Abstract 1505: Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition

Abstract

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis and lacks targeted therapies. Basal-like TNBC exhibits hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation of different cancers. However, it is unknown whether JNK signaling is a clinically relevant target in TNBC. Here, we tested the hypotheses that JNK signaling plays a fundamental role in TNBC pathogenesis by promoting self-renewal of cancer stem cells (CSCs). Methods: The role of JNK signaling in TNBC pathogenesis was determined by examining the effects of JNK signaling inhibition using siRNA or JNK-IN-8 (an ATP-competitive JNK inhibitor) and the effects of JNK overexpression on growth, migration, and invasion of TNBC cells using cell proliferation, migration, and invasion assays. CSC regulation by JNK signaling was examined using mammosphere-forming assay. The effect of JNK signaling inhibition on tumor growth was assessed using a xenograft mouse model. The downstream molecules involved in JNK signaling-mediated CSC regulation were identified using RT-PCR stem array analysis. Association between JNK and c-Jun was analyzed using a reverse phase protein array dataset of TNBC samples (n = 81). Disease-free survival (DFS) probability in TNBC patients was analyzed by the Kaplan-Meier method using a cDNA microarray dataset (n = 79). Results: Knockdown of JNK1 or JNK2 led to significant reductions in growth, migration, and invasion of TNBC cells, mammosphere formation, and ALDH1+ CSC subpopulation. Similar results were obtained when JNK signaling was inhibited using JNK-IN-8. In contrast, overexpression of JNK1 or JNK2 enhanced these cellular activities. Importantly, JNK signaling inhibition by JNK-IN-8 (25 mg/kg) reduced tumor growth by 60.88% (P<0.001) in a xenograft mouse model. RT-PCR stem array analysis indicated the involvement of Notch1 in JNK signaling-mediated CSC regulation; luciferase reporter assay showed that JNK regulated Notch1 expression at the transcriptional level via c-Jun, a transcription factor and a substrate of JNK. Knockdown of c-Jun or Notch1 reduced cell growth, mammosphere formation, and ALDH1+ CSC subpopulation; overexpression of c-Jun or Notch1 blocked the inhibitory effect of JNK-IN-8 on mammosphere formation. These results suggest that JNK signaling promotes CSC self-renewal through up-regulation of Notch1 via c-Jun. Furthermore, in TNBC patients, a high level of phosphorylated JNK was associated with a high level of phosphorylated c-Jun (P = 3×10-8); low c-Jun mRNA level was associated with better DFS (P = 0.03); and there was a tendency (P = 0.17) toward better DFS with low JNK1 mRNA level but not with low JNK2 mRNA level. Conclusion: JNK signaling regulates TNBC tumorigenesis by promoting CSC self-renewal via Notch1 signaling. JNK-IN-8 is a promising therapeutic agent for TNBC by targeting JNK/Notch1 signaling. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Dionysios N. Giannoukos, Yuan Qi, Debu Tripathy, Nathanael S. Gray, Kevin N. Dalby, Chandra Bartholomeusz, Naoto T. Ueno. Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2015-1505