Two Isoforms of the RNA Binding Protein, Coding Region Determinant-binding Protein (CRD-BP/IGF2BP1), Are Expressed in Breast Epithelium and Support Clonogenic Growth of Breast Tumor Cells

Saja A Fakhraldeen,Rod J Clark,Avtar Roopra,Emily N Chin,Wei Huang,John Castorino,Kari B Wisinski,Taewon Kim,Vladimir S Spiegelman,Caroline M Alexander

doi:10.1074/jbc.m115.655175

Abstract

CRD-BP/IGF2BP1 has been characterized as an "oncofetal" RNA binding protein typically highly expressed in embryonic tissues, suppressed in normal adult tissues, but induced in many tumor types. In this study, we show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. Although CRD-BP mRNA expression is induced in breast tumor cells, levels remain ∼1000-fold lower than in embryonic tissues. Despite low expression levels, CRD-BP is required for clonogenic growth of breast cancer cells. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is therefore missing antibody epitopes, CRD-BP expression has been under-reported by previous studies. We show that a CRD-BP mutant mouse strain retains expression of the shorter transcript (ΔN-CRD-BP), which originates in intron 2, suggesting that the impact of complete ablation of this gene in mice is not yet known. Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform. In summary, although CRD-BP expression levels are low in breast cancer cells, this protein is necessary for clonogenic activity.

Highlights

The RNA binding protein, coding region determinant-binding protein (CRD-BP), is expressed by tumor cells and protects key mRNAs
Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform
Identification and Characterization of the Novel ⌬N-CRD-BP Isoform in Mouse and Human Cells—We identified a discrepancy between the results of different primer sets designed to assess CRD-BP mRNA expression

Summary

Background

The RNA binding protein, coding region determinant-binding protein (CRD-BP), is expressed by tumor cells and protects key mRNAs. We show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is missing antibody epitopes, CRD-BP expression has been underreported by previous studies. In adult tissues, CRD-BP mRNA is expressed at only 0.1–1% of the levels found in embryonic tissues, and this has led to under-reporting of mRNA expression Despite this relatively low expression level, we show that CRD-BP is required for clonogenic growth and that either the full-length or N-terminally truncated isoform of CRD-BP can rescue clonogenicity of CRD-BP knockdown breast cancer cells

Experimental Procedures

Results

70 Vinculin 50

Discussion

Empty vector