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

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.

Dasatinib inhibition of cSRC prevents the migration and metastasis of canine mammary cancer cells with enhanced Wnt and HER signalling

“…The TKI Src‐I1, which is an inhibitor of cSRC, also decreased cell proliferation but only at a rather high concentration (20 μM) of Src‐I1 . According to Bain et al , the IC50 value of Src‐I1 is approximately 0.18 μM, which is far below the concentration that we used . Therefore, we investigated a panel of related TKIs to find a connection between cSRC, Wnt and EGFR signalling.…”

“…Therefore, we investigated the underlying cause of the high basal Wnt activity in the CMT‐U27 metastatic canine mammary cell line, which was selected from a panel of canine mammary cell lines where 3 out of 12 cell lines were characterized by high ligand‐independent Wnt signalling . Additional studies showed that these cell lines had HER2 /3 overexpression, absent PTEN mRNA , and sensitivity to Src‐I1, indicating a possible relationship between HER signalling and Wnt activity . However, in the present study, we were unable to show a direct relation by silencing HER protein expression with siRNAs.…”

“…Therefore, we investigated a panel of related TKIs to find a connection between cSRC, Wnt and EGFR signalling. Afatinib, a small molecule inhibitor of HER signalling, upregulated Wnt activity, most likely through inhibition of PI3K/mTOR activity, and we have also observed this with the mTOR inhibitor everolimus in the same cell line . Therefore, it is highly unlikely that HER signalling plays a role in the activation of the Wnt pathway.…”

“…In our studies, a panel of 12 different canine mammary cell lines were used as described previously . For detailed studies, the well‐described canine cell lines CMT‐U27 (a generous gift from Prof. Dr. Hellmen, SLU, Uppsala, Sweden) and CIPm (a generous gift from Prof. Dr. Sasaki, Laboratory of Veterinary Surgery, University of Tokyo, Japan) were used, representing a cell line with ligand‐independent high Wnt signalling (CMT‐U27) and absent Wnt signalling (CIPm) . The cells were cultured in DMEM/F12 (Invitrogen, Bleiswijk, The Netherlands) supplemented with 10% foetal bovine serum (FBS) (Invitrogen).…”

“…However, inhibition of this pathway with the mTOR inhibitor everolimus or the dual PI3K/mTOR inhibitor BEZ325 further upregulated the already high basal activated Wnt activity . Wnt activity could only be inhibited by Src‐I1, a tyrosine kinase inhibitor that targets cSRC, among others . Recently, SRC has been shown to promote resistance to trastuzumab in HER2‐positive breast cancer.…”