Dipartimento di Farmacia e Scienze della Salute e della Nutrizione - Tesi di Dottorato

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Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento di Farmacia e Scienze della Salute e della Nutrizione dell'Università della Calabria.

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Author: search.filters.author.Bonofiglio, Daniela

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    Stromal cells in breast cancer microenvironment: molecular mechanisms involved in tumor progression and potential therapeutic targets
    (Università della Calabria, 2021-03-26) Augimeri, Giuseppina; Andò, Sebastiano; Bonofiglio, Daniela; Kleer, Celina
    Stromal cells in the tumor microenvironment (TME) play an important role in breast cancer progression, metastasis and therapeutic outcome. Among stromal cells, Tumor-Associated Macrophages (TAMs) and Mesenchymal Stem Cells (MSCs) have been shown to sustain breast tumor progression and worsen breast cancer prognosis. Elucidating the molecular mechanisms of epithelial/stromal cell interactions and discovering new therapeutic targets within the breast TME represent the main challenge of current research to increase the chances of successful treatment of breast cancer patients. Here, we firstly investigated the role of ligand-activated Peroxisome Proliferator Activator Receptor γ (PPARγ), a well-known tumor suppressor gene, to modulate breast TAM functional phenotype. We found that the treatment with natural and synthetic PPARγ ligands reduced the cytokine secretion by TAMs generated by exposure of conditioned media (CM) from breast cancer cells (BCCs). Interestingly, this effect was reversed by the PPARγ antagonist GW9662, suggesting the potential involvement of PPARγ in the attenuation of TAM polarization. Next, since it has been reported that soluble factors released in the TME mediate the tumor/stroma interactions, we mainly focused on the role of leptin which has been reported to sustain macrophage recruitment. Thus, we explored the impact of the leptin receptor knockdown (ObR sh) on BCCs in mediating the interaction between tumor cells and macrophages. In co-culture experiments between monocytes and BCCs, the absence of ObR reduced the recruitment of macrophages and affected their cytokine mRNA expression profile toward a less aggressive phenotype. We confirmed a decreased macrophage infiltration and reduced breast cancer growth in xenograft tumors of mice injected with ObR sh BCC. Furthermore, we explored the interaction between BCCs and MSCs within the breast TME. To this aim, we generated BCCs engulfing MSCs which result in hybrid cancer cells characterized by a multinucleated phenotype with increased dormancy and chemoresistance. In mouse models of breast cancer metastasis, hybrid cells had a reduced ability to form metastasis, but upon doxorubicin treatment they acquired resistance, inducing the metastatic spread of breast cancer. Collectively, our findings provide novel insights into the role of PPARγ and leptin signaling in modulating TAM polarization, opening new avenues for therapeutic intervention in breast cancer. Moreover, we identified and characterized a hybrid cell population, generated through. MSC engulfment by BBCs, with phenotypic features of malignancy, highlighting the potential of targeting stromal cells, to overcome drug resistance and metastasis in breast cancer.
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    Role of PPARy in the complex interplay between brest cancer cells and tumor microenvironment
    (2019-03-21) Gionfriddo, Giulia; Andò, Sebastiano; Bonofiglio, Daniela
    Stromal Derived Factor-1α (SDF-1α) and its cognate receptor CXCR4 play a key role in mediating breast cancer cell invasion and metastasis. Therefore, drugs able to inhibit CXCR4 activation may add critical tools to reduce tumor progression, especially in the most aggressive form of the breast cancer disease. Peroxisome Proliferator-Activated Receptor (PPAR) γ, a member of the nuclear receptor superfamily, has been found to downregulate CXCR4 gene expression in different cancer cells, however the molecular mechanism underlying this effect is not fully understood. Here, we identified a novel PPARγ-mediated mechanism that negatively regulates CXCR4 expression in both epithelial and stromal breast cancer cells. We found that ligand-activated PPARγ downregulated CXCR4 transcriptional activity through the recruitment of the silencing mediator of retinoid and thyroid hormone receptor (SMRT) corepressor onto a newly identified PPAR response element (PPRE) within the CXCR4 promoter in breast cancer cell lines. As a consequence, the PPARγ agonist rosiglitazone (BRL) significantly inhibited cell migration and invasion and this effect was PPARγ-mediated, since it was reversed in the presence of the PPARγ antagonist GW9662. According to the ability of Cancer-Associated Fibroblasts (CAFs), the most abundant component of breast cancer stroma, to secrete high levels of SDF-1 α, BRL reduced migratory promoting activities induced by conditioned media (CM) derived from CAFs and affected CXCR4 downstream signaling pathways activated by CAF-CM. In addition, CAFs exposed to BRL showed a decreased expression of CXCR4, a reduced motility and invasion along with a phenotype characterized by an altered morphology. A further component of the tumor microenvironment, that contributes to breast cancer progression and metastasis, is represented by Tumor Associated-Macrophages (TAMs), which phenotype is shaped by complex interactions with breast cancer cells. We found that the PPARγ ligand BRL, as well as DHA conjugates to ethanolamine and serotonin DHEA and DHA-5-HT respectively, were able to counteract the effects of CM derived from breast cancer cells on macrophage polarization. Collectively, our findings provide novel insights into the role of PPARγ in inhibiting breast cancer progression and further highlight the utility of PPARγ ligands for future therapies aimed at targeting both cancer and surrounding stromal cells in breast cancer patients
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    PPARγ ligands as novel agents able to inhibit breast tumor growth and progression
    (2011) Vizza, Donatella; Sisci, Diego; Bonofiglio, Daniela
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    Role of PPAR γ in breast cancer cell death and in adipocyte differentiation
    (2013-12-02) Rovito, Daniela; Sisci, Diego; Bonofiglio, Daniela
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    Molecular mechanism of PPARγ mediated apoptosis in different cancer celles
    (2008-11-26) Qi, Hongyan; Sisci, Diego; Bonofiglio, Daniela
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    Mechanisms of RA action in steroidogenic tissues and pro-apoptotic effects of combined treatment of breast tumoral cell lines with 9-cis retinoic acid and rosiglitazione
    (2008-11-20) Pingitore, Attilio; Bonofiglio, Daniela; Sisci, Diego
    Vitamin A (Retinol) plays a central role in many essential biological processes such as vision, immunity, reproduction, growth, development, control of cellular proliferation and differentiation. The main active forms of retinol, not primary involved in vision, are alltrans retinoic acid and 9-cis retinoic acid, both able to act at nuclear level by binding their receptors RAR and RXR and modulating many physiological processes. However, the nuclear action of vitamin A derivatives is not the only mechanism of retinoic acid (RA) acting on cells. RA is able to modify covalently proteins via a post-translational modification, named retinoylation that has been shown to occur at physiological concentration on pre-existing proteins and localized mainly in the mitochondrial compartment. The present study has been focused on the non genomic action of RA on steroidogenic tissues, testes and adrenal glands, giving further details on the ability of RA to influence protein activity and therefore cell physiology. In particular RA effects on mitochondria from the adrenal glands and the 2-oxoglutarate carrier protein from testes and TM-3 Leydig cell line were studied, providing new data on the peculiarity of steroidogenic tissues to incorporate RA at dietary levels and demonstrating how the shuttling of reducing equivalent across the mitochondrial membrane is influenced by RA treatment. Looking for the biochemical mechanism of RA action on the Adenine Nucleotide Translocator, that exchanges ATP for ADP between mitochondria and cytosol, for the first time, it was possible to demonstrate how the activity of this carrier protein is positively modulated by the Coenzyme A, a fundamental component of the retynoilating buffer. At pharmacological levels, retinoids are also active compounds in the treatment of cancer due to the capability to promote cell differentiation and their pro-apoptotic activity. In this latter concern, the mechanisms of nutriceutical concentration of 9-cis RA, together with nanomolar concentration of the selective PPARγ ligand, rosiglitazone, to promote apoptosis in breast cancer cell lines, have been investigated. The data lay the basis for a potential use of the combined therapy with low doses of both BRL and 9-cis RA as novel therapeutic tool particularly for breast cancer patients who develop resistance to antiestrogen therapy
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    Dynamic effects of retinoic acid and its isomers on cancer and physiology
    (2010-11-23) Perri, Mariarita; Bonofiglio, Daniela; Sisci, Diego
    In the search for new cancer chemo-preventive compounds, hundreds of naturally occurring molecules have been evaluated. Among these, antioxidants appear to be very promising. In this contest, over the last decade retinoids, natural and synthetic substances structurally related to vitamin A, are often used as part of a combined therapy and have been object of intense investigation. However, clinical trials have shown that retinoids can also be deleterious and are associated with the activation of proto-oncogenes, leading to an increased incidence of neoplasias. In fact, retinoic acid (RA) partition is regulated by cognate intracellular lipid binding proteins (iLBPs): cellular retinoic acid binding protein II (CRABP-II) delivers RA to RARs, while fatty acid binding protein 5 (FABP5) shuttles the RA to PPARβ/δ. In cells with high CRABP-II/FABP5 ratio, RA functions through RAR acting as a pro-apoptotic agent, while signaling through PPARβ/δ promotes survival in those cells highly expressing FABP5. So that, in some tissues RA promotes cell survival and hyperplasia. The apparently conflicting data regarding the pro-oxidant/ anti-oxidant and proliferative/anti-proliferative potential of different retinoids molecules, stimulated us to investigate the effect of RA on cell proliferation and its mechanisms in two different tumor Leydig cell lines (MLTC-1 and R2C) using as normal phenotype counterpart the Leydig TM-3 cell line. Our previous data demonstrated how pharmacological doses of RA induce cell death via the apoptotic pathway in Leydic TM-3 cell line. Recently dose-response treatment of TM-3, MLTC-1 and R2C with RA at nutraceutic/physiological doses, promotes cell proliferation accompanied by stimulation of antioxidant enzymes activity (CAT, GST), decreases p21 levels and fosters cell cycle progression via activation of the IP3K/Akt pathway in the cancer cell line, while administration of pharmacological doses of RA still results in apoptosis. Interestingly treatment with 500 nM of RA resulted in cytosolic vacuolization, hallmark of the autophagic process. Autophagy is a major cellular pathway for the degradation of long-lived proteins and organelles in eukaryotic cells. A large number of intracellular/extracellular stimuli, including amino acid starvation, testosterone production and invasion of microorganisms are able to induce autophagic response. In addition, retinoic acid is also implicated in a post-translation modification called retinoylation that modify, in vitro, the activity of the mitochondrial carrier oxo-chetoglutarate (OCG). Moreover, retinoids are often used as part of a combined therapy, their action is prevalently mediated by two types of receptor RAR and RXR. This latter, is also called master coordinator due to its versatility to heterodimering with several nuclear receptor. Thus, we have elucidated the molecular mechanism by which combined treatment with rosiglitazone (BRL) and 9 cis retinoic acid (9cRA) at nanomolar doses triggers apoptotic events in breast cancer cells, suggesting potential therapeutic uses for these compounds, demonstrating an up-regulation of tumor suppressor gene p53 and its activity is due to the NFkB site, giving emphasis to the potential use of the combined therapy with low doses of both BRL and 9cRA as novel therapeutic tool particularly for breast cancer patients who develop resistance to anti-estrogen therapy. Recently, 9cRA was found as endogenous in pancreas highlighted its rule in both glucose stimulated insulin secretion (GSIS) mechanism and glucose homeostasis, establishing it as autocoid hormone with a unique physiological function among retinoids, and broaden insight into mechanisms of GSIS