title: Luteolin is a novel p90 ribosomal S6 kinase (RSK) inhibitor that  suppresses Notch4 signaling by blocking the activation of Y-box  binding protein-1 (YB-1).
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.
authors.affiliation: Laboratory for Oncogenomic Research, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Reipas
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Law
authors.forename: Nicole
authors.initials: N
authors.lastname: Couto
authors.forename: Sumaiya
authors.initials: S
authors.lastname: Islam
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Li
authors.forename: Huifang
authors.initials: H
authors.lastname: Li
authors.forename: Artem
authors.initials: A
authors.lastname: Cherkasov
authors.forename: Karen
authors.initials: K
authors.lastname: Jung
authors.forename: Amarpal S
authors.initials: AS
authors.lastname: Cheema
authors.forename: Steven J M
authors.initials: SJ
authors.lastname: Jones
authors.forename: John A
authors.initials: JA
authors.lastname: Hassell
authors.forename: Sandra E
authors.initials: SE
authors.lastname: Dunn
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D006801:Humans; D047311:Luteolin / Q000494:pharmacology*; D008958:Models, Molecular; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051840:Y-Box-Binding Protein 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D051840:Y-Box-Binding Protein 1; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D047311:Luteolin