Of the most important clinical needs for
bladder cancer (BC) management is the identification of
biomarkers for disease aggressiveness. Urine is a "
gold mine" for
biomarker discovery, nevertheless, with multiple
proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary
proteins based on the use of immobilized
metal affinity chromatography for the discovery of
biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized
metal affinity chromatography fractionation and eluted
proteins analyzed by 1D-SDS-PAGE, band excision and liquid chromatography tandem MS. Among the identified
proteins, multiple corresponded to
proteins with affinity for metals and/or reported to be phosphorylated and included
proteins with demonstrated association with BC such as MMP9,
fibrinogen forms, and
clusterin. In agreement to the immobilized
metal affinity chromatography results,
aminopeptidase N,
profilin 1, and
myeloblastin were further found to be differentially expressed in urine from patients with invasive compared with non invasive BC and benign controls, by Western blot or Elisa analysis, nevertheless exhibiting high interindividual variability. By tissue microarray analysis,
profilin 1 was found to have a marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3)
tumors with occasional expression in stroma; importantly, this pattern strongly correlated with poor prognosis and increased mortality. The functional relevance of
profilin 1 was investigated in the T24 BC cells where blockage of the
protein by the use of
antibodies resulted in decreased cell motility with concomitant decrease in actin polymerization. Collectively, our study involves the application of a fractionation method of urinary
proteins and as one main result of this analysis reveals the association of
profilin 1 with BC paving the way for its further investigation in BC stratification.