Showing papers in "Journal of Molecular Medicine in 2013"

Exosomes in tumor microenvironment influence cancer progression and metastasis

Abstract: Exosomes are small membrane vesicles of endocytic origin with a size of 50–100 nm. They can contain microRNAs, mRNAs, DNA fragments, and proteins, which are shuttled from a donor cell to recipient cells. Many different cell types including immune cells, mesenchymal cells, and cancer cells release exosomes. There is emerging evidence that cancer-derived exosomes contribute to the recruitment and reprogramming of constituents associated with tumor environment. Here, we discuss different mechanisms associated with biogenesis, payload, and transport of exosomes. We highlight the functional relevance of exosomes in cancer, as related to tumor microenvironment, tumor immunology, angiogenesis, and metastasis. Exosomes may exert an immunosuppressive function as well as trigger an anti-tumor response by presenting tumor antigens to dendritic cells. Exosomes may serve as cancer biomarkers and aid in the treatment of cancer.

MALAT1 -- a paradigm for long noncoding RNA function in cancer

Abstract: The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a bona fide long noncoding RNA (lncRNA). MALAT1, also known as nuclear-enriched transcript 2 (NEAT2), was discovered as a prognostic marker for lung cancer metastasis but also has been linked to several other human tumor entities. Recent work established a critical regulatory function of this lncRNA in lung cancer metastasis and cell migration. Moreover, MALAT1 is an interesting target for antimetastatic therapy in non-small cell lung carcinoma. Two alternative modes of action have been proposed for MALAT1: regulation of gene expression or alternative splicing. Although the exact mechanism of action in different physiological and pathological conditions still needs to be elucidated, MALAT1 acts as a regulator of gene expression. Although MALAT1 is highly evolutionary conserved in mammals and plays an important role in cancer and metastasis, MALAT1 is not essential for development in a knockout mouse model under normal physiological conditions. Hence, one central question for the future is finding the right stressor and the pathological or environmental condition which requires MALAT1 expression in vivo and entailing its strong evolutionary conservation. Here, we summarize the current knowledge about this important lncRNA. We introduce its discovery, biogenesis, and regulation and describe its known functions, mechanisms of action, and interaction partners.

The metastasis-promoting roles of tumor-associated immune cells

Abstract: Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells, have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors, and proteases that remodel the tumor microenvironment. Invasion and metastasis require neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment and examines the factors allowing these cells to promote each stage of metastasis.

A metabolic remodeling in right ventricular hypertrophy is associated with decreased angiogenesis and a transition from a compensated to a decompensated state in pulmonary hypertension.

Abstract: Right ventricular (RV) failure is an important clinical problem with no available therapies, largely because its molecular mechanisms are unknown. Mitochondrial remodeling resulting to a metabolic shift toward glycolysis has been described in RV hypertrophy (RVH), but it is unknown whether this is beneficial or detrimental. While clinically RV failure follows a period of compensation, the transition from a compensated (cRVH) to a decompensated hypertrophied RV (dRVH) is not studied in animal models. We modeled the natural history of RVH and failure in the monocrotaline rat model of pulmonary hypertension by serially assessing clinically relevant parameters in the same animal. We defined dRVH as the stage in which RV systolic pressure started decreasing, along with the cardiac output, while the RV continued to remodel. dRVH was characterized by ascites, weight loss, and high mortality, compared to cRVH. A cRVH myocardium had hyperpolarized mitochondria and low production of mitochondria-derived reactive oxygen species (mROS), activated hypoxia-inducible factor 1α (HIF1α), and increased levels of glucose transporter 1, vascular endothelial growth factor, and stromal-derived factor 1, promoting increased glucose uptake (measured by positron emission tomography–computed tomography) and angiogenesis measured by lectin imaging in vivo. The transition to dRVH was marked by a sharp rise in mROS, inhibition of HIF1α, and activation of p53, both of which contributed to down-regulation of pyruvate dehydrogenase kinase and decreased glucose uptake. This transition was also associated with a sharp decrease in angiogenic factors and angiogenesis. We show that the previously described metabolic shift, promoting HIF1α activation and angiogenesis, is not sustained during the progression of RV failure. The loss of this beneficial remodeling may be triggered by a rise in mROS resulting in HIF1α inhibition and suppressed angiogenesis. The resultant ischemia may contribute to the rapid deterioration of RV function upon entrance to a decompensation phase. The use of clinical criteria and techniques to define and study dRVH facilitates clinical translation of our findings with direct implications for RV therapeutic and biomarker discovery programs.

Vascular remodeling in pulmonary hypertension

Abstract: Pulmonary hypertension is a complex, progressive condition arising from a variety of genetic and pathogenic causes. Patients present with a spectrum of histologic and pathophysiological features, likely reflecting the diversity in underlying pathogenesis. It is widely recognized that structural alterations in the vascular wall contribute to all forms of pulmonary hypertension. Features characteristic of the remodeled vasculature in patients with pulmonary hypertension include increased stiffening of the elastic proximal pulmonary arteries, thickening of the intimal and/or medial layer of muscular arteries, development of vaso-occlusive lesions, and the appearance of cells expressing smooth muscle-specific markers in normally non-muscular small diameter vessels, resulting from proliferation and migration of pulmonary arterial smooth muscle cells and cellular transdifferentiation. The development of several animal models of pulmonary hypertension has provided the means to explore the mechanistic underpinnings of pulmonary vascular remodeling, although none of the experimental models currently used entirely replicates the pulmonary arterial hypertension observed in patients. Herein, we provide an overview of the histological abnormalities observed in humans with pulmonary hypertension and in preclinical models and discuss insights gained regarding several key signaling pathways contributing to the remodeling process. In particular, we will focus on the roles of ion homeostasis, endothelin-1, serotonin, bone morphogenetic proteins, Rho kinase, and hypoxia-inducible factor 1 in pulmonary arterial smooth muscle and endothelial cells, highlighting areas of cross-talk between these pathways and potentials for therapeutic targeting.

Inflammation and oxidative stress in angiogenesis and vascular disease

Abstract: Recent evidence suggests that processes of inflammation and angiogenesis are interconnected, especially in human pathologies. Newly formed blood vessels enable the continuous recruitment of inflammatory cells, which release a variety of proangiogenic cytokines, chemokines, and growth factors and further promote angiogenesis. These series of positive feedback loops ultimately create a vicious cycle that exacerbates inflammation, transforming it into the chronic process. Recently, this concept of reciprocity of angiogenesis and inflammation has been expanded to include oxidative stress as a novel mechanistic connection between inflammation-driven oxidation and neovascularization. Production of reactive oxygen species results from activation of immune cells by proinflammatory stimuli. As oxidative stress can lead to chronic inflammation by activating a variety of transcription factors including NF-κB, AP-1, and PPAR-γ, inflammation itself has a reciprocal relationship with oxidative stress. This review discusses the recent findings in the area bridging neovascularization and oxidation and highlights novel mechanisms of inflammation- and oxidative stress-driven angiogenesis.

A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma

Abstract: Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2A (F374Y) (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2A (F374Y) may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.

Imaging ROS signaling in cells and animals.

Abstract: Reactive oxygen species (ROS) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. Understanding such complexity of the ROS signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ROS dynamics at high selectivity, sensitivity, and spatiotemporal resolution. The past decade has witnessed significant progress in ROS imaging at levels of intact cells, whole organs or tissues, and even live organisms. In particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein-based ROS indicators, the use of protein indicator-expressing animal models, and the advent of in vivo imaging technology. Innovative ROS imaging has led to important discoveries in ROS signaling—for example, mitochondrial superoxide flashes as elemental ROS signaling events and hydrogen peroxide transients for wound healing. This review aims at providing an update of the current status in ROS imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions.

Mechanisms of Neovascularization and Resistance to Anti-angiogenic Therapies in Glioblastoma Multiforme

Abstract: Glioblastoma multiforme (GBM) is the most malignant brain tumor and highly resistant to intensive combination therapies. GBM is one of the most vascularized tumors and vascular endothelial growth factor (VEGF) produced by tumor cells is a major factor regulating angiogenesis. Successful results of preclinical studies of anti-angiogenic therapies using xenograft mouse models of human GBM cell lines encouraged clinical studies of anti-angiogenic drugs, such as bevacizumab (Avastin), an anti-VEGF antibody. However, these clinical studies have shown that most patients become resistant to anti-VEGF therapy after an initial response. Recent studies have revealed some resistance mechanisms against anti-VEGF therapies involved in several types of cancer. In this review, we address mechanisms of angiogenesis, including unique features in GBMs, and resistance to anti-VEGF therapies frequently observed in GBM. Enhanced invasiveness is one such resistance mechanism and recent works report the contribution of activated MET signaling induced by inhibition of VEGF signaling. On the other hand, tumor cell-originated neovascularization including tumor-derived endothelial cell-induced angiogenesis and vasculogenic mimicry has been suggested to be involved in the resistance to anti-VEGF therapy. Therefore, these mechanisms should be targeted in addition to anti-angiogenic therapies to achieve better results for patients with GBM.

Cardiac glutaminolysis: a maladaptive cancer metabolism pathway in the right ventricle in pulmonary hypertension.

Abstract: The rapid growth of cancer cells is permitted by metabolic changes, notably increased aerobic glycolysis and increased glutaminolysis. Aerobic glycolysis is also evident in the hypertrophying myocytes in right ventricular hypertrophy (RVH), particularly in association with pulmonary arterial hypertension (PAH). It is unknown whether glutaminolysis occurs in the heart. We hypothesized that glutaminolysis occurs in RVH and assessed the precipitating factors, transcriptional mechanisms, and physiological consequences of this metabolic pathway. RVH was induced in two models, one with PAH (Monocrotaline-RVH) and the other without PAH (pulmonary artery banding, PAB-RVH). Despite similar RVH, ischemia as determined by reductions in RV VEGFα, coronary blood flow, and microvascular density was greater in Monocrotaline-RVH versus PAB-RVH. A sixfold increase in 14C-glutamine metabolism occurred in Monocrotaline-RVH but not in PAB-RVH. In the RV working heart model, the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) decreased glutaminolysis, caused a reciprocal increase in glucose oxidation, and elevated cardiac output. Consistent with the increased glutaminolysis in RVH, RV expressions of glutamine transporters (SLC1A5 and SLC7A5) and mitochondrial malic enzyme were elevated (Monocrotaline-RVH > PAB-RVH > control). Capillary rarefaction and glutamine transporter upregulation also occurred in RVH in patients with PAH. cMyc and Max, known to mediate transcriptional upregulation of glutaminolysis, were increased in Monocrotaline-RVH. In vivo, DON (0.5 mg/kg/day × 3 weeks) restored pyruvate dehydrogenase activity, reduced RVH, and increased cardiac output (89 ± 8, vs. 55 ± 13 ml/min, p < 0.05) and treadmill distance (194 ± 71, vs. 36 ±7 m, p < 0.05) in Monocrotaline-RVH. Glutaminolysis is induced in the RV in PAH by cMyc–Max, likely as a consequence of RV ischemia. Inhibition of glutaminolysis restores glucose oxidation and has a therapeutic benefit in vivo.

MiR-142-3p functions as a tumor suppressor by targeting CD133, ABCG2, and Lgr5 in colon cancer cells

Abstract: Studies have shown that the expression of CD133, leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), and ATP binding cassette (ABC)G2 proteins is associated with malignancy and poor prognosis in colon cancer However, molecular regulation mechanism of the three proteins has not been elucidated Here, we report that microRNA-142-3p (miR-142-3p) inhibits the expression of CD133, Lgr5, and ABCG2 in colon cancer cells by binding to both the 3′-untranslated region and the coding sequences of the three genes The miR-142-3p was markedly decreased in colon cancer specimens, in which it was negatively correlated with the expression of CD133, Lgr5, and ABCG2 Reduction of miR-142-3p corresponds to poor differentiation and bigger tumor size in colon cancers Moreover, miR-142-3p levels were reduced in cells that formed spheres compared to cells that were cultured in regular media Transfection of miR-142-3p mimics in colon cancer cells downregulated cyclin D1 expression, induced G1 phase cell cycle arrest, and elevated the sensitivity of the cells to 5-fluorouracil Furthermore, OCT4 suppressed miR-142-3p, and hypomethylation of the OCT4 promoter was associated with a reduction in miR-142-3p Finally, the miR-142-3p inhibited the growth of colon cancer cells in vivo, which was accompanied by the downregulation of CD133, Lgr5, and ABCG2 in tumor tissues Our results elucidate a novel regulation pathway in colon cancer cells and suggest a potential therapeutic approach for colon cancer therapy

The maternal embryonic leucine zipper kinase (MELK) is upregulated in high-grade prostate cancer

Abstract: Loss of cell cycle control is a prerequisite for cancer onset and progression. In prostate cancer, increased activity of cell cycle genes has been associated with prognostic parameters such as biochemical relapse and survival. The identification of novel oncogenic and druggable targets in patient subgroups with poor prognosis may help to develop targeted therapy approaches. We analyzed prostate cancer and corresponding benign tissues (n = 98) using microarrays. The comparison of high- and low-grade tumors (Gleason score ≥ 4 + 3 vs. ≤ 3 + 4) revealed 144 differentially expressed genes (p < 0.05). Out of these, 15 genes were involved in the cell cycle process. The gene maternal embryonic leucine zipper kinase (MELK) was identified to be highly correlated with cell cycle genes like UBE2C, TOP2A, CCNB2, and AURKB. Increased MELK gene expression in high-risk prostate cancer was validated by qPCR in an independent patient cohort (p < 0.005, n = 79). Immunohistochemistry analysis using a tissue microarray (n = 94) revealed increased MELK protein expression in prostate cancer tissues of high Gleason scores. RNAi-based inhibition of MELK in PC3 and LNCaP cells suggested putative function in chromatin modification, embryonic development and cell migration. The concerted inhibition of MELK and other cell cycle targets by the antibiotic siomycin A strongly impaired cell viability of prostate cancer cells, and may point to a novel therapy approach for a subset of high-risk prostate cancer patients.

FOXO1-mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) decreases glucose oxidation and impairs right ventricular function in pulmonary hypertension: therapeutic benefits of dichloroacetate

Abstract: Pyruvate dehydrogenase kinase (PDK) is activated in right ventricular hypertrophy (RVH), causing an increase in glycolysis relative to glucose oxidation that impairs right ventricular function. The stimulus for PDK upregulation, its isoform specificity, and the long-term effects of PDK inhibition are unknown. We hypothesize that FOXO1-mediated PDK4 upregulation causes bioenergetic impairment and RV dysfunction, which can be reversed by dichloroacetate. Adult male Fawn-Hooded rats (FHR) with pulmonary arterial hypertension (PAH) and right ventricular hypertrophy (RVH; age 6–12 months) were compared to age-matched controls. Glucose oxidation (GO) and fatty acid oxidation (FAO) were measured at baseline and after acute dichloroacetate (1 mM × 40 min) in isolated working hearts and in freshly dispersed RV myocytes. The effects of chronic dichloroacetate (0.75 g/L drinking water for 6 months) on cardiac output (CO) and exercise capacity were measured in vivo. Expression of PDK4 and its regulatory transcription factor, FOXO1, were also measured in FHR and RV specimens from PAH patients (n = 10). Microarray analysis of 168 genes related to glucose or FA metabolism showed >4-fold upregulation of PDK4, aldolase B, and acyl-coenzyme A oxidase. FOXO1 was increased in FHR RV, whereas HIF-1α was unaltered. PDK4 expression was increased, and the inactivated form of FOXO1 decreased in human PAH RV (P < 0.01). Pyruvate dehydrogenase (PDH) inhibition in RVH increased proton production and reduced GO’s contribution to the tricarboxylic acid (TCA) cycle. Acutely, dichloroacetate reduced RV proton production and increased GO’s contribution (relative to FAO) to the TCA cycle and ATP production in FHR (P < 0.01). Chronically dichloroacetate decreased PDK4 and FOXO1, thereby activating PDH and increasing GO in FHR. These metabolic changes increased CO (84 ± 14 vs. 69 ± 14 ml/min, P < 0.05) and treadmill-walking distance (239 ± 20 vs. 171 ± 22 m, P < 0.05). Chronic dichloroacetate inhibits FOXO1-induced PDK4 upregulation and restores GO, leading to improved bioenergetics and RV function in RVH.

Adenosine signaling during acute and chronic disease states

Abstract: Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal,and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine-based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes, such as inflammatory cell regulation, vascular barrier function, and tissue fibrosis.

Endothelial progenitor cell: a blood cell by many other names may serve similar functions.

Abstract: The first reports of circulating cells that displayed the capacity to repair and regenerate damaged vascular endothelial cells as progenitor cells for the endothelial lineage (EPC) were met with great enthusiasm. However, the cell surface antigens and colony assays used to identify the putative EPC were soon found to overlap with those of the hematopoietic lineage. Over the past decade, it has become clear that specific hematopoietic subsets play important roles in vascular repair and regeneration. This review will provide some overview of the hematopoietic hierarchy and methods to segregate distinct subsets that may provide clarity in identifying the proangiogenic hematopoietic cells. This review will not discuss those circulating viable endothelial cells that play a role as EPC and are called endothelia colony-forming cells. The review will conclude with identification of some roadblocks to progress in the field of identification of circulating cells that participate in vascular repair and regeneration.

Pathologies at the nexus of blood coagulation and inflammation: thrombin in hemostasis, cancer, and beyond

Abstract: Thrombin is the protease involved in blood coagulation. Its deregulation can lead to hemostatic abnormalities, which range from subtle subclinical to serious life-threatening coagulopathies, i.e., during septicemia. Additionally, thrombin plays important roles in many (patho)physiological conditions that reach far beyond its well-established role in stemming blood loss and thrombosis, including embryonic development and angiogenesis but also extending to inflammatory processes, complement activation, and even tumor biology. In this review, we will address thrombin’s broad roles in diverse (patho)physiological processes in an integrative way. We will also discuss thrombin as an emerging major target for novel therapies.

Transcriptional control of adenosine signaling by hypoxia-inducible transcription factors during ischemic or inflammatory disease

Abstract: Inflammatory lesions, ischemic tissues, or solid tumors are characterized by the occurrence of severe tissue hypoxia within the diseased tissue. Subsequent stabilization of hypoxia-inducible transcription factors-particularly of hypoxia-inducible factor 1α (HIF1A)--results in significant alterations of gene expression of resident cells or inflammatory cells that have been recruited into such lesions. Interestingly, studies of hypoxia-induced changes of gene expression identified a transcriptional program that promotes extracellular adenosine signaling. Adenosine is a signaling molecule that functions through the activation of four distinct adenosine receptors--the ADORA1, ADORA2A, ADORA2B, and ADORA3 receptors. Extracellular adenosine is predominantly derived from the phosphohydrolysis of precursor nucleotides, such as adenosine triphosphate or adenosine monophosphate. HIF1A-elicited alterations in gene expression enhance the enzymatic capacity within inflamed tissues to produce extracellular adenosine. Moreover, hypoxia-elicited induction of adenosine receptors--particularly of ADORA2B--results in increased signal transduction. Functional studies in genetic models for HIF1A or adenosine receptors implicate this pathway in an endogenous feedback loop that dampens excessive inflammation and promotes injury resolution, while at the same time enhancing ischemia tolerance. Therefore, pharmacological strategies to enhance HIF-elicited adenosine production or to promote adenosine signaling through adenosine receptors are being investigated for the treatment of acute inflammatory or ischemic diseases characterized by tissue hypoxia.

Aptamers: multifunctional molecules for biomedical research.

Abstract: Aptamers are single-stranded oligonucleotides that fold into well-defined three-dimensional shapes, allowing them to bind their targets with high affinity and specificity. They can be generated through an in vitro process called “Systemic Evolution of Ligands by Exponential Enrichment” and applied for specific detection, inhibition, and characterization of various targets like small organic and inorganic molecules, proteins, and whole cells. Aptamers have also been called chemical antibodies because of their synthetic origin and their similar modes of action to antibodies. They exhibit significant advantages over antibodies in terms of their small size, synthetic accessibility, and ability to be chemically modified and thus endowed with new properties. The first generation of aptamer drug “Macugen” was available for public use within 25 years of the discovery of aptamers. With others in the pipeline for clinical trials, this emerging field of medical biotechnology is raising significant interest. However, aptamers pose different problems for their development than for antibodies that need to be addressed to achieve practical applications. It is likely that current developments in aptamer engineering will be the basis for the evolution of improved future bioanalytical and biomedical applications. The present review discusses the development of aptamers for therapeutics, drug delivery, target validation and imaging, and reviews some of the challenges to fully realizing the promise of aptamers in biomedical applications.

Roles of Wnt signals in bone resorption during physiological and pathological states.

Abstract: Osteoclasts, multinucleated giant cells, are responsible for bone resorption in physiological and pathological conditions such as osteoporosis and rheumatoid arthritis. Osteoclasts develop from the monocyte/macrophage lineage under the strict control of bone-forming osteoblasts. Osteoblast-lineage cells express two cytokines essential for osteoclast differentiation, colony-stimulating factor-1, and receptor activator of nuclear factor κB ligand (RANKL) and also express osteoprotegerin, a soluble decoy receptor for RANKL. The signaling molecule Wnt has been shown to be important for the differentiation of osteoblasts through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Recent studies have established that Wnt-mediated signals are also crucial for bone resorption in both physiological and pathological conditions. In this review, we introduce recent advances in roles of Wnt signaling in bone formation and bone resorption.

Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma

Abstract: Angiogenesis inhibitors have long been considered desirable anticancer agents. However, it was found that many tumors could develop resistance to antiangiogenesis inhibitors. Antiangiogenic therapy results in metabolic stress. Autophagy is an important survival mechanism in cancer cells under metabolic stress; however, it remains unknown if autophagy contributes to antiangiogenesis resistance. In this study, we reported that bevacizumab treatment reduced the development of new blood vessels and inhibited cell growth in xenografts of hepatocellular carcinoma (HCC) tumors. Bevacizumab treatment also upregulated expression of the autophagy-related genes (Beclin1 and LC3) and increased autophagosome formation. Our in vitro studies demonstrated that autophagy inhibition significantly increased apoptosis of HCC cells during nutrient starvation or hypoxia. In addition, the combined treatment of an autophagy inhibitor and bevacizumab markedly inhibited the tumor growth of HCC xenografts, led to enhanced apoptosis, and impaired the proliferation of tumor cells compared with treatment with either drug alone. Furthermore, autophagy inhibition led to enhanced reactive oxygen species (ROS) generation in HCC cells exposed to nutrient starvation or hypoxia in vitro and increased DNA oxidative damage in vivo. Antioxidants reduced nutrient starvation or the hypoxia-induced cell death of HCC cells after autophagy inhibition. Our results suggest that autophagy modulates ROS generation and contributes to cell survival under metabolic stress. Therefore, autophagy inhibition may be a novel way of increasing the efficicacy of antiangiogenic agents in the treatment of HCC.

Nucleolar activity in neurodegenerative diseases: a missing piece of the puzzle?

Abstract: Nucleoli are the sites where synthesis of rRNA and ribosomal assembly take place Along with these “traditional” roles, the nucleolus controls cellular physiology and homeostasis The cellular and molecular alterations associated with impaired nucleolar activity (“nucleolar stress”) have just started to be systematically explored in the nervous system taking advantage of newly available animal models lacking rRNA synthesis in specific neurons These studies showed that nucleolar function is necessary for neuronal survival and that its modality of action differs between and within cell types Nucleolar function is also crucial in pathology as it controls mitochondrial activity and critical stress signaling pathways mimicking hallmarks of human neurodegenerative diseases This mini-review will focus on the modes of action of nucleolar stress and discuss how the manipulation of nucleolar activity might underscore novel strategies to extend neuronal function and survival

Down-regulation of miR-124/-214 in cutaneous squamous cell carcinoma mediates abnormal cell proliferation via the induction of ERK

Abstract: Squamous cell carcinoma (SCC) is one of the most common skin cancers. Because its potential to recur and metastasize leads to a poor prognosis and significant mortality, it is necessary to develop new early diagnostic tools and new therapeutic approaches. In this study, we found protein levels of ERK1 and ERK2 were increased in SCC cell lines without changing mRNA levels and that ERK1/2 mediates abnormal cell proliferation in these cells. Then, mechanisms underlying the overexpression of ERK1/2 in SCC were investigated focusing on microRNA. We found that miR-214 is the regulator of ERK1, whereas ERK2 is regulated by miR-124 and miR-214. Expression of miR-124 and miR-214 was significantly down-regulated in SCC in vitro and in vivo. Treatment with 5-aza-deoxycytidine and trichostatin A synergistically recovered the miR-124/-214 down-regulation in SCC cell line. However, bisulphite sequencing revealed the methylation status of miR-124/-214 promoter was not increased in the SCC cell line and tumor tissue. Taken together, the down-regulation of miR-124/-214 in SCC is most likely caused, at least in part, by hypermethylation of other promoter regions rather than the miR-124/-214 promoter. Supplementation of these microRNAs in the SCC cell line reduced the abnormal cell proliferation by normalizing ERK1/2 levels. Additionally, serum concentration of miR-124 was correlated with miR-124 expression levels in the tumor tissues and inversely correlated with tumor progression. On the other hand, miR-214 was not detected in the serum. Investigation of the regulatory mechanisms of keratinocyte proliferation by microRNA may lead to develop new biomarkers and treatments using microRNA.

Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

Abstract: Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.

Platinum-resistance in ovarian cancer cells is mediated by IL-6 secretion via the increased expression of its target cIAP-2

Abstract: Ovarian carcinoma patients are initially responsive to platinum-based therapy, but eventually become refractory to treatment due to the development of platinum chemoresistance. Elevated levels of interleukin-6 (IL-6) in the sera and ascites of these patients predict poor clinical outcome. Our goal was to analyze the interaction between cisplatin and cisplatin-resistant ovarian cancer cells, and to identify means of circumventing platinum resistance. We studied ovarian carcinoma cell lines and cells drawn from ovarian carcinoma patients. Gene array analyses were performed on ovarian carcinoma cells upon treatment with cisplatin, and the results were validated by ELISA and Western blotting (WB). Cytotoxicity assays were performed on anti-IL-6 Ab-, IL-6-, and cellular inhibitor of apoptosis 2 (cIAP-2) siRNA-treated cells, following cisplatin addition. Our results revealed a highly significant increase in IL-6 and cIAP-2 mRNA and protein levels upon treatment with cisplatin. WB analysis of cisplatin-treated cells exhibited decreased cIAP-2 expression level following anti-IL-6 Ab addition. Furthermore, IL-6 by itself, significantly increased cIAP-2 levels in ovarian carcinoma cells. Finally, cytotoxicity assays showed sensitization to cisplatin following the addition of IL-6 and cIAP-2 inhibitors. In conclusion, cisplatin treatment of ovarian carcinoma cells upregulates IL-6 and cIAP-2 levels while their inhibition significantly sensitizes them to cisplatin. Here, we present cIAP-2 as a novel inducer of platinum resistance in ovarian carcinoma cells, and suggest an axis beginning with an encounter between cisplatin and these cells, mediated sequentially by IL-6 and cIAP-2, resulting in cisplatin resistance. Consequently, we propose that combining IL-6/cIAP-2 inhibitors with cisplatin will provide new hope for ovarian carcinoma patients by improving the current treatment.

Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo.

Abstract: Direct gene transfer strategies are of promising value to treat articular cartilage defects. Here, we tested the ability of a recombinant adeno-associated virus (rAAV) SOX9 vector to enhance the repair of cartilage lesions in vivo. The candidate construct was provided to osteochondral defects in rabbit knee joints vis-a-vis control (lacZ) vector treatment and to cells relevant of the repair tissue (mesenchymal stem cells, chondrocytes). Efficient, long-term transgene expression was noted within the lesions (up to 16 weeks) and in cells in vitro (21 days). Administration of the SOX9 vector was capable of stimulating the biological activities in vitro and over time in vivo. SOX9 treatment in vivo was well tolerated, leading to improved cartilage repair processes with enhanced production of major matrix components. Remarkably, application of rAAV SOX9 delayed premature terminal differentiation and hypertrophy in the newly formed cartilage, possible due to contrasting effects of SOX9 on RUNX2 and β-catenin osteogenic expression in this area. Most strikingly, SOX9 treatment improved the reconstitution of the subchondral bone in the defects, possibly due to an increase in RUNX2 expression in this location. These findings show the potential of direct rAAV gene delivery as an efficient tool to treat cartilage lesions.

DNA methylation biomarkers as diagnostic and prognostic tools in colorectal cancer

Abstract: Colorectal cancer (CRC) is the third most common type of cancer and is responsible for 9 % of cancer deaths in both men and women in the USA for 2013. It is a heterogenous disease, and its three classification types are microsatellite instability, chromosomal instability, and CpG island methylator phenotype. Biomarkers are molecules, which can be used as indicators of cancer. They have the potential to achieve great sensitivities and specificities in diagnosis and prognosis of CRC. DNA methylation biomarkers are epigenetic markers, more specifically genes that become silenced after aberrant methylation of their promoter in CRC. Some methylation biomarkers like SEPT9 (ColoVantage®) and vimentin (ColoSureTM) are already commercially available. Other blood and fecal-based biomarkers are currently under investigation and clinical studies so that they can be used in the near future. Biomarker panels are also currently being studied since they show great potential in diagnosis as they can combine robust biomarkers to achieve even greater sensitivities than single markers. Finally, methylation-sensitive microRNAs (miRNAs) are very promising markers, and their investigation as biomarkers, is only at primitive stage.

LRRK2 interactions with α-synuclein in Parkinson’s disease brains and in cell models

Abstract: Mutations in the genes encoding leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are associated with both autosomal dominant and idiopathic forms of Parkinson’s disease (PD). α-Synuclein is the main protein in Lewy bodies, hallmark inclusions present in both sporadic and familial PD. We show that in PD brain tissue, the levels of LRRK2 are positively related to the increase in α-synuclein phosphorylation and aggregation in affected brain regions (amygdala and anterior cingulate cortex), but not in the unaffected visual cortex. In disease-affected regions, we show co-localization of these two proteins in neurons and Lewy body inclusions. Further, in vitro experiments show a molecular interaction between α-synuclein and LRRK2 under endogenous and over-expression conditions. In a cell culture model of α-synuclein inclusion formation, LRRK2 co-localizes with the α-synuclein inclusions, and knocking down LRRK2 increases the number of smaller inclusions. In addition to providing strong evidence for an interaction between LRRK2 and α-synuclein, our results shed light on the complex relationship between these two proteins in the brains of patients with PD and the underlying molecular mechanisms of the disease.

NF-κB in colorectal cancer.

Abstract: Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide, responsible for more than half a million deaths annually. CRC is a multistep process that entails the accumulation of genetic/epigenetic aberrations, which lead to the simultaneous failure of protective mechanisms and the activation of tumorigenic pathways. In most cases of CRC a deregulation of the Wnt-signaling pathway is required. The transcription factor nuclear factor κB (NF-κB) has been recognized as a key player in the initiation and propagation of CRC. Under physiological conditions, NF-κB orchestrates the inflammatory process and participates in the modulation of various steps of cell cycle and survival. It is normally kept in an inactive state in the cytoplasm by binding to a group of inhibitory proteins. Upon receipt of a signal, its inhibitor is phosphorylated and proteolytically degraded and NF-κB is actively translocated to the nucleus, where it facilitates target-gene transcription. Recent experimental data reveal the important role of NF-κB in tumor cells as well as in the surrounding “cancerous” and reactive microenvironment. Various tumor cell-derived and contextual cues feed constantly this vicious circuitry sustaining inflammation and promoting proliferation, angiogenesis, invasion and eventually metastasis. Therefore NF-κB along with its upstream and downstream network presents a rational target for therapeutic interventions. Numerous small molecules, inhibitory peptides, antisense RNAs, natural compounds, as well as gene therapy strategies interfere with multiple steps of the NF-κΒ signaling cascade. The design of NF-κΒ-targeted treatment may aid the efforts towards the pursuit of more efficient therapeutic measures devoid of severe systemic side-effects.

Single-stranded nucleic acids promote SAMHD1 complex formation

Abstract: SAM domain and HD domain-containing protein 1 (SAMHD1) is a dGTP-dependent triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs) thereby limiting the intracellular dNTP pool. Mutations in SAMHD1 cause Aicardi–Goutieres syndrome (AGS), an inflammatory encephalopathy that mimics congenital viral infection and that phenotypically overlaps with the autoimmune disease systemic lupus erythematosus. Both disorders are characterized by activation of the antiviral cytokine interferon-α initiated by immune recognition of self nucleic acids. Here we provide first direct evidence that SAMHD1 associates with endogenous nucleic acids in situ. Using fluorescence cross-correlation spectroscopy, we demonstrate that SAMHD1 specifically interacts with ssRNA and ssDNA and establish that nucleic acid-binding and formation of SAMHD1 complexes are mutually dependent. Interaction with nucleic acids and complex formation do not require the SAM domain, but are dependent on the HD domain and the C-terminal region of SAMHD1. We finally demonstrate that mutations associated with AGS exhibit both impaired nucleic acid-binding and complex formation implicating that interaction with nucleic acids is an integral aspect of SAMHD1 function.

Neuromuscular junction abnormalities in DNM2-related centronuclear myopathy

Abstract: Dynamin-2-related centronuclear myopathy (DNM2-CNM) is a clinically heterogeneous muscle disorder characterized by muscle weakness and centralized nuclei on biopsy. There is little known about the muscle dysfunction underlying this disorder, and there are currently no treatments. In this study, we establish a novel zebrafish model for DNM2-CNM by transiently overexpressing a mutant version of DNM2 (DNM2-S619L) during development. We show that overexpression of DNM2-S619L leads to pathological changes in muscle and a severe motor phenotype. We further demonstrate that the muscle weakness seen in these animals can be significantly alleviated by treatment with an acetylcholinesterase inhibitor. Based on these results, we reviewed the clinical history of five patients with two different DNM2-CNM mutations (S619L and E368K) and found electrophysiological evidence of abnormal neuromuscular transmission in two of the individuals. All five patients showed improved muscle strength and motor function, and/or reduced fatigability following acetylcholinesterase inhibitor treatment. Together, our results suggest that deficits at the neuromuscular junction may play an important role in the pathogenesis of DNM2-CNM and that treatments targeting this dysfunction can provide an effective therapy for patients with this disorder.