Abstract
BackgroundAnnexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis.MethodsThe expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays.ResultsIn all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.ConclusionThis study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.
Highlights
Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis
The aim of our study is to investigate the role of ANXA1 in human PC progression, in particular we focused on its involvement in cancer cell migration and invasiveness
As previously described [26], more aggressive features for MIA PaCa-2 and PANC-1 as, differently from BxPC-3 and CAPAN-2, these cancer cells possess a marked mesenchymal phenotype characterized by up-regulation of the mesenchymal marker vimentin and down-regulation of the epithelial marker E-cadherin (Figure 1B,C,D) [28]
Summary
Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. Pancreatic carcinoma (PC) is one of the most aggressive gastrointestinal malignancies worldwide, with poor prognosis [1] and a 5-year survival rate of 3-5% [2]. Tumour metastases are the most common causes of death in cancer patients and represent the utmost challenge for cancer treatment. In particular cell migration and invasion play a crucial role in the progression of cancer since their deregulation causes tumour metastasis [6]. ANXA1 exhibits calcium-mediated phospholipid binding properties and participates in many physiopathological processes, including inhibition of cell proliferation, antiinflammatory effects, regulation of cell migration, differentiation and death [7]
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