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2.  Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans 
Scientific Reports  2016;6:34537.
Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.
PMCID: PMC5046109  PMID: 27694851
3.  Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells – Evidence of unique microRNA cargos 
RNA Biology  2015;12(8):810-823.
Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.
PMCID: PMC4615768  PMID: 26176991
cancer; extracellular RNA; malignant melanoma; membrane vesicles; non-coding RNA; next-generation sequencing
4.  Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts 
Oncotarget  2016;7(17):23801-23811.
Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.
PMCID: PMC5029664  PMID: 27009863
melanoma; miR210; hypoxia; xenografts; MEK inhibitor
5.  Synthesis aided structural determination of amyloid-β (1–15) glycopeptide, a new biomarker for Alzheimer’s disease† 
Unique tyrosine glycosylated amyloid-β (1–15) glycopeptides were synthesized with well-defined stereochemistry at the glycosidic linkages. Aided by these glycopeptides and tandem mass spectrometry analysis, the naturally existing amyloid-β glycopeptides, isolated from Alzheimer’s disease patients, were determined to contain an α-linked N-acetyl galactosamine at the modified tyrosine 10 residue. Glycosylation can significantly impact the property of amyloid-β as the glycopeptide has much lower affinity for Cu+ ions.
PMCID: PMC4221429  PMID: 25329175
6.  Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants 
Nature  2005;436(7052):807-811.
The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitt’s lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis.
PMCID: PMC4599579  PMID: 16094360
8.  Ecology, evolution, and management strategies of northern pike populations in the Baltic Sea 
Ambio  2015;44(Suppl 3):451-461.
Baltic Sea populations of the northern pike (Esox lucius) have declined since the 1990s, and they face additional challenges due to ongoing climate change. Pike in the Baltic Sea spawn either in coastal bays or in freshwater streams and wetlands. Pike recruited in freshwater have been found to make up about 50 % of coastal pike stocks and to show natal homing, thus limiting gene flow among closely located spawning sites. Due to natal homing, sub-populations appear to be locally adapted to their freshwater recruitment environments. Management actions should therefore not involve mixing of individuals originating from different sub-populations. We offer two suggestions complying with this advice: (i) productivity of extant freshwater spawning populations can be boosted by modifying wetlands such that they promote spawning and recruitment; and (ii) new sub-populations that spawn in brackish water can potentially be created by transferring fry and imprinting them on seemingly suitable spawning environments.
PMCID: PMC4447694  PMID: 26022327
Climate change; Conservation; Esox lucius; Habitat restoration; Homing; Population divergence
9.  Burden of herpes zoster and post-herpetic neuralgia in Sweden 
BMC Infectious Diseases  2015;15:215.
The societal economic burden of herpes zoster in Sweden is not well described today. This study is a top-down analysis of Swedish registers with the objective to describe the burden of herpes zoster and post-herpetic neuralgia in Sweden during 2011.
Data for inpatient care; outpatient primary and specialized cares; the prescriptions of drugs, sick leave and the number or diagnostic tests were collected from Swedish national databases. The incidence of the disease was estimated based on the number of prescriptions of antiviral drugs.
The incidence of herpes zoster was estimated to 315 and 577 cases per 100,000 people for patients at all ages and > = 50 years, respectively. Almost 30,000 patients at all ages were diagnosed with herpes zoster and the societal cost to treat these patients, including the cost to treat those patients who later developed post-herpetic neuralgia, added up to nearly 227 MSEK (31.6 M€) which corresponds to 7,600 SEK (€870) per patient. The main contributors to the total cost for the treatment of HZ patients were primary care (43 %); sick leave (28 %); hospitalization (10 %) and specialist care (7 %). Medication was a relatively small contributor with 8.5 MSEK (4 %; 1.0 M€) to the overall costs for patients at all ages. The corresponding total cost including only patients 50 years and older was 168 MSEK (19.2 M€) or 8,200 SEK (€939) per patient.
The current study demonstrates that the burden of herpes zoster is significant in Sweden. The society, the health care payers and the patients potentially have a lot to gain by introducing a vaccination program to patients 50 years and older and as a consequence reduce the economic and clinical burden of herpes zoster and post-herpetic neuralgia.
PMCID: PMC4493830  PMID: 26002038
10.  Melanoma patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions 
Oncotarget  2014;5(20):9609-9618.
The development of novel therapies against melanoma would benefit from individualized tumor models to ensure the rapid and accurate identification of biomarkers of therapy response. Previous studies have suggested that patient-derived xenografts (PDXes) could be useful. However, the utility of PDXes in guiding real-time treatment decisions has only been reported in anecdotal forms. Here tumor biopsies from patients with stage III and IV metastatic malignant melanoma were transplanted into immunocompromised mice to generate PDXes. 23/26 melanoma biopsies generated serially transplantable PDX models, and their histology, mutation status and expression profile resembled their corresponding patient biopsy. The potential treatment for one patient was revealed by an in vitro drug screen and treating PDXes with the MEK inhibitor trametinib. In another patient, the BRAF mutation predicted the response of both the patient and its corresponding PDXes to MAPK-targeted therapy. Importantly, in this unselected group of patients, the time from biopsy for generation of PDXes until death was significantly longer than the time required to reach the treatment phase of the PDXes. Thus, it could be clinically meaningful to use this type of platform for melanoma patients as a pre-selection tool in clinical trials.
PMCID: PMC4259423  PMID: 25228592
melanoma; mouse models; patient-derived xenografts
11.  Isolated hepatic perfusion as a treatment for uveal melanoma liver metastases (the SCANDIUM trial): study protocol for a randomized controlled trial 
Trials  2014;15:317.
Uveal melanoma is the most common primary intraocular malignancy in adults. Despite successful control of the primary tumor, metastatic disease will ultimately develop in approximately 50% of patients, with the liver being the most common site for metastases. The median survival for patients with liver metastases is between 6 and 12 months, and no treatment has in randomized trials ever been shown to prolong survival. A previous phase II trial using isolated hepatic perfusion (IHP) has suggested a 14-month increase in overall survival compared with a historic control group consisting of the longest surviving patients in Sweden during the same time period (26 versus 12 months).
This is the protocol for a multicenter phase III trial randomizing patients with isolated liver metastases of uveal melanoma to IHP or best alternative care (BAC). Inclusion criteria include liver metastases (verified by biopsy) and no evidence of extra-hepatic tumor manifestations by positron emission tomography–computed tomography (PET-CT). The primary endpoint is overall survival at 24 months, with secondary endpoints including response rate, progression-free survival, and quality of life. The planned sample size is 78 patients throughout five years.
Patients with isolated liver metastases of uveal melanoma origin have a short expected survival and no standard treatment option exists. This is the first randomized clinical trial to evaluate IHP as a treatment option with overall survival being the primary endpoint.
Trial registration registration number: NCT01785316 (registered 1 February 2013). EudraCT registration number: 2013-000564-29.
PMCID: PMC4138407  PMID: 25106493
Uveal melanoma; Liver metastases; Isolated hepatic perfusion; Regional treatment
12.  Functional multiplex reporter assay using tagged Gaussia luciferase 
Scientific Reports  2013;3:1046.
We have developed a multiplex reporter system to monitor multiple biological variables in real-time. The secreted Gaussia luciferase was fused to ten different epitope tags (Gluctag), each expressed in different tumor cells. By immunobinding of the tags followed by Gluctag detection, this system allowed the independent and real-time monitoring of mixed cell cultures in vitro and of mixed subcutaneous and intracranial tumor subpopulations in vivo.
PMCID: PMC3541509  PMID: 23308339
13.  Cost-effectiveness of dronedarone and standard of care compared with standard of care alone: US results of an ATHENA lifetime model 
The first antiarrhythmic drug to demonstrate a reduced rate of cardiovascular hospitalization in atrial fibrillation/flutter (AF/AFL) patients was dronedarone in a placebo-controlled, double-blind, parallel arm Trial to assess the efficacy of dronedarone 400 mg bid for the prevention of cardiovascular Hospitalization or death from any cause in patiENts with Atrial fibrillation/atrial flutter (ATHENA trial). The potential cost-effectiveness of dronedarone in this patient population has not been reported in a US context. This study assesses the cost-effectiveness of dronedarone from a US health care payers’ perspective.
Methods and results
ATHENA patient data were applied to a patient-level health state transition model. Probabilities of health state transitions were derived from ATHENA and published data. Associated costs used in the model (2010 values) were obtained from published sources when trial data were not available. The base-case model assumed that patients were treated with dronedarone for the duration of ATHENA (mean 21 months) and were followed over a lifetime. Cost-effectiveness, from the payers’ perspective, was determined using a Monte Carlo microsimulation (1 million fictitious patients). Dronedarone plus standard care provided 0.13 life years gained (LYG), and 0.11 quality-adjusted life years (QALYs), over standard care alone; cost/QALY was $19,520 and cost/LYG was $16,930. Compared to lower risk patients, patients at higher risk of stroke (Congestive heart failure, history of Hypertension, Age ≥ 75 years, Diabetes mellitus, and past history of Stroke or transient ischemic attack (CHADS2) scores 3–6 versus 0) had a lower cost/QALY ($9580–$16,000 versus $26,450). Cost/QALY was highest in scenarios assuming lifetime dronedarone therapy, no cardiovascular mortality benefit, no cost associated with AF/AFL recurrence on standard care, and when discounting of 5% was compared with 0%.
By extrapolating the results of a large, multicenter, randomized clinical trial (ATHENA), this model suggests that dronedarone is a cost-effective treatment option for approved indications (paroxysmal/persistent AF/AFL) in the US.
PMCID: PMC3544268  PMID: 23326201
cost-effectiveness; dronedarone; ATHENA
14.  Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway 
Neuro-Oncology  2011;13(11):1213-1224.
Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.
PMCID: PMC3199161  PMID: 21757445
cardiac glycoside; glioblastoma; lanatoside C; non-apoptotic cell death; TRAIL
15.  Targeting the glycoproteome 
Glycoconjugate Journal  2012;30(2):119-136.
Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.
PMCID: PMC3552370  PMID: 22886069
Glycoproteomics; Glycopeptide; Attachment sites; Liquid chromatography; Tandem mass spectrometry; Enrichment; Lectin affinity; Hydrazide chemistry
16.  Cks1 Is Required for Tumor Cell Proliferation but Not Sufficient to Induce Hematopoietic Malignancies 
PLoS ONE  2012;7(5):e37433.
The Cks1 component of the SCFSkp2 complex is necessary for p27Kip1 ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27Kip1. To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.
PMCID: PMC3356264  PMID: 22624029
17.  Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis 
PLoS Genetics  2012;8(3):e1002573.
c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, ApcMin mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes.
Author Summary
Cancer occurs when cells change their behavior and start to divide in an uncontrolled manner. To achieve this altered behavior, cells need to change their metabolism to be able to grow even when nutrient and oxygen supplies are limiting. Therefore, targeting metabolic pathways could be used to treat patients suffering from cancer. Here we studied a gene called MYC, which can regulate many metabolic pathways. By using genetically modified mice we can show that tumors have a remarkable ability to change their metabolism, even if key enzymes are removed. Taken together, our data suggest that metabolic disturbance by drugs in the clinic may present a future challenge.
PMCID: PMC3305401  PMID: 22438825
18.  Chk2 deficiency in Myc-overexpressing lymphoma cells elicits a synergistic lethal response in combination with PARP inhibition 
Cell Cycle  2011;10(20):3598-3607.
Myc is a transcription factor frequently found deregulated in human cancer. The Myc-mediated cellular transformation process is associated with fast proliferative cells and inherent genomic instability, giving rise to malignant, invasive neoplasms with poor prognosis for survival. Transcription-independent functions of Myc include stimulation of replication. Excessive Myc expression stimulates a replication-associated DNA damage response that signals via the phosphoinositide-3-kinase (PI3K)-related protein kinases (PIKKs) ATM and ATR. These, in turn, activate the DNA damage transducers Chk1 and Chk2. Here, we show that Myc can stimulate Chek2 transcript indirectly in vitro as well as in B cells of λ-Myc transgenic mice or in the intestine of ApcMin mice. However, Chk2 is dispensable for Myc's ability to transform cells in vitro and for the survival of established lymphoma cells from λ-Myc transgenic mice. Chk2 deficiency induces polyploidy and slow growth, but the cells are viable and protected against DNA damage. Furthermore, inhibition of both Chk1/Chk2 with AZD7762 induces cell death and significantly delays disease progression of transplanted lymphoma cells in vivo. DNA damage recruits PARP family members to sites of DNA breaks that, in turn, facilitate the induction of DNA repair. Strikingly, combining Chk2 and PARP inhibition elicits a synergistic lethal response in the context of Myc overexpression. Our data indicates that only certain types of chemotherapy would give rise to a synergistic lethal response in combination with specific Chk2 inhibitors, which will be important if Chk2 inhibitors enter the clinic.
PMCID: PMC3266184  PMID: 22030621
lymphoma; Myc; Chk1; Chk2; PARP; DNA damage; AZD-7762; ABT-888
19.  miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis 
Oncotarget  2010;1(8):710-720.
Glioblastoma (GBM) is a malignant brain tumor with dismal prognosis. GBM patients have a median survival of less than 2 years. GBM is characterized by fast cell proliferation, infiltrative migration, and by the induction of angiogenesis. MicroRNAs and polycomb group (PcG) proteins have emerged as important regulators of gene expression.
Here we determined that miR-101 is down-regulated in GBM, resulting in overexpression of the miR-101 target PcG protein EZH2, a histone methyltransferase affecting gene expression profiles in an epigenetic manner.
Inhibition of EZH2 in vitro by pre-miR-101, EZH2 siRNA, or small molecule DZNep, attenuated GBM cell growth, migration/invasion, and GBM-induced endothelial tubule formation. In addition, for each biological process we identified ontology-associated transcripts that significantly correlate with EZH2 expression. Inhibition of EZH2 in vivo by systemic DZNep administration in a U87-Fluc-mCherry GBM xenograft mouse imaging model resulted in reduced tumor growth.
Our results indicate that EZH2 has a versatile function in GBM progression and that its overexpression is at least partly due to decreased miR-101 expression. Inhibition of EZH2 may be a potential therapeutic strategy to target GBM proliferation, migration, and angiogenesis.
PMCID: PMC3124376  PMID: 21321380
cancer; microRNA; Policomb group; glioblastoma; angiogenesis
20.  The direct Myc target Pim3 cooperates with other Pim kinases in supporting viability of Myc-induced B-cell lymphomas 
Oncotarget  2011;2(6):448-460.
The Pim kinases are weak oncogenes. However, when co-expressed with a strong oncogene, such as c-Myc, Pim kinases potentiate the oncogenic effect resulting in an acceleration of tumorigenesis. In this study we show that the least studied Pim kinase, Pim-3, is encoded by a gene directly regulated by c-Myc via binding to one of the conserved E-boxes within the Pim3 gene. Accordingly, lymphomas arising in Myc-transgenic mice and Burkitt lymphoma cell lines exhibit elevated levels of Pim-3. Interestingly, inhibition of Pim kinases by a novel pan-Pim kinase inhibitor, Pimi, in Myc-induced lymphoma results in cell death that appears independent of caspases. The data indicate that Pim kinase inhibition could be a viable treatment strategy in certain human lymphomas that rely on Pim-3 kinase expression.
PMCID: PMC3248204  PMID: 21646687
cancer; lymphoma; oncogenes; c-Myc; Pim-3
21.  Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis 
Molecular cancer research : MCR  2010;8(3):353-362.
The universal cyclin-Cdk inhibitor p27Kip1 functions as a tumor suppressor and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E-Cdk2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the impact of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared to the effects of Cks1 loss. Collectively these findings suggest that Cks1 targets in addition to p27Kip1 are critical for Myc-driven proliferation and tumorigenesis.
PMCID: PMC3095030  PMID: 20197382
Myc; Skp2; p27Kip1; lymphomagenesis
22.  miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis 
Oncotarget  2011;1(8):710-720.
Glioblastoma (GBM) is a malignant brain tumor with dismal prognosis. GBM patients have a median survival of less than 2 years. GBM is characterized by fast cell proliferation, infiltrative migration, and by the induction of angiogenesis. MicroRNAs and polycomb group (PcG) proteins have emerged as important regulators of gene expression.
Here we determined that miR-101 is down-regulated in GBM, resulting in overexpression of the miR-101 target PcG protein EZH2, a histone methyltransferase affecting gene expression profiles in an epigenetic manner. Results: Inhibition of EZH2 in vitro by pre-miR-101, EZH2 siRNA, or small molecule DZNep, attenuated GBM cell growth, migration/invasion, and GBM-induced endothelial tubule formation. In addition, for each biological process we identified ontology-associated transcripts that significantly correlate with EZH2 expression. Inhibition of EZH2 in vivo by systemic DZNep administration in a U87-Fluc-mCherry GBM xenograft mouse imaging model resulted in reduced tumor growth.
Our results indicate that EZH2 has a versatile function in GBM progression and that its overexpression is at least partly due to decreased miR-101 expression. Inhibition of EZH2 may be a potential therapeutic strategy to target GBM proliferation, migration, and angiogenesis.
PMCID: PMC3124376  PMID: 21321380
cancer; microRNA; Policomb group; glioblastoma; angiogenesis
23.  Myc suppression of Nfkb2 accelerates lymphomagenesis 
BMC Cancer  2010;10:348.
Deregulated c-Myc expression is a hallmark of several human cancers where it promotes proliferation and an aggressive tumour phenotype. Myc overexpression is associated with reduced activity of Rel/NF-κB, transcription factors that control the immune response, cell survival, and transformation, and that are frequently altered in cancer. The Rel/NF-κB family member NFKB2 is altered by chromosomal translocations or deletions in lymphoid malignancies and deletion of the C-terminal ankyrin domain of NF-κB2 augments lymphocyte proliferation.
Precancerous Eμ-Myc-transgenic B cells, Eμ-Myc lymphomas and human Burkitt lymphoma samples were assessed for Nfkb2 expression. The contribution of Nfkb2 to Myc-driven apoptosis, proliferation, and lymphomagenesis was tested genetically in vivo.
Here we report that the Myc oncoprotein suppresses Nfkb2 expression in vitro in primary mouse fibroblasts and B cells, and in vivo in the Eμ-Myc transgenic mouse model of human Burkitt lymphoma (BL). NFKB2 suppression by Myc was also confirmed in primary human BL. Promoter-reporter assays indicate that Myc-mediated suppression of Nfkb2 occurs at the level of transcription. The contribution of Nfkb2 to Myc-driven lymphomagenesis was tested in vivo, where Nfkb2 loss was shown to accelerate lymphoma development in Eμ-Myc transgenic mice, by impairing Myc's apoptotic response.
Nfkb2 is suppressed by c-Myc and harnesses Myc-driven lymphomagenesis. These data thus link Myc-driven lymphomagenesis to the non-canonical NF-κB pathway.
PMCID: PMC2902445  PMID: 20598117
24.  Selection against PUMA Gene Expression in Myc-Driven B-Cell Lymphomagenesis▿ †  
Molecular and Cellular Biology  2008;28(17):5391-5402.
The p53 tumor suppressor pathway limits oncogenesis by inducing cell cycle arrest or apoptosis. A key p53 target gene is PUMA, which encodes a BH3-only proapoptotic protein. Here we demonstrate that Puma deletion in the Eμ-Myc mouse model of Burkitt lymphoma accelerates lymphomagenesis and that ∼75% of Eμ-Myc lymphomas naturally select against Puma protein expression. Furthermore, approximately 40% of primary human Burkitt lymphomas fail to express detectable levels of PUMA and in some tumors this is associated with DNA methylation. Burkitt lymphoma cell lines phenocopy the primary tumors with respect to DNA methylation and diminished PUMA expression, which can be reactivated following inhibition of DNA methyltransferases. These findings establish that PUMA is silenced in human malignancies, and they suggest PUMA as a target for the development of novel chemotherapeutics.
PMCID: PMC2519737  PMID: 18573879
25.  The Novel ETS Factor TEL2 Cooperates with Myc in B Lymphomagenesis†  
Molecular and Cellular Biology  2005;25(6):2395-2405.
The human ETS family gene TEL2/ETV7 is highly homologous to TEL1/ETV6, a frequent target of chromosome translocations in human leukemia and specific solid tumors. Here we report that TEL2 augments the proliferation and survival of normal mouse B cells and dramatically accelerates lymphoma development in Eμ-Myc transgenic mice. Nonetheless, inactivation of the p53 pathway was a hallmark of all TEL2/Eμ-Myc lymphomas, indicating that TEL2 expression alone is insufficient to bypass this apoptotic checkpoint. Although TEL2 is infrequently up-regulated in human sporadic Burkitt's lymphoma, analysis of pediatric B-cell acute lymphocytic leukemia (B-ALL) samples showed increased coexpression of TEL2 and MYC and/or MYCN in over one-third of B-ALL patients. Therefore, TEL2 and MYC also appear to cooperate in provoking a cadre of human B-cell malignancies.
PMCID: PMC1061619  PMID: 15743832

Results 1-25 (29)