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High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.

Timmermans-Sprang EP, Gracanin A, Mol JA - BMC Cancer (2015)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584, CM, Utrecht, The Netherlands. e.p.m.timmermans@uu.nl.

ABSTRACT

Background: Elevated 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.

Methods: Twelve 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.

Results: High 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.

Conclusions: High 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.

No MeSH data available.


Related in: MedlinePlus

Effects of drugs on Wnt signaling. PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt reporter activity. Three cell lines with a high TOP/FOP ratio (>30) (CMT1, CMT-U27, and CMT9) and CIPm cells with a low TOP/FOP ratio (<2) were grown for 40 h in the presence of PI3K/mTor inhibitor BEZ235 (50nM), mTor inhibitor Everolimus (100nM) and cSRC inhibitor Src-l1 (20 μM). After 40 h the TOP/FOP ratio was measured with a Dual-Luciferase assay. The mean ratio is expressed of three independent experiments (±SEM). *P < 0.01 versus appropriate cell line control (DMSO). PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt activity in the basal activated Wnt cell lines
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Fig4: Effects of drugs on Wnt signaling. PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt reporter activity. Three cell lines with a high TOP/FOP ratio (>30) (CMT1, CMT-U27, and CMT9) and CIPm cells with a low TOP/FOP ratio (<2) were grown for 40 h in the presence of PI3K/mTor inhibitor BEZ235 (50nM), mTor inhibitor Everolimus (100nM) and cSRC inhibitor Src-l1 (20 μM). After 40 h the TOP/FOP ratio was measured with a Dual-Luciferase assay. The mean ratio is expressed of three independent experiments (±SEM). *P < 0.01 versus appropriate cell line control (DMSO). PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt activity in the basal activated Wnt cell lines

Mentions: This inhibitory effect on viability was clearly dose-dependent and present in both the high basal Wnt cell line CMT-U27 and the CIPm cell line (Fig. 3 and Table 2). Comparable concentrations of BEZ235 were also used in human studies with different cell lines [33, 34]. In human MCF7 cells, Everolimus (Rad001) was tested in a range from 0-1000 nM and only the 1000 nM concentration showed a 50 % reduction in cell number [35]. Incubation of these cells with BEZ235 and Everolimus resulted in unexpected further significant enhancement of Wnt activity of CMT1, CMT-U27 and CMT9, with a more than 2-fold increase in the TOP/FOP ratios (Fig. 4).Fig. 3


High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.

Timmermans-Sprang EP, Gracanin A, Mol JA - BMC Cancer (2015)

Effects of drugs on Wnt signaling. PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt reporter activity. Three cell lines with a high TOP/FOP ratio (>30) (CMT1, CMT-U27, and CMT9) and CIPm cells with a low TOP/FOP ratio (<2) were grown for 40 h in the presence of PI3K/mTor inhibitor BEZ235 (50nM), mTor inhibitor Everolimus (100nM) and cSRC inhibitor Src-l1 (20 μM). After 40 h the TOP/FOP ratio was measured with a Dual-Luciferase assay. The mean ratio is expressed of three independent experiments (±SEM). *P < 0.01 versus appropriate cell line control (DMSO). PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt activity in the basal activated Wnt cell lines
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4513708&req=5

Fig4: Effects of drugs on Wnt signaling. PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt reporter activity. Three cell lines with a high TOP/FOP ratio (>30) (CMT1, CMT-U27, and CMT9) and CIPm cells with a low TOP/FOP ratio (<2) were grown for 40 h in the presence of PI3K/mTor inhibitor BEZ235 (50nM), mTor inhibitor Everolimus (100nM) and cSRC inhibitor Src-l1 (20 μM). After 40 h the TOP/FOP ratio was measured with a Dual-Luciferase assay. The mean ratio is expressed of three independent experiments (±SEM). *P < 0.01 versus appropriate cell line control (DMSO). PI3K/mTor inhibitor BEZ235 and mTor inhibitor Everolimus stimulates the TOP/FOP ratios. Incubation with the cSRC inhibitor Src-l1 shows a down regulation of the Wnt activity in the basal activated Wnt cell lines
Mentions: This inhibitory effect on viability was clearly dose-dependent and present in both the high basal Wnt cell line CMT-U27 and the CIPm cell line (Fig. 3 and Table 2). Comparable concentrations of BEZ235 were also used in human studies with different cell lines [33, 34]. In human MCF7 cells, Everolimus (Rad001) was tested in a range from 0-1000 nM and only the 1000 nM concentration showed a 50 % reduction in cell number [35]. Incubation of these cells with BEZ235 and Everolimus resulted in unexpected further significant enhancement of Wnt activity of CMT1, CMT-U27 and CMT9, with a more than 2-fold increase in the TOP/FOP ratios (Fig. 4).Fig. 3

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584, CM, Utrecht, The Netherlands. e.p.m.timmermans@uu.nl.

ABSTRACT

Background: Elevated 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.

Methods: Twelve 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.

Results: High 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.

Conclusions: High 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.

No MeSH data available.


Related in: MedlinePlus