Valentina Bizzarro

A Critical Requirement for Notch Signaling in Maintenance of the Quiescent Skeletal Muscle Stem Cell State

Notch signaling plays a key role in virtually all tissues and organs in metazoans; however, limited examples are available for the regulatory role of this pathway in adult quiescent stem cells. We performed a temporal and ontological assessment of effectors of the Notch pathway that indicated highest activity in freshly isolated satellite cells and, unexpectedly, a sharp decline before the first mitosis, and subsequently in proliferating, satellite cell‐derived myoblasts. Using genetic tools to conditionally abrogate canonical Notch signaling during homeostasis, we demonstrate that satellite cells differentiate spontaneously and contribute to myofibers, thereby resulting in a severe depletion of the stem cell pool. Furthermore, whereas loss of Rbpj function provokes some satellite cells to proliferate before fusing, strikingly, the majority of mutant cells terminally differentiate unusually from the quiescent state, without passing through S‐phase. This study establishes Notch signaling pathway as the first regulator of cellular quiescence in adult muscle stem cells. STEM CELLS 2012; 30:243–252.

Annexin A1: Novel roles in skeletal muscle biology

Annexin A1 (ANXA1, lipocortin‐1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca2+‐dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti‐inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. New insights show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics. J. Cell. Physiol. 227: 3007–3015, 2012.

Structure-based design, synthesis and preliminary anti-inflammatory activity of bolinaquinone analogues

As a part of our drug discovery efforts we developed a series of simplified derivatives of bolinaquinone (BLQ), a hydroxyquinone marine metabolite, showing potent anti-inflammatory activity. Thirteen new hydroxyquinone derivatives closely related to BLQ were synthesized and tested on mouse macrophage-like RAW 264.7 cell line in order to investigate their ability to modulate the production of Prostaglandin E2 (PGE2). This optimization process led to the identification of three strictly correlated compounds with comparable and higher inhibitory potency than BLQ on PGE2 production. To evaluate the affinity of BLQ and its analogues for hsPLA2, surface plasmon resonance (SPR) experiments were performed.

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.

Inhibition of Wnt/ β -Catenin pathway and Histone acetyltransferase activity by Rimonabant: a therapeutic target for colon cancer

In a high percentage (≥85%) of both sporadic and familial adenomatous polyposis forms of colorectal cancer (CRC), the inactivation of the APC tumor suppressor gene initiates tumor formation and modulates the Wnt/β-Catenin transduction pathways involved in the control of cell proliferation, adhesion and metastasis. Increasing evidence showed that the endocannabinoids control tumor growth and progression, both in vitro and in vivo. We evaluated the effect of Rimonabant, a Cannabinoid Receptor 1 (CB1) inverse agonist, on the Wnt/β-Catenin pathway in HCT116 and SW48 cell lines carrying the genetic profile of metastatic CRC poorly responsive to chemotherapies. In these models, Rimonabant inhibited the Wnt/β-Catenin canonical pathway and increased β-Catenin phosphorylation; in HCT116 cells, but not in SW48, the compound also triggered the Wnt/β-Catenin non canonical pathway activation through induction of Wnt5A and activation of CaMKII. The Rimonabant-induced downregulation of Wnt/β-Catenin target genes was partially ascribable to a direct inhibition of p300/KAT3B histone acetyltransferase, a coactivator of β-Catenin dependent gene regulation. Finally, in HCT116 xenografts, Rimonabant significantly reduced tumor growth and destabilized the nuclear localization of β-Catenin. Obtained data heavily supported the rationale for the use of cannabinoids in combined therapies for metastatic CRC harbouring activating mutations of β-Catenin.

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.