Raffaella Belvedere

Annexin A1 induces skeletal muscle cell migration acting through formyl peptide receptors.

Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca2+-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca2+ perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca2+. These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.

Evaluation of in situ injectable hydrogels as controlled release device for ANXA1 derived peptide in wound healing.

In this paper, for the first time, hydrogels containing Annexin A1 N-terminal derived peptide, Ac2-26, as a novel dressing were successfully developed for dermal wound repair application. High mannuronic (M) content alginate and low molecular weight chitosan have been used as hydrogel carrier. Peptide recovery analyses, FTIR studies and molecular modelling highlighted chemical interactions between peptide and hydrogel polymers. Ac2-26 resulted entrapped into chitosan hydrogel matrix that prevented its release, whereas such interaction in alginate hydrogel slowed down peptide diffusion enabling its sustained release till 72 h. In vivo wound healing studies conducted on mice dorsal wounds indicate that after the 9th day of post wounding Ac2-26/alginate hydrogels could significantly accelerate wound healing, with complete closure of the wound on day 14th. Therefore, these results suggest that the developed of Ac2-26 high M content alginate hydrogel could be a promising wound dressing with potential application in dermal wound healing.

Annexin A1 N-terminal derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions.

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG) or high glucose (HG) we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i) Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii) N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.

Annexin A1 is involved in the acquisition and maintenance of a stem cell-like/aggressive phenotype in prostate cancer cells with acquired resistance to zoledronic acid

In this study, we have characterized the role of annexin A1 (ANXA1) in the acquisition and maintenance of stem-like/aggressive features in prostate cancer (PCa) cells comparing zoledronic acid (ZA)-resistant DU145R80 with their parental DU145 cells. ANXA1 is over-expressed in DU145R80 cells and its down-regulation abolishes their resistance to ZA. Moreover, ANXA1 induces DU145 and DU145R80 invasiveness acting through formyl peptide receptors (FPRs). Also, ANXA1 knockdown is able to inhibit epithelial to mesenchymal transition (EMT) and to reduce focal adhesion kinase (FAK) and metalloproteases (MMP)-2/9 expression in PCa cells. DU145R80 show a cancer stem cell (CSC)-like signature with a high expression of CSC markers including CD44, CD133, NANOG, Snail, Oct4 and ALDH7A1 and CSC-related genes as STAT3. Interestingly, ANXA1 knockdown induces these cells to revert from a putative prostate CSC to a more differentiated phenotype resembling DU145 PCa cell signature. Similar results are obtained concerning some drug resistance-related genes such as ATP Binding Cassette G2 (ABCG2) and Lung Resistant Protein (LRP). Our study provides new insights on the role of ANXA1 protein in PCa onset and progression.

Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway

Annexin A1 (ANXA1) is a Ca2+-binding protein over-expressed in pancreatic cancer (PC). We recently reported that extracellular ANXA1 mediates PC cell motility acting on Formyl Peptide Receptors (FPRs). Here, we describe other mechanisms by which intracellular ANXA1 could mediate PC progression. We obtained ANXA1 Knock-Out (KO) MIA PaCa-2 cells using the CRISPR/Cas9 genome editing technology. LC-MS/MS analysis showed altered expression of several proteins involved in cytoskeletal organization. As a result, ANXA1 KO MIA PaCa-2 partially lost their migratory and invasive capabilities with a mechanism that appeared independent of FPRs. The acquisition of a less aggressive phenotype has been further investigated in vivo. Wild type (WT), PGS (scrambled) and ANXA1 KO MIA PaCa-2 cells were engrafted orthotopically in SCID mice. No differences were found about PC primary mass, conversely liver metastatization appeared particularly reduced in ANXA1 KO MIA PaCa-2 engrafted mice. In summary, we show that intracellular ANXA1 is able to preserve the cytoskeleton integrity and to maintain a malignant phenotype in vitro. The protein has a relevant role in the metastatization process in vivo, as such it appears attractive and suitable as prognostic and therapeutic marker in PC progression.

Hypoxia regulates ANXA1 expression to support prostate cancer cell invasion and aggressiveness

ABSTRACT Annexin A1 (ANXA1) is a Ca2+-binding protein overexpressed in the invasive stages of prostate cancer (PCa) development; however, its role in this tumor metastatization is largely unknown. Moreover, hypoxic conditions in solid tumors have been related to poor prognosis in PCa patients. We have previously demonstrated that ANXA1 is implicated in the acquisition of chemo-resistant features in DU145 PCa cells conferring them a mesenchymal/metastatic phenotype. In this study, we have investigated the mechanisms by which ANXA1 regulates metastatic behavior in LNCaP, DU145 and PC3 cells exposed to hypoxia. ANXA1 was differentially expressed by PCa cell lines in normoxia whereas hypoxic stimuli resulted in a significant increase of protein expression. Additionally, in low oxygen conditions ANXA1 was extensively secreted out-side the cells where its binding to formyl peptide receptors (FPRs) induced cell invasion. Loss and gain of function experiments performed by using the RNA interfering siANXA1 and an ANXA1 over-expressing plasmid (MF-ANXA1), also confirmed the leading role of the protein in modulating LNCaP, DU145 and PC3 cell invasiveness. Finally, ANXA1 played a crucial role in the regulation of cytoskeletal dynamics underlying metastatization process, such as the loss of adhesion molecules and the occurrence of the epithelial to mesenchymal transition (EMT). ANXA1 expression increased inversely to epithelial markers such as E-cadherin and cytokeratins 8 and 18 (CKs) and proportionally to mesenchymal ones such as vimentin, ezrin and moesin. Our results indicated that ANXA1 may be a key mediator of hypoxia-related metastasis-associated processes in PCa.

The Pharmaceutical Device Prisma® Skin Promotes in Vitro Angiogenesis through Endothelial to Mesenchymal Transition during Skin Wound Healing

Glycosaminoglycans are polysaccharides of the extracellular matrix supporting skin wound closure. Mesoglycan is a mixture of glycosaminoglycans such as chondroitin-, dermatan-, heparan-sulfate and heparin and is the main component of Prisma® Skin, a pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. Here, we show the in vitro effects of this device in the new vessels formation by endothelial cells, since angiogenesis represents a key moment in wound healing. We found a strong increase of migration and invasion rates of these cells treated with mesoglycan and Prisma® Skin which mediate the activation of the pathway triggered by CD44 receptor. Furthermore, endothelial cells form longer capillary-like structures with a great number of branches, in the presence of the same treatments. Thus, the device, thanks to the mesoglycan, leads the cells to the Endothelial-to-Mesenchymal Transition, suggesting the switch to a fibroblast-like phenotype, as shown by immunofluorescence assays. Finally, we found that mesoglycan and Prisma® Skin inhibit inflammatory reactions such as nitric oxide secretion and NF-κB nuclear translocation in endothelial cells and Tumor Necrosis Factor-α production by macrophages. In conclusion, based on our data, we suggest that Prisma® Skin may be able to accelerate angiogenesis in skin wound healing, and regulate inflammation avoiding chronic, thus pathological, responses.

Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts

ABSTRACT Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

TFF1 Promotes EMT-Like Changes through an Auto-Induction Mechanism

Trefoil factor 1 (TFF1) is a small secreted protein expressed in the gastrointestinal tract where, together with the other two members of its family, it plays an essential role in mucosal protection and repair against injury. The molecular mechanisms involved in the protective function of all three TFF proteins are not fully elucidated. In this paper, we investigated the role of TFF1 in epithelial to mesenchymal transition (EMT) events. The effects of TFF1 on cellular models in normoxia and/or hypoxia were evaluated by western blot, immunofluorescence, qRT-PCR and trans-well invasion assays. Luciferase reporter assays were used to assess the existence of an auto-regulatory mechanism of TFF1. The methylation status of TFF1 promoter was measured by high-resolution melting (HRM) analysis. We demonstrate a TFF1 auto-induction mechanism with the identification of a specific responsive element located between −583 and −212 bp of its promoter. Our results suggest that TFF1 can regulate its own expression in normoxic, as well as in hypoxic, conditions acting synergistically with the hypoxia-inducible factor 1 (HIF-1α) pathway. Functionally, this auto-induction mechanism seems to promote cell invasion and EMT-like modifications in vitro. Additionally, exogenously added human recombinant TFF1 protein was sufficient to observe similar effects. Together, these findings suggest that the hypoxic conditions, which can be induced by gastric injury, promote TFF1 up-regulation, strengthened by an auto-induction mechanism, and that the trefoil peptide takes part in the epithelial-mesenchymal transition events eventually triggered to repair the damage.

miR-196a Is Able to Restore the Aggressive Phenotype of Annexin A1 Knock-Out in Pancreatic Cancer Cells by CRISPR/Cas9 Genome Editing

Annexin A1 (ANXA1) is a Ca2+-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.