Abstract B065: The RSK/YB-1 pathway represents an opportunity for targeting TNBC and holds promise of treating metastases
Authors: Stratford Anna L. , Berns Rachel , So Pauline , Pambid Mary R. , Abbas Fotovati , Abu-Ali Samah , Hu Kaiji , Bennewith Kevin , Dullaghan Edie , Dunn Sandra E. 
Published: May 2015

Abstract
Triple-negative breast cancers (TNBC) account for 15-25% of all breast cancers, have poor outcomes and high rates of relapse. Along with metastatic disease treatment options for TNBC are limited to that of conventional chemotherapy. The lack of targeted therapies for these cancers is a distinct unmet clinical need. YB-1, a transcription factor, is associated with 70% of TNBC and poor prognosis. While YB-1 is not easily druggable, p90 ribosomal S6 kinase (RSK), which lies upstream of YB-1 and activates it by phosphorylation of serine 102, is an ideal candidate. We recently reported that RSK inhibition decreases TNBC cell growth. Also RSK and YB-1 are implicated in invasion and therefore may play a role in metastatic spread. Herein, we asked whether RSK inhibitors would be beneficial for patients with metastatic disease. Our concern for treating metastatic disease was inspired by a study we conducted in 2222 patients with breast cancer. Women who had local, regional or distant metastases were at a much higher risk of dying. Remarkably those with distant metastases were 100 times for likely to die from breast cancer as compared to those without disseminated disease. Further, women with TNBC specifically had the worst outcomes and their time to death was the shortest of any breast cancer subtype. We therefore conducted a screen of 128 compounds, which are in clinical trials, in SUM149 TNBC cells and compared them to the RSK inhibitor BI-D1870. Most of these drugs failed to inhibit TNBC growth; however, BI-D1870 was highly active. These promising results point towards RSK as a potential molecular target for TNBC yet there are no inhibitors available for use in patients at this time.
To further validate RSK as a target for TNBC we asked which of the four RSK isoforms (RSK1-4) are expressed. Only RSK 1 and 2 are expressed in breast cancer cell lines. Inhibiting RSK by siRNA or BI-D1870 suppressed the growth of TNBC cells by ~90%; however, there was less of an effect on non-TNBC cells where growth was attenuated by ~50%. RSK inhibition with siRNA also triggered apoptosis to a greater degree in TNBC cell lines. There was no growth inhibitory effect on normal mammary epithelial cells (184htrt). Further, RSK inhibition suppressed the growth of TNBC colonies in soft agar by ~90%. Kinexus antibody arrays were used to understand why loss of RSK suppressed the growth of TNBC. Of note, cell proliferation, invasion and apoptosis proteins were suppressed indicating the multifaceted benefit of inhibiting RSK.
Next we asked whether the RSK/YB-1 pathway was active in metastases. We assessed activated RSK in a panel of metastatic murine breast cancer cell lines. The RSK pathway was more active in the metastatic cell lines compared to the non-metastatic cells. Taking this further, we compared the non-metastatic 67NR to the metastatic 4T1 cells where RSK 1 and 2 were more highly expressed in the latter. Likewise, P-YB-1S102 was more active. Transfecting 67NR cells with activated RSK1 increased their invasion. Conversely inhibiting RSK with BI-D1870 decreased the growth and invasion of the 4T1 cells. In a mouse xenograft model, P-YB-1S102 was highly expressed in the lung and liver metastases obtained from the 4T1 cells. Similarly, P-YB-1S102 was active in a distant metastases obtained from a patient with TNBC. Subsequently, we identified three RSK inhibitors from a chemical library screen. The most active agent, compound 2, had an in vitro IC50=10nM which was comparable to BI-D1870. Accordingly compound 2 inhibited TNBC growth and signaling through YB-1. In conclusion we identify RSK as a promising therapeutic target for TNBC that has the potential to suppress the growth of metastases.
Citation Format: Anna L. Stratford, Rachel Berns, Pauline So, Mary R. Pambid, Fotovati Abbas, Samah Abu-Ali, Kaiji Hu, Kevin Bennewith, Edie Dullaghan, Sandra E. Dunn. The RSK/YB-1 pathway represents an opportunity for targeting TNBC and holds promise of treating metastases. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B065.