The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

Abstract

BackgroundThe discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).MethodsThe ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.ResultsWe used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.ConclusionThis is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.

Conditional Knockout of the RNA-Binding Protein HuR in CD4+ T Cells Reveals a Gene Dosage Effect on Cytokine Production

“…Additionally, the data obtained with our conditional knockout mouse model suggest that HuR-dependent and -independent control mechanisms of GATA-3 and Th2 cytokines may coexist. Consistent with this idea, we have recently demonstrated that HuR can positively or negatively control expression of the same genes in breast cancer cells depending on the cellular milieu (59,60).…”

Identification of miR-193b Targets in Breast Cancer Cells and Systems Biological Analysis of Their Functional Impact

“…The discrepancies here could partly be due to different time-points used in the microarray and proteomics analyses, 24 h and 48 h, respectively. Furthermore, the steady-state-levels of mRNA and protein rarely correlate directly because of complicated post-transcriptional control mechanisms (12,13). Although proteomic strategies are currently limited to the identification of only a fraction of the cell's proteome, they complement the microarray experiments as they offer a read-out of the functional component of the transcript activity.…”

Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis

“…Our work involving targets discovered by RIP-Chip in cell lines under normoxic conditions indicates that HuR may indeed regulate the same targets in opposite fashion, dependent upon cellular milieu, though the reasons are not presently clear. 42 There are obvious differences when performing these experiments under the hypoxic conditions which operate in animals in which tumors form. The published data has both indicated that HuR overexpression correlates with increased as well as decreased aggressiveness in breast cancer.…”