Emerging Targeted Therapeutic Strategies to Overcome Imatinib Resistance of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common malignant mesenchymal neoplasms of the gastrointestinal tract. The gold standard for the diagnosis of GISTs is morphologic analysis with an immunohistochemical evaluation plus genomic profiling to assess the mutational status of lesions. The majority of GISTs are driven by gain-of-function mutations in the proto-oncogene c-KIT encoding the tyrosine kinase receptor (TKR) known as KIT and in the platelet-derived growth factor-alpha receptor (PDGFRA) genes. Approved therapeutics are orally available as tyrosine kinase inhibitors (TKIs) targeting KIT and/or PDGFRA oncogenic activation. Among these, imatinib has changed the management of patients with unresectable or metastatic GISTs, improving their survival time and delaying disease progression. Nevertheless, the majority of patients with GISTs experience disease progression after 2-3 years of imatinib therapy due to the development of secondary KIT mutations. Today, based on the identification of new driving oncogenic mutations, targeted therapy and precision medicine are regarded as the new frontiers for GISTs. This article reviews the most important mutations in GISTs and highlights their importance in the current understanding and treatment options of GISTs, with an emphasis on the most recent clinical trials.

The Role of Nutrients in Prevention, Treatment and Post-Coronavirus Disease-2019 (COVID-19).

SARS-CoV-2 virus, infecting human cells via its spike protein, causes Coronavirus disease 2019 (COVID-19). COVID-19 is characterized by shortness of breath, fever, and pneumonia and is sometimes fatal. Unfortunately, to date, there is still no definite therapy to treat COVID-19. Therefore, the World Health Organization (WHO) approved only supportive care. During the COVID-19 pandemic, the need to maintain a correct intake of nutrients to support very weakened patients in overcoming disease arose. The literature available on nutrient intake for COVID-19 is mainly focused on prevention. However, the safe intake of micro- and/or macro-nutrients can be useful either for preventing infection and supporting the immune response during COVID-19, as well as in the post-acute phase, i.e., "long COVID", that is sometimes characterized by the onset of various long lasting and disabling symptoms. The aim of this review is to focus on the role of nutrient intake during all the different phases of the disease, including prevention, the acute phase, and finally long COVID.

Therapeutic Strategies Targeting Urokinase and Its Receptor in Cancer.

Several studies have ascertained that uPA and uPAR do participate in tumor progression and metastasis and are involved in cell adhesion, migration, invasion and survival, as well as angiogenesis. Increased levels of uPA and uPAR in tumor tissues, stroma and biological fluids correlate with adverse clinic-pathologic features and poor patient outcomes. After binding to uPAR, uPA activates plasminogen to plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme able to facilitate tumor cell invasion and dissemination to distant sites. Moreover, uPAR activated by uPA regulates most cancer cell activities by interacting with a broad range of cell membrane receptors. These findings make uPA and uPAR not only promising diagnostic and prognostic markers but also attractive targets for developing anticancer therapies. In this review, we debate the uPA/uPAR structure-function relationship as well as give an update on the molecules that interfere with or inhibit uPA/uPAR functions. Additionally, the possible clinical development of these compounds is discussed.

The Emerging Role of Neutrophil Extracellular Traps (NETs) in Tumor Progression and Metastasis.

Neutrophil Extracellular Traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils. In addition to their key role in the neutrophil innate immune response, NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Interestingly, a close correlation between cancer cell recruitment of neutrophils in the tumor microenvironment (Tumor Associated Neutrophils. TANs) and NET formation has been also observed either in primary tumors and metastatic sites. Moreover, NETs can also catch circulating cancer cells and promote metastasis. Furthermore, it has been reported that wake dormant cancer cells, causing tumor relapse and metastasis. This review will primarily focus on the pro-tumorigenic activity of NETs in tumors highlighting their ability to serve as a potential target for cancer therapy.

Tumor Associated Neutrophils. Their Role in Tumorigenesis, Metastasis, Prognosis and Therapy.

Tumor Associated Neutrophils (TANs) are engaged into the tumor microenvironment by cytokines and chemokines, can be distinguished according to their activation and cytokine status and effects on tumor cell growing in N1 and N2 TANs. N1 TANs exert an antitumor activity, by direct or indirect cytotoxicity. N2 TANs stimulate immunosuppression, tumor growth, angiogenesis and metastasis by DNA instability, or by cytokines and chemokines release. In tumor patients, either a high number of TANs and Neutrophil-to-Lymphocyte Ratio (NLR) do correlate with poor prognosis, and, so far, TAN counts and NLR can be regarded as biomarkers. Owing to the pivotal role of TANs in stimulating tumor progression, therapeutic strategies to target TANs have been suggested, and two major approaches have been proposed: (a) targeting the CXCL-8/CXCR-1/CXCR-2 axis, thereby blocking TANs or (b) targeting substances produced by polymorpho-nuclear cells that promote tumor growth. Many studies have been accomplished either in vitro and in animal models, whereas clinical studies are restrained, presently, due to the risk of inducing immunosuppression. In this review, we deeply discuss the anti-tumorigenic or pro-tumorigenic activity of TANs. In particular, TANs relevance in tumor prognosis and in vitro therapeutic strategies are widely described. On-going clinical trials, aimed to inhibit neutrophil recruitment into the tumor are also accurately debated.

A urokinase receptor-derived peptide inhibiting VEGF-dependent directional migration and vascular sprouting.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR88₋92 is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR88₋92 chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR88₋92 sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR88₋92. In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of αvß3 integrin at focal adhesions, and αvß3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer.

Single amino acid substitutions in the chemotactic sequence of urokinase receptor modulate cell migration and invasion.

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR(88-92) chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR(S90P) exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR(S90P) cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR(S90E) cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR(S90P). In conclusion, our findings indicate that Ser(90) is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR(S90E) and uPAR(S90P) are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

Regulation of cell migration and invasion by specific modules of uPA: mechanistic insights and specific inhibitors.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Involvement of the soluble urokinase receptor in chondrosarcoma cell mobilization.

High levels of urokinase receptor (uPAR) in tissue and serum of patients with chondrosarcoma correlate with poor prognosis. First, we analyzed the uPAR levels in tissues and plasma of five patients affected by chondrosarcoma. Interestingly, very high levels of uPAR and its soluble forms (SuPAR) were found on tumor cell surfaces and plasma, respectively, of two patients with lung metastases. Therefore, to investigate the role of SuPAR in chondrosaromas, we generated a primary cell culture from a chondrosarcoma tissue overexpressing uPAR on cell surfaces. We found that chondrosarcoma-like primary culture cells release a large amount of SuPAR in the medium. In vitro, SuPAR elicits chondrosarcoma cell migration likely through its uPAR(88-92) sequence, since the DII(88-183) or DIIDIIR(88-284) uPAR domains retain motogen effect whereas DI(1-87) or DIII(184-284) domains, both lacking the uPAR(88-92) sequence, are ineffective. Chondrosarcoma cells cross matrigel in response to SuPAR, and their invasion capability is abrogated by RERF peptide which inhibits uPAR(88-92) signalling. These findings assign a role to uPAR in mobilizing chondrosarcoma cells and suggest that RERF peptide may be regarded as a prototype to generate new therapeutics for the chondrosarcoma treatment.

Structure-based design of an urokinase-type plasminogen activator receptor-derived peptide inhibiting cell migration and lung metastasis.

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser(88)-Arg-Ser-Arg-Tyr(92) is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH(2) (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe-dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an alphav integrin-dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/alphav association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein-expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy.