title: 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 phosphorylation-dependent 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.
authors.affiliation: Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Astanehe
authors.forename: M R
authors.initials: MR
authors.lastname: Finkbeiner
authors.forename: M
authors.initials: M
authors.lastname: Krzywinski
authors.forename: A
authors.initials: A
authors.lastname: Fotovati
authors.forename: J
authors.initials: J
authors.lastname: Dhillon
authors.forename: I M
authors.initials: IM
authors.lastname: Berquin
authors.forename: G B
authors.initials: GB
authors.lastname: Mills
authors.forename: M A
authors.initials: MA
authors.lastname: Marra
authors.forename: S E
authors.initials: SE
authors.lastname: Dunn
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011621:Pteridines / Q000494:pharmacology; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D000068878:Trastuzumab; D051840:Y-Box-Binding Protein 1 / Q000378:metabolism*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; C516541:BI D1870; D047908:Intracellular Signaling Peptides and Proteins; D011621:Pteridines; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; C491395:MKNK1 protein, human; D017346:Protein Serine-Threonine Kinases; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D000068878:Trastuzumab