Effect of suppressing the p38 pathway through upregulation of Wip1 on Her2 early dissemination of breast cancer.

Abstract

e13029 Background: Metastasis is the leading cause of cancer-related deaths. While mechanisms for metastasis were extensively studied in cancer cells from patients with detectable tumors, pathways underlying metastatic dissemination from early lesions are poorly understood. Her2 promotes breast cancer early dissemination by suppressing p38, but how it downregulates p38 is unclear. In this study, we investigated the mechanism underlying suppression of p38 by Her2 in breast cancer early dissemination using cell models of early lesions of breast cancer. Methods: We studied the role of Wip1 in disseminating phenotypes in cell line models of breast cancer early lesions. These disseminating phenotypes include cell motility measured in transwell assays, cell invasion measured by percentage of organoids with outwards in organoid-forming medium, and epithelial-to-mesenchymal transition (EMT) measured by E-cadherin and Vimentin levels in Western blotting analysis, percentage of organoids with High E-cadherin junctions and nuclear translocation and activation of β-catenin in immunofluorescence (IF) staining assays. Results: We demonstrate that high levels of Wip1, a p38 phosphatase, correlated with Her2+ status, and reduced phosphorylation of p38 and its downstream effectors MK2 and Hsp27 in tissue samples and cell models of breast cancer early lesions. We found that Wip1 promoted migration, invasion and EMT, while Wip1 knockdown abrogated the ability of Her2 to induce these disseminating phenotypes, in cell line models of early lesion of breast cancer. In contrast to the wild type Wip1, the catalytically inactive mutants of Wip1 failed to induce the disseminating phenotypes. Thus, Wip1 induction is both sufficient, and necessary for Her2, to promote migration, invasion and EMT in breast cancer early lesion cells. In an attempt to identify the Wip1 downstream effectors involved in disseminating phenotypes, we found that ectopic expression of the constitutively active mutants of p38γ and δ, but not that of p38α and β, reversed the ability of Wip1 to suppress MK2 and Hsp27 phosphorylation and to induce migration, invasion and EMT. It suggests that Wip1 promotes the early dissemination by suppressing p38γ and δ. We investigate the mechanism by which Her2 induces Wip1 expression. Ectopic expression of Her2 did not alter the level of Wip1 mRNA or the Wip1 protein stability, suggesting that Her2 may promote Wip1 expression by regulating its translation. Moreover, inhibitors of Her2, Wip1 and Skp2 can each reduce the disseminating phenotypes in cells models of breast cancer early lesions ectopically expressing Her2, Wip1 or Skp2. Conclusions: Our findings identify the Her2-Wip1-p38-Mk2-Hsp27 cascade as a novel mechanism mediating breast cancer early dissemination and provide a basis for new therapies targeting early metastatic dissemination in Her2+ breast cancer using inhibitors for Her2, Wip1 or Skp2.