Trop-2 is up-regulated in invasive prostate cancer and displaces FAK from focal contacts

Abstract

In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa.

Keywords: TRAMP; exosome; gleason grade; metastasis; pT2/pT3/pT4 prostate cancer.

Figure 1. Trop-2 localization and expression in PCa

A. Localization of Trop-2 as investigated by IF staining and confocal microscopy in human PCa (pT3 stage, Gleason Score 9). B. Representative IHC staining for Trop-2 using specimens from patients at pT3 (left) and pT2 (middle) stages of PCa is shown. A non-immune IgG was used as negative control on a stage pT3 section (right). Bars, 100 μm.

Figure 2. Analysis of Trop-2 expression in metastatic PCa from TRAMP mice

A. IF analysis of Trop-2 expression in metastatic prostate tumors from TRAMP mice (top). Cell nuclei were counterstained with DAPI. A non-immune goat IgG was used as a negative control Ab (bottom). B. Representative images of a dissected normal genito-urinary (GU) system (top left), primary prostate tumor (bottom left), and lung (top right) and liver (bottom right) macroscopic metastases. Seminal vesicles (black arrow); metastases (yellow arrowheads). C. H&E staining of non-metastatic (top left), metastatic primary prostate tumors (bottom left), and of metastases in lungs (top right) and liver (bottom right).

Figure 3. Correlation of Trop-2 and α5β1 integrin expression in murine PCa

A. Analysis of α5, β1 (top), αv and β5 (bottom) integrin subunits, as well as of Trop-2 (bottom) expression by IB using protein lysates from non-metastatic (left) and metastatic (right) prostate tumors collected from TRAMP mice. ERK1, control of protein loading. B. Protein lysates of PC3 cells endogenously expressing Trop-2 were immunoprecipitated using an Ab targeting Trop-2; a non-immune mouse IgG was used as a negative control Ab (Neg. Ctr.). The immunoprecipitates were then separated by SDS-PAGE and analyzed by IB for detection of the α5 integrin subunit and Trop-2. C. Protein lysates of PC3 cells were immunoprecipitated using Abs targeting β1 integrins or Trop-2; a non-immune mouse IgG was used as a negative control Ab (Neg. Ctr.). The immunoprecipitates were then analyzed by IB for detection of the α3 integrin subunit.

Figure 4. Trop-2-dependent modulation of FAK localization

Localization of vinculin and FAK in PC3/Ctr.shRNA and PC3/Trop-2 shRNA cells seeded on FN was analyzed by IF (left). Vinculin (Vin)- and FAK-containing FAs were counted, and the average numbers per cell are shown in the bar graph (right). Error bars, SEM. **, Student's t-test P < 0.001.

Figure 5. PC3 exosome uptake by PCa cells enhances cell migration on FN in a Trop-2-dependent manner

A. Analysis of Trop-2 levels in purified PC3 exosomal lysates separated by SDS-PAGE in non-reducing conditions and immunoblotted using an Ab to Trop-2; CD63 and CD81 were used as positive exosomal markers while calnexin (CANX) was used as a negative marker for exosomes. Exo, exosomes; TCL, total cell lysates. B. IB analysis of Trop-2 expression in exosomes secreted by PC3 cells (Ctr.shRNA and Trop-2 shRNA) using an Ab to Trop-2; CD63 was used as positive exosomal markers while calnexin (CANX) was used as a negative marker for exosomes. Exo, exosomes; TCL, total cell lysates. C. Migration assays of PC3^Trop-2- (left) or LNCaP (right) cells either untreated or treated with 10 μg/ml of PC3 exosomes (Exo) in which Trop-2 is expressed (Parental and Ctr.shRNA) or down-regulated (Trop-2 shRNA). Left, χ² test ; right, Student's t-test. *, P ≤ 0.05.