Abstract
BackgroundInsulin-like growth factor binding protein 5 (IGFBP5) has been shown to be associated with breast cancer metastasis in clinical marker studies. However, a major difficulty in understanding how IGFBP5 functions in this capacity is the paradoxical observation that ectopic overexpression of IGFBP5 in breast cancer cell lines results in suppressed cellular proliferation. In cancer tissues, IGFBP5 resides mainly in the cytoplasm; however, in transfected cells, IGFBP5 is mainly located in the nucleus. We hypothesized that subcellular localization of IGFBP5 affects its functions in host cells.MethodsTo test this hypothesis, we generated wild-type and mutant IGFBP5 expression constructs. The mutation occurs within the nuclear localization sequence (NLS) of the protein and is generated by site-directed mutagenesis using the wild-type IGFBP5 expression construct as a template. Next, we transfected each expression construct into MDA-MB-435 breast cancer cells to establish stable clones overexpressing either wild-type or mutant IGFBP5.ResultsFunctional analysis revealed that cells overexpressing wild-type IGFBP5 had significantly lower cell growth rate and motility than the vector-transfected cells, whereas cells overexpressing mutant IGFBP5 demonstrated a significantly higher ability to proliferate and migrate. To illustrate the subcellular localization of the proteins, we generated wild-type and mutant IGFBP5-pDsRed fluorescence fusion constructs. Fluorescence microscopy imaging revealed that mutation of the NLS in IGFBP5 switched the accumulation of IGFBP5 from the nucleus to the cytoplasm of the protein.ConclusionTogether, these findings imply that the mutant form of IGFBP5 increases proliferation and motility of breast cancer cells and that mutation of the NLS in IGFBP5 results in localization of IGFBP5 in the cytoplasm, suggesting that subcellular localization of IGFBP5 affects its cell growth and migration functions in the breast cancer cells.
Highlights
Insulin-like growth factor binding protein 5 (IGFBP5) has been shown to be associated with breast cancer metastasis in clinical marker studies
To determine whether the low cytoplasmic level of mutant IGFBP5 was the result of increased secretion of the IGFBP5 into the media, we collected both cell lysate and conditional media from the stable cell lines and performed western blot analysis
We observed that the majority of the secreted wild-type IGFBP5 was cleaved whereas that majority of the mutant IGFBP5 remained intact in the conditioned media (Figure 4)
Summary
Insulin-like growth factor binding protein 5 (IGFBP5) has been shown to be associated with breast cancer metastasis in clinical marker studies. The NLS contains a heparin-binding motif (HBM, consensus sequence BBBXXB, where B is a basic amino acid and X is any amino acid) at amino acids 206 to 211 (KRKQCK) and carries alternative IGF-binding sites [9,10]. This region seems to be critical in determining the diverse functions of IGFBP5. IGFBP5 has been shown to determine cell fates by regulating apoptotic molecules (bax, bcl-2) [13] and activating p38 MAP kinase and Erk 1/2 signal transduction pathways [14].
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