Publications by authors named "Francesca Bianchini"

84 Publications

CRISPR/Cas9 uPAR Gene Knockout Results in Tumor Growth Inhibition, EGFR Downregulation and Induction of Stemness Markers in Melanoma and Colon Carcinoma Cell Lines.

Front Oncol 2021 14;11:663225. Epub 2021 May 14.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Firenze, Italy.

uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR "rescue" expression cell lines as well as we promoted the expression of only its 3'UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3'UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a "molecular sponge". In particular miR146a, by binding PLAUR 3' UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.
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http://dx.doi.org/10.3389/fonc.2021.663225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163229PMC
May 2021

Synthesis and biological studies of c(RGDyK) conjugates of cucurbitacins.

Future Med Chem 2021 May 16;13(10):877-895. Epub 2021 Apr 16.

Department of Chemistry, Aristotle University of Thessaloniki, University Campus, Thessaloniki, 54124, Greece.

Cucurbitacins (CUCUs) are triterpenoids known to display potent cytotoxic effects; however, their clinical application is limited due to poor pharmacokinetics and systemic toxicity. This work focuses on the development of c(RGDyK)-CUCU conjugates for the selective delivery of CUCUs to integrin-overexpressing cancer cells. The activity of the conjugates against various cancer cells was studied. They exhibited a mild cytostatic effect to six cancer cell lines and a cytotoxic effect against integrin-overexpressing MCF-7 and A549 cells. Their chemical and metabolic stability was extensively studied using LC-MS analysis. The conjugates maintained high affinity for αβ integrin receptors. c(RGDyK) conjugation via a PEG linker was beneficial for CUCU-D and the resulting conjugate was approximately three-times more active than the free CUCU-D in MCF7 cells.
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http://dx.doi.org/10.4155/fmc-2020-0309DOI Listing
May 2021

Enhanced Antitumoral Activity and Photoacoustic Imaging Properties of AuNP-Enriched Endothelial Colony Forming Cells on Melanoma.

Adv Sci (Weinh) 2021 Feb 21;8(4):2001175. Epub 2020 Dec 21.

Department of Experimental and Clinical Biomedical Sciences University of Florence Florence 50134 Italy.

Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.
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http://dx.doi.org/10.1002/advs.202001175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887578PMC
February 2021

Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis.

Rheumatology (Oxford) 2021 Jan 20. Epub 2021 Jan 20.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology, University of Florence, Florence, Italy.

Objectives: Systemic sclerosis (SSc) is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease.

Methods: Seahorse XF96 Extracellular Flux Analyzer was exploited to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (EC). Immunofluorescence, flow cytometer and real time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients.

Results: SSc fibroblasts, compared with control, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification, that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic EC up-regulated MMP-12 which cleaves and inactivates uPAR, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in EC, thus promoting the endothelial-to-mesenchymal transition (EndoMT) process that contributes to both capillary rarefaction and tissue fibrosis in SSc.

Conclusion: This study disclosed a liaison among the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.
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http://dx.doi.org/10.1093/rheumatology/keab022DOI Listing
January 2021

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells.

Cell Mol Life Sci 2021 Mar 25;78(6):3057-3072. Epub 2020 Nov 25.

Department of Experimental and Clinical Biomedical Sciences, Section of Experimental Pathology and Oncology, University of Florence, Viale G.B. Morgagni, 50, 50134, Florence, Italy.

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.
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http://dx.doi.org/10.1007/s00018-020-03707-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004497PMC
March 2021

The acidic tumor microenvironment drives a stem-like phenotype in melanoma cells.

J Mol Med (Berl) 2020 10 15;98(10):1431-1446. Epub 2020 Aug 15.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology, University of Florence, Florence, Italy.

Acidosis characterizes the microenvironment of most solid tumors and is considered a new hallmark of cancer. It is mainly caused by both "aerobic" and "anaerobic" glycolysis of differently adapted cancer cells, with the final product lactic acid being responsible of the extracellular acidification. Many evidences underline the role of extracellular acidosis in tumor progression. Among the different findings, we demonstrated that acidosis-exposed cancer cells are characterized by an epithelial-to-mesenchymal transition phenotype with high invasive ability, high resistance to apoptosis, anchorage-independent growth, and drug therapy. Acidic melanoma cells over-express SOX2, which is crucial for the maintenance of their oxidative metabolism, and carbonic anhydrase IX, that correlates with poor prognosis of cancer patients. Considering these evidences, we realized that the profile outlined for acid cancer cells inevitably remind us the stemness profile. Therefore, we wondered whether extracellular acidosis might induce in cancer cells the acquisition of stem-like properties and contribute to the expansion of the cancer stem cell sub-population. We found that a chronic adaptation to acidosis stimulates in cancer cells the expression of stem-related markers, also providing a high in vitro/in vivo clonogenic and trans-differentiating ability. Moreover, we observed that the acidosis-induced stem-like phenotype of melanoma cells was reversible and related to the EMT induction. These findings help to characterize a further aspect of stem cell niche, contributing to the sustainment and expansion of cancer stem cell subpopulation. Thus, the usage of agents controlling tumor extracellular acidosis might acquire great importance in the clinic for the treatment of aggressive solid tumor. KEY MESSAGES: • Extracellular acidosis up-regulates EMT and stem-related markers in melanoma cells • Acidic medium up-regulates in vitro self-renewal capacity of melanoma cells • Chronic acidosis adaptation induces trans-differentiation ability in melanoma cells • Melanoma cells adapted to acidosis show higher tumor-initiating potential than control cells • Extracellular acidosis promotes a stem-like phenotype in prostate and colorectal carcinoma cells.
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http://dx.doi.org/10.1007/s00109-020-01959-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525286PMC
October 2020

Th17 lymphocyte-dependent degradation of joint cartilage by synovial fibroblasts in a humanized mouse model of arthritis and reversal by secukinumab.

Eur J Immunol 2021 01 9;51(1):220-230. Epub 2020 Aug 9.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

How T-helper (Th) lymphocyte subpopulations identified in synovial fluid from patients with juvenile idiopathic arthritis (JIA) (Th17, classic Th1, or nonclassic Th1) drive joint damage is of great interest for the possible use of biological drugs that inhibit the specific cytokines. Our objective was to clarify the role of such Th subpopulations in the pathogenesis of articular cartilage destruction by synovial fibroblasts (SFbs), and the effect of Th17 blockage in an animal model. SFbs were isolated from healthy subjects and patients with JIA, and peripheral blood Th lymphocytes subsets were obtained from healthy subjects. Fragments of human cartilage from healthy subjects in a collagen matrix containing JIA or normal SFbs grafted underskin in SCID mice were used to measure cartilage degradation under the effects of Th supernatants. JIA SFbs overexpress MMP9 and MMP2 and Th17 induce both MMPs in normal SFbs, while nonclassic Th1 upregulate urokinase plasminogen activator (uPA) activity. In vitro invasive phenotype of normal SFbs is stimulated with conditioned medium of Th17 and nonclassic-Th1. In the in vivo "inverse wrap" model, normal SFbs stimulated with supernatants of Th17-lymphocytes and nonclassic Th1 produced a cartilage invasion and degradation similar to JIA SFbs. Secukinumab inhibits the cartilage damage triggered by factors produced by Th17.
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http://dx.doi.org/10.1002/eji.202048773DOI Listing
January 2021

The Carbonic Anhydrase IX inhibitor SLC-0111 as emerging agent against the mesenchymal stem cell-derived pro-survival effects on melanoma cells.

J Enzyme Inhib Med Chem 2020 Dec;35(1):1185-1193

Department of Clinical and Experimental Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology, University of Florence, Florence, Italy.

Mesenchymal stem cells (MSC) take part to solid tumour-associated stroma and critically influence progression of malignancy. Our study represents a striking example of melanoma progression to a more malignant and resistant phenotype promoted by MSC and the possibility to contrast this diabolic liaison using CAIX inhibitors. In particular, we demonstrated that melanoma cells exposed to a MSC-conditioned medium switch to a more malignant phenotype, characterised by resistance to programmed cell death and endowed with an epithelial-to-mesenchymal transition and stem cell characteristics. These effects were reversed abrogating MSC CAIX activity using SLC-0111, a CAIX inhibitor. Moreover, the acquisition by melanoma cells of a Vemurafenib-resistant phenotype upon MSC-conditioned medium exposure was removed when MSC were treated with SLC-0111. Therefore, MSC may profoundly reprogramme melanoma cells towards a wide resistant phenotype through CAIX involvement, as the use of SLC-0111 is able to contrast the development of this highly risky adaptation for disease progression.
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http://dx.doi.org/10.1080/14756366.2020.1764549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269050PMC
December 2020

FDG uptake in cancer: a continuing debate.

Theranostics 2020 6;10(7):2944-2948. Epub 2020 Feb 6.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology.

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http://dx.doi.org/10.7150/thno.40599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053207PMC
March 2021

β3-Adrenoreceptor Blockade Induces Stem Cells Differentiation in Melanoma Microenvironment.

Int J Mol Sci 2020 Feb 20;21(4). Epub 2020 Feb 20.

Hematology-Oncology Department, "Anna Meyer Children's Hospital", 50139 Florence, Italy.

Although there is an increasing evidence that cancer stem cell (CSC) niches in the tumor microenvironment (TME) plays a crucial role in sustaining solid tumors progression, several molecular players involved in this regulation still remain unknown. The role of β-adrenergic signaling in enhancing tumor growth through β2-adrenoreceptors (β2-ARs) has been confirmed in different cancer models, but the role played by the β3-adrenergic receptor (β3-AR) has recently emerged. Previous studies showed that β3-AR promotes cancer growth through the activation of different stromal cells in the TME, and leads to melanoma malignancy progression through inflammation, angiogenesis, and immunotolerance. Here we show that in B16 melanoma-bearing mice, the pharmacological β3-AR blockade is able to reduce the expression of CSC markers, and to induce a differentiated phenotype of hematopoietic subpopulations in TME. In particular, cytofluorimetric analysis (FACS) of the tumor mass shows that β3-AR antagonist SR59230A promotes hematopoietic differentiation as indicated by increased ratios of lymphoid/hematopoietic stem cells (HSCs) and of myeloid progenitor cells/HSCs, and increases the number of Ter119 and natural killer (NK) precursor cells, and of granulocyte precursors, indicating active hematopoiesis within the tumor tissue. Moreover, pharmacological antagonism of β3-AR induces mesenchymal stem cell (MSC) differentiation into adipocytes subtracting a potential renewal of the stem compartment by these cells. Here we demonstrate that β3-AR blockade in the TME by inducing the differentiation of different stromal cells at the expense of stemness traits could possibly have a favorable effect on the control of melanoma progression.
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http://dx.doi.org/10.3390/ijms21041420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073111PMC
February 2020

Cancer Glycolytic Dependence as a New Target of Olive Leaf Extract.

Cancers (Basel) 2020 Jan 29;12(2). Epub 2020 Jan 29.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50134 Florence, Italy.

Oleuropein (Ole), the main bioactive phenolic component of L. has recently attracted the scientific attention for its several beneficial properties, including its anticancer effects. This study is intended to investigate whether an olive leaf extract enriched in Ole (OLEO) may counteract the aerobic glycolysis exploited by tumor cells. We found that OLEO decreased melanoma cell proliferation and motility. OLEO was also able to reduce the rate of glycolysis of human melanoma cells without affecting oxidative phosphorylation. This reduction was associated with a significant decrease of glucose transporter-1, protein kinase isoform M2 and monocarboxylate transporter-4 expression, possible drivers of such glycolysis inhibition. Extending the study to other tumor histotypes, we observed that the metabolic effects of OLEO are not confined to melanoma, but also confirmed in colon carcinoma, breast cancer and chronic myeloid leukemia. In conclusion, OLEO represents a natural product effective in reducing the glycolytic metabolism of different tumor types, revealing an extended metabolic inhibitory activity that may be well suited in a complementary anti-cancer therapy.
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http://dx.doi.org/10.3390/cancers12020317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072393PMC
January 2020

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer.

J Enzyme Inhib Med Chem 2020 Dec;35(1):391-397

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.
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http://dx.doi.org/10.1080/14756366.2019.1706090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968260PMC
December 2020

Potential Role of HLA Class I Antigens in the Glycolytic Metabolism and Motility of Melanoma Cells.

Cancers (Basel) 2019 Aug 26;11(9). Epub 2019 Aug 26.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology, Viale G.B. Morgagni, 50-50134, Florence, Italy.

Besides playing a crucial role in immune surveillance, human leukocyte antigens (HLA) possess numerous non-immune functions involved in cell communication. In the present study, screening of a panel of HLA class I- and HLA class II-specific monoclonal antibodies (mAbs) for their effects on the metabolism of human melanoma cells showed for the first time that the HLA-B,C-specific mAb B1.23.2 reduced the expression level of key glycolytic enzymes, but did not affect that of mitochondrial respiration effectors. As a result, the metabolism of melanoma cells shifted from a Warburg metabolism to a more oxidative phosphorylation. In addition, the HLA-B,C-specific mAb B1.23.2 downregulated the expression of glutamine transporter and glutaminase enzyme participating in the reduction of tricarboxylic acid cycle. The HLA-B,C-specific mAb B1.23.2-mediated reduction in energy production was associated with a reduction of melanoma cell motility. On the whole, the described results suggest that HLA class I antigens, and in particular the gene products of HLA-B and C loci play a role in the motility of melanoma cells by regulating their metabolism.
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http://dx.doi.org/10.3390/cancers11091249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770395PMC
August 2019

Sequencing of NOTCH1 gene in an Italian population with bicuspid aortic valve: Preliminary results from the GISSI OUTLIERS VAR study.

Gene 2019 Oct 19;715:143970. Epub 2019 Jul 19.

Heart Care Foundation, Florence.

Background: Bicuspid aortic valve (BAV) formation is genetically determined, with reduced penetrance and variable expressivity. NOTCH1 is a proven candidate gene and its mutations have been found in familial and sporadic cases of BAV.

Methods: 66 BAV patients from the GISSI VAR study were genotyped for the NOTCH1 gene.

Results: We identified 63 variants, in heterozygous and homozygous states. Fifty-two are common polymorphisms present in almost all patients. Eleven variants are new and never yet reported: two are non-synonymous substitutions, Gly540Asp in exon 10 and Glu851Gln in exon 16; one is in the 3'UTR region and seven in introns, one corresponds to a T allele insertion in intron 27. We selected four statistically noteworthy and seven new variants identified in six BAV patients and correlated them with clinical and demographic variables and with imaging and histological parameters. Preliminary data show that four were BAV patients with isolated stenosis in patients over 60 aged. These variants may correlate with a later need for surgery for the presence of stenosis and not aortic valve regurgitation or ascending aortic aneurysm.

Conclusions: Completing the genotyping of 62 BAV patients we found 11 new variants in the NOTCH1 gene never yet reported. These findings confirm that the identification of new, clinically remarkable biomarkers for BAV requires a deeper genetic understanding of the NOTCH1 gene variants, which could be targeted by future diagnostic and therapeutic strategies.
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http://dx.doi.org/10.1016/j.gene.2019.143970DOI Listing
October 2019

Anoikis Resistance as a Further Trait of Acidic-Adapted Melanoma Cells.

J Oncol 2019 2;2019:8340926. Epub 2019 Jun 2.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio," Section of Experimental Pathology and Oncology, Italy.

Melanoma is characterized by a low extracellular pH, which contributes to the development of an aggressive phenotype characterized by several properties as the switch to an epithelial-to-mesenchymal program, the increase of apoptotic resistance, and the migratory ability together with the development of drug resistance. Here, we demonstrate that melanoma cells grown in low pH medium (pH 6.7) for a short (24 hours) or long (at least 3 months) period equally express an anoikis resistance profile. Anoikis is a form of apoptosis prompted by loss of adhesion, particularly requested by aggressive cancer cells to metastasize. Anoikis resistance was ascertained in acidic melanoma cells either grown in agarose-coated plates or incubated in rocking conditions. Both analyses indicate that acidic cells were more able to survive in a nonadherent condition than cells grown in standard pH, an effect resulting in a more cloning efficiency and migratory ability. Ability to survive during rocking was inhibited using mTOR/NF-kB inhibitors. Finally, we checked whether characteristics related to the anoikis resistance acquired by acidic melanoma cells might be also suitable for challenge. We injected acidic melanoma cells into blood stream, and then we verify how many cells survived in blood after 15 min from the injection. Only acidic cells, transient and chronic, survived, whereas melanoma cells grown in standard pH medium did not. Overall, we have had the opportunity to demonstrate that low extracellular pH represents an additional mechanism able to promote an anoikis resistance in solid tumors.
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http://dx.doi.org/10.1155/2019/8340926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582804PMC
June 2019

Reprogramming of Amino Acid Transporters to Support Aspartate and Glutamate Dependency Sustains Endocrine Resistance in Breast Cancer.

Cell Rep 2019 07;28(1):104-118.e8

Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence 50134, Italy. Electronic address:

Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER breast cancers.
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http://dx.doi.org/10.1016/j.celrep.2019.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616584PMC
July 2019

Identification of highly potent and selective MMP2 inhibitors addressing the S1' subsite with d-proline-based compounds.

Bioorg Med Chem 2019 05 22;27(9):1891-1902. Epub 2019 Mar 22.

Department of Chemistry "Ugo Schiff", University of Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy; Interdepartmental Center for Preclinical Development of Molecular Imaging (CISPIM), University of Florence, Viale Morgagni 85, 50134 Florence, Italy. Electronic address:

MMP2 and MMP9, also called gelatinases, play a primary role in the angiogenic switch, as a fundamental step of tumor progression, and show high degree of structural similarity. Clinically successful gelatinase inhibitors need to be highly selective as opposite effects have been found for the two enzymes, and the S1' subsite is the major driver to attain selective and potent inhibitors. The synthesis of d-proline-derived hydroxamic acids containing diverse appendages at the amino group, varying in length and decoration allowed to give insight on the MMP2/MMP9 selectivity around the S1' subsite, resulting in the identification of sub-nanomolar compounds with high selectivity up to 730. Molecular docking studies revealed the existence of an additional hydrophobic channel at the bottom of S1' subsite for MMP2 enzyme useful to drive selectivity towards such gelatinase.
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http://dx.doi.org/10.1016/j.bmc.2019.03.043DOI Listing
May 2019

β -Adrenoceptor as a potential immuno-suppressor agent in melanoma.

Br J Pharmacol 2019 07 9;176(14):2509-2524. Epub 2019 May 9.

Neonatal Intensive Care Unit, Medical Surgical Fetal-Neonatal Department, Meyer University Children's Hospital, Florence, Italy.

Background And Purpose: Stress-related catecholamines have a role in cancer and β-adrenoceptors; specifically, β -adrenoceptors have been identified as new targets in treating melanoma. Recently, β -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which β -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β -adrenoceptors in immune-tolerance regulation.

Experimental Approach: A mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of β-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β-blockers (propranolol and SR59230A) and specific β-adrenoceptor siRNAs targeting β - or β -adrenoceptors were used.

Key Results: Only β -, but not β -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and β -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and β -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes.

Conclusions And Implications: Our data suggest that β -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth.

Linked Articles: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
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http://dx.doi.org/10.1111/bph.14660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592854PMC
July 2019

Integrin-targeted AmpRGD sunitinib liposomes as integrated antiangiogenic tools.

Nanomedicine 2019 06 6;18:135-145. Epub 2019 Mar 6.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parma, Italy. Electronic address:

We report here the preparation, physico-chemical characterization, and biological evaluation of a new liposome formulation as a tool for tumor angiogenesis inhibition. Liposomes are loaded with sunitinib, a tyrosine kinase inhibitor, and decorated with cyclo-aminoprolineRGD units (cAmpRGD), efficient and selective ligands for integrin αβ. The RGD units play multiple roles since they target the nanovehicles at the integrin αβ-overexpressing cells (e.g. activated endothelial cells), favor their active cell internalization, providing drug accumulation in the cytoplasm, and likely take part in the angiogenesis inhibition by interfering in the αβ-VEGFR2 cross-talk. Both in vitro and in vivo studies show a better efficacy of this integrated antiangiogenic tool with respect to the free sunitinib and untargeted sunitinib-loaded liposomes. This system could allow a lower administration of the drug and, by increasing the vector specificity, reduce side-effects in a prolonged antiangiogenic therapy.
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http://dx.doi.org/10.1016/j.nano.2019.02.015DOI Listing
June 2019

Cell-targeted c(AmpRGD)-sunitinib molecular conjugates impair tumor growth of melanoma.

Cancer Lett 2019 04 11;446:25-37. Epub 2019 Jan 11.

Food and Drug Department, University of Parma, Parco Area delle Scienze 27A, 43124, Parma, Italy. Electronic address:

Drug resistance and off-organ toxicity remain unsolved issues in chemotherapy of advanced-stage melanoma patients. Thus, the creation of new molecular conjugates able to combine a selective accumulation, high ability of internalization and signaling pathway inhibition, are highly requested. Recently, we reported a new class of molecular conjugates, compounds 1-3, where the anti-αβ integrin peptidomimetic c(AmpRGD), which is a selective ligand for αβ integrin, was covalently bound to the tyrosine kinase inhibitor sunitinib. Here, we report that these c(AmpRGD)-sunitinib conjugates and, in particular, compound 3, are selectively internalized by human melanoma cells through αβ receptor-mediated endocytosis. Compound 3 is more effective than sunitinib in reducing in vitro melanoma cells proliferation, cloning efficiency, migration, and invasion. More interestingly, compound 3 is able to significantly reduce the growth of xenografted melanoma tumor developed in immune-compromised mice, more efficiently than an equimolar dose of sunitinib. Indeed, its targeting ability was demonstrated by the selective localization at the tumor level with respect to healthy tissues. Thus, c(AmpRGD)-sunitinib conjugates such as compound 3 could serve as intriguing multiple-target agents to selectively reach melanoma cells and interfere with the progression of the disease.
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http://dx.doi.org/10.1016/j.canlet.2018.12.021DOI Listing
April 2019

Oleuropein, the Main Polyphenol of Leaf Extract, Has an Anti-Cancer Effect on Human BRAF Melanoma Cells and Potentiates the Cytotoxicity of Current Chemotherapies.

Nutrients 2018 Dec 8;10(12). Epub 2018 Dec 8.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50134 Florence, Italy.

Oleuropein (Ole), a secoiridoid glucoside present in leaves, gained scientific interest thanks to its several biological properties, including the anticancer one. We verified whether Ole might potentiate the cytotoxicity of conventional drugs used to treat melanoma, disclosing a potentially new therapeutic strategy. We tested the cytotoxic action of Ole alone or in combination with chemotherapeutics on A375 human melanoma cells. We found that Ole was able, at a dose of 500 µM, to stimulate apoptosis, while at a non-toxic dose of 250 µM, it affected cell proliferation and induced the downregulation of the pAKT/pS6 pathway. A dose of 250 µM Ole did not potentiate the effect of Vemurafenib (PLX4032), but it succeeded in increasing the cytotoxic effect of Dacarbazine (DTIC). The major effect was found in the association between Ole and Everolimus (RAD001), also on PLX4032-resistant BRAF melanoma cells, which possibly cooperate in the inhibition of the pAKT/pS6 pathway. Of interest, an olive leaf extract enriched in equimolar Ole was more effective and able to further improve DTIC and RAD001 efficacy on BRAF melanoma cells with respect to Ole alone. Therefore, Ole represents a natural product able to potentiate a wide array of chemotherapeutics against BRAF melanoma cells affecting the pAKT/pS6 pathway.
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http://dx.doi.org/10.3390/nu10121950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316801PMC
December 2018

3-Adrenoreceptors Control Mitochondrial Dormancy in Melanoma and Embryonic Stem Cells.

Oxid Med Cell Longev 2018 13;2018:6816508. Epub 2018 Nov 13.

Neonatal Intensive Care Unit, Medical Surgical Fetal-Neonatal Department, A. Meyer Children's University Hospital, Florence, Italy.

The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that 3-adrenergic receptor (3-AR) is involved in tumor progression, playing an important role in metastasis. Among -adrenergic receptors, 3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. 3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, 3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that 3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The 3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific 3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of 3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.
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http://dx.doi.org/10.1155/2018/6816508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258109PMC
January 2019

SOX2 as a novel contributor of oxidative metabolism in melanoma cells.

Cell Commun Signal 2018 11 22;16(1):87. Epub 2018 Nov 22.

Department of Clinical and Experimental Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology, University of Florence, Viale G.B. Morgagni, 50, 50134, Florence, Italy.

Background: Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or "Warburg effect"), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma.

Methods: To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay.

Results: In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption.

Conclusions: SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.
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http://dx.doi.org/10.1186/s12964-018-0297-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249961PMC
November 2018

The carbonic anhydrase IX inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy.

J Enzyme Inhib Med Chem 2019 Dec;34(1):117-123

a Department of Clinical and Experimental Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology , University of Florence , Florence , Italy.

Drug combination represents one of the most accredited strategies of cancer therapy able to improve drug efficacy and possibly overcome drug resistance. Among the agents used to complement conventional chemotherapy, carbonic anhydrase IX (CAIX) inhibitors appear as one of the most suitable, as markers of hypoxic and acidic cancer cells which do not respond to chemo- and radiotherapy. We performed preclinical in vitro assays to evaluate whether the SLC-0111 CAIX inhibitor co-operates and potentiates the cytotoxic effects of conventional chemotherapeutic drugs in A375-M6 melanoma cells, MCF7 breast cancer cells, and HCT116 colorectal cancer cells. Here, we demonstrate that the SLC-0111 CAIX inhibitor potentiates cytotoxicity of Dacarbazine and Temozolomide currently used for advanced melanoma treatment. SLC-0111 also increases breast cancer cell response to Doxorubicin and enhances 5-Fluorouracil cytostatic activity on colon cancer cells. These findings disclose the possibility to extend the use of CAIX inhibitors in the combination therapy of various cancer histotypes.
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http://dx.doi.org/10.1080/14756366.2018.1532419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211231PMC
December 2019

Gold Nanoparticles Functionalized with RGD-Semipeptides: A Simple yet Highly Effective Targeting System for α β Integrins.

Chemistry 2018 Aug 16;24(46):12093-12100. Epub 2018 Jul 16.

Center for Biomolecular [email protected], Istituto Italiano di Tecnologia (IIT), Via Barsanti, 73010, Arnesano, Lecce, Italy.

Effective and selective targeting of the α β integrin subtype is of high relevance in cancer research for the development of therapeutic systems with improved efficacy and of diagnostic imaging probes. We report here a new class of highly selective, α β -targeted gold nanoparticles (AuNPs), which carry cyclic 4-aminoproline-RGD semipeptides (cAmpRGD) as the targeting moiety immobilized at low surface density on the poly(ethylene glycol) (PEG)-based nanoparticle coating. We show that these nanoparticles are potent inhibitors of the integrin-mediated melanoma tumor cell adhesion to vitronectin and are selectively internalized via receptor-mediated endocytosis. Furthermore, we have developed bifunctional cAmpRGD-functionalized AuNPs by conjugation of a fluorophore (FAM or TAMRA) to a separate set of reactive groups on the PEG-based coating. These bifunctional AuNPs not only recapitulate the binding properties of cAmpRGD-AuNPs but also can be visualized via confocal laser microscopy, allowing direct observation of nanoparticle internalization. The peculiar molecular design of these nanoparticles and their precisely defined architecture at the molecular level accounts for their selective integrin binding with very low nonspecific background.
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http://dx.doi.org/10.1002/chem.201801823DOI Listing
August 2018

Stereodivergent synthesis of 5-aminopipecolic acids and application in the preparation of a cyclic RGD peptidomimetic as a nanomolar αβ integrin ligand.

Org Biomol Chem 2018 05;16(18):3402-3414

Dipartimento di Chimica "U. Schiff", Università degli Studi di Firenze, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy.

A stereodivergent strategy was devised to obtain enantiopure cis and trans 5-aminopipecolic acids (5-APAs) in suitably protected forms to be employed in peptide synthesis as conformationally constrained α- and δ-amino acids. The cis isomer was used as a δ-amino acid to construct a cyclic RGD-containing peptidomimetic, the ability of which to compete with biotinylated vitronectin for binding with the isolated αVβ3 integrin was measured (IC50 = 4.2 ± 0.9 nM). A complete 1H NMR and computational conformational analysis was performed to elucidate the reasons for the high affinity of this cyclic peptidomimetic in comparison with cilengitide.
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http://dx.doi.org/10.1039/c8ob00534fDOI Listing
May 2018

Carbonic anhydrase IX inhibition affects viability of cancer cells adapted to extracellular acidosis.

J Mol Med (Berl) 2017 12 19;95(12):1341-1353. Epub 2017 Sep 19.

Department of Clinical and Experimental Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

Among the players of the adaptive response of cancer cells able to promote a resistant and aggressive phenotype, carbonic anhydrase IX (CAIX) recently has emerged as one of the most relevant drug targets. Indeed, CAIX targeting has received a lot of interest, and selective inhibitors are currently under clinical trials. Hypoxia has been identified as the master inductor of CAIX, but, to date, very few is known about the influence that another important characteristic of tumor microenvironment, i.e., extracellular acidosis, exerts on CAIX expression and activity. In the last decades, acidic microenvironment has been associated with aggressive tumor phenotype endowed with epithelial-to-mesenchymal transition (EMT) profile, high invasive and migratory ability, apoptosis, and drug resistance. We demonstrated that melanoma, breast, and colorectal cancer cells transiently and chronically exposed to acidified medium (pH 6.7 ± 0.1) showed a significantly increased CAIX expression compared to those grown in standard conditions (pH 7.4 ± 0.1). Moreover, we observed that the CAIX inhibitor FC16-670A (also named SLC-0111, which just successfully ended phase I clinical trials) not only prevents such increased expression under acidosis but also promotes apoptotic and necrotic programs only in acidified cancer cells. Thus, CAIX could represent a selective target of acidic cancer cells and FC16-670A inhibitor as a useful tool to affect this aggressive subpopulation characterized by conventional therapy escape.

Key Messages: Cancer cells overexpress CAIX under transient and chronic extracellular acidosis. Acidosis-induced CAIX overexpression is NF-κB mediated and HIF-1α independent. FC16-670A prevents CAIX overexpression and induces acidified cancer cell death.
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http://dx.doi.org/10.1007/s00109-017-1590-9DOI Listing
December 2017

Everolimus selectively targets vemurafenib resistant BRAF melanoma cells adapted to low pH.

Cancer Lett 2017 11 18;408:43-54. Epub 2017 Aug 18.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Section of Experimental Pathology and Oncology, University of Florence, Italy; Istituto Toscano Tumori, Center of Excellence for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies (DENOTHE), Italy. Electronic address:

Vemurafenib, a BRAF inhibitor, elicits in ∼80% of BRAF-mutant melanoma patients a transient anti-tumor response which precedes the emergence of resistance. We tested whether an acidic tumor microenvironment may favor a BRAF inhibitor resistance. A375M6 BRAF melanoma cells, either exposed for a short period or chronically adapted to an acidic medium, showed traits compatible with an epithelial-mesenchymal transition, reduced proliferation and high resistance to apoptosis. Both types of acidic cells treated with vemurafenib did not change their proliferation, distribution in cell cycle and level of p-AKT, in contrast to cells grown at standard pH, which showed reduced proliferation, cell cycle arrest and ERK/AKT inhibition. Even after treatment with trametinib (MEK inhibitor) acidic cell features did not change. Then, since both types of acidic cells exhibited high p-p70S6K, i.e. active mTOR signaling, we tested everolimus, an mTOR inhibitor, which was efficient in inducing apoptosis in acidic cells without affecting melanoma cells grown at standard pH. Our results indicate that an acidic microenvironment may cooperate in inducing a BRAF inhibitor resistance in melanoma cells and a combined therapy with everolimus could be used to overcome that resistance.
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http://dx.doi.org/10.1016/j.canlet.2017.08.010DOI Listing
November 2017

Synthesis and conformational analysis of peptides embodying 2,3-methanopipecolic acids.

Org Biomol Chem 2017 Aug;15(32):6826-6836

Department of Chemistry "U. Schiff", University of Florence, Via della Lastruccia 13, I-50019, Sesto Fiorentino, Italy.

The conformational analysis of linear and cyclic peptides incorporating 2,3-methanopipecolic acids (or Cyclopropane Pipecolic Acids, CPAs) as conformationally constrained α-amino acids is reported. Compared to peptides containing proline or pipecolic acid, a striking increase of the cis isomer (42-92%) around the CPA amide bond is observed, both in water and organic solvents, when these unnatural amino acids are embodied in linear amino acid sequences. The rotational barrier around the same bond in water was calculated, giving results comparable to that for the prolyl cis/trans isomerization. In organic solvents, CPAs at the i + 2 position of a peptide induce the formation of a type VIa β-turn secondary structure. When incorporated into a cyclic peptide, the cis geometry around the 2,3-methanopipecolic amide bond still prevails and, in the example studied herein (a cyclic RGD-containing ligand of αβ integrin mimicking Cilengitide), conservation of the backbone geometry and side chain spatial orientation of the native peptide is also found. Given the importance of the proline cis/trans isomerism in many biological processes, CPAs could be useful as proline mimetics for probing protein-ligand interactions and generating novel bioactive compounds.
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http://dx.doi.org/10.1039/c7ob01617dDOI Listing
August 2017

uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells.

Int J Cancer 2017 09 21;141(6):1190-1200. Epub 2017 Jun 21.

Department of Experimental and Clinical Biomedical Sciences, Section of Experimental Pathology and Oncology, University of Florence, Florence, 50134, Italy.

In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma.
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http://dx.doi.org/10.1002/ijc.30817DOI Listing
September 2017