uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

Biagioni, Alessio; Laurenzana, Anna; Chillà, Anastasia; Rosso, Mario Del; Andreucci, Elena; Poteti, Martina; Bani, Danièle; Guasti, Daniele; Fibbi, Gabriella; Margheri, Francesca

doi:10.3390/cells9020308

Cited by 19 publications

(24 citation statements)

References 42 publications

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“…To further validate the evidence that uPAR is critical for the Exos-mediated angiogenesis, we have exploited the CRISPR–CAS9 technology to obtain a robust irreversible uPAR gene knockout in A375 and M6 (Fig. 7 a, left panel), as recently published [ 29 ]. In parallel, to directly demonstrate the role of uPAR, we performed an experiment of expression rescue (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…At the same time, we have also shown that the MMP12-dependent uPAR cleavage is responsible for an angiogenesis impairment in HMVECs [ 42 ]. Here, to study the uPAR function in Exos, we exploited the CRISPR–Cas 9 technology to obtain a complete uPAR knockout, as recently published [ 29 ]. In this study, we reported that the uPAR silencing and, even more, the CRISPR-mediated knockout abrogate the pro-angiogenic potential of melanoma Exos both in vitro and in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…A complete PLAUR gene knockout was obtained, as previously described [ 29 ], by transfection of A375 and A375-M6 with two CRISPR/Cas9 D10A plasmids, each one bearing a specific sgRNA designed by the manufacturer to generate a double strand break in uPAR exon 3. For uPAR expression rescue experiments, cells were stably transfected using an Okayama–Berg vector containing uPAR cDNA and selected with G418 as resistance marker (0.5 mg/ml) as previously reported [ 29 , 30 ].…”

Section: Methodsmentioning

confidence: 99%

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

Biagioni

Laurenzana

Menicacci

et al. 2020

Cell. Mol. Life Sci.

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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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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Biagioni

Laurenzana

Menicacci

et al. 2020

Cell. Mol. Life Sci.

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“…More recently, uPAR knockout by (CRISPR)/Cas9 approach has been shown to induce a glycolytic and OXPHOS reprogramming in melanoma and colon carcinoma cell lines. In particular, in uPAR KO cells, authors observe an increased number of mitochondria in two melanoma cell lines and an immature biogenesis of mitochondria in the colon carcinoma line, accompanied by a significant enhancement of the mitochondrial respiratory capacity and a decreased glycolysis, even though with an increased secretion of lactate [ 103 ].…”

Section: Upar and Cancer Hallmarksmentioning

confidence: 99%

The Urokinase Receptor: A Multifunctional Receptor in Cancer Cell Biology. Therapeutic Implications

Santi

Napolitano

Montuori

et al. 2021

IJMS

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Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.

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“…Many studies, including our own, had focused on the features and the behavior of cancer cells after uPAR cleavage or downregulation, both in vitro and in vivo, using anti-uPAR oligodeoxynucleotide (ODN) (13,14) and miRNA (15), exploiting uPAR inactivation specific cleavage systems such as MMP-12 (16), or inhibiting its interaction with the complex "uPAR interactome" by a specific uncoupler peptide (17). In one of our previous studies, we decided to exploit the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 technique to establish two melanoma and one colon carcinoma cell lines with a complete uPAR KO (18), to better understand its role in tumor progression, examining the typical cancer hallmarks. This wide-spreading technology, based on a naturally-occurring system that protects bacteria from phages infections (19), is particularly useful being uPAR commonly modulated by many extracellular factors such as hypoxia, cytokines and transcription factors such as NF-kB and TCF/ LEF (20) but also by cell-cell contact (21).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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. In vivo 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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uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

Cited by 19 publications

References 42 publications

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

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

The Urokinase Receptor: A Multifunctional Receptor in Cancer Cell Biology. Therapeutic Implications

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

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