“…In human and mouse mammary glands, the activated Wnt/β-Catenin is related to enhanced HER2 expression [ 25 ], something also found in 30 % of canine malignant mammary tumors [ 26 ]. Combining the up regulation of both HER2 and HER3 in our canine mammary cell lines and the fact that the HER2/HER3 complex is the strongest activator for downstream signaling pathways makes this an interesting area for further research [ 15 , 27 , 28 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Leaving aside the question of how EGFR/HER2/HER3 signaling is involved in high basal Wnt activity, it is clear that the upregulation of the MUC1 and EGFR/HER2 expression have a relationship with the increased Wnt activity that occurs after inhibition of the PI3K/mTor pathway. The presence of glycosylated HER3 protein in the highly activated Wnt cell line is new, and because our results suggest that activation of cSRC could play a role [ 15 , 46 ], we next studied the influence of cSRC inhibitors on the Wnt activity and HER3 levels.…”
Section: Resultsmentioning
confidence: 99%
“…Activation of EGFR initiates the activation of PI3K/AKT/mTor and MAPK as well as various other signaling pathways, including the PLC-Υ1, JAK/STAT, and cSRC pathway [ 15 , 47 ]. Because the EGFR can form a complex with cSRC, FAK (focal adhesion kinase), integrins and β-Catenin resulting in growth, viability, survival and migration [ 46 ], we tested the effect of the cSRC inhibitor 1 (Src-I1) and a FAK inhibitor [ 48 , 49 ].…”
Section: Resultsmentioning
confidence: 99%
“…Binding of EGF or related growth factors to the EGF receptors induces homo- and heterodimers leading to phosphorylation on specific tyrosine residues; these residues serve as docking sites for a variety of signaling molecules, leading to activation of intracellular pathways such as the mitogen-activated protein kinase (MAPK), the phosphatidylinositol-3-kinase (PI3K), Stats, RAS and cSRC pathways. Although not binding any ligand, HER2 plays a central role together with the HER3 protein that lacks proper tyrosine kinase activity, with this complex being the strongest activator for downstream signaling pathways [ 14 , 15 ]. HER3 can also signal ligand independent and its activation is associated with resistance to HER2 targeting tyrosine kinase inhibitors in breast cancer [ 16 ].…”
BackgroundElevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.MethodsTwelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.ResultsHigh basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.ConclusionsHigh basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.
“…In human and mouse mammary glands, the activated Wnt/β-Catenin is related to enhanced HER2 expression [ 25 ], something also found in 30 % of canine malignant mammary tumors [ 26 ]. Combining the up regulation of both HER2 and HER3 in our canine mammary cell lines and the fact that the HER2/HER3 complex is the strongest activator for downstream signaling pathways makes this an interesting area for further research [ 15 , 27 , 28 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Leaving aside the question of how EGFR/HER2/HER3 signaling is involved in high basal Wnt activity, it is clear that the upregulation of the MUC1 and EGFR/HER2 expression have a relationship with the increased Wnt activity that occurs after inhibition of the PI3K/mTor pathway. The presence of glycosylated HER3 protein in the highly activated Wnt cell line is new, and because our results suggest that activation of cSRC could play a role [ 15 , 46 ], we next studied the influence of cSRC inhibitors on the Wnt activity and HER3 levels.…”
Section: Resultsmentioning
confidence: 99%
“…Activation of EGFR initiates the activation of PI3K/AKT/mTor and MAPK as well as various other signaling pathways, including the PLC-Υ1, JAK/STAT, and cSRC pathway [ 15 , 47 ]. Because the EGFR can form a complex with cSRC, FAK (focal adhesion kinase), integrins and β-Catenin resulting in growth, viability, survival and migration [ 46 ], we tested the effect of the cSRC inhibitor 1 (Src-I1) and a FAK inhibitor [ 48 , 49 ].…”
Section: Resultsmentioning
confidence: 99%
“…Binding of EGF or related growth factors to the EGF receptors induces homo- and heterodimers leading to phosphorylation on specific tyrosine residues; these residues serve as docking sites for a variety of signaling molecules, leading to activation of intracellular pathways such as the mitogen-activated protein kinase (MAPK), the phosphatidylinositol-3-kinase (PI3K), Stats, RAS and cSRC pathways. Although not binding any ligand, HER2 plays a central role together with the HER3 protein that lacks proper tyrosine kinase activity, with this complex being the strongest activator for downstream signaling pathways [ 14 , 15 ]. HER3 can also signal ligand independent and its activation is associated with resistance to HER2 targeting tyrosine kinase inhibitors in breast cancer [ 16 ].…”
BackgroundElevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.MethodsTwelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.ResultsHigh basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.ConclusionsHigh basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.
“…Binding of EGF also stimulates the rapid internalization of EGFR [13]. EGFR endocytosis and EGFR-mediated cell signaling are mutually regulated [14,15].…”
The overactivation of epidermal growth factor (EGF) receptor (EGFR) is implicated in various cancers. Endocytosis plays an important role in EGFR-mediated cell signaling. We previously found that EGFR endocytosis during mitosis is mediated differently from interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. By transfecting HeLa, MCF-7, and 293T cells with CBL siRNA or dominant-negative 70z-CBL, we found that at high EGF doses, CBL is required for EGFR endocytosis in mitotic cells, but not in interphase cells. In addition, the endocytosis of mutant EGFR Y1045F-YFP (mutation at the direct CBL binding site) is strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL is completely inhibited in mitosis. Moreover, EGF induces stronger ubiquitination of mitotic EGFR than interphase EGFR, and mitotic EGFR is trafficked to lysosomes for degradation. Furthermore, we showed that, different from interphase, low doses of EGF still stimulate EGFR endocytosis by non-clathrin mediated endocytosis (NCE) in mitosis. Contrary to interphase, CBL and the CBL-binding regions of EGFR are required for mitotic EGFR endocytosis at low doses. This is due to the mitotic ubiquitination of the EGFR even at low EGF doses. We conclude that mitotic EGFR endocytosis exclusively proceeds through CBL-mediated NCE.
Endocytosis plays important roles in regulating EGFR signaling. We previously found that EGFR endocytosis during mitosis is mediated differently than during interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we studied the mechanisms regulating mitotic EGFR endocytosis. We found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. At high EGF doses, inhibition of inhibited EGFR endocytosis of mitotic cells, but not of interphase cells. Moreover, the endocytosis of mutant EGFR Y1045F-YFP was strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL was completely inhibited. EGF induced stronger ubiquitination of mitotic EGFR than interphase EGFR and mitotic EGFR is trafficked to lysosome for degradation. Furthermore, during mitosis low doses of EGF also stimulate EGFR endocytosis by NCE. Contrary to interphase, CBL and the CBL-binding regions of EGFR were required for mitotic EGFR endocytosis at low doses. This may be due to the mitotic ubiquitination of the EGFR even at low EGF doses. In conclusion, mitotic EGFR endocytosis solely proceed through CBL-mediated NCE.
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