Abstract 3241: Preclinical development of JNK-targeted therapy for triple-negative breast cancer.
Authors: Xie Xuemei , Kaoud Tamer S. , Edupuganti Ramakrishna , Sammons Rachel M. , Hortobagyi Gabriel N. , Ueno Naoto T. , Dalby Kevin N. , Bartholomeusz Chandra 
Published: August 2014

Patients with triple-negative breast cancer (TNBC), which is negative for estrogen receptor, progesterone receptor, and HER2, have a very poor prognosis due to metastasis and limited targeted therapies. JNK (c-Jun N-terminal kinase) signaling promotes tumorigenesis and metastasis and regulates cancer stem cell self-renewal and maintenance in glioma, and the JNK pathway is hyperactivated in basal-like TNBC. Therefore, we hypothesized that suppressing JNK activity inhibits tumorigenesis and metastasis of TNBC cells.

To test our hypothesis, we generated several JNK-targeted JIP (JNK-interacting protein) mimetic peptides as well as the small molecule JNK inhibitor JNK-In-8 and assessed their therapeutic potential in TNBC cells. JIP mimetic peptides were designed and synthesized on the basis of the D-site of JIP, which is critical for JNK binding.

All the peptides and JNK-In-8 specifically inhibited JNK kinase activity. Because of high JNK specificity, JIP2 (JIP peptide) and JNK-In-8 were chosen for bioactivity studies. JIP2 (10 μM) inhibited phosphorylation of JNK and its substrate c-Jun in HEK293 cells. Similar results were observed for JNK-In-8 (0.5 μM) in SUM149 and MDA-MB-231 cells. However, whereas JIP2 was not cytotoxic against SUM149 and MDA-MB-231 cells, JNK-In-8 (IC50=4.0 μM) significantly inhibited proliferation of these cells by inducing G1 cell-cycle arrest at low doses and apoptosis at high doses. Furthermore, SUM149 and MDA-MB-231 cells treated with JNK-In-8 (2.5 μM, P&lt;0.05) exhibited significant inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity.

JIP2 (10 μM) strongly inhibited SUM149 cell migration (42.02%, P&lt;0.01) and invasion (42.11%, P&lt;0.01). Similarly, JNK-In-8 (5 μM) blocked migration (36.26%, P&lt;0.01) and invasion (77.96%, P&lt;0.001) of SUM149 cells as well as migration (62.48%, P&lt;0.001) and invasion (61.49%, P&lt;0.001) of MDA-MB-231 cells. These results indicate the capability of JIP2 and JNK-In-8 to suppress metastasis of TNBC cells.

Interestingly, JNK-In-8 (2.5 μM) also reduced the proportion of CD44+ CD24- cells (SUM149: DMSO 50.23% vs JNK-In-8 40.41%) and that of ALDH1+ cells (SUM149: DMSO 38.25% vs JNK-In-8 25.84%; MDA-MB-231: DMSO 19.94% vs JNK-In-8 9.21%) as well as mammosphere formation (SUM149: 65.95%, P&lt;0.001; MDA-MB-231, 65.7%, P&lt;0.001), suggesting the potential of JNK-In-8 to inhibit JNK-mediated cancer stem cell maintenance.

Our data demonstrate that JNK inhibitors may inhibit tumorigenesis and metastasis of TNBC cells by suppressing cancer stem cell maintenance and that JNK may serve as a promising target for TNBC therapy. Further studies will be conducted to determine the molecular mechanisms of JIP2- and JNK-In-8-mediated antitumor action in TNBC and the therapeutic efficacy of JIP2 and JNK-In-8 in a TNBC xenograft mouse model.

Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupuganti, Rachel M. Sammons, Gabriel N. Hortobagyi, Naoto T. Ueno, Kevin N. Dalby, Chandra Bartholomeusz. Preclinical development of JNK-targeted therapy for triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3241. doi:10.1158/1538-7445.AM2013-3241