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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.

Background/Rationale. While some patients with triple negative breast cancer achieve long-term remission with chemotherapy, many have cancers that are chemotherapy resistant. The lack of targeted therapies for this subtype also makes some triple negative cancers difficult to treat and control. PRKCQ, a member of the novel protein kinase C family, is preferentially expressed in triple negative breast cancers compared to ER+/Luminal cancers. We previously reported that PRKCQ expression drives growth-factor independent growth, anoikis resistance and migration of breast epithelial cells. In addition, PRKCQ is required for in vivo growth of triple negative breast cancers tumor xenografts. We sought to determine if PRKCQ expression modulates sensitivity of triple negative breast cancer cells to standard of care chemotherapy and whether PRKCQ inhibition could be a strategy to induce death of chemotherapy-resistant triple negative breast cancer cells. Methods. We determined the effects of modulating PRKCQ expression, using PRKCQ cDNA or shRNA vectors, on Doxorubicin or Taxol treatment-induced effects on triple negative breast cancer cells, including those that are relatively chemotherapy resistant at baseline. We determined the mechanisms by which PRKCQ expression regulates sensitivity to Taxol. Results. Increased PRKCQ expression in MCF-10A breast epithelial cells suppresses the apoptosis-inducing effects of Doxorubicin or Taxol treatment. PRKCQ-induced Taxol resistance is dependent on PRKCQ kinase activity. PRKCQ-expressing MCF-10A cells secrete enhanced levels of IL-6, leading to the autocrine activation of Stat3; IL-6/Stat3 activation is necessary for PRKCQ-induced resistance to Taxol. Finally, downregulation of PRKCQ sensitized MDA-231-Luc cells to Taxol treatment and induced apoptosis of these cells which are relatively resistant to Taxol at baseline. Conclusions. PRKCQ regulates sensitivity to standard of care chemotherapies used in the treatment of triple negative breast cancer. IL-6/Stat3 signaling induced by PRKCQ kinase activity is responsible for resistance to the effects of Taxol treatment. Targeting PRKCQ therefore could be an attractive strategy to overcome chemotherapy resistance of a subset of triple negative breast cancers. Citation Format: Irie HY, Byerly J. PRKCQ regulates taxol sensitivity of triple negative breast cancer cells via IL-6/Stat3 signaling. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-07.

Background: EMT in cancer promotes resistance to chemotherapy and radiotherapy, as well as immune suppression in the tumor microenvironment. EMT is also associated with enhanced tumor dissemination to other organs. EMT is a dynamic cell re-programming process whereby cancer cells lose epithelial markers and acquire mesenchymal markers, enhanced cell migration, and anoikis resistance. EMT is promoted by transcriptional and epigenetic regulators, as well as by signaling pathways. TNBCType and 101-gene model have identified distinct subsets of TNBC that exhibit mesenchymal gene signatures and phenotypes. This particular subset may be associated with chemotherapy resistance and metastatic recurrence in patients with TNBC. We identified protein tyrosine kinase 6 (PTK6) as a promoter of EMT in mesenchymal TNBC through its ability to prevent degradation of SNAIL, a key EMT transcriptional factor. A higher level of SNAIL expression is associated with poor TNBC patient prognosis. We investigated whether SNAIL suppression and EMT reversal by PTK6 small molecule inhibitor treatment enhance efficacy of chemotherapeutic agents that are part of standard of care treatment for patients with TNBC. Methods: Mesenchymal TNBC cell lines or organoids generated from TNBC PDX tumors were treated with varying concentrations of PTK6 small molecule inhibitor alone or in combination with chemotherapeutic agents in 3D cell cultures. The cell viability was assessed using 3D CellTiter-Glo or alamar blue. The combination Index was calculated to examine potential synergistic effects (CI<1: synergism, CI=1: additive, CI>1: antagonism). The in vivo combination effects of PTK6 inhibitor and paclitaxel were also assessed in two TNBC PDX models. Gene ontology analysis, targeted RT-PCR gene expression profiling and protein array were performed to identify potential mechanisms for chemosensitization effects of PTK6 inhibitor treatment. Results: Pre-treatment with PTK6 inhibitor increases sensitivity to paclitaxel or doxorubicin in 3D matrigel culture of TNBC cell lines, as well as in TNBC PDX organoids. The Combination Index suggested synergies between PTK6 inhibitor and chemotherapy treatment (paclitaxel or doxorubicin). While administration of PTK6 inhibitor or paclitaxel alone only modestly suppressed growth of PDX tumors in vivo, PTK6 inhibitor treatment sensitized tumors to paclitaxel treatment, as evidenced by the dramatic suppression of tumor volume and rate of growth. Gene ontology analysis identified gene sets that are significantly differentially expressed in PTK6 inhibitor-treated TNBC tumors, including extracellular matrix, cell migration, cell cycle and microtubule activity. A targeted RT-PCR profiling and protein array found that PTK6 inhibitor modulates expression of molecules that are associated with chemotherapy resistance and immune regulation; decrease in MMP3, PLEK2, osteopontin, IL-6 and CCL5 and increase in CD40. We are currently investigating whether these changes are dependent on Snail downregulation/EMT reversal caused by PTK6 inhibition. Conclusion: PTK6 inhibition may sensitize TNBC to chemotherapy treatment by suppressing Snail and reversing EMT. We will further validate these effects of PTK6 inhibitor treatment with other chemotherapies and immunotherapies. Citation Format: Ito K, Lee E, Sato K, Byerly JH, Zhu J, Irie HY. PTK6 small molecule inhibitor enhances efficacies of chemotherapy in mesenchymal TNBC [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-02.

Background/Rationale: The non-receptor tyrosine kinase PTK6/Brk is highly expressed in the ER+/Luminal breast cancer subtypes. PTK6 expression has prognostic significance for patients with ER+ disease; higher transcript levels are associated with poorer survival. The functions of PTK6 in the context of ER+ breast cancer and sensitivity to endocrine therapy have not been explored. We sought to determine the functional roles of PTK6 in ER+ breast cancer cells, including those that are relatively resistant to current endocrine therapies. Methods/Results. We modulated the expression of PTK6 using both gain- and loss-of-function approaches. Enhanced expression of PTK6 in Tamoxifen-sensitive ER+ breast cancer cells was sufficient to confer relative Tamoxifen resistance. Furthermore, downregulation of PTK6 in ER+ breast cancer cells, including those that have acquired resistance to Tamoxifen, induced apoptosis, as evidenced by an increase in AnnexinV+ cells and increased levels of cleaved PARP. PTK6 downregulation impaired growth of Tamoxifen-resistant variants of ER+ MCF7 and T47D cells (MCF7TamR and T47DTamR) in 3D Matrigel culture, and virtually abrogated primary tumor growth of MCF7TamR xenografts. Mechanistically, p38MAPK activation is critical for PTK6 downregulation-induced apoptosis of ER+ breast cancer cells, as p38 inhibition partially rescues cells from PTK6 shRNA-associated apoptosis. Conclusions: Our studies highlight the critical role that PTK6 plays in the survival of ER+ breast cancer cells, including those that are resistant to endocrine therapy. Enhanced PTK6 expression in ER+ breast cancer cells is sufficient to promote endocrine therapy resistance, which could contribute to the poorer prognosis associated with higher PTK6 expression in ER+ patient tumors. As small molecule PTK6 inhibitors are becoming available, our studies support further evaluation of PTK6 as a candidate therapeutic target for endocrine therapy resistant ER+ breast cancers. Citation Format: Irie HY, Park SH, Katsyv I, Zhang W, Nayak A. Protein tyrosine kinase 6 (PTK6) promotes survival of endocrine therapy-resistant ER+ breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-13.