RanBPM interacts with psoriasin in vitro and their expression correlates with specific clinical features in vivo in breast cancer

Abstract

Background: Psoriasin has been identified as a gene that is highly expressed in pre-invasive breast cancer, but is often downregulated with breast cancer progression. It is currently unknown whether psoriasin influences epithelial cell malignancy directly or by affecting the surrounding environment. However the protein is found in the nucleus, cytoplasm as well as extracellularly. In the present study we have sought to identify potential psoriasin-binding proteins and to describe their expression profile in breast tumors.

Methods: The yeast two-hybrid method was used to identify potential binding partners for psoriasin. The interaction of psoriasin with RanBPM was confirmed in-vitro by co-immunoprecipitation. The expression of RanBPM and psoriasin was measured by RT-PCR in a series of breast cell lines, breast tumors and primary lymphocytes.

Results: We have identified RanBPM as an interacting protein by the yeast two-hybrid assay and confirmed this interaction in-vitro by co-immunoprecipitation. RT-PCR analysis of RanBPM mRNA expression in cell lines (n = 13) shows that RanBPM is widely expressed in different cell types and that expression is higher in tumor than in normal breast epithelial cell lines. RanBPM expression can also be induced in peripheral blood mononuclear cells by treatment with PHA. RanBPM mRNA is also frequently expressed in invasive breast carcinomas (n = 64) and a higher psoriasin/RanBPM ratio is associated with both ER negative (p < 0.0001) and PR negative status (p < 0.001), and inflammatory cell infiltrates (p < 0.0001) within the tumor.

Conclusions: These findings support the hypothesis that psoriasin may interact with RanBPM and this may influence both epithelial and stromal cells and thus contribute to breast tumor progression.

Figure 1

Confirmation of specificity of interactions observed in yeast 2-hybrid assay. Panels show yeast transformed with a) psoriasin and RanBPM expression plasmids (left) and RanBPM alone (right), and yeast transformed with b) Psoriasin alone, c) psoriasin and empty prey vector, and d) psoriasin with a control gene not isolated in the primary screen. In panels b-d, plates on left are Histidine + (control) and plates on the right are Histidine – (test). Panel a) shows activation of LacZ reporter gene only occurs in yeast transformed with both expression plasmids (left) and no activation in yeast transformed with RanBPM alone (right). Panel b) shows that psoriasin alone cannot activate the Histidine reporter gene as demonstrated by absence of growth on Histidine – plate. Panels c) and d) show that there is a specific interaction necessary for activation of the reporter gene.

Figure 2

In vitro interaction of psoriasin and RanBPM as determined by co-immunoprecipitation.³⁵S-Met labelled proteins were generated. Psoriasin (lane 1) and RanBPM (lane 2) were electrophoresed through a denaturing polyacrylamide gel and detected by autoradiography. Psoriasin binds to and co-immunoprecipitates with RanBPM (lane 4). RanBPM and psoriasin do not bind to protein G-beads on their own, (lane 3 and 5 respectively). RanBPM does not interact with mouse submaxillary gland protein when either RanBPM (lane 6) or mouse submaxillary gland protein (lane 7) is precipitated.

Figure 3

RanBPM mRNA expression in selected cell lines. RanBPM mRNA expression in fibroblasts (CRL and 125), normal mammary epithelia (MCF10ATB, MCF10A1, MCF10AT1), and epithelial carcinoma cell lines from breast (T47D5, MCF-7, 126, MDA-MB-231, BT20) and cervix (HeLa). RanBPM is expressed at higher levels in cells derived from tumors compared to those derived from normal epithelia and stroma. Columns and bars represent means and standard deviations from duplicate experiments. Mann Whitney test was used to compare levels between normal epithelial cells and fibroblasts (*) or neoplastic epithelial cells (**).

Figure 4

RanBPM mRNA expression in peripheral blood mononuclear cells (PBMCs). RanBPM mRNA expression is strongly induced by stimulation of cells with PHA, weakly induced by TSST-1, but not anti-CD3, LPS or SAC. Columns and bars represent means and standard deviations from duplicate experiments performed on samples from 5 donors.