Abstract
Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44(+) cells. Luteolin's efficacy against drug-resistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched populations. ChIP-on-ChIP experiments in SUM149 cells revealed a 12-fold enrichment of YB-1 binding to the Notch4 promoter. We chose to pursue this because there are several reports indicating that Notch4 maintains cells in an undifferentiated, TIC state. Herein we report that silencing YB-1 with siRNA decreased Notch4 mRNA. Conversely, transient expression of Flag:YB-1(WT) or the constitutively active mutant Flag:YB-1(D102) increased Notch4 mRNA. The levels of Notch4 transcript and the abundance of the Notch4 intracellular domain (N4ICD) correlated with activation of P-RSK(S221/7) and P-YB-1(S102) in a panel of TNBC cell lines. Silencing YB-1 or RSK reduced Notch4 mRNA and this corresponded with loss of N4ICD. Likewise, the RSK inhibitors, luteolin and BI-D1870, suppressed P-YB-1(S102) and thereby reduced Notch4. In conclusion, inhibiting the RSK/YB-1 pathway with luteolin is a novel approach to blocking Notch4 signaling and as such provides a means of inhibiting TICs.
Highlights
Therapeutic intervention relies on conventional chemotherapeutics for patients with triple-negative breast cancer (TNBC)
We screened the Prestwick Library consisting of 1120 chemicals at 10 or μM or BI-D1870 (10 μM) in an in vitro RSK1 kinase assay against the Y-box binding protein-1 (YB-1) peptide containing the S102 site
The YB-1 peptide was selected because it was previously characterized for binding to RSK1 using in vitro kinase assays [5] and through molecular docking [41]
Summary
Therapeutic intervention relies on conventional chemotherapeutics for patients with triple-negative breast cancer (TNBC). Since this subtype does not express estrogen receptor (ER), progesterone receptor (PR) or Her-2 patients are ineligible for targeted agents to these molecules such as tamoxifen or trastuzumab. The p90 ribosomal S6 kinases (RSK), RSK1 and RSK2, are associated with breast cancer growth This family of serine/threonine kinases is part of the MAPK pathway and is responsible for activating a wide range of substrates involved in cell proliferation, motility and survival [5, 6]. Genome-wide screen for breast cancer subtype-specific inhibitors, RSK2 was one of only three molecules found to be important for sustaining the growth of TNBC [11]. Considering the poor prognosis for patients with TNBC, this new information indicating that RSK2 inhibitors could improve treatment of this disease makes a focus in this area timely
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