as in melanomas. This is known as vasculogenic mimicry (VM), a phenomenon in which epithelial tumor cells form vascular channel-like structures to obtain nutrients without the participation of endothelial cells. These laminin-rich channel-like spaces are lined by tumor cells and contain erythrocytes and plasma. These channels are thought to provide a mechanism of perfusion and a dissemination route within the tumor that functions either independently of or simultaneously with angiogenesis. A connection has been suggested between VM and angiogenesis based on the existence of blood flow in the vascular channels. Thus, VM might be an important factor to consider in the design of antivascular therapies. Importantly, it has been shown that breast cancer patients who exhibit VM in their resected tumors have a lower 5-year survival than do patients without VM.
Although a correlation has been found between the presence of VM and poor clinical outcome, little is known regarding the molecular composition and regulation of these channels. Based on microarray analysis of melanoma cells, the biologically relevant proteins in VM were vascular endothelial cadherin (VE-cadherin), erythropoietin-producing hepatocellular carcicnoma-A2 (EPHA2), matrix metalloproteinases (MMPs), and laminin 5-γ-2-chain (LAMC2). Independently reducing their levels of expression resulted in complete inability of aggressive melanoma cells to execute VM in three-dimensional culture conditions. Furthermore, xenografts generated from inflammatory breast cancer (IBC) cells that execute VM in vitro expressed higher levels of angiogenic factors than did xenografts from non-IBC cells that did not execute VM. These angiogenic factors included angiogenin-1, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), flt-1, integrin-ß3, and CD31. It has been suggested that upregulation of angiogenesis-related genes may result in the observed vascular phenotype of IBC tumor cells. Therefore, it is important to investigate whether known antiangiogenic agents might prevent VM. A recent report suggested that specific antiangiogenic agents such as anginex, TNP-470, and endostatin had minimal effect on VM in human melanoma MUM-2B and C8161 cells, suggesting differential response of endothelial cell dependent angiogenesis and VM. It is therefore of great importance to investigate additional factors that may regulate vascular channel formation and determine whether inhibiting those factors might prevent VM.
Over-expression of cyclo-oxygenase (COX)-2 is known to correlate with the aggressive and invasive potential of tumor cells by several mechanisms. One of the mechanisms modulated by COX-2 during carcinogenesis is angiogenesis, presumably through increased production of proangiogenic factors such as VEGF and IL-8. Similarly, COX-2-specific inhibitors reduce angiogenesis by inhibiting mitogen activated protein kinase (extracellular signal regulated kinase [ERK]2) activity and by interfering with nuclear translocation of ERK. To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation. The aims of our study were to compare the ability of human breast cancer cells expressing high and low levels of COX-2 to form vascular channels on three-dimensional matrigel cultures, and to assess the effect of therapeutically targeting COX-2 in vitro and in vivo on VM. In additional, we sought to identify candidate genes that are involved in COX-2-induced channel formation by microarray analysis and, finally, to correlate the cell line data with findings in surgically resected human breast cancer specimens.
Abstract and Introduction
Abstract
Introduction: Cyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor.
Methods: To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2.
Results: We found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E2. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation.
Conclusion: Antivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.
Introduction
Tumor growth and metastasis are thought to be angiogenesis-related processes. However, it has recently been reported that an angiogenesis-independent pathway, in which tumors can feed themselves without the use of classical blood vessels, exists in very aggressive tumors of the lung and breast, as well as in melanomas. This is known as vasculogenic mimicry (VM), a phenomenon in which epithelial tumor cells form vascular channel-like structures to obtain nutrients without the participation of endothelial cells. These laminin-rich channel-like spaces are lined by tumor cells and contain erythrocytes and plasma. These channels are thought to provide a mechanism of perfusion and a dissemination route within the tumor that functions either independently of or simultaneously with angiogenesis. A connection has been suggested between VM and angiogenesis based on the existence of blood flow in the vascular channels. Thus, VM might be an important factor to consider in the design of antivascular therapies. Importantly, it has been shown that breast cancer patients who exhibit VM in their resected tumors have a lower 5-year survival than do patients without VM.
Although a correlation has been found between the presence of VM and poor clinical outcome, little is known regarding the molecular composition and regulation of these channels. Based on microarray analysis of melanoma cells, the biologically relevant proteins in VM were vascular endothelial cadherin (VE-cadherin), erythropoietin-producing hepatocellular carcicnoma-A2 (EPHA2), matrix metalloproteinases (MMPs), and laminin 5-γ-2-chain (LAMC2). Independently reducing their levels of expression resulted in complete inability of aggressive melanoma cells to execute VM in three-dimensional culture conditions. Furthermore, xenografts generated from inflammatory breast cancer (IBC) cells that execute VM in vitro expressed higher levels of angiogenic factors than did xenografts from non-IBC cells that did not execute VM. These angiogenic factors included angiogenin-1, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), flt-1, integrin-ß3, and CD31. It has been suggested that upregulation of angiogenesis-related genes may result in the observed vascular phenotype of IBC tumor cells. Therefore, it is important to investigate whether known antiangiogenic agents might prevent VM. A recent report suggested that specific antiangiogenic agents such as anginex, TNP-470, and endostatin had minimal effect on VM in human melanoma MUM-2B and C8161 cells, suggesting differential response of endothelial cell dependent angiogenesis and VM. It is therefore of great importance to investigate additional factors that may regulate vascular channel formation and determine whether inhibiting those factors might prevent VM.
Over-expression of cyclo-oxygenase (COX)-2 is known to correlate with the aggressive and invasive potential of tumor cells by several mechanisms. One of the mechanisms modulated by COX-2 during carcinogenesis is angiogenesis, presumably through increased production of proangiogenic factors such as VEGF and IL-8. Similarly, COX-2-specific inhibitors reduce angiogenesis by inhibiting mitogen activated protein kinase (extracellular signal regulated kinase [ERK]2) activity and by interfering with nuclear translocation of ERK. To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation. The aims of our study were to compare the ability of human breast cancer cells expressing high and low levels of COX-2 to form vascular channels on three-dimensional matrigel cultures, and to assess the effect of therapeutically targeting COX-2 in vitro and in vivo on VM. In additional, we sought to identify candidate genes that are involved in COX-2-induced channel formation by microarray analysis and, finally, to correlate the cell line data with findings in surgically resected human breast cancer specimens.