Induction of Stemness Markers in Melanoma Cells by CRISPR/Cas9 uPAR Gene Knockout results in Tumor Growth Inhibition

The CRISPR/Cas9 technology, based on a naturally immune system that protects bacteria and archaea from viral infections is now widely used to manipulate genes in cells and living organisms. This system allowed us to perform a specific gene knockout, hypothetically applicable for treatments of genetic diseases and cancer. Here we have exploited the CRISPR/Cas9 gene knockdown approach to investigate the function of the urokinase plasminogen activator (uPA) receptor (uPAR) in human melanoma and the consequences of its irreversible clearing. uPAR is composed by a three-domains protein and a glycosylphosptatidyl anchor exposed on the external surface of the cell membrane. Together with its ligand uPA and the specific inhibitor Plasminogen Activator Inhibitor-1 (PAI-1), uPAR forms the so-called membrane-bound Plasminogen Activation System (PAS). Its pro-tumoral role has been shown in all the malignant tumors where uPAR is over-expressed and for this reason, PAS overexpression has reached the level of evidence 1 as an indicator of poor prognosis in node-negative breast cancer. Therefore, controlling and regulating its activity is relevant in clinical practice. Indeed, PAS acts at two distinct levels: a) by regulation of cell invasion and motility, and b) by orchestrating receptor tyrosine kinases signalling that results into cell proliferation and survival. uPAR-dependent invasion requires binding of the serine protease uPA or of its zymogen pro-uPA. uPA, in turn, activates plasminogen to plasmin, that amplifies pro-uPA activation. Plasminogen anchored to the cell surface by several low-affinity high capacity receptors, amplifies cell invasion machinery directly on the cell surface. Plasmin directly cleaves fibrin and some components of the extracellular matrix and activates the zymogens of several matrix metalloproteinases (MMPs), including gelatinases MMP9 and MMP2, directly involved in invasion and metastatization. Invasion also requires a “grip” for invasive cell conferred by uPAR binding to Vitronectin, an event that induces the formation of uPAR dimers and promotes cell anchorage. The studies on uPAR-integrin interaction have also enlightened cues on uPAR-dependent signalling, which entails motility, survival and proliferation. Many signalling pathways are triggered following uPA-driven activation of uPAR, independent of its catalytic activity, but mediated by several lateral partners, including integrins, EGFR, PDGFR-β, IGF-1R and c-Met. This complex interactome gives rise to a net of signalling ranging from MAPK, FAK, Src, small GTPase Rac pathways, to JAK, STAT and PI3K-AKT. Upon interaction of their α-chain with uPAR, integrins α5β1 and αvβ3 assume a conformation capable of activating Receptor Tyrosine Kinases (RTKs). Such interaction is potentiated by catalytic and non-catalytic uPA binding to uPAR and can induce RTKs activity even in the absence of the relevant ligands (EGF, PDGF, ILGF and HGF) in a FAK-dependent manner. It is also important to remark that either uPAR-bound or soluble uPA can stimulate cell proliferation by activation of latent growth factors or release of ECM-bound growth factors. We have shown that uPAR is strongly over-expressed in human melanoma cells and controls both the mesenchymal (protease-dependent) and the amoeboid (protease-independent) movements, fundamental for cell invasion. We have also cleaved uPAR expressed by human melanoma cells by the uPAR-degrading enzyme MMP12 properly engineered within shuttle Endothelial Progenitor Cells (EPCs) (EPC-MMP12). Once delivered into human melanomas xenografted in nude mice, such EPC-MMP12 inhibited tumor growth, angiogenesis and lung metastases. Further, we have observed that uPAR expression is proportional to the phase of tumor progression of melanoma cells and that the TGFβ of conditioned medium of mesenchymal stem cells (MSC) induces the acquisition of an epithelial-to-mesenchymal (EMT) profile in melanoma cells, characterized by uPAR and TGFβ/TGFβ-receptor upregulation: TGFβ gene silencing in MSCs downregulates uPAR expression and EMT in melanoma. On these bases, we have knocked-out the uPAR gene from A375, a human melanoma cell line, by the CRISP/Cas9 technique with the aim to inhibit melanoma aggressiveness. Surprisingly, we have observed that upon uPAR KO, a high percentage of melanoma cells acquired a stem cell profile that was unrelated to cell proliferation and survival. They also gained a more aggressive phenotype, in terms of chemoresistance and resistance to UV-induced apoptosis, and changed their metabolism by increasing glycolysis and strengthening the Warburg phenotype that characterizes almost all cancer cells. However, melanoma cells were incapable of growing, in both canonical 2D cultures and 3D structures commonly called spheroids. On the basis of such observations, we speculate that lack of uPAR could be one of the responsible for the minimal residual disease after chemo- and radiotherapy. Cells that in vivo do not express uPAR could in fact resist to these therapies and, after an unspecified period of time, give rise to a new tumor. Further studies in vivo using xenografts will be used to better understand whether uPAR KO gene therapy is effective or if it would be preferable to adopt a standard chemotherapy coupled with peptide, like M25, unpairing uPAR from its lateral partners, abrogating in this way all the uPAR-mediated signal cascade.