Profilin 1 is a potential biomarker for bladder cancer aggressiveness

Abstract

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.

Fig. 1.

Urine fractionation using IMAC (nickel). SDS-PAGE (5% stacking-10% separating, silver staining) analysis of fractions received during IMAC. A, Representative fractions following the analysis of a cancer pooled urine sample (in this case corresponding to invasive BC); SM: starting material, FT: flow-through, W: washes, E: eluted fractions. B, Excised bands from the eluted fractions subsequently processed by tryptic digestion and LC-MS/MS analysis. The band numbers shown here correspond to those provided in Table I and supplemental Table S1.

Fig. 2.

Western blot analysis of myeloblastin and aminopeptidase N in individual urine samples and respective quantification data of the specific protein band following normalization according to total protein content. A total of 108 samples (28 urine samples from patients with benign diseases of the urogenital tract, 30 Ta, 24T1, and 26T2+ cancers with similar age and sex distribution) in the case of aminopeptidase N and 97 samples (23 urine samples from patients with benign diseases of the urogenital tract, 26Ta, 23T1, and 25T2+) were analyzed supporting a significant difference in the invasive BC versus non invasive cancers or benign diseases (* p < 0.05; t test). A large variation of values is nevertheless observed within each group.

Fig. 3.

Representative expression patterns of profilin 1 in bladder tumors (sections at 20× and respective 40× views, as indicated) spotted on tissue microarrays. A–D, Negative cytoplasmic expression of profilin 1 in cancer cells with very low (scored as negative) (A–B), and positive (C–D) profilin stromal expression; E–H, low (+) cytoplasmic profilin 1 with negative (E–F) and positive (G–H) profilin stromal staining; I–J, intermediate (++) cytoplasmic with negative stoma staining; K–L, high (+++) cytoplasmic with positive stroma expression of profilin 1.

Fig. 4.

Survival analyses -Kaplan-Meier curves displaying the associations of profilin 1 expression in epithelial and stroma cells with tumor recurrence and disease outcome (survival). A, Kaplan-Meier indicating that in patients with non-invasive disease, profilin expression in the stroma was significantly associated with a high recurrence rate (p = 0.032), and B, with a poor overall survival (0.005). C–D, Kaplan-Meier curves indicating that in patients with invasive disease, the presence of low intensity of the cytoplasmic profilin expression was significantly associated with poor survival (C, disease-specific; p = 0.014, and D, overall survival; p = 0.016). E–F, Kaplan-Meier indicating that taking together all tumors under analysis, a low intensity of profilin cytoplasmic expression was significantly associated with poor survival (E, disease-specific (p < 0.0005), and F, overall survival (p < 0.0005)).

Fig. 5.

In vitro effects of profilin 1 on bladder cancer cell motility and actin polymerization. A, Expression of profilin 1 in T24 conditioned media (CM, i) and cell extracts (ii); Profilin 1 found in the CM is not a result of contamination from necrotic cells as shown by respective blots for tubulin (iii, iv); the mean intensity of the tubulin band in CM is 20 times lower than the ones of profilin 1 in CM. The results from the analysis of three different (A–C) cell culture preparations and cell extracts are depicted. B, In vitro blockage of profilin 1 results in decrease of bladder cancer cell motility. i) Diagram showing the number of migrated cells following incubation of T24 cells with specific rabbit anti-human profilin 1 or control Ab. Nontreated cells were also used as control. In all cases 50,000 cells were initially plated, migration was allowed for 6 h toward T24 CM. The nonmigrated cells were then removed from the top of the insert and migrated cells were fixed and stained (Ral Kit). Migration was quantified by counting the nuclei that passed through the filter from a minimum of 10 fields of view (20×) (Leica CTR MIC microscope, Image J software). Two independent experiments were performed including two replicates each. Data are presented as the mean ± S.D. and were analyzed by Student's t test. (ii-iv): Representative optical images of migrated T24 untreated cells (ii), T24 cells incubated with control Ab (iii), and T24 cells following incubation with specific profilin Ab (iv). C, Blocking of profilin 1 results in decrease in polymerized actin. Decreased Phalloidin-FITC uptake from T24 cells after incubation with specific profilin 1 Ab (i) compared with control Ab (ii). Immunofluorescent staining was merged with DAPI nuclear staining. Images were taken from a Leica CTR-MIC Fluorescent microscope (20x). iii) T24 cells incubated with profilin 1 Ab present a decrease in median fluorescence intensity compared with cells incubated with control Ab, as shown by FACS analysis. iv) Representative bar plot denoting a 20% reduction of median fluorescence intensity of T24 cells after incubation with profilin 1 Ab compared with the cells that were incubated with a control Ab.