Correction: Hypoxia-inducible factor 1 upregulation of both VEGF and ANGPTL4 is required to promote the angiogenic phenotype in uveal melanoma.

[This corrects the article DOI: 10.18632/oncotarget.6868.].

PD-L1 Expression in Pediatric Low-Grade Gliomas Is Independent of BRAF V600E Mutational Status.

To evaluate a potential relationship between BRAF V600E mutation and PD-L1 expression, we examined the expression of PD-L1 in pediatric high- and low-grade glioma cell lines as well as a cohort of pediatric low-grade glioma patient samples. Half of the tumors in our patient cohort were V600-wildtype and half were V600E mutant. All tumors expressed PD-L1. In most tumors, PD-L1 expression was low (<5%), but in some cases over 50% of cells were positive. Extent of PD-L1 expression and immune cell infiltration was independent of BRAF V600E mutational status. All cell lines evaluated, including a BRAF V600E mutant xenograft, expressed PD-L1. Transient transfection of cell lines with a plasmid expressing mutant BRAF V600E had minimal effect on PD-L1 expression. These findings suggest that the PD-1 pathway is active in subsets of pediatric low-grade glioma as a mechanism of immune evasion independent of BRAF V600E mutational status. Low-grade gliomas that are unresectable and refractory to traditional therapy are associated with significant morbidity and continue to pose a treatment challenge. PD-1 pathway inhibitors may offer an alternative treatment approach. Clinical trials will be critical in determining whether PD-L1 expression indicates likely therapeutic benefit with immune checkpoint inhibitors.

ADAM3A copy number gains occur in a subset of conjunctival squamous cell carcinoma and its high grade precursors.

Conjunctival squamous cell carcinoma (cSCC) and its precursors are among the most frequent ocular surface neoplasms worldwide. Copy gain of 8p11.22 and ADAM3A overexpression have been recently identified in invasive cSCC. We sought to study copy number gains using fluorescent in situ hybridization (FISH) in cSCC and the spectrum of precursor lesions. A total of 54 cases conjunctival squamous intraepithelial neoplasia (CIN), carcinoma in situ (CIS), or cSCC were studied using FISH with an ADAM3A (8p11 locus) probe and a chromosome 8 (Chr 8) centromere reference probe. Eighty one percent (44/54) of the cases presented in men and 19% (10/54) in women. The age at presentation ranged from 12 to 94 years (mean 65.5 years). Severe CIN was diagnosed in 45% (24/54) of the cases, followed by CIS in 31% (17/54), moderate CIN in 15% (8/54), invasive cSCC in 7% (4/54), and mild CIN in 2% (1/54). Nine (of 54) (17%) cases harbored ADAM3A or Chr 8 gains, with one of these cases demonstrating high level amplification. All ADAM3A alterations were restricted to high-grade lesions, including 2/17 (12%) cCIS, 1/4 (24%) cSCC, 5/24 (20%) severe CIN and 1/8 (12%) moderate CIN. Monosomy 8 was detected in 2 (4%) cases. No ADAM3A alterations were detected in non-neoplastic controls. Gains of ADAM3A/chromosome 8 occur in a subset of cSCC and its precursors. Alterations were present in high-grade lesions, sparing non-neoplastic conjunctiva and absent in tested controls. Thus, the specificity of this alteration as a biomarker for ocular SCC deserves further study.

Downregulation of Nodal inhibits metastatic progression in retinoblastoma.

Retinoblastoma is the most common intraocular malignancy in children. We previously found that the ACVR1C/SMAD2 pathway is significantly upregulated in invasive retinoblastoma samples from patients. Here we studied the role of an ACVR1C ligand, Nodal, in regulating growth and metastatic dissemination in retinoblastoma. Inhibition of Nodal using multiple short hairpin (shRNAs) in WERI Rb1 and Y79 retinoblastoma cell cultures reduced growth by more than 90%, as determined by CCK-8 growth assay. Proliferation was also significantly inhibited, as found by Ki67 assay. These effects were paralleled by inhibition in the phosphorylation of the downstream effector SMAD2, as well as induction of apoptosis, as we observed more than three-fold increase in the percentage of cells positive for cleaved-caspase-3 or expressing cleaved-PARP1. Importantly, we found that downregulation of Nodal potently suppressed invasion in vitro, by 50 to 80%, as determined by transwell invasion assay (p = 0.02). Using an orthotopic model of retinoblastoma in zebrafish, we found 34% reduction in the ability of the cells to disseminate outside the eye, when Nodal was knocked down by shRNA (p = 0.0003). These data suggest that Nodal plays an important role in promoting growth, proliferation and invasion in retinoblastoma, and can be considered a new therapeutic target for both primary tumor growth and metastatic progression.

ACVR1C/SMAD2 signaling promotes invasion and growth in retinoblastoma.

Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-ß family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.

Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing.

Purpose: We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis. Methods: This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool. Results: Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge. Conclusions: Next generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

HIF-1α- Targeting Acriflavine Provides Long Term Survival and Radiological Tumor Response in Brain Cancer Therapy.

Tumor progression, limited efficacy of current standard treatments, and the rise in patient mortality are associated with gene expression caused by the synergistic action of intratumoral hypoxia and HIF-1α activation. For this reason, recent investigations have focused on HIF-targeting therapeutic agents, with encouraging preclinical and clinical results in solid tumors. Here we describe the efficacy of a HIF-1α inhibitor, Acriflavine, and demonstrate its potency against brain cancer. This safe antibacterial dye induces cell death and apoptosis in several glioma cell lines, targets HIF-1α-mediated pathways, and decreases the level of PGK1, VEGF and HIF-1α in vitro and in vivo. Administered locally via biodegradable polymers, Acriflavine provides significant benefits in survival resulting in nearly 100% long term survival, confirmed by MRI and histological analyses. This study reports preclinical evidence that this safe, small molecule can contribute to brain tumor therapy and highlights the significance of HIF-1α-targeting molecules.

Hypoxia inhibits growth, proliferation, and increases response to chemotherapy in retinoblastoma cells.

Retinoblastoma is a malignant tumor of the retina and the most frequent intraocular cancer in children. Low oxygen tension (hypoxia) is a common phenomenon in advanced retinoblastomas, but its biological effect on retinoblastoma growth is not clearly understood. Here we studied how hypoxia altered retinoblastoma gene expression and modulated growth and response to chemotherapy. The hypoxic marker lysyl oxidase (LOX) was expressed in 8 of 12 human retinoblastomas analyzed by immunohistochemistry, suggesting that a hypoxic microenvironment is present in up to two thirds of the cases. WERI Rb1 and Y79 retinoblastoma lines were exposed to 1% or 5% pO2, cobalt chloride (CoCl2), or to normoxia (21% pO2) for up to 8 days. Both 1% and 5% pO2 inhibited growth of both lines by more than 50%. Proliferation was reduced by 25-50% when retinoblastoma cells were exposed to 1% vs 21% pO2, as determined by Ki67 assay. Surprisingly, Melphalan, Carboplatin, and Etoposide produced greater reduction in growth and survival of hypoxic cells than normoxic ones. Gene expression profile analysis of both lines, exposed for 48 h to 1%, 5%, or 21% pO2, showed that glycolysis and glucose transport were the most up-regulated pathways, whereas oxidative phosphorylation was the most down-regulated pathway in hypoxia as compared to normoxia. These data support a role for hypoxia in suppressing growth, proliferation, and enhancing response of retinoblastoma cells to chemotherapy, possibly by impairing energy production through activation of glycolysis and inhibition of mitochondrial respiration. Targeting glucose metabolism or enhancing delivery of chemotherapeutic agents to hypoxic regions may improve treatment of advanced retinoblastomas.

The dual mTOR kinase inhibitor TAK228 inhibits tumorigenicity and enhances radiosensitization in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKT, and PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type. Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis. TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p = 0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.

Targeting Notch signaling as a novel therapy for retinoblastoma.

Retinoblastoma is the most common intraocular malignancy of childhood. Notch plays a key role in retinal cells from which retinoblastomas arise, and we therefore studied the role of Notch signaling in promoting retinoblastoma proliferation. Moderate or strong nuclear expression of Hes1 was found in 10 of 11 human retinoblastoma samples analyzed immunohistochemically, supporting a role for Notch in retinoblastoma growth. Notch pathway components were present in WERI Rb1 and Y79 retinoblastoma lines, with Jag2 and DLL4 more highly expressed than other ligands, and Notch1 and Notch2 more abundant than Notch3. The cleaved/active form of Notch1 was detectable in both lines. Inhibition of the pathway, achieved using a γ-secretase inhibitor (GSI) or by downregulating Jag2, DLL4 or CBF1 using short hairpin RNA, potently reduced growth, proliferation and clonogenicity in both lines. Upregulation of CXCR4 and CXCR7 and downregulation of PI3KC2ß were identified by microarray upon Jag2 suppression. The functional importance of PI3KC2ß was confirmed using shRNA. Synergy was found by combining GSI with Melphalan at their IC50. These findings indicate that Notch pathway is active in WERI Rb1 and Y79, and in most human retinoblastoma samples, and suggest that Notch antagonists may represent a new approach to more effectively treat retinoblastoma.

Altered gene expression in conjunctival squamous cell carcinoma.

Conjunctival squamous cell carcinoma is a malignancy of the ocular surface. The molecular drivers responsible for the development and progression of this disease are not well understood. We therefore compared the transcriptional profiles of eight snap-frozen conjunctival squamous cell carcinomas and one in situ lesion with normal conjunctival specimens in order to identify diagnostic markers or therapeutic targets. RNA was analyzed using oligonucleotide microarrays, and a wide range of transcripts with altered expression identified, including many dysregulated in carcinomas arising at other sites. Among the upregulated genes, we observed more than 30-fold induction of the matrix metalloproteinases, MMP-9 and MMP-11, as well as a prominent increase in the mRNA level of a calcium-binding protein important for the intracellular calcium signaling, S100A2, which was induced over 20-fold in the tumor cohort. Clusterin was the most downregulated gene, with an approximately 180-fold reduction in the mRNA expression. These alterations were all confirmed by qPCR in the samples used for initial microarray analysis. In addition, immunohistochemical analysis confirmed the overexpression of MMP-11 and S100A2, as well as reductions in clusterin, in several independent in situ carcinomas of conjunctiva. These data identify a number of alterations, including upregulation of MMP-9, MMP-11, and S100A2, as well as downregulation of clusterin, associated with epithelial tumorigenesis in the ocular surface.

Hypoxia-inducible factor 1 upregulation of both VEGF and ANGPTL4 is required to promote the angiogenic phenotype in uveal melanoma.

PURPOSE: Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM). While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated. Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM. EXPERIMENTAL DESIGN: UM cells were examined for expression of HIF-1α, VEGF, and ANGPTL4. Their contribution to the angiogenic potential of UM cells was assessed using the endothelial cell tubule formation and directed in vivo angiogenesis assays. These results were corroborated in tissue from UM animal models and in tissue from patients with UM. RESULTS: Inhibition of VEGF partially reduced tubule formation promoted by conditioned medium from UM cells. Inhibition of ANGPTL4, which was highly expressed in hypoxic UM cells, a UM orthotopic transplant model, a UM tumor array, and vitreous samples from UM patients, inhibited the angiogenic potential of UM cells in vitro and in vivo; this effect was additive to VEGF inhibition. CONCLUSIONS: Targeting both ANGPTL4 and VEGF may be required for the effective inhibition of angiogenesis in UM.

Atracurium Besylate and other neuromuscular blocking agents promote astroglial differentiation and deplete glioblastoma stem cells.

Glioblastoma multiforme (GBM) are the most common primary malignant brain tumor in adults, with a median survival of about one year. This poor prognosis is attributed primarily to therapeutic resistance and tumor recurrence after surgical removal, with the root cause suggested to be found in glioblastoma stem cells (GSCs). Using glial fibrillary acidic protein (GFAP) as a reporter of astrocytic differentiation, we isolated multiple clones from three independent GSC lines which express GFAP in a remarkably stable fashion. We next show that elevated expression of GFAP is associated with reduced clonogenicity in vitro and tumorigenicity in vivo. Utilizing this in vitro cell-based differentiation reporter system we screened chemical libraries and identified the non-depolarizing neuromuscular blocker (NNMB), Atracurium Besylate, as a small molecule which effectively induces astroglial but not neuronal differentiation of GSCs. Functionally, Atracurium Besylate treatment significantly inhibited the clonogenic capacity of several independent patient-derived GSC neurosphere lines, a phenomenon which was largely irreversible. A second NNMB, Vecuronium, also induced GSC astrocytic differentiation while Dimethylphenylpiperazinium (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, significantly blocked Atracurium Besylate pro-differentiation activity. To investigate the clinical importance of nAChRs in gliomas, we examined clinical outcomes and found that glioma patients with tumors overexpressing CHRNA1 or CHRNA9 (encoding for the AChR-α1 or AChR-α9) exhibit significant shorter overall survival. Finally, we found that ex-vivo pre-treatment of GSCs, expressing CHRNA1 and CHRNA9, with Atracurium Besylate significantly increased the survival of mice xenotransplanted with these cells, therefore suggesting that tumor initiating subpopulations have been reduced.

EMT-associated factors promote invasive properties of uveal melanoma cells.

PURPOSE: Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors. METHODS: ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed. RESULTS: ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth. CONCLUSIONS: The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

Identification of multiple DNA copy number alterations including frequent 8p11.22 amplification in conjunctival squamous cell carcinoma.

PURPOSE: Little is known about the molecular alterations that drive formation and growth of conjunctival squamous cell carcinoma (cSCC). We therefore sought to identify genetic changes that could be used as diagnostic markers or therapeutic targets. METHODS: The DNA extracted from 10 snap-frozen cSCC tumor specimens and 2 in situ carcinomas was analyzed using array-based comparative genomic hybridization (aCGH), and further examined with NanoString and quantitative PCR. RESULTS: The number of regions of DNA loss ranged from 1 to 23 per tumor, whereas gains and amplifications ranged from 1 to 15 per tumor. Most large regions of chromosomal gain and loss were confirmed by NanoString karyotype analysis. The commonest alteration was amplification of 8p11.22 in 9 tumors (75%), and quantitative PCR analysis revealed 100-fold or greater overexpression of ADAM3A mRNA from 8p11.22 locus. In addition, recurring losses were observed at 14q13.2 and 22q11.23, both lost in 5 (42%) of the 12 tumors, and at 12p13.31, lost in 4 (33%) of the 12 samples. Of the eight loci associated with the DNA damage repair syndrome xeroderma pigmentosum, three showed loss of at least one allele in our aCGH analysis, including XPA (9q22.33, one tumor), XPE/DDB2 (11p11.2, one tumor) and XPG/ERCC5 (13q33.1, three tumors). CONCLUSIONS: Conjunctival SCC contains a range of chromosomal alterations potentially important in tumor formation and growth. Amplification of 8p11.22 and overexpression of ADAM3A suggests a potential role for this protease. Our findings also suggest that defects in DNA repair loci are important in sporadic cSCC.

The demethylating agent 5-Aza reduces the growth, invasiveness, and clonogenicity of uveal and cutaneous melanoma.

PURPOSE: Uveal melanoma is the most common primary intraocular malignancy in adults. Although local disease can be controlled with radiation therapy or enucleation, many cases are complicated by metastases, which account for the significant mortality from this disease. To date, no chemotherapeutic regimens effectively treat local or metastatic disease. Epigenetic silencing of tumor suppressor genes has been shown to be an important factor in the growth and metastasis of many cancers. One form of epigenetic alteration is DNA methylation, which often occurs at promoter elements resulting in the silencing of target gene transcription. METHODS: We used 5-aza-2'-deoxycytidine (5-Aza), a well characterized demethylating agent that is US Food and Drug Administration approved to decrease DNA methylation in multiple uveal and cutaneous melanoma cell lines. RESULTS: Demethylation of melanoma cell lines using 5-Aza causes significant decreases in growth, invasion, and clonogenicity. Treatment of melanoma cells with combined 5-Aza therapy and irradiation showed an even more pronounced effect on cell viability. In addition, treatment with 5-Aza decreased the number of metastases from the eye to the lung in a murine cutaneous melanoma xenograft model. CONCLUSIONS: We demonstrate in vitro and in vivo that demethylating agents such as 5-Aza may be promising chemotherapeutic agents for treating melanoma and decreasing progression to metastatic disease. These results provide proof of concept for an exciting potential therapy to reduce mortality from this disease. Future work will focus on identifying pathways that mediate these changes.

Hypoxia promotes uveal melanoma invasion through enhanced Notch and MAPK activation.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

Induced pluripotent stem cells from familial Alzheimer's disease patients differentiate into mature neurons with amyloidogenic properties.

Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in ß-amyloid (Aß)42/Aß40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.

A role for Jag2 in promoting uveal melanoma dissemination and growth.

PURPOSE: Controlling the spread of uveal melanoma is key to improving survival of patients with this common intraocular malignancy. The Notch ligand Jag2 has been shown to be upregulated in primary tumors that metastasize, and we therefore investigated its role in promoting invasion and clonogenic growth of uveal melanoma cells. METHODS: mRNA and protein expression of Notch pathway components were measured using qPCR and Western blot in uveal melanoma cell lines. Expression of Jag2 ligand was upregulated using Jag2-GFP-MSCV constructs or downregulated by sh-Jag2 in the uveal melanoma cell lines Mel285, Mel290, 92.1, and OMM1, and the effects on growth and invasion were assessed. RESULTS: Jag2 was introduced into Mel285 and Mel290 cells, which have low baseline levels of both this ligand and Notch activity. Overall growth of the Jag2-expressing cultures increased somewhat, and a significant 3-fold increase in clonogenic growth in soft agar was also noted. Introduction of Jag2 increased motility in both wound-healing and transwell invasion assays. We also observed a significant increase in Jag2 and Hes1 mRNA in invasive OMM1 cells that had passed through a Matrigel-coated filter in the transwell assay when compared with noninvading cells. Loss-of-function studies performed in 92.1 and OMM1 lines using Jag2 shRNAs showed that downregulation of the ligand significantly suppressed cellular growth, invasion, and migration. CONCLUSIONS: Our data suggest that Jag2 may play an important role in promoting Notch activity, growth, and metastasis in uveal melanoma.