Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry.

Tumour vascularisation is vital for cancer sustainment representing not only the main source of nutrients and oxygen supply but also an escape route for single or clustered cancer cells that, once detached from the primary mass, enter the blood circulation and disseminate to distant organs. Among the mechanisms identified to contribute to tumour vascularisation, vasculogenic mimicry (VM) is gaining increasing interest in the scientific community representing an intriguing target for cancer treatment. VM indeed associates with highly aggressive tumour phenotypes and strongly impairs patient outcomes. Differently from vessels of healthy tissues, tumour vasculature is extremely heterogeneous and tortuous, impeding efficient chemotherapy delivery, and at the meantime hyperpermeable and thus extremely accessible to metastasising cancer cells. Moreover, tumour vessel disorganisation creates a self-reinforcing vicious circle fuelling cancer malignancy and progression. Because of the inefficient oxygen delivery and metabolic waste removal from tumour vessels, many cells within the tumour mass indeed experience hypoxia and acidosis, now considered hallmarks of cancer. Being strong inducers of vascularisation, therapy resistance, inflammation and metastasis, hypoxia and acidosis create a permissive microenvironment for cancer progression and dissemination. Along with these considerations, we decided to focus our attention on the relationship between hypoxia/acidosis and VM. Indeed, besides tumour angiogenesis, VM is strongly influenced by both hypoxia and acidosis, which could potentiate each other and fuel this vicious circle. Thus, targeting hypoxia and acidosis may represent a potential target to treat VM to impair tumour perfusion and cancer cell sustainment.

Schwann cell insulin-like growth factor receptor type-1 mediates metastatic bone cancer pain in mice.

Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to metastatic bone cancer pain (MBCP), but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve, and IGF-1-dependent stimulus/non-stimulus-evoked pain-like behaviors. Adeno-associated virus-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in Schwann cells, but not in dorsal root ganglion (DRG) neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. Schwann cell IGF-1R signaling promoted an endothelial nitric oxide synthase-mediated transient receptor potential ankyrin 1 (TRPA1) activation and release of reactive oxygen species that, via macrophage-colony stimulating factor-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment.

Nintedanib-αVß6 Integrin Ligand Conjugates Reduce TGFß-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFß is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvß6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvß6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvß6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvß6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvß6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvß6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

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

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.

Modular synthesis of 2,4-diaminoanilines as CNS drug-like non-covalent inhibitors of asparagine endopeptidase.

Asparagine endopeptidase (AEP), also called legumain, is a pH-dependent endolysosomal cysteine protease that cleaves its substrates after asparagine residues. Recent studies showed that it possesses δ-secretase activity and that it is implicated in numerous neurological diseases such as Alzheimer's disease (AD). Following evidence of aryl-morpholines as useful asparagine endopeptidase inhibitors, a series of morpholinoanilines with diverse substituents at ortho position were synthesized in view of improving the potency and scope of this molecular scaffold, allowing to identify ethyl 2-isonipecotate-4-morpholinoaniline possessing inhibition potency in the nanomolar range. CNS MPO (CNS MultiParameter Optimization) calculations revealed that most of the compounds developed in this work show physicochemical parameters in the desirable range for CNS drug candidates.

Design, synthesis and evaluation of RGD peptidomimetic - Gold nanostar conjugates as M21 cell adhesion inhibitors.

Effective targeting of αvß3 integrin is of high relevance in cancer research as this protein is overexpressed on several types of tumor cells, making such receptor ideal for the development of therapeutics and of diagnostic imaging agents. In this paper, the synthesis of a novel functionalized triazole-based RGD peptidomimetic and its covalent conjugation on pegylated gold nanostars is reported. These highly stable nanoconstructs showed a multivalent effect in binding αvß3 integrin receptors and proved to inhibit M21 cell adhesion at 25 pM concentration. Thanks to their peculiar surface plasmon resonance in the "NIR transparent window", targeted gold nanostars may represent a promising agent for anticancer multi-modality treatments. 2009 Elsevier Ltd. All rights reserved.

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

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.

Extracellular Acidosis Differentially Regulates Estrogen Receptor ß-Dependent EMT Reprogramming in Female and Male Melanoma Cells.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERß, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERß in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERß in male cells and EMT was strongly promoted. An inverse relationship between ERß expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERß expressing cell subpopulations and ERß receptor silencing. Finally, we found that ERß regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERß regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

Identification of a Common Pharmacophore for Binding to MMP2 and RGD Integrin: Towards a Multitarget Approach to Inhibit Cancer Angiogenesis and Metastasis.

During tumor angiogenesis different growth factors, cytokines and other molecules interact closely with each other to facilitate tumor cell invasion and metastatic diffusion. The most intensively studied as molecular targets in anti-angiogenic therapies are vascular endothelial growth factor (VEGF) and related receptors, integrin receptors and matrix metalloproteinases (MMPs). Considering the poor efficacy of cancer angiogenesis monotherapies, we reasoned combining the inhibition of αvß3 and MMP2 as a multitarget approach to deliver a synergistic blockade of tumor cell migration, invasion and metastasis. Accordingly, we identified a common pharmacophore in the binding cavity of MMP2 and αvß3, demonstrating such approach with the design, synthesis and bioassays of tyrosine-derived peptidomimetics carrying the necessary functional groups to bind to key pharmacophoric elements of MMP2 and αvß3 RGD integrin.

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

OBJECTIVES: 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: A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients. RESULTS: SSc fibroblasts, compared with controls, 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 ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, 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 ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc. CONCLUSION: This study showed the relationship of 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.

New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4ß1 Integrin.

Integrin α4ß1 belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α4ß1 integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α4ß1-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.

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

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.

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

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.

ß3-Adrenoreceptor Blockade Induces Stem Cells Differentiation in Melanoma Microenvironment.

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 ß-adrenergic signaling in enhancing tumor growth through ß2-adrenoreceptors (ß2-ARs) has been confirmed in different cancer models, but the role played by the ß3-adrenergic receptor (ß3-AR) has recently emerged. Previous studies showed that ß3-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 ß3-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 ß3-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 ß3-AR induces mesenchymal stem cell (MSC) differentiation into adipocytes subtracting a potential renewal of the stem compartment by these cells. Here we demonstrate that ß3-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.

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

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.

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

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.

Integrin-targeted AmpRGD sunitinib liposomes as integrated antiangiogenic tools.

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 αVß3. The RGD units play multiple roles since they target the nanovehicles at the integrin αVß3-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 αVß3-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.

Gold Nanoparticles Functionalized with RGD-Semipeptides: A Simple yet Highly Effective Targeting System for αV ß3 Integrins.

Effective and selective targeting of the αV ß3 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, αV ß3 -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.

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

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.

Stereodivergent synthesis of 5-aminopipecolic acids and application in the preparation of a cyclic RGD peptidomimetic as a nanomolar αVß3 integrin ligand.

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 αVß3 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.