Metalloproteinases (MMPs) are a cluster of at least 23 enzymes belonging to the more wide family of endopeptidases called Metzincins, whose structure is characterized by the presence of a zinc ion at ...the catalytic site. Although the general view of MMPs as physiologic scissors involved in extracellular matrix (ECM) degradation and tissue remodeling is still valid, additional functions have recently emerged, including the ability to cleave non ECM molecules such as growth factors, cytokines and chemokines from their membrane-anchored proforms. These functions are utilized by tumor cells and are fundamental in the determination of tumor progression and invasion. The effect of MMPs activity in cancer progression has been traditionally associated with the acquisition by tumor cells of an invasive phenotype, an indispensable requisite for the metastatic spreading of cancer cells. In addition to the traditional view, a new role for MMPs in creating a favourable microenvironment has been proposed, so that MMPs are not only involved in cell invasion, but also in signaling pathways that control cell growth, inflammation, or angiogenesis. Finally, recent evidence suggest a role of MMPs in the so called "pre-metastatic niche" that is the hypothesis of an early distant modification of the premetastatic site by primary cancer cells. This new hypothesis is changing our traditional view about MMPs and provides important insights into the effective time window for the therapeutic use of MMP inhibitors. In this review we provide the main available data about the ability of MMPs in creating a suitable microenvironment for tumor growth in metastatic sites and we indicate the implication of these data on the potential use of MMP inhibitors in the metastatic therapy.
Background Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported ...research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. Methods To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl.sup.-/-) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). Results We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl.sup.-/- mice as compared to wild-type. Rankl.sup.-/- mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl.sup.-/- SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. Conclusions We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance. Keywords: RANKL, Skeletal stem cells, Differentiation, Osteopetrosis, Denosumab, Therapy
The modification of gene expression profile, a first step in adaptation to exercise, leads to changes in the level of molecules associated with skeletal muscle activity and energy metabolism—such as ...myokines—as well as those involved in their transcriptional regulation, like microRNA. This study aimed to investigate the influence of strenuous exercise on circulating microRNAs and their possible association with myokine response. Pre-competition and post-competition plasma samples were collected from 14 male athletes participating in a vertical run (+1,000 m gain, 3,600 m length). Circulating total (t-miRNA) and extracellular vesicle-associated (EV-miRNA) miRNAs were extracted from the pooled plasma. Nanoparticle tracking analysis was performed to investigate pre- and post-competition EV concentration and size distribution. A panel of 179 miRNAs was assayed by qPCR and analyzed by Exiqon GenEx v6 normalized on the global mean. t-miRNA and EV-miRNAs whose level was ≥5-fold up- or down-regulated were validated for each single subject. Target prediction on MirWalk v3.0, Gene-Ontology, and pathway enrichment analysis on Panther v17.0 were performed to define the potential biological role of the identified miRNAs. A panel of 14 myokines was assayed in each sample by a multiplex immunoassay. In whole plasma, five miRNAs were upregulated and two were downregulated; in the EV fraction, five miRNAs were upregulated and three were downregulated. Nanoparticle tracking analysis revealed a similar EV size distribution in pre- and post-competition samples and a decreased concentration in post-competition samples related to pre-competition samples. Gene-Ontology and pathway enrichment analysis revealed that the identified t-miRNAs and EV-miRNAs were potentially involved in metabolism regulation in response to exercise. Correlation between fold-change of the post-competition relative to pre-competition plasma level of both t-miRNAs and EV-miRNAs and myokines further confirmed these results. This study provides an example of a systemic response to acute endurance exercise, in which circulating miRNAs play a pivotal role.
Bone metastases contribute to morbidity in patients with common cancers, and conventional therapy provides only palliation and can induce systemic side effects. The development of nanostructured ...delivery systems that combine carriers with bone-targeting molecules can potentially overcome the drawbacks presented by conventional approaches. We have recently developed biodegradable, biocompatible nanoparticles (NP) made of a conjugate between poly (D,L-lactide-co-glycolic) acid and alendronate, suitable for systemic administration, and directly targeting the site of tumor-induced osteolysis. Here, we loaded NP with doxorubicin (DXR), and analyzed the in vitro and in vivo activity of the drug encapsulated in the carrier system. After confirming the intracellular uptake of DXR-loaded NP, we evaluated the anti-tumor effects in a panel of human cell lines, representative for primary or metastatic bone tumors, and in an orthotopic mouse model of breast cancer bone metastases. In vitro, both free DXR and DXR-loaded NP, (58-580 ng/mL) determined a significant dose-dependent growth inhibition of all cell lines. Similarly, both DXR-loaded NP and free DXR reduced the incidence of metastases in mice. Unloaded NP were ineffective, although both DXR-loaded and unloaded NP significantly reduced the osteoclast number at the tumor site (P = 0.014, P = 0.040, respectively), possibly as a consequence of alendronate activity. In summary, NP may act effectively as a delivery system of anticancer drugs to the bone, and deserve further evaluation for the treatment of bone tumors.
Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal ...recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.
Summary
Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory disease. Presently, no treatment regimens are available for these defects in juvenile ...diseases. We identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in a mouse model overexpressing IL-6.
Introduction
Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory diseases and have a significant impact on patients’ quality of life. Presently, no treatment regimens are available for these defects in juvenile diseases. To test a new therapeutic approach, we used growing mice overexpressing the pro-inflammatory cytokine IL-6 (TG), which show a generalized bone loss and stunted growth.
Methods
Since TG mice present increased bone resorption and impaired bone formation, we tested a combined therapy with the antiresorptive modified osteoprotegerin, Fc-OPG, and the anabolic PTH. We injected TG mice with Fc-OPG once at the 4th day of life and with hPTH(1–34) everyday from the 16th to the 30th day of age.
Results
A complete prevention of growth and bone defects was observed in treated mice due to normalization of osteoclast and osteoblast parameters. Re-establishment of normal bone turnover was confirmed by RT-PCR analysis and by in vitro experiments that revealed the full rescue of osteoclast and osteoblast functions. The phenotypic recovery of TG mice was due to the sequential treatment, because TG mice treated with Fc-OPG or hPTH alone showed an increase of body weight, tibia length, and bone volume to intermediate levels between those observed in vehicle-treated WT and TG mice.
Conclusions
Our results identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in IL-6 overexpressing mice, thus providing the proof of principle for a therapeutic approach to correct these defects in juvenile inflammatory diseases.
Bone is the most frequent site of metastasis of the most common cancers in men and women. Bone metastasis incidence has been steadily increasing over the years, mainly because of higher life ...expectancy in oncologic patients. Although bone metastases are sometimes asymptomatic, their consequences are most often devastating, impairing both life quality and expectancy, due to the occurrence of the skeletal-related events, including bone fractures, hypercalcemia and spinal cord compression. Up to 75% of patients endure crippling cancer-induced bone pain (CIBP), against which we have very few weapons. This review's purpose is to discuss the molecular and cellular mechanisms that lead to CIBP, including how cancer cells convert the bone "virtuous cycle" into a cancer-fuelling "vicious cycle", and how this leads to the release of molecular mediators of pain, including protons, neurotrophins, interleukins, chemokines and ATP. Preclinical tests and assays to evaluate CIBP, including the incapacitance tester (in vivo), and neuron/glial activation in the dorsal root ganglia/spinal cord (ex vivo) will also be presented. Furthermore, current therapeutic options for CIBP are quite limited and nonspecific and they will also be discussed, along with up-and-coming options that may render CIBP easier to treat and let patients forget they are patients.
An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by ...activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.
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