Abstract 5501: Withaferin A-mediated inhibition of breast carcinoma involves concomitant activation of CHOP and Elk1 via ERK/ribosomal S6 kinase (RSK) signaling axis regulating death receptor 5 expression. .
Authors: Nagalingam Arumugam , BOK Joseph H , Kuppusamy Panjamurthy , Saxena Neeraj , Sharma Dipali 
Published: August 2014

Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells.

Methods: Efficacy of WFA treatment on cell growth, invasion and, migration potential was evaluated by using clonogenicity, anchorage-independent growth, matrigel-invasion and spheroid-migration assays. Western blot and immunofluorescence analysis were used to examine activation of ERK/RSK (extracellular signal-regulated kinase/ ribosomal S6 kinase) and downstream signaling axes. Functional importance of ERK/RSK and CHOP (C/EBP homologous protein) in the biologic effects of WFA was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immunohistochemical, RT-PCR and western blot analysis of tumors were used.

Results: Analysis of the underlying molecular mechanisms revealed that WFA treatment induced phosphorylation of ERK which in turn activated RSK. Further, WFA-induced ERK/RSK signaling resulted in concomitant upregulation of CHOP and Elk-1(ETS-like transcription factor 1). By using ERK1/2 siRNA and FMK-MEA inhibitor, we found that ERK/RSK signaling axis is required for WFA-mediated modulation of CHOP and Elk-1. Intriguingly, we discovered that WFA triggers the nuclear translocation of CHOP and Elk1 resulting in transcriptional upregulation of Death Receptor protein (DR5). CHOP and Elk1 overexpression potentiated while phospho-deficient Elk-1 inhibited WFA-induced DR5 expression exhibiting that CHOP/Elk-1 cooperatively regulate DR5 expression. Furthermore, using nontoxic doses of WFA, we showed that WFA treatment effectively inhibited breast tumorigenesis in vivo. Analysis of WFA-treated breast tumors showed increased activation of ERK/RSK axis and DR5 along with higher nuclear accumulation of CHOP and Elk-1.

Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk-1 axes in breast tumor growth inhibition. WFA treatment could potentially be a rational therapeutic strategy for breast carcinoma.

Citation Format: Arumugam Nagalingam, Joseph H BOK, Panjamurthy Kuppusamy, Neeraj Saxena, Dipali Sharma. Withaferin A-mediated inhibition of breast carcinoma involves concomitant activation of CHOP and Elk1 via ERK/ribosomal S6 kinase (RSK) signaling axis regulating death receptor 5 expression. . [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 5501. doi:10.1158/1538-7445.AM2013-5501