Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells

Abstract

IntroductionBasal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1.MethodsInhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation.ResultsUsing an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not.ConclusionsWe therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention.

The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1

“…Indeed, we have recently demonstrated that RSK, similar to AKT, can phosphorylate YB-1 on its S102 residue in BLBC (Stratford et al, 2008). Our findings suggest the presence of a positive feedback loop by which P-YB-1 (S102) induces p110a, increasing PI3K activity, thereby leading to further increases in P-RSK.…”

“…Once p110a is induced its kinase activity is upregulated leading to the activation of signaling through either AKT or RSK depending on whether PIK3CA is mutated. Activated AKT (Sutherland et al, 2005) or RSK (Stratford et al, 2008) phosphorylates YB-1 on its S102 site thereby allowing it to shuttle into the nucleus where it binds to the PIK3CA promoter to induce more p110a. This feedforward loop also causes an activation of signaling through RSK that enhances cellular invasion through phosphorylation of IkBa (Ghoda et al, 1997), allowing NFkB to enter into the nucleus where it can induce uPA production (Sliva et al, 2002).…”

“…This followed a smaller study indicating that 73% of BLBC primary tumors overexpress YB-1 . Both serine/threonine kinases AKT (Sutherland et al, 2005) and the p90 ribosomal S6 kinase (RSK; Stratford et al, 2008) phosphorylate YB-1 on its S102 residue, thereby altering its nuclear trafficking. Within the nucleus, YB-1 binds to the inverted CCAAT elements known as YB-1-responsive elements (YREs) to affect transcription (Didier et al, 1988).…”

MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition

“…However, there being no specific inhibitors of MNK1 available, we sought to identify other strategies to inhibit MNK1. RSK is the major kinase responsible for phosphorylating YB-1 at S102 (Stratford et al, 2008). Therefore, we addressed whether its inhibition would affect MNK1 levels.…”

“…YB-1 is an oncogenic transcription/translation factor whose expression is associated with relapse and poor overall survival in various malignancies, including breast cancer (Wu et al, 2006;Habibi et al, 2008). The p90 ribosomal S6 kinase (RSK) (Stratford et al, 2008), and to a lesser extent AKT (Sutherland et al, 2005), can phosphorylate YB-1 on S102, promoting its nuclear translocation. Within the nucleus, YB-1 binds to inverted CCAAT boxes of genes (Didier et al, 1988) including MDR1 (Bargou et al, 1997), EGFR (Wu et al, 2006), PIK3CA , MET ) and CD44 to increase their transcription and thereby promote tumor progression.…”