Abstract P1-01-02: Targeting PTK6 to treat mesenchymal triple negative breast cancer
Authors: Ito K , Park SH , Nayak A , Byerly J , Irie HY 
Published: February 2018

Abstract
Background/Rational: Patients with triple negative breast cancers (TNBC) have limited therapeutic options beyond conventional chemotherapy. Unfortunately, high-risk for metastatic recurrence and chemotherapy resistant diseases cause the worst 5-year survival rate in patients with TNBC, which have been significant clinical challenges. Novel therapeutic targets or strategies to combat metastasis and chemotherapy resistance are necessary to improve quality of life and outcomes for patients with high risk TNBC. Epithelial-to-mesenchymal transition (EMT) and anoikis resistance are processes recognized as contributing to enhanced metastatic potential and treatment resistance. A subset of TNBC exhibits mesenchymal gene signatures and phenotypes that may be associated with high metastatic recurrence, chemotherapy resistance and immunosuppression. In a functional genomic screen, we identified several candidates as novel regulators of EMT and anoikis sensitivity of TNBC cells. We have focused on roles of one highly validated candidate, protein tyrosine kinase 6 (PTK6) on EMT, anoikis resistance and metastatic capacity in TNBC. Methods: We analyzed expression of PTK6 and mesenchymal markers in patient triple negative tumors by immunohistochemistry. In breast epithelial and TNBC cell lines, the levels of PTK6 were genetically modulated, and determined effects on growth, migration and EMT. In vivo mouse models were used to show effects of PTK6 inhibition on metastatic capacity of TNBC cells. We have also validated effects of PTK6 specific small molecule inhibitor on TNBC growth and metastases. In order to dissect specific mechanisms by which PTK6 inhibition regulates TNBC mesenchymal phenotypes, we used a siRNA library screening and identified novel E3 ligases that may be responsible for PTK6 inhibition-induced EMT regulation. Results: Overexpression of PTK6 in MCF10A cells is sufficient to promote an EMT; promotes migration, suppresses epithelial markers (E-cadherin/claudin-1) and increases mesenchymal markers (N-cadherin and fibronectin). In contrast, PTK6 inhibition either PTK6 shRNAs or treatment with a specific kinase inhibitor enhances E-cadherin expression and suppresses migration, anoikis resistance and lung colonization of TNBC cells. PTK6-dependent E-cadherin regulation is specifically dependent on levels of SNAIL, a transcriptional repressor that is associated with poor TNBC patient prognosis. SNAIL down-regulation by PTK6 inhibition is directly responsible for the modulation of anoikis sensitivity, which is in turn causally linked to lung colonization potential. PTK6 inhibition promotes the proteasome-dependent degradation of SNAIL via a novel mechanism independent of GSK3β/β-TRCP pathway or Fbox E3ligases (FBXO5, FBXO11, FBXL14) that are known to regulate SNAIL ubiquitination. Using a siRNA library screening approach, we identified novel E3 ligase candidates that may be responsible for SNAIL ubiquitination and degradation downstream of PTK6 inhibition. Conclusion/Future direction: PTK6 is a representative novel regulator of EMT and anoikis resistance that can be targeted to prevent metastases of TNBC. Modulation of mesenchymal phenotypes of TNBC cells may be able to regulate chemotherapy resistance and/or immunosuppressive microenvironment.
Citation Format: Ito K, Park SH, Nayak A, Byerly J, Irie HY. Targeting PTK6 to treat mesenchymal triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-02.