Abstract P2-07-05: A potential role for Janus protein tyrosine kinases in the regulation of epithelial-mesenchymal transition in a model of epidermal growth factor induced breast cancer epithelial-mesenchymal transition
Authors: Teneale A Stewart, Iman Azimi, Felicity M Davis, Erik W Thompson, Andrew J Brooks, Sarah J Roberts-Thomson, Gregory R Monteith
Journal: Cancer Research (2015)

Abstract Background. Epithelial-mesenchymal transition (EMT), a process whereby tumorigenic epithelial cells acquire an invasive and migratory phenotype, is an important event in the invasion-metastasis cascade. As such, intracellular signaling pathways involved in the regulation of EMT represent potential therapeutic targets in the treatment and prevention of invasive cancer subtypes. The calcium ion, a highly versatile intracellular messenger, plays an important role in processes important in tumorigenesis including invasion and metastasis, and altered calcium signaling has been identified in various cancers. We recently identified that activation of signal transducer and activator of transcription 3 (STAT3) and expression of specific EMT markers in the MDA-MB-468 cell line model of epidermal growth factor (EGF) induced EMT display some calcium dependence. While the calcium permeable ion channel TRPM7 was shown to partially regulate this STAT3 activation and vimentin expression, the precise mechanisms of their regulation are not yet fully understood. The aim of this research was to investigate the upstream intracellular signaling pathway involved in EGF stimulated STAT3 activation and the subsequent induction of EMT in this model. Methods. MDA-MB-468 basal-like breast cancer cells were pre-treated for 1 hr with the Janus protein tyrosine kinase (JAK) inhibitor, JAK inhibitor I (1 and 10 μM), or the Src family tyrosine kinase inhibitor, PP2 (0.1, 1 and 10 μM), followed by stimulation with EGF (50 ng/mL) for 10 or 20 min, and 24 h to assess effects on STAT3 activation and/or EMT marker expression, respectively. Total cellular protein was isolated following inhibitor treatment ± EGF stimulation, and the level of phosphorylated STAT3 (10 or 20 min) or vimentin protein expression (24 h), was analyzed using Western blotting. Cellular RNA was isolated following inhibitor treatment ± EGF stimulation and levels of vimentin mRNA (24 h) were assessed using real time RT-PCR. Results. Treatment of MDA-MB-468 breast cancer cells with JAK inhibitor I resulted in a significant decrease in EGF stimulated STAT3 phosphorylation, while inhibition of Src family tyrosine kinases with PP2 also significantly decreased EGF stimulated STAT3 phosphorylation. In addition to its effects on STAT3 phosphorylation, pre-treatment of MDA-MB-468 cells with JAK inhibitor I also appeared to decrease EGF-induced vimentin protein and mRNA expression, indicating a potential role for Janus protein tyrosine kinases in the induction of EMT in this model. Conclusions. Janus protein tyrosine kinase signaling appears to play a role in the regulation of STAT3 activation, and the induction of the EMT marker vimentin in the MDA-MB-468 cell line model of EGF-induced EMT. Future studies will focus on investigating the specific JAK family member(s) involved in the EGF-STAT3 signaling pathway, as well as the nexus between calcium and identified regulators of EGF stimulated STAT3 activation, and EMT marker expression, in this model of breast cancer EMT. Citation Format: Teneale A Stewart, Iman Azimi, Felicity M Davis, Erik W Thompson, Andrew J Brooks, Sarah J Roberts-Thomson, Gregory R Monteith. A potential role for Janus protein tyrosine kinases in the regulation of epithelial-mesenchymal transition in a model of epidermal growth factor induced breast cancer epithelial-mesenchymal transition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-05.