Cancer is one of the most detrimental diseases all over the world. Especially, it has been well known that breast cancer patients show low survival rate after diagnosis. Moreover, incidence rate of breast cancer has been consistently increased among Korean women. Human epidermal growth factor recept...
Cancer is one of the most detrimental diseases all over the world. Especially, it has been well known that breast cancer patients show low survival rate after diagnosis. Moreover, incidence rate of breast cancer has been consistently increased among Korean women. Human epidermal growth factor receptor 2 (HER2) is overexpressed in about 30% of breast cancer patients who show poor prognosis. Therapeutics for targeting HER2 improves survival of breast cancer patients. However, resistance to therapeutics causes troubles to overcome cancer. Here, I established trastuzumab, a HER2 target therapeutical antibody, resistant breast cancer cell lines and analyzed protein expression profiles with two dimensional digest-LC-MS/MS. Increased level of extracellular matrix protein 1 (ECM1) was detected in trastuzumab resistant (TR) cells and this increased level of ECM1 was shown to be responsible for cell proliferation and trastuzumab resistance. The ECM1 is a secreted glycoprotein and plays an important role in angiogenesis in blood vessel endothelium. Recently, it was reported that ECM1 induces tumorigenesis through promoting development of blood vessel. Moreover, ECM1 is highly expressed in various types of cancer patients and predicted as a marker for tumor development. ECM1-induced Galectin-3 cleavage through activation of matrix metallopeptidase-9 (MMP-9) not only regulated Mucin 1 (MUC1) expression but also increased epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) protein stability. This stabilization of receptor tyrosine kinases led to ERK activation and increased cell proliferation. Interestingly, ECM1 was shown to enhance the phosphorylation of EGFR and ERK upon EGF treatment through physical association with EGFR. These data provide evidence that ECM1 has an important role in both cancer development and trastuzumab resistance in breast cancer. I also provide evidence that targeting ECM1 with antibody against ECM1 could be new therapeutic approaches for breast cancer patients. The Warburg effect is known as oncogenic metabolism switch to allow cancer cells take up more glucose than normal cells and favor anaerobic glycolysis. I found that the expression of proteins related with Warburg effect was up-regulated in trastuzumab resistant (TR) BT-474 cells overexpressing ECM1 as compared to control cells by two dimensional digest-LC-MS/MS. I demonstrated that ECM1 induced the gene expression promoting Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA) and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of PKM-2 at ser 37 which is responsible for expression of genes promoting Warburg effect was affected by the modulation of ECM1 expression. Indeed, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting Warburg effect mediated by PKM2. ECM1 is also correlated with invasiveness and poor prognosis in various type of cancer. However, the functional role of ECM1 in metastasis is unclear. Here, I found that high ECM1 level was detected in breast cancer patients sera and associated with recurrence of tumor. The modulation of ECM1 expression affected not only cell migration and invasion but also sphere forming ability and drug resistance. In addition, ECM1 regulated the gene expression related with epithelial to mesenchymal transition (EMT) progression and cancer stem cells (CSC) maintenance. Interestingly, ECM1 increased β-catenin expression at post translational level through induction of MUC1 which was physically associated with β-catenin. Indeed, forced expression of β-catenin caused the gene expression that potentiated EMT progression and CSC phenotype maintenance. These data provide evidence that ECM1 has a critical role in cancer metastasis through β-catenin stabilization. CD24 is the one of the markers for CSC. To determine the role of CD24 in breast cancer cells, I knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-β1-mediated EMT were also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin β1 bound with CD24 and subsequent destabilization of integrin β1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin β1 function, which is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.
Cancer is one of the most detrimental diseases all over the world. Especially, it has been well known that breast cancer patients show low survival rate after diagnosis. Moreover, incidence rate of breast cancer has been consistently increased among Korean women. Human epidermal growth factor receptor 2 (HER2) is overexpressed in about 30% of breast cancer patients who show poor prognosis. Therapeutics for targeting HER2 improves survival of breast cancer patients. However, resistance to therapeutics causes troubles to overcome cancer. Here, I established trastuzumab, a HER2 target therapeutical antibody, resistant breast cancer cell lines and analyzed protein expression profiles with two dimensional digest-LC-MS/MS. Increased level of extracellular matrix protein 1 (ECM1) was detected in trastuzumab resistant (TR) cells and this increased level of ECM1 was shown to be responsible for cell proliferation and trastuzumab resistance. The ECM1 is a secreted glycoprotein and plays an important role in angiogenesis in blood vessel endothelium. Recently, it was reported that ECM1 induces tumorigenesis through promoting development of blood vessel. Moreover, ECM1 is highly expressed in various types of cancer patients and predicted as a marker for tumor development. ECM1-induced Galectin-3 cleavage through activation of matrix metallopeptidase-9 (MMP-9) not only regulated Mucin 1 (MUC1) expression but also increased epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) protein stability. This stabilization of receptor tyrosine kinases led to ERK activation and increased cell proliferation. Interestingly, ECM1 was shown to enhance the phosphorylation of EGFR and ERK upon EGF treatment through physical association with EGFR. These data provide evidence that ECM1 has an important role in both cancer development and trastuzumab resistance in breast cancer. I also provide evidence that targeting ECM1 with antibody against ECM1 could be new therapeutic approaches for breast cancer patients. The Warburg effect is known as oncogenic metabolism switch to allow cancer cells take up more glucose than normal cells and favor anaerobic glycolysis. I found that the expression of proteins related with Warburg effect was up-regulated in trastuzumab resistant (TR) BT-474 cells overexpressing ECM1 as compared to control cells by two dimensional digest-LC-MS/MS. I demonstrated that ECM1 induced the gene expression promoting Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA) and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of PKM-2 at ser 37 which is responsible for expression of genes promoting Warburg effect was affected by the modulation of ECM1 expression. Indeed, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting Warburg effect mediated by PKM2. ECM1 is also correlated with invasiveness and poor prognosis in various type of cancer. However, the functional role of ECM1 in metastasis is unclear. Here, I found that high ECM1 level was detected in breast cancer patients sera and associated with recurrence of tumor. The modulation of ECM1 expression affected not only cell migration and invasion but also sphere forming ability and drug resistance. In addition, ECM1 regulated the gene expression related with epithelial to mesenchymal transition (EMT) progression and cancer stem cells (CSC) maintenance. Interestingly, ECM1 increased β-catenin expression at post translational level through induction of MUC1 which was physically associated with β-catenin. Indeed, forced expression of β-catenin caused the gene expression that potentiated EMT progression and CSC phenotype maintenance. These data provide evidence that ECM1 has a critical role in cancer metastasis through β-catenin stabilization. CD24 is the one of the markers for CSC. To determine the role of CD24 in breast cancer cells, I knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-β1-mediated EMT were also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin β1 bound with CD24 and subsequent destabilization of integrin β1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin β1 function, which is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.
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