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

Abstract

Trastuzumab (Herceptin) resistance is a major obstacle in the treatment of patients with HER2-positive breast cancers. We recently reported that the transcription factor Y-box binding protein-1 (YB-1) leads to acquisition of resistance to trastuzumab in a phosphorylationdependent manner that relies on p90 ribosomal S6 kinase (RSK). To explore how this may occur we compared YB-1 target genes between trastuzumab-sensitive cells (BT474) and those with acquired resistance (HR5 and HR6) using genome-wide chromatin immunoprecipitation sequencing (ChIP-sequencing), which identified 1391 genes uniquely bound by YB-1 in the resistant cell lines. We then examined differences in protein expression and phosphorylation between these cell lines using the Kinexus Kinex antibody microarrays. Cross-referencing these two data sets identified the mitogen-activated protein kinase-interacting kinase (MNK) family as potentially being involved in acquired resistance downstream from YB-1. MNK1 and MNK2 were subsequently shown to be overexpressed in the resistant cell lines; however, only the former was a YB-1 target based on ChIP-PCR and small interfering RNA (siRNA) studies. Importantly, loss of MNK1 expression using siRNA enhanced sensitivity to trastuzumab. Further, MNK1 overexpression was sufficient to confer resistance to trastuzumab in cells that were previously sensitive. We then developed a de novo model of acquired resistance by exposing BT474 cells to trastuzumab for 60 days (BT474LT). Similar to the HR5/HR6 cells, the BT474LT cells had elevated MNK1 levels and were dependent on it for survival. In addition, we demonstrated that RSK phosphorylated MNK1, and that this phosphorylation was required for ability of MNK1 to mediate resistance to trastuzumab. Furthermore, inhibition of RSK with the small molecule BI-D1870 repressed the MNK1-mediated trastuzumab resistance. In conclusion, this unbiased integrated approach identified MNK1 as a player in mediating trastuzumab resistance as a consequence of YB-1 activation, and demonstrated RSK inhibition as a means to overcome recalcitrance to trastuzumab.

Targeting the Y/CCAAT box in cancer: YB-1 (YBX1) or NF-Y?

“…Interestingly, the preferred target logo, derived from the analysis of ChIP-Seq experiments, is consistent with the RNA-binding features of YB-1, and indeed resembles Kozak sequences. 127 Thus, we offer an alternative explanation for the many reports of YB-1 binding in ChIP experiments, and indeed ChIP-Seq, in transformed cells: 126 YB-1 could be transitorily located in an area physically 'close' to promoters, or other important regulatory regions, where transcriptional initiation decisions are made, but loaded on partially synthesized primary RNAs ( Figure 5). One finding consistent with this interpretation is that chromatin association of YB-1 is apparently lost upon treatment with ribonucleases, which destroys pre-mRNAs.…”

“…YB-1-regulated genes, as summarized by Eliseeva et al, 71 were analyzed for the presence of YB-1 peaks in the YB-1 ChIP-Seq data reported by Astanehe et al, 126 and for NF-Y peaks present in ENCODE data of K562, HeLa-S3 and GM12878 cells (Wang et al 60 and Fleming and Struhl, submitted for publication)…”

“…121,126 We analyzed the data for 430 000 ChIP-Seq peaks in three cancer cell types and could not identify a Y/CCAAT sequence, either searching for known TFBS or with de novo motif discovery tools. 127 This is uncommon for a sequence-specific TF, whose logo is usually recognizable within the bound peaks, but not unheard of: 60 in many cases, one can identify either a new logo or one of those characterized for other TFs.…”

Dual Abrogation of Mnk and Mtor: A Novel Therapeutic Approach for the Treatment of Aggressive Cancers

“…These upstream kinases activate mouse MNK1 via phosphorylation on Thr 197/202 [21]. Recent work shows that MNK1 can also be phosphorylated by p90 ribosomal protein S6 kinase (p90 rsk ), in a mechanism that confers trastuzumab resistance in HER-2 positive breast cancers [38]. The MNKs associate with the C-terminus of eIF4G, a vital interaction that brings the kinase into close enough proximity to phosphorylate eIF4E on Ser209 [5,39].…”

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

“…The oncogenic effect of eIF4E overexpression in mouse models of lymphoma (28) and prostate cancer (22) is strictly dependent on the phosphorylation status of eIF4E. Activation of MNK contributes to cell proliferation, transformation, and metastasis (25,26); growth and survival during cancer development (19, 29 -33); or serum deprivation (34,35) and also contributes to cancer chemoresistance (36,37) (also reviewed in Ref. 38).…”