title: Granzyme B is expressed in urothelial carcinoma and promotes cancer cell invasion.
abstract: Granzyme B (GrB) is a serine proteinase known to be expressed by cytotoxic lymphocytes and to induce, in presence of perforin (Pf), apoptosis in target cells. Recently, GrB expression has been shown (often in absence of Pf) in nonlymphoid cells, but its function is not defined. In our study, we investigated GrB and Pf expression in bladder cancer cell lines and in urothelial carcinoma (UC) tissues by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, ELISA, immunofluorescence and immunohistochemistry. We also assessed the function of GrB in UC cells; the in vitro function of GrB was examined by loss-of-function experiments. Our results revealed that GrB is expressed, in absence of Pf, in UC cells. Significant differences were found between GrB expression and both increasing pathological tumor spreading and high-grade vs. low-grade pTa tumors. Notably, GrB in UC tissues was concentrated at the cancer invasion front and was expressed in neoplastic cells undergoing epithelial-mesenchymal transition, a key event in carcinoma invasion. Indeed, GrB-positive cells also expressed Snail, N-cadherin or were negative for E-cadherin. GrB expressed in tumor cell lines was enzymatically active and capable of vitronectin cleavage, implying extracellular matrix (ECM) remodeling by GrB. Inhibition of GrB activity or Stealth RNA interference-mediated GrB gene silencing markedly suppressed bladder cancer cell invasion through matrigel. This data provides the first evidence for a role of GrB in promoting cancer cell invasion. Taken together, our findings suggest that GrB, via ECM degradation, contributes to the establishment of the UC invasive phenotype.
authors.affiliation: Department of Ecology and Sustainable Economic Development DECOS, Tuscia University, Viterbo, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: D'Eliseo
authors.forename: Paola
authors.initials: P
authors.lastname: Pisu
authors.forename: Chiara
authors.initials: C
authors.lastname: Romano
authors.forename: Andrea
authors.initials: A
authors.lastname: Tubaro
authors.forename: Cosimo
authors.initials: C
authors.lastname: De Nunzio
authors.forename: Stefania
authors.initials: S
authors.lastname: Morrone
authors.forename: Angela
authors.initials: A
authors.lastname: Santoni
authors.forename: Antonella
authors.initials: A
authors.lastname: Stoppacciaro
authors.forename: Francesca
authors.initials: F
authors.lastname: Velotti
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D009361:Neoplasm Invasiveness; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000201:enzymology* / Q000473:pathology
chemical_list: D017931:DNA Primers; D053804:Granzymes