Abstract
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.
Highlights
Elevated basal, ligand-independent, Wingless-type MMTV integration site family (Wnt) signaling in some canine breast cancer cells is not caused by classical mutations in adenomatous polyposis coli (APC), β-Catenin or GSK3β but, at least partially, by enhanced Lymphoid enhancer-binding factor 1 (LEF1) expression
Canine mammary cell lines and culture Canine mammary tumor cell lines used in this study were CMT1, CMT-U229, CMT-U335, CMT-U27, CMT9, P114, CHMp, CHMm, CNMp, CNMm, CIPp and CIPm [19,20,21]
Phosphatase and tensin homolog (PTEN) deficiency occurs in 5 to 14 % of primary human breast cancers and PTEN is lost in human T47D and MCF7 cell lines [13]
Summary
Canine mammary cell lines and culture Canine mammary tumor cell lines used in this study were CMT1, CMT-U229, CMT-U335, CMT-U27, CMT9, P114, CHMp, CHMm, CNMp, CNMm, CIPp and CIPm [19,20,21]. Cells were tested to be free from mycoplasma with a Mycosensor QPCR assay according to manufacturer’s protocol (Agilent technologies, Middelburg, The Netherlands). Relative target gene expression was normalized to a set of eight reference genes (tested in Genorm with a pairwise variation (PV) of 0.07) for the transfection experiments. Cells were seeded in 96 wells plates (Primaria, BD Biosciences, Breda, The Netherlands) and after 24 h incubation to attach and stretch, treated with the different compounds for 40 h. Protein extraction and Western blot Cells were seeded in 75 cm bottles and after 24 h incubation to attach and stretch, treated with the different compounds. Differences in TOP/FOP activities, RNA, cell viability and protein expression levels were statistically assessed using unpaired, two tailed Student’s t test, a P value less than 0.01 was considered significant
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