Identification of Y-Box Binding Protein 1 As a Core Regulator of MEK/ERK Pathway-Dependent Gene Signatures in Colorectal Cancer Cells

Abstract

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

Author Summary

The simultaneous analysis of gene expression in cancer using microarrays is a standard approach for monitoring disease-related modifications involved in tumorigenesis, triggering malignant properties and clinical behavior. However, the factors that drive these alterations most often remain elusive. We sought to identify transcription factors that mediate the transcriptional effects of the receptor tyrosine kinase/RAS oncoprotein pathway, a frequently activated oncogenic signaling system, in cultured colorectal cancer cells. We used an integrated approach combining molecular and functional assays, as well as computational tools, to identify regulatory factors that trigger the alterations of gene expression and modulate cellular growth. We identified the YBX1 protein, a member of the highly conserved family of cold shock domain transcription factors, as a regulator of signaling effects triggered by the RAS cancer gene. Then we assayed the messenger RNA expression of YBX1 and YBX1-responsive target genes by interrogating microarrays, and also expression of the YBX1 protein by immunohistochemistry in colorectal tumors. We found that YBX1 expression is correlated with a bad clinical outcome in colon cancer patients.