The interaction between the canonical Wnt pathway and the mTOR pathway in canine mammary tumor cells

Abstract

Background: Mammary tumors are the most common neoplasms in intact bitches with an estimated life-time risk to develop of 1:4. About 50% of these tumors are malignant and one-third of these may form life-threatening distant metastases. Also in humans breast cancer is a very common disease with a life-time risk of about 1:8. The initial hormone dependency and the common pathways involved in cell proliferation and migration, such as Wnt and PI3K signaling in both human and canine mammary carcinomas underscore the relevance of the research in canine mammary cancer. The main problem in breast cancer treatment is the recurrence of tumor growth and metastases. Tumor cells with stem cell properties such as a phenotypical epithelial mesenchymal transition (EMT) and elevated activity of the canonical Wnt pathway play an important role in regrowth and metastasis. In previous research a subset of canine mammary cell lines were shown to have elevated ligand-independent canonical Wnt activity. This was associated with enhanced expression of EGFR, HER2 and HER3 receptors and loss of PTEN expression suggesting a role for activation of the PI3K/mTor pathway. However, inhibition with a specific mTOR inhibitor or a dual PI3K/mTOR inhibitor resulted in further increased Wnt activity that appeared to be sensitive to inhibition of the also through EGFR and HER2/3 activated SRC.

Aim: The aim of this research project was to further study the role of SRC inhibition on basal Wnt activity and inhibition of in vitro migration as a surrogate marker for metastasis. In a second part the role of HER2 and HER3 were further investigated by selective inhibition of HER2 and HER3 expression.

Methods: Using a panel of specific inhibitors related to the previously used SRC inhibitor SRC-I1 their effect was studied on two canine mammary cell lines, one with elevated basal Wnt activity and one with absent Wnt activity. The effects of the inhibitors on cell viability were measured by MTT assay, on the basal Wnt activity using TCF-reporter constructs and on cell migration using scratch assays. In the second study expression of HER2 or HER3 was selectively inhibited using specific siRNAs. The effects of these siRNAs were studied on protein expression by Western blot, on Wnt activity using reporter assays, and on target gene expression using qPCR.

Results: In general no differences in sensitivity for a decrease in viability were found between the two cell lines with the exception of a higher sensitivity of the high basal Wnt cell line for Aurora kinase A inhibition. This was, however, not associated with a decreased Wnt activity which was sensitive to SRC inhibitors SRC-I1 and PP2 but also to an IGF-1R inhibitor. In addition, migration appeared to be sensitive to the SRC inhibitors SRC-I1 and PP2. These data confirm the central role of SRC activation in the enhanced basal Wnt activity and metastatic properties. The remaining question was if the overexpression of HER2 and HER3 were causing the enhanced Wnt activity. Selective inhibition of HER2 or HER3 expression, using specific siRNAs, however did not unequivocally show overexpression of HER2 or HER3 as the cause of Wnt pathway activation.

Conclusion: Further evidence is presented for a central role of SRC in Wnt activation and migratory properties of the canine mammary cell line used. The role of HER2 and HER3 remain, however, inconclusive. This may be caused by incomplete knockdown or the necessity of inactivating both genes at the same time. In addition, a role for the EGFR may exist and needs further research.