USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis.

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

Germline RET variants underlie a subset of paediatric osteosarcoma.

BACKGROUND: Although considerable effort has been put into decoding of the osteosarcoma genome, very little is known about germline mutations that underlie this primary malignant tumour of bone. METHODS AND RESULTS: We followed here a coincidental finding in a multiple endocrine neoplasia family in which a 32-year-old patient carrying a germline pathogenic RET mutation developed an osteosarcoma 2 years after the resection of a medullary thyroid carcinoma. Sequencing analysis of additional 336 patients with osteosarcoma led to the identification of germline activating mutations in the RET proto-oncogene in three cases and somatic amplifications of the gene locus in five matched tumours (4%, n=5/124 tumours). Functional analysis of the pathogenic variants together with an integrative analysis of osteosarcoma genomes confirmed that the mutant RET proteins couple functional kinase activity to dysfunctional ligand binding. RET mutations further co-operated with alterations in TP53 and RB1, suggesting that osteosarcoma pathogenesis bears reminiscence to the stepwise model of medullary thyroid carcinoma. CONCLUSIONS: After Li-Fraumeni-predisposing mutations in TP53, RET becomes the second most mutated cancer-predisposing gene in the germline of patients with osteosarcoma. Hence, early identification of RET mutation carriers can help to identify at-risk family members and carry out preventive measures.

LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function.

Autophagy perturbation represents an emerging therapeutic strategy in cancer. Although LATS1 and LATS2 kinases, core components of the mammalian Hippo pathway, have been shown to exert tumor suppressive activities, here we report a pro-survival role of LATS1 but not LATS2 in hepatocellular carcinoma (HCC) cells. Specifically, LATS1 restricts lethal autophagy in HCC cells induced by sorafenib, the standard of care for advanced HCC patients. Notably, autophagy regulation by LATS1 is independent of its kinase activity. Instead, LATS1 stabilizes the autophagy core-machinery component Beclin-1 by promoting K27-linked ubiquitination at lysine residues K32 and K263 on Beclin-1. Consequently, ubiquitination of Beclin-1 negatively regulates autophagy by promoting inactive dimer formation of Beclin-1. Our study highlights a functional diversity between LATS1 and LATS2, and uncovers a scaffolding role of LATS1 in mediating a cross-talk between the Hippo signaling pathway and autophagy.

Gain Fat-Lose Metastasis: Converting Invasive Breast Cancer Cells into Adipocytes Inhibits Cancer Metastasis.

Cancer cell plasticity facilitates the development of therapy resistance and malignant progression. De-differentiation processes, such as an epithelial-mesenchymal transition (EMT), are known to enhance cellular plasticity. Here, we demonstrate that cancer cell plasticity can be exploited therapeutically by forcing the trans-differentiation of EMT-derived breast cancer cells into post-mitotic and functional adipocytes. Delineation of the molecular pathways underlying such trans-differentiation has motivated a combination therapy with MEK inhibitors and the anti-diabetic drug Rosiglitazone in various mouse models of murine and human breast cancer in vivo. This combination therapy provokes the conversion of invasive and disseminating cancer cells into post-mitotic adipocytes leading to the repression of primary tumor invasion and metastasis formation.

Ultrasound-Guided Percutaneous Radiofrequency for the Treatment of Morton's Neuroma.

PURPOSE: Morton's neuroma (MN) is a leading cause of disability. The purpose of this study was to investigate the effectiveness of radiofrequency (RF) in patients with chronic pain refractory to conservative therapies. METHODS: Between September 2012 and December 2016, RF was performed on 52 patients. A sterile RF needle probe (22G, 5-cm needle with 10-mm electrode active tip) was inserted between the toes into the intermetatarsal space at the center of the lesion. Potential complications of the intervention and post-procedure care were examined. All patients provided written, informed consent. All percutaneous procedures were performed with the NeuroTherm® NT1100 (Neurotherm Inc., M.) device and using ultrasound guidance (Philips IU22). Continuous RF was performed with one cycle of 90 s and with the probe tip maintaining a temperature of 85 °C (impedance 95-210 mΩ). RESULTS: Mean VAS score before the procedures was 9.0 ± 0.6. A reduction of pain intensity was achieved after 1 week by RF (mean VAS scores 3.7 ± 0.9; p < 0.05), with a stabilization of the painful symptomatology in the following months and after 1 year of treatments (mean VAS scores 2.0 ± 0.4 after 12 months, p < 0.05). The FHSQ scores showed improved quality of life (QOL) in all patients at 6 months (p < 0.05) and 1 year (p < 0.05) of RF. No patients developed complications. CONCLUSIONS: RF is a safe, efficient, and minimally invasive technique for the treatment of symptomatic MN.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

RATIONALE: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ß-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/ß-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Pulse-Dose Radiofrequency in Athletic Pubalgia: Preliminary Results.

CONTEXT: Chronic pubalgia affects around 10% of athletes. OBJECTIVE: To determine the role of pulse-dose radiofrequency (PDRF) in athletes with chronic pubalgia and investigate the causes with imaging. DESIGN: Prospective nonrandomized single-group study. PATIENTS: PDRF was performed on 32 patients with a chronic pain that had been refractory to conservative therapies during the last 3 mo. INTERVENTION: The genital branches of the genitofemoral, ilioinguinal, and iliohypogastric nerves and the obturator nerve were the goals of treatment. A 10-cm, 20-gauge cannula was inserted with a percutaneous access on the upper and lower edges of the iliopubic branch. After the spindle was removed, a radiofrequency needle with a 10-mm "active tip" was inserted. The radiofrequency technique was performed with 1200 pulses at 45 V and 20-ms duration, followed by a 480-ms silent phase. MAIN OUTCOME MEASURES: The follow-up with a clinical examination was performed at 1, 3, 6, and 9 mo after the procedure. During the follow-up visits, the patients were asked to rate their pain on a 0-10 VAS scale. RESULTS: All of the enrolled patients completed the study. Mean VAS score before the treatment was 8.4 ± 0.6. Twenty-four patients had a reduction of pain VAS scores more than 50% during all follow-up visits and started training and physiotherapy in the days after the radiofrequency procedure. Six patients, each treated 2 times, had a reduction more than 50% of VAS scores and could start training and physiotherapy only after the 2nd procedure. One patient had no pain relief with 2 treatments. Pain intensity decreased up to 9 mo in 31 patients (mean VAS scores 3.4 ± 0.5 at 6 mo and 3.8 ± 0.9 at 9 mo). No complications were observed. CONCLUSIONS: PDRF is an effective and safe technique in management of chronic pubalgia in athletes.

KLF4 is a key determinant in the development and progression of cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss-of-function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFß/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFß/BMP-dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3-MEK5-ERK5-MEF2 signaling axis that induces a strong increase in Kruppel-like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1-null ECs. KLF4 promotes TGFß/BMP signaling through the production of BMP6. Importantly, in endothelial-specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.

Signaling pathways in the specification of arteries and veins.

The establishment of arterial and venous identity of endothelial cells is critical for the proper anatomic configuration and function of the vascular tree. Arterial and venous specification of endothelial cells is determined by genetic factors, although surrounding cells and hemodynamic forces may also contribute to vascular remodeling. This review provides an overview of the signaling pathways and related transcription factors implicated in differentiation of endothelial cells. We will discuss, in particular, the role of upstream and downstream effectors of Wnt, Sox, and Notch pathways. The understanding of the molecular mechanisms that orchestrate endothelial differentiation may have therapeutic relevance for diseases such as atherosclerosis, arteriovenous malformations, aneurysms, and others.

Transcriptional regulation of arterial differentiation via Wnt, Sox and Notch.

PURPOSE OF REVIEW: The development of a functionally and anatomically correct vascular network is a complex phenomenon that requires the combined activity of different signaling pathways and transcription factors. Notch signaling activation, for instance, is crucial for arterial specification. Here, we discuss the current knowledge on how other signaling pathways cooperate with Notch to orchestrate arterial differentiation of embryonic and postnatal vasculature. RECENT FINDINGS: The role of Notch in vascular development and arterial differentiation is well known. However, it was found that canonical Wnt signaling may act upstream of Notch, upregulating Dll4 and inducing endothelial cells to acquire arterial characteristics. Furthermore, the transcription factor Sox17 may act as a link between Wnt and Notch in the induction of a correct arterio/venous differentiation. SUMMARY: In the past years, the research on vascular development was mostly focused on the mechanisms that regulate vessel growth. We now understand that in order to interfere with several vascular diseases (e.g. aneurysm, cerebral ischemia and stroke) or tumor vascularization, we need to understand the signals that direct arterio/venous specification. Here, we discuss the interplay between Notch, Wnt and Sox that exert a combined positive action on arterial differentiation.

Thoraco-lumbar traumatic vertebral fractures augmentation by osteo-conductive and osteo-inductive bone substitute containing strontium-hydroxyapatite: our experience.

INTRODUCTION: The aim of our study was to evaluate the effectiveness of osteointegrable strontium-hydroxyapatite (Sr-HA) bone cement in the treatment of thoracolumbar traumatic vertebral fractures by percutaneous vertebroplasty (VTP). METHODS: We treated 35 patients [29 (82.85 %) men, 6 (17.14 %) women, mean age 34.05 ± 8.36 years (range 21-54 years)] with single type A1.1 and A1.2 thoracolumbar traumatic vertebral fracture without endospecal bone fragments. Pain intensity was evaluated before and at 1 day; 1 week; and 1, 6, and 12 months after VTP by a 10-point visual analog scale (VAS) score (0 = no pain, 10 = unbearable pain). Physical status and quality of life were evaluated by Oswestry Disability Index (ODI) questionnaire before and 1, 6, and 12 months after VTP. RESULTS: Procedural technical success was achieved in all patients with no deaths observed during follow-up. In three patients (8.57 %), postprocedural CT showed cement leakages: one intradiscal and two in venous plexus. No adjacent vertebral body fractures nor intrasomatic recollapse was detected. The VAS and ODI scores showed a statistically significant reduction 1 week after procedure (P value <0.0001) with a progressive statistically significant reduction during follow-up (P value <0.0001). Twenty-three patients (65.71 %) assigned a value of 0 to the VAS scale at 1 year after treatment. CONCLUSIONS: Strontium-hydroxyapatite bone cement is an effective and safe bone filler in percutaneous vertebroplasty with low leakage rate and absence of major complications when performed by a skilled equipe. It allows an immediate and long-lasting stabilization with a significant pain reduction and quality of life improvement.

CT-guided percutaneous pulse-dose radiofrequency for pudendal neuralgia.

PURPOSE: The purpose of our study was to evaluate the efficacy of computed tomography (CT)-guided percutaneous pulse-dose radiofrequency (PDRF) for the treatment of chronic pain in patients with pudendal neuralgia (PN) unresponsive to conservative approaches. METHODS: From March 2010 to January 2012, 30 patients with a diagnosis of PN were prospectively enrolled in the study, 18 women and 12 men (mean age 47 years). A 20-gauge cannula with a 10-cm length was placed under CT guide in the pudendal (Alcock's) canal. After the spindle was removed, a radiofrequency needle with a 5-mm active tip was introduced. The appropriate needle placement near the pudendal nerve, without an involvement of the vessels, was confirmed with an injection of 1-2 ml of contrast agent. PDRF was performed with 1,200 pulses at high voltage (45 V) with 20 ms duration followed by 480 ms silent phases. RESULTS: Twenty-six patients completed the study. Procedural success was achieved in all patients. Mean VAS scores before PDRF was 9 ± 0.7. Patients had a great improvement in pain intensity after 1 week by PDRF (mean VAS scores 3.8 ± 1.7, p < 0.05), with a stabilization of the symptomatology in the following months (mean VAS scores 1.5 ± 1.1 at 6 months by PDRF, p < 0.05) and excellent results after 1 year by the procedure (mean VAS scores 1.9 ± 0.7, p < 0.05). CONCLUSIONS: In our preliminary experience, CT-guided percutaneous PDRF should be recommended for treatment of PN because we evaluated the tolerability of this procedure with satisfactory and encouraging results.

Pulse-dose radiofrequency for knee osteoartrithis.

PURPOSE: Knee osteoarthritis (OA) is a leading cause of disability in the elderly. The purpose of this study was to investigate the effectiveness of pulse-dose radiofrequency (PDRF) in patients with chronic pain refractory to conservative therapies. MATERIAL AND METHODS: Between January 2011 and November 2012, PDRF was performed on 40 patients. A 20-gauge cannula, 10 cm in length, was introduced in the antero-lateral region of the interested knee, and its tip was placed in the joint space under fluoroscopic guidance. After the spindle was removed, an RF needle with a 10-mm "active tip" was introduced. PDRF was performed with 1,200 pulses at high voltage (45 V) with 20-ms duration followed by a 480-ms silent phase. RESULTS: Mean VAS scores before the procedures was 6.8 ± 0.8. A great decrease of pain intensity was achieved 1 week after PDRF (mean VAS scores 1.8 ± 0.4 [p < 0.05]) with stabilization of painful symptomatology in the following months and excellent results 1 year after treatment (mean VAS scores 2.3 ± 0.6 after 12 months [p < 0.05]). Western Ontario and McMaster Universities Index of Osteoarthritis scores showed improved quality of life in all patients at 6 months (p < 0.05) and at 1 year (p < 0.05) after PDRF. No patients developed complications. CONCLUSION: This study successfully investigated the effectiveness of PDRF in patients with chronic pain unresponsive to conservative therapies. PDRF seems to be an effective and reliable technique for palliative management of chronic pain in patients with knee OA.

Sox17 is indispensable for acquisition and maintenance of arterial identity.

The functional diversity of the arterial and venous endothelia is regulated through a complex system of signalling pathways and downstream transcription factors. Here we report that the transcription factor Sox17, which is known as a regulator of endoderm and hemopoietic differentiation, is selectively expressed in arteries, and not in veins, in the mouse embryo and in mouse postnatal retina and adult. Endothelial cell-specific inactivation of Sox17 in the mouse embryo is accompanied by a lack of arterial differentiation and vascular remodelling that results in embryo death in utero. In mouse postnatal retina, abrogation of Sox17 expression in endothelial cells leads to strong vascular hypersprouting, loss of arterial identity and large arteriovenous malformations. Mechanistically, Sox17 acts upstream of the Notch system and downstream of the canonical Wnt system. These data introduce Sox17 as a component of the complex signalling network that orchestrates arterial/venous specification.

Iron handling in hippocampal neurons: activity-dependent iron entry and mitochondria-mediated neurotoxicity.

The characterization of iron handling in neurons is still lacking, with contradictory and incomplete results. In particular, the relevance of non-transferrin-bound iron (NTBI), under physiologic conditions, during aging and in neurodegenerative disorders, is undetermined. This study investigates the mechanisms underlying NTBI entry into primary hippocampal neurons and evaluates the consequence of iron elevation on neuronal viability. Fluorescence-based single cell analysis revealed that an increase in extracellular free Fe(2+) (the main component of NTBI pool) is sufficient to promote Fe(2+) entry and that activation of either N-methyl-d-aspartate receptors (NMDARs) or voltage operated calcium channels (VOCCs) significantly potentiates this pathway, independently of changes in intracellular Ca(2+) concentration ([Ca(2+) ](i) ). The enhancement of Fe(2+) influx was accompanied by a corresponding elevation of reactive oxygen species (ROS) production and higher susceptibility of neurons to death. Interestingly, iron vulnerability increased in aged cultures. Scavenging of mitochondrial ROS was the most powerful protective treatment against iron overload, being able to preserve the mitochondrial membrane potential and to safeguard the morphologic integrity of these organelles. Overall, we demonstrate for the first time that Fe(2+) and Ca(2+) compete for common routes (i.e. NMDARs and different types of VOCCs) to enter primary neurons. These iron entry pathways are not controlled by the intracellular iron level and can be harmful for neurons during aging and in conditions of elevated NTBI levels. Finally, our data draw the attention to mitochondria as a potential target for the treatment of the neurodegenerative processes induced by iron dysmetabolism.