SKCa- and Kv1-type potassium channels and cancer: Promising therapeutic targets?

Ion channels are transmembrane structures that allow the passage of ions across cell membranes such as the plasma membrane or the membranes of various organelles like the nucleus, endoplasmic reticulum, Golgi apparatus or mitochondria. Aberrant expression of various ion channels has been demonstrated in several tumor cells, leading to the promotion of key functions in tumor development, such as cell proliferation, resistance to apoptosis, angiogenesis, invasion and metastasis. The link between ion channels and these key biological functions that promote tumor development has led to the classification of cancers as oncochannelopathies. Among all ion channels, the most varied and numerous, forming the largest family, are the potassium channels, with over 70 genes encoding them in humans. In this context, this review will provide a non-exhaustive overview of the role of plasma membrane potassium channels in cancer, describing 1) the nomenclature and structure of potassium channels, 2) the role of these channels in the control of biological functions that promotes tumor development such as proliferation, migration and cell death, and 3) the role of two particular classes of potassium channels, the SKCa- and Kv1- type potassium channels in cancer progression.

Advances in Osteosarcoma.

PURPOSE OF REVIEW: This article gives a brief overview of the most recent developments in osteosarcoma treatment, including targeting of signaling pathways, immune checkpoint inhibitors, drug delivery strategies as single or combined approaches, and the identification of new therapeutic targets to face this highly heterogeneous disease. RECENT FINDINGS: Osteosarcoma is one of the most common primary malignant bone tumors in children and young adults, with a high risk of bone and lung metastases and a 5-year survival rate around 70% in the absence of metastases and 30% if metastases are detected at the time of diagnosis. Despite the novel advances in neoadjuvant chemotherapy, the effective treatment for osteosarcoma has not improved in the last 4 decades. The emergence of immunotherapy has transformed the paradigm of treatment, focusing therapeutic strategies on the potential of immune checkpoint inhibitors. However, the most recent clinical trials show a slight improvement over the conventional polychemotherapy scheme. The tumor microenvironment plays a crucial role in the pathogenesis of osteosarcoma by controlling the tumor growth, the metastatic process and the drug resistance and paved the way of new therapeutic options that must be validated by accurate pre-clinical studies and clinical trials.

Pharmacological options in the treatment of osteogenesis imperfecta: A comprehensive review of clinical and potential alternatives.

Osteogenesis imperfecta (OI) is a genetically heterogeneous connective tissue disorder characterized by bone fragility and different extra-skeletal manifestations. The severity of these manifestations makes it possible to classify OI into different subtypes based on the main clinical features. This review aims to outline and describe the current pharmacological alternatives for treating OI, grounded on clinical and preclinical reports, such as antiresorptive agents, anabolic agents, growth hormone, and anti-TGFß antibody, among other less used agents. The different options and their pharmacokinetic and pharmacodynamic properties will be reviewed and discussed, focusing on the variability of their response and the molecular mechanisms involved to attain the main clinical goals, which include decreasing fracture incidence, improving pain, and promoting growth, mobility, and functional independence.

Inhibiting Endothelin Receptors with Macitentan Strengthens the Bone Protective Action of RANKL Inhibition and Reduces Metastatic Dissemination in Osteosarcoma.

Current treatments for osteosarcoma, combining conventional polychemotherapy and surgery, make it possible to attain a five-year survival rate of 70% in affected individuals. The presence of chemoresistance and metastases significantly shorten the patient's lifespan, making identification of new therapeutic tools essential. Inhibiting bone resorption has been shown to be an efficient adjuvant strategy impacting the metastatic dissemination of osteosarcoma, tumor growth, and associated bone destruction. Unfortunately, over-apposition of mineralized matrix by normal and tumoral osteoblasts was associated with this inhibition. Endothelin signaling is implicated in the functional differentiation of osteoblasts, raising the question of the potential value of inhibiting it alone, or in combination with bone resorption repression. Using mouse models of osteosarcoma, the impact of macitentan, an endothelin receptor inhibitor, was evaluated regarding tumor growth, metastatic dissemination, matrix over-apposition secondary to RANKL blockade, and safety when combined with chemotherapy. The results showed that macitentan has no impact on tumor growth or sensitivity to ifosfamide, but significantly reduces tumoral osteoid tissue formation and the metastatic capacity of the osteosarcoma. To conclude, macitentan appears to be a promising therapeutic adjuvant for osteosarcoma alone or associated with bone resorption inhibitors.

ICG-001, an Inhibitor of the ß-Catenin and cAMP Response Element-Binding Protein Dependent Gene Transcription, Decreases Proliferation but Enhances Migration of Osteosarcoma Cells.

High-grade osteosarcomas are the most frequent malignant bone tumors in the pediatric population, with 150 patients diagnosed every year in France. Osteosarcomas are associated with low survival rates for high risk patients (metastatic and relapsed diseases). Knowing that the canonical Wnt signaling pathway (Wnt/ß-catenin) plays a complex but a key role in primary and metastatic development of osteosarcoma, the aim of this work was to analyze the effects of ICG-001, a CBP/ß-catenin inhibitor blocking the ß-catenin dependent gene transcription, in three human osteosarcoma cell lines (KHOS, MG63 and 143B). The cell proliferation and migration were first evaluated in vitro after ICG-001 treatment. Secondly, a mouse model of osteosarcoma was used to establish the in vivo biological effect of ICG-001 on osteosarcoma growth and metastatic dissemination. In vitro, ICG-001 treatment strongly inhibits osteosarcoma cell proliferation through a cell cycle blockade in the G0/G1 phase, but surprisingly, increases cell migration of the three cell lines. Moreover, ICG-001 does not modulate tumor growth in the osteosarcoma mouse model but, rather significantly increases the metastatic dissemination to lungs. Taken together, these results highlight, despite an anti-proliferative effect, a deleterious pro-migratory role of ICG-001 in osteosarcoma.

The twin cytokines interleukin-34 and CSF-1: masterful conductors of macrophage homeostasis.

Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as "twin" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.

Sonic Hedgehog Signature in Pediatric Primary Bone Tumors: Effects of the GLI Antagonist GANT61 on Ewing's Sarcoma Tumor Growth.

Osteosarcoma (OS) and Ewing's sarcoma (ES) are the most common malignant bone tumors in children and adolescents. In many cases, the prognosis remains very poor. The Sonic hedgehog (SHH) signaling pathway, strongly involved in the development of many cancers, regulate transcription via the transcriptional factors Gli1-3. In this context, RNAseq analysis of OS and ES cell lines reveals an increase of some major compounds of the SHH signaling cascade in ES cells, such as the transcriptional factor Gli1. This increase leads to an augmentation of the transcriptional response of Gli1 in ES cell lines, demonstrating a dysregulation of Gli1 signaling in ES cells and thus the rationale for targeting Gli1 in ES. The use of a preclinical model of ES demonstrates that GANT61, an inhibitor of the transcriptional factor Gli1, reduces ES primary tumor growth. In vitro experiments show that GANT61 decreases the viability of ES cell, mainly through its ability to induce caspase-3/7-dependent cell apoptosis. Taken together, these results demonstrates that GANT61 may be a promising therapeutic strategy for inhibiting the progression of primary ES tumors.

Ribosomopathies: New Therapeutic Perspectives.

Ribosomopathies are a group of rare diseases in which genetic mutations cause defects in either ribosome biogenesis or function, given specific phenotypes. Ribosomal proteins, and multiple other factors that are necessary for ribosome biogenesis (rRNA processing, assembly of subunits, export to cytoplasm), can be affected in ribosomopathies. Despite the need for ribosomes in all cell types, these diseases result mainly in tissue-specific impairments. Depending on the type of ribosomopathy and its pathogenicity, there are many potential therapeutic targets. The present manuscript will review our knowledge of ribosomopathies, discuss current treatments, and introduce the new therapeutic perspectives based on recent research. Diamond-Blackfan anemia, currently treated with blood transfusion prior to steroids, could be managed with a range of new compounds, acting mainly on anemia, such as L-leucine. Treacher Collins syndrome could be managed by various treatments, but it has recently been shown that proteasomal inhibition by MG132 or Bortezomib may improve cranial skeleton malformations. Developmental defects resulting from ribosomopathies could be also treated pharmacologically after birth. It might thus be possible to treat certain ribosomopathies without using multiple treatments such as surgery and transplants. Ribosomopathies remain an open field in the search for new therapeutic approaches based on our recent understanding of the role of ribosomes and progress in gene therapy for curing genetic disorders.

Bisphosphonates in common pediatric and adult bone sarcomas.

The therapeutic strategies proposed currently for bone sarcomas are based on neo-adjuvant chemotherapy, delayed en-bloc wide resection, and adjuvant chemotherapy. Unfortunately, bone sarcomas are characterized by high rates of poor drug response, with a high risk of drug resistance, local recurrence and/or a high propensity for induced metastases. The pathogenesis of bone sarcomas is strongly associated with dysregulation of local bone remodeling and increased osteolysis that plays a part in tumor development. In this context, bisphosphonates (BPs) have been proposed as a single agent or in combination with conventional drugs to block bone resorption and the vicious cycle established between bone and sarcoma cells. Pre-clinical in vitro studies revealed the potential "anti-tumor" activities of nitrogen-bisphosphonates (N-BPs). In pre-clinical models, N-BPs reduced significantly primary tumor growth in osteosarcoma and Ewing sarcoma, and the installation of lung metastases. In chondrosarcoma, N-BPs reduced the recurrence of local tumors after intralesional curettage, and increased overall survival. In pediatric and adult osteosarcoma patients, N-BPs have been assessed in combination with conventional chemotherapy and surgery in randomized phase 3 studies with no improvement in clinical outcome. The lack of benefit may potentially be explained by the biological impact of N-BPs on macrophage differentiation/recruitment which may alter CD8+-T lymphocyte infiltration. Thanks to their considerable affinity for the mineralized extracellular matrix, BPs are an excellent platform for drug delivery in malignant bone sites with reduced systemic toxicity, which opens up new opportunities for their future use.

Origins of Alterations to Rankl Null Mutant Mouse Dental Root Development.

The purpose of the present study was to assess the early stages of development of mouse first molar roots in the osteopetrotic context of RANKL invalidation in order to demonstrate that the radicular phenotype observed resulted not only from defective osteoclasts, but also from loss of cell-to-cell communication among dental, periodontium and alveolar bone cells involving RANKL signaling. Two experimental models were used in this study: Rankl mutants with permanent RANKL invalidation, and C57BL/6J mice injected during the first postnatal week with a RANKL neutralizing antibody corresponding to a transient RANKL invalidation. The dento-alveolar complex was systematically analyzed using micro-CT, and histological and immunohistochemical approaches. These experiments showed that the root elongation alterations observed in the Rankl-/- mice were associated with reduced proliferation of the RANK-expressing HERS cells with a significant decrease in proliferating cell nuclear antigen (PCNA) expression and a significant increase in P21 expression. The phenotypic comparison of the adult first molar root at 35 days between permanent and transitory invalidations of RANKL made it possible to demonstrate that alterations in dental root development have at least two origins, one intrinsic and linked to proliferation/differentiation perturbations in dental-root-forming cells, the other extrinsic and corresponding to disturbances of bone cell differentiation/function.

Primary Retention of Molars and RANKL Signaling Alteration during Craniofacial Growth.

The primary retention of molars observed in clinic corresponds to a still-unexplained absence of molar eruption despite the presence of an eruption pathway, resembling the experimental transient inhibition of RANKL signaling in mice. The aim of the present study was to confront the hypothesis according to which the primary retention of molars is associated with transitory perturbations to RANKL signaling during growth as part of a wider craniofacial skeleton pattern. The experimental strategy was based on combining a clinical study and an animal study corresponding to the characterization of the craniofacial phenotypes of patients with primary retention of molars and analyses in mice of the consequences of transient inhibition of RANKL signaling on molar eruption and craniofacial growth. The clinical study validated the existence of a particular craniofacial phenotype in patients with primary retention of molars: a retromandibular skeletal class II typology with reduced mandibular dimensions which manifests itself at the dental level by a class II/2 with palatoversion of the upper incisors and anterior overbite. The animal study demonstrated that transient invalidation of RANKL signaling had an impact on the molar eruption process, the severity of which was dependent on the period of inhibition and was associated with a reduction in two craniofacial morphometric parameters: total skull length and craniofacial vault length. In conclusion, primary retention of molars may be proposed as part of the craniofacial skeleton phenotype associated with a transitory alteration in RANKL signaling during growth.

SHH Signaling Pathway Drives Pediatric Bone Sarcoma Progression.

Primary bone tumors can be divided into two classes, benign and malignant. Among the latter group, osteosarcoma and Ewing sarcoma are the most prevalent malignant primary bone tumors in children and adolescents. Despite intensive efforts to improve treatments, almost 40% of patients succumb to the disease. Specifically, the clinical outcome for metastatic osteosarcoma or Ewing sarcoma remains poor; less than 30% of patients who present metastases will survive 5 years after initial diagnosis. One common and specific point of these bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Over the past years, considerable interest in the Sonic Hedgehog (SHH) pathway has taken place within the cancer research community. The activation of this SHH cascade can be done through different ways and, schematically, two pathways can be described, the canonical and the non-canonical. This review discusses the current knowledge about the involvement of the SHH signaling pathway in skeletal development, pediatric bone sarcoma progression and the related therapeutic options that may be possible for these tumors.

Genetically-achieved disturbances to the expression levels of TNFSF11 receptors modulate the effects of zoledronic acid on growing mouse skeletons.

Zoledronic acid (ZOL), a nitrogen bisphosphonate (N-BP), is currently used to treat and control pediatric osteolytic diseases. Variations in the intensity of the effects and side effects of N-BPs have been reported with no clear explanations regarding their origins. We wonder if such variations could be associated with different levels of RANKL signaling activity in growing bone during and after the treatment with N-BPs. To answer this question, ZOL was injected into neonate C57BL/6J mice with different genetically-determined RANKL signaling activity levels (Opg+/+\RankTg-, Opg+/+\RankTg+, Opg+/-\RankTg-, Opg+/-\RankTg+, Opg-/-\RankTg- and Opg-/-\RankTg+ mice) following a protocol (4 injections from post-natal day 1 to 7 at the dose of 50 µg/kg) that mimics those used in onco-pediatric patients. At the end of pediatric growth (1 and half months) and at an adult age (10 months), the bone morphometric and mineral parameters were measured using µCT in the tibia and skull for the different mice. A histologic analysis of the dental and periodontal tissues was also performed. At the end of pediatric growth, a delay in long bone and skull bone growth, a blockage of tooth eruption, some molar root alterations and a neoplasia-like structure associated with incisor development were found. Interestingly, the magnitude of these side effects was reduced by Opg deficiency (Opg-/-) but increased by Rank overexpression (RankTg). Analysis of the skeletal phenotype at ten months confirmed respectively the beneficial and harmful effects of Opg deficiency and Rank overexpression. These results validated the hypothesis that the RANKL signaling activity level in the bone microenvironment is implicated in the modulation of the response to ZOL. Further studies will be necessary to understand the underlying molecular mechanisms, which will help decipher the variability in the effects of N-BPs reported in the human population. SIGNIFICANT STATEMENTS: The present study establishes that in mice the RANKL signaling activity level is a major modulator of the effects and side-effects of bisphosphonates on the individual skeleton during growth. However, the modulatory actions are dependent on the ways in which this level of activity is increased. A decrease in OPG expression is beneficial to the skeletal phenotype observed at the end of growth, while RANK overexpression deteriorates it. Far removed from pediatric treatment, in adults, the skeletal phenotypes initially observed at the end of growth for the different levels of RANKL signaling activity were maintained, although significant improvement was associated only with reductions in OPG expression.

[Effects of post-natal inhibition of RANKL on molar eruption and root formation in C57BL/6 mice]. / Effets de l'inhibition post-natale de RANKL sur l'éruption et la formation radiculaire des molaires de souris C57BL/6.

INTRODUCTION: Recent observations performed in the orthodontic department of La Pitié-Salpêtrière hospital in Paris reported an increase of non-familial eruption defects of permanent molars. Our recent data have evidenced the involvement of osteoclasts (OC) in both the eruption and the dental retention processes through the RANKL/RANK/OPG signaling pathway. These facts are at the origin of the hypothesis of the existence of an environmental etiology for those eruption defects that would correspond to the perturbation of cellular autocrine/paracrine signaling pathways as the RANKL/ RANK/OPG. MATERIALS AND METHODS: C57BL/6 mice were submitted to repeated injections with anti-RANKL neutralizing antibody during the nine days following birth. A phenotypic comparison with transgenic mice overexpressing RANK was performed for the functional characterization of the RANKL/RANK/OPG pathway. The dento-alveolar complex was analyzed using micro-CT for bone density and Masson's trichrome staining for histological examination. RESULTS: The RANKL transient invalidation of RANKL stopped the molar root development and tooth eruption contrary to transgenic mice overexpressing RANK. The recruitment and the OC activity were strongly impacted. DISCUSSION: This research is of direct clinical interest in understanding the pathology of eruption as indirect in establishing orthodontic treatment protocols for particular cases.

The contribution of immune infiltrates and the local microenvironment in the pathogenesis of osteosarcoma.

Osteosarcoma is a rare primary bone cancer characterized by cancer cells producing calcified osteoid extracellular matrix and inducing lung metastases with a high frequency. The local microenvironment defined several tumor niches controlling the tumor growth and cell extravasation. The immune infiltrate composes one of these niches. The immune environment of osteosarcoma is mainly composed by T-lymphocytes and macrophages but also contains other subpopulations including B-lymphocytes and mast cells. Osteosarcoma cells control the recruitment and differentiation of immune infiltrating cells and establish a local immune tolerant environment favorable to the tumor growth, drug resistance and the occurrence of metastases. Osteosarcoma cells are able to affect the balance between M1 and M2 macrophage subtypes and so could control the T-lymphocyte responses via the PD-1/PDL-1 system. In addition, mesenchymal stem cells may also contribute to this immune tolerance and strengthen the immune evasion. The present review gives a brief overview of the immune components of osteosarcoma and their most recent therapeutic interests.

Dormant, quiescent, tolerant and persister cells: Four synonyms for the same target in cancer.

Although many drugs/treatments are now available for most diseases, too often, resistance to these treatments impedes complete therapeutic success. Acquired resistance is a major problem in many pathologies but it is an acute one in cancers and infections. This is probably because these diseases often require long durations of treatment, which ascribe to the selection of resistant cells. However, the actual mechanisms implicated in the selection process are still under debate. It is becoming increasingly clear that resistance is associated with the heterogeneity of cancer cells or micro-organisms and that multiple mechanisms underlie the emergence of drug-resistant subpopulations. Recently, it has been suggested that a subpopulation of drug tolerant cells present in cancer populations and called "persisters" play a major role in this resistance. Recent studies have shown that microorganisms share similar properties. Still, how persister/tolerant cells intervene in the development of resistance is not completely elucidated but seems to be related to epigenetic changes in treated cells and the capacity of persisters to modulate and/or highjack their microenvironment. Due to the complexity of this process, the input from mathematicians, as well as new methods of bioinformatics and statistics, is necessary to fully comprehend the acquisition of resistance/tolerance deriving from and leading to the heterogeneous cell populations. The present review will give a brief overview of the most recent data available on drug tolerant cells in cancers and their similarities with microorganisms.

Maternal RANKL Reduces the Osteopetrotic Phenotype of Null Mutant Mouse Pups.

RANKL signalization is implicated in the morphogenesis of various organs, including the skeleton. Mice invalidated for Rankl present an osteopetrotic phenotype that was less severe than anticipated, depending on RANKL's implication in morphogenesis. The hypothesis of an attenuated phenotype, as a result of compensation during gestation by RANKL of maternal origin, was thus brought into question. In order to answer this question, Rankl null mutant pups from null mutant parents were generated, and the phenotype analyzed. The results validated the presence of a more severe osteopetrotic phenotype in the second-generation null mutant with perinatal lethality. The experiments also confirmed that RANKL signalization plays a part in the morphogenesis of skeletal elements through its involvement in cell-to-cell communication, such as in control of osteoclast differentiation. To conclude, we have demonstrated that the phenotype associated with Rankl invalidation is attenuated through compensation by RANKL of maternal origin.

The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases.

Background: Osteosarcoma is the most frequent form of malignant pediatric bone tumor. Despite the current therapeutic arsenal, patient life-expectancy remains low if metastases are detected at the time of diagnosis, justifying research into better knowledge at all stages of osteosarcoma ontogenesis and identification of new therapeutic targets. Receptor Activator of Nuclear factor κB (RANK)expression has been reported in osteosarcoma cells, raising the question of Receptor Activator of Nuclear factor κB Ligand (RANKL)/RANK signaling implications in these tumor cells (intrinsic), in addition to previously reported implications through osteoclast activation in the tumor microenvironment (extrinsic). Methods: Based on in vitro and in vivo experimentations using human and mouse osteosarcoma cell lines, the consequences on the main cellular processes of RANK expression in osteosarcoma cells were analyzed. Results: The results revealed that RANK expression had no impact on cell proliferation and tumor growth, but stimulated cellular differentiation and, in an immune-compromised environment, increased the number of lung metastases. The analysis of RANKL, RANK and osteoprotegerin (OPG) expressions in biopsies of a cohort of patients revealed that while RANK expression in osteosarcoma cells was not significantly different between patients with or without metastases at the time of diagnosis, the OPG/RANK ratio decreased significantly. Conclusion: Altogether, these results are in favor of RANKL-RANK signaling inhibition as an adjuvant for the treatment of osteosarcoma.