Developing a Quality Assurance Framework for Neuro-Ophthalmology Using a Multisite Data Registry.

BACKGROUND: Quality assurance (QA) in neuro-ophthalmology (NOPH) is often lacking. We aimed to assess the quality of referral assessment and time to consult for common neuro-ophthalmological conditions by implementing a quality-assurance registry, NODE (Neuro-ophthalmology Database), in a tertiary neuro-ophthalmology clinic. Australian standardized triage categories, namely, P1 (consult ≤30 days), P2 (consult ≤30-60 days), and P3 (consult ≤60-90 days), were developed and validated for neuro-ophthalmological conditions. METHODS: We collected data from NODE on 676 patients at the Alfred Hospital, Melbourne and developed a consensus on the assignation of NOPH conditions to triage categories using a modified Delphi approach. A panel of 7 experienced neuro-ophthalmologists scored conditions and assignation to triage categories. Consensus was considered when ≥75% of the panel strongly agreed or agreed. We analyzed the mean days from referral to triage and from triage to the initial consultation and compared that with the developed triage category standard. RESULTS: Most patients presenting to the service were female (64%). Common diagnoses were idiopathic intracranial hypertension (IIH) (19%), optic neuropathy (ON) (14%), nonspecific headaches (11%), cranial nerve defects (CND) (8%), and papilledema (7%). Consensus on triage category assignment was reached after 2 rounds of scoring from expert panel members. The mean time from referral to triage was performed in <5 days for all the common diagnosis at the NOPH clinic. The mean days (±SD) from P1 category triage to initial consult for IIH was 15 (±12) days, acute ON 16 (±14) days, CND was 20 (±15) days, and papilledema was 20 (±19) days. The mean days from P2 triage to initial consultant for nonspecific headaches was 22 (±20) days and for EOMD was 48 (±22) days. The mean time (days) from P3 triage to initial consultant for nonocular myasthenia gravis was 38 days (±29) days and for visual snow was 54 (±31) days. CONCLUSIONS: We have established a consensus agreement on triage categories for neuro-ophthalmological conditions, which can be further validated using a larger panel of experts. We established a NOPH registry that will serve as a framework to benchmark quality of care between NOPH services. Data from our NOPH registry demonstrated that most conditions are appropriately triaged and seen.

ERK5 signalling pathway is a novel target of sorafenib: Implication in EGF biology.

Sorafenib is a multikinase inhibitor widely used in cancer therapy with an antitumour effect related to biological processes as proliferation, migration or invasion, among others. Initially designed as a Raf inhibitor, Sorafenib was later shown to also block key molecules in tumour progression such as VEGFR and PDGFR. In addition, sorafenib has been connected with key signalling pathways in cancer such as EGFR/EGF. However, no definitive clue about the molecular mechanism linking sorafenib and EGF signalling pathway has been established so far. Our data in HeLa, U2OS, A549 and HEK293T cells, based on in silico, chemical and genetic approaches demonstrate that the MEK5/ERK5 signalling pathway is a novel target of sorafenib. In addition, our data show how sorafenib is able to block MEK5-dependent phosphorylation of ERK5 in the Ser218/Tyr220, affecting the transcriptional activation associated with ERK5. Moreover, we demonstrate that some of the effects of this kinase inhibitor onto EGF biological responses, such as progression through cell cycle or migration, are mediated through the effect exerted onto ERK5 signalling pathway. Therefore, our observations describe a novel target of sorafenib, the ERK5 signalling pathway, and establish new mechanistic insights for the antitumour effect of this multikinase inhibitor.

p38ß and Cancer: The Beginning of the Road.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is implicated in cancer biology and has been widely studied over the past two decades as a potential therapeutic target. Most of the biological and pathological implications of p38MAPK signaling are often associated with p38α (MAPK14). Recently, several members of the p38 family, including p38γ and p38δ, have been shown to play a crucial role in several pathologies including cancer. However, the specific role of p38ß (MAPK11) in cancer is still elusive, and further investigation is needed. Here, we summarize what is currently known about the role of p38ß in different types of tumors and its putative implication in cancer therapy. All evidence suggests that p38ß might be a key player in cancer development, and could be an important therapeutic target in several pathologies, including cancer.

Identification of non-coding genetic variants in samples from hypoxemic respiratory disease patients that affect the transcriptional response to hypoxia.

A wide range of diseases course with an unbalance between the consumption of oxygen by tissues and its supply. This situation triggers a transcriptional response, mediated by the hypoxia inducible factors (HIFs), that aims to restore oxygen homeostasis. Little is known about the inter-individual variation in this response and its role in the progression of disease. Herein, we sought to identify common genetic variants mapping to hypoxia response elements (HREs) and characterize their effect on transcription. To this end, we constructed a list of genome-wide HIF-binding regions from publicly available experimental datasets and studied the genetic variability in these regions by targeted re-sequencing of genomic samples from 96 chronic obstructive pulmonary disease and 144 obstructive sleep apnea patients. This study identified 14 frequent variants disrupting potential HREs. The analysis of the genomic regions containing these variants by means of reporter assays revealed that variants rs1009329, rs6593210 and rs150921338 impaired the transcriptional response to hypoxia. Finally, using genome editing we confirmed the functional role of rs6593210 in the transcriptional regulation of EGFR. In summary, we found that inter-individual variability in non-coding regions affect the response to hypoxia and could potentially impact on the progression of pulmonary diseases.

Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor-mediated upregulation of caveolin-1.

Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygen-labile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factor-dependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.

Deregulation of E2-EPF ubiquitin carrier protein in papillary renal cell carcinoma.

Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC.

SATB2 augments ΔNp63α in head and neck squamous cell carcinoma.

ΔNp63α is a critical pro-survival protein overexpressed in 80% of head and neck squamous cell carcinomas (HNSCCs) where it inhibits TAp73ß transcription of p53-family target genes, which is thought to increase HNSCC resistance to chemotherapy-induced cell death. However, the mechanisms governing ΔNp63α function are largely unknown. In this study, we identify special AT-rich-binding protein 2 (SATB2) as a new ΔNp63α-binding protein that is preferentially expressed in advanced-stage primary HNSCC and show that SATB2 promotes chemoresistance by enhancing ΔNp63α-mediated transrepression by augmenting ΔNp63α engagement to p53-family responsive elements. Furthermore, SATB2 expression positively correlates with HNSCC chemoresistance, and RNA interference-mediated knockdown of endogenous SATB2 re-sensitizes HNSCC cells to chemotherapy- and γ-irradiation-induced apoptosis, irrespective of p53 status. These findings unveil SATB2 as a pivotal modulator of ΔNp63α that governs HNSCC cell survival.

ERK5 Is a Major Determinant of Chemical Sarcomagenesis: Implications in Human Pathology.

Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5-KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology.

VHL promotes E2 box-dependent E-cadherin transcription by HIF-mediated regulation of SIP1 and snail.

The product of the von Hippel-Lindau gene (VHL) acts as the substrate-recognition component of an E3 ubiquitin ligase complex that ubiquitylates the catalytic alpha subunit of hypoxia-inducible factor (HIF) for oxygen-dependent destruction. Although emerging evidence supports the notion that deregulated accumulation of HIF upon the loss of VHL is crucial for the development of clear-cell renal cell carcinoma (CC-RCC), the molecular events downstream of HIF governing renal oncogenesis remain unclear. Here, we show that the expression of a homophilic adhesion molecule, E-cadherin, a major constituent of epithelial cell junctions whose loss is associated with the progression of epithelial cancers, is significantly down-regulated in primary CC-RCC and CC-RCC cell lines devoid of VHL. Reintroduction of wild-type VHL in CC-RCC (VHL(-/-)) cells markedly reduced the expression of E2 box-dependent E-cadherin-specific transcriptional repressors Snail and SIP1 and concomitantly restored E-cadherin expression. RNA interference-mediated knockdown of HIFalpha in CC-RCC (VHL(-/-)) cells likewise increased E-cadherin expression, while functional hypoxia or expression of VHL mutants incapable of promoting HIFalpha degradation attenuated E-cadherin expression, correlating with the disengagement of RNA polymerase II from the endogenous E-cadherin promoter/gene. These findings reveal a critical HIF-dependent molecular pathway connecting VHL, an established "gatekeeper" of the renal epithelium, with a major epithelial tumor suppressor, E-cadherin.

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis.

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.

Hypoxia-Inducible Factor 2-Dependent Pathways Driving Von Hippel-Lindau-Deficient Renal Cancer.

The most common type of the renal cancers detected in humans is clear cell renal cell carcinomas (ccRCCs). These tumors are usually initiated by biallelic gene inactivation of the Von Hippel-Lindau (VHL) factor in the renal epithelium, which deregulates the hypoxia-inducible factors (HIFs) HIF1α and HIF2α, and provokes their constitutive activation irrespective of the cellular oxygen availability. While HIF1α can act as a ccRCC tumor suppressor, HIF2α has emerged as the key HIF isoform that is essential for ccRCC tumor progression. Indeed, preclinical and clinical data have shown that pharmacological inhibitors of HIF2α can efficiently combat ccRCC growth. In this review, we discuss the molecular basis underlying the oncogenic potential of HIF2α in ccRCC by focusing on those pathways primarily controlled by HIF2α that are thought to influence the progression of these tumors.

Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion.

OBJECTIVES: To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines. MATERIALS AND METHODS: An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA. RESULTS: Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches. CONCLUSION: The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.

E1a is an exogenous in vivo tumour suppressor.

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches.

p38MAPK and Chemotherapy: We Always Need to Hear Both Sides of the Story.

The p38MAPK signaling pathway was initially described as a stress response mechanism. In fact, during previous decades, it was considered a pathway with little interest in oncology especially in comparison with other MAPKs such as ERK1/2, known to be target of oncogenes like Ras. However, its involvement in apoptotic cell death phenomena makes this signaling pathway more attractive for many cancer research laboratories. This apoptotic role allows to establish a link between p38MAPK and regular chemotherapeutic agents such as Cisplatin or base analogs (Cytarabine, Gemcitabine or 5-Fluorouracil) which are currently used in hospitals across the world. In fact, and more recently, p38MAPK has also been connected with targeted therapies like tyrosine kinase inhibitors (vg. Imatinib, Sorafenib) and, to a lesser extent, with monoclonal antibodies. In addition, the oncogenic or tumor suppressor potential of this signaling pathway has aroused the interest of the scientific community in evaluating p38MAPK as a novel target for cancer therapy. In this review, we will summarize the role of p38MAPK in chemotherapy as well as the potential that p38MAPK inhibition can bring to cancer therapy. All the evidences suggest that p38MAPK could be a double-edged sword and that the search for the most appropriate candidate patients, depending on their pathology and treatment, will lead to a more rational use of this new therapeutic tool.

Hereditary angioedema: the mutation spectrum of SERPING1/C1NH in a large Spanish cohort.

Hereditary angioedema (HAE) is a disease caused by defects in the C1 inhibitor gene (SERPING1/C1NH). We screened the entire C1NH gene for mutations in a large series of 87 Spanish families (77 with type I, and 10 with type II HAE) by SSCP, sequencing, Southern blotting, and quantitative multiplex PCR of short fluorescent fragments (QMPSF), and we characterized several defects at the mRNA level. We found large rearrangements in 13 families, and point mutations or microdeletions/insertions in 74 families. The 13 large rearrangements included nine exon deletions, of which at least eight were distinct, two were distinct exon duplications, and two were rearrangements whose precise nature could not be determined. We confirmed that exon 4 is particularly prone to rearrangements. Thirty-six mutations were unreported, and included 10 microdeletions/insertions, 10 missense, five nonsense, eight splicing, and three splicing or missense mutations. Moreover, we detected six novel uncharacterized sequence variants (USV). RT-PCR studies showed that in addition to several intronic splice site mutations tested, the exonic mutations c.882C>G and c.884T>G, located near the 3' end of exon 5, also produced exon skipping. This is the first evidence of SERPING1/C1NH mutations in coding regions that differ from the canonical splice sites that affect splicing, which suggests the presence of an exonic splicing enhancer (ESE) in exon 5.

Detection of C1 inhibitor (SERPING1/C1NH) mutations in exon 8 in patients with hereditary angioedema: evidence for 10 novel mutations.

Hereditary angioedema (HAE) is caused by mutations in the C1 inhibitor gene (SERPING1, C1NH) and the result is C1 inhibitor deficiency, either in levels or function. We have searched exon 8 for mutations by direct sequencing and analyzed the rest of the exons by SSCP in 87 Spanish families affected by HAE. Out of 87 screened families, we have detected exon 8 mutations in 26. Among these, 17 different mutations were identified: 14 point mutations and 3 frameshift. Seven of the point mutations and the three frameshift were not previously reported. Mutations were: S438P; R444P; V451G; W460X; V468D; G471E; X479R; S417fsX427; I440fsX450; E429fsX450. The rest of the families presented previously reported mutations, 5 missense and two nonsense. In none of the 26 families was an additional change identified in the rest of the exons by SSCP, and, in 20 out of the 22 families with point mutation, we verified that the mutation did not affect a healthy relative. Seven of these families had no history of the disease, and in five of them we were able to verify that the progenitors did not have the mutation. Therefore, they were de novo mutations.

Transcriptional regulation of genes via hypoxia-inducible factor.

Hypoxia-inducible factor (HIF) is the principal transcription factor that regulates adaptive physiologic responses to compromised oxygen tension. von Hippel-Lindau (VHL) tumor-suppressor protein binds and ubiquitylates the catalytic α subunit of HIF in an oxygen-dependent manner for rapid destruction via the 26S proteasome, thereby establishing VHL as a critical negative regulator of HIF. Mutations in VHL cause VHL disease, which is frequently characterized by the overexpression of HIFα and the development of tumors in multiple organ systems, including the central nervous system and the kidney. Here, we describe classical experimental approaches to demonstrate and validate HIF-responsive transcriptional regulation of genes.

SERPING1 mutations in 59 families with hereditary angioedema.

Hereditary angioedema due to C1 Inhibitor (C1Inh) deficiency (HAE types I and II) is a rare, life-threatening disease causing spontaneous edema of the submucosal layers. A cohort of 127 individuals with symptoms of recurrent familial angioedema from 59 non-related families was studied. All the patients included fulfilled the diagnostic and biochemical criteria of HAE, including low C1Inh function and/or concentration. Genetic studies were carried out by PCR and sequencing of the C1NH locus followed, in the negative cases, by MLPA, long-range PCR and restriction enzyme analysis of genomic DNA to detect potential large rearrangements. Mutations located in consensus splicing sequences or nearby positions were studied by RT-PCR. The study identified 52 different mutations (25 missense, 15 frameshift, 7 splicing defects and 5 large deletions) responsible for the disease in 56 HAE families. In the remaining three families no molecular alteration could be detected. Twenty-seven of the mutations in this cohort are novel and 10 are confirmed de novo cases. The pathologic effect of the 5 splicing defects first reported here was assessed at the RNA and protein levels. Large deletions affecting exons 4 and 7, ranging from approximately 1500 to 2500 bp, were partially characterized by their altered restriction patterns upon long-range amplification. These results highlight the heterogeneity of mutations in the C1NH gene causing C1Inh deficiency and HAE. An approach to the molecular effects associated to each of the mutations reported here was made when possible based on the available data of pathological variants of serpins.

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia.

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.

Oxygen-independent degradation of HIF-alpha via bioengineered VHL tumour suppressor complex.

Tumour hypoxia promotes the accumulation of the otherwise oxygen-labile hypoxia-inducible factor (HIF)-alpha subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen-dependent degradation of HIF-alpha is carried out by the von Hippel-Lindau (VHL) protein-containing E3 that directly binds and ubiquitylates HIF-alpha for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL-HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF-alpha or HIF-downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF-alpha while preserving its E3 enzymatic activity. Using speckle variance-optical coherence tomography (sv-OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus-mediated delivery of the bioengineered VHL protein in a dorsal skin-fold window chamber model. These findings introduce the concept and feasibility of 'bio-tailored' enzymes in the treatment of HIF-overexpressing tumours.