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1.  Major vault protein supports glioblastoma survival and migration by upregulating the EGFR/PI3K signalling axis 
Oncotarget  2013;4(11):1904-1918.
Despite their ubiquitous expression and high conservation during evolution, precise cellular functions of vault ribonucleoparticles, mainly built of multiple major vault protein (MVP) copies, are still enigmatic. With regard to cancer, vaults were shown to be upregulated during drug resistance development as well as malignant transformation and progression. Such in a previous study we demonstrated that human astrocytic brain tumours including glioblastoma are generally high in vault levels while MVP expression in normal brain is comparably low. However a direct contribution to the malignant phenotype in general and that of glioblastoma in particular has not been established so far. Thus we address the questions whether MVP itself has a pro-tumorigenic function in glioblastoma. Based on a large tissue collection, we re-confirm strong MVP expression in gliomas as compared to healthy brain. Further, the impact of MVP on human glioblastoma aggressiveness was analysed by using gene transfection, siRNA knock-down and dominant-negative genetic approaches. Our results demonstrate that MVP/vaults significantly support migratory and invasive competence as well as starvation resistance of glioma cells in vitro and in vivo. The enhanced aggressiveness was based on MVP-mediated stabilization of the epidermal growth factor receptor (EGFR)/phosphatidyl-inositol-3-kinase (PI3K) signalling axis. Consequently, MVP overexpression resulted in enhanced growth and brain invasion in human glioblastoma xenograft models. Our study demonstrates, for the first time, that vaults have a tumour-promoting potential by stabilizing EGFR/PI3K-mediated migration and survival pathways in human glioblastoma.
PMCID: PMC3875758  PMID: 24243798
major vault protein; glioblastoma multiforme; invasion; EGFR; PI3K
2.  Fibroblast Growth Factor Receptors as Therapeutic Targets in Human Melanoma: Synergism with BRAF Inhibition 
Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1–4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.
doi:10.1038/jid.2011.177
PMCID: PMC3383623  PMID: 21753785
3.  Impact of terminal dimethylation on the resistance profile of α-N-heterocyclic thiosemicarbazones 
Biochemical Pharmacology  2012;83(12):1623-1633.
Graphical abstract
Triapine is an α-N-heterocyclic thiosemicarbazone with promising anticancer activity against hematologic malignancies but widely ineffective against solid tumor types in clinical trials. The anticancer activity of thiosemicarbazones can be dramatically increased by terminal dimethylation. KP1089 is a gallium compound containing two terminal dimethylated thiosemicarbazone ligands. To gain insights on the vulnerability of this highly active terminal dimethylated thiosemicarbazone to drug resistance mechanisms, a new cell model with acquired resistance against the lead compound KP1089 was established. Subsequent genomic analyses (arrayCGH and FISH) revealed amplification of the ABCC1 gene on double minute chromosomal DNA in KP1089-resistant cells as well as overexpression of ABCC1 and ABCG2 on the protein level. KP1089 was further confirmed as a substrate of ABCC1 and ABCG2 but not of ABCB1 using a panel of ABC transporter-overexpressing cell models as well as ABC transporter inhibitors. Moreover, glutathione depletion strongly enhanced KP1089 activity, although no glutathione conjugate formation by glutathione-S-transferase was observed. Thus, a co-transport of KP1089 together with glutathione is suggested. Finally, a panel of thiosemicarbazone derivatives was tested on the new KP1089-resistant cell line. Notably, KP1089-resistant cells were not cross-resistant against thiosemicarbazones lacking terminal dimethylation (e.g. Triapine) which are less active than KP1089. This suggests that terminal dimethylation of thiosemicarbazones – linked with distinctly enhanced anticancer activity – leads to altered resistance profiles compared to classical thiosemicarbazones making this compound class of interest for further (pre)clinical evaluation.
doi:10.1016/j.bcp.2012.03.004
PMCID: PMC3342514  PMID: 22426010
ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; CSA, cyclosporin A; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; ESI-MS, electrospray ionization mass spectrometry; GSH, glutathione; GST, glutathione-S-transferase; KP1089, [bis(2-acetylpyridine 4,4-dimethylthiosemicarbazonato-N,N,S)gallium(III)] tetrachloridogallate; KP1550, 2-acetylpyridine 4,4-dimethylthiosemicarbazone; KP1657, [bis(2-acetylpyridine thiosemicarbazonato-N,N,S)gallium(III)] nitrate; KP1719, [bis(3-aminopyridine-2-carboxaldehyde 4,4-dimethylthiosemicarbazonato-N,N,S)gallium(III)] hexafluorophosphate; KP1740, [bis(2-formylpyridine 4,4-dimethylthiosemicarbazonato-N,N,S)gallium(III)] hexafluorophosphate; LRP, lung resistance protein; MDR, multidrug resistance; MRP, multidrug resistance-related protein; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; TD, terminal dimethylation; Triapine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone; Resistance; Thiosemicarbazones; Multidrug resistance; Triapine; ABC transporter; Glutathione
4.  Aggressiveness of human melanoma xenograft models is promoted by aneuploidy-driven gene expression deregulation 
Oncotarget  2012;3(4):399-413.
Melanoma is a devastating skin cancer characterized by distinct biological subtypes. Besides frequent mutations in growth- and survival-promoting genes like BRAF and NRAS, melanomas additionally harbor complex non-random genomic alterations. Using an integrative approach, we have analysed genomic and gene expression changes in human melanoma cell lines (N=32) derived from primary tumors and various metastatic sites and investigated the relation to local growth aggressiveness as xenografts in immuno-compromised mice (N=22). Although the vast majority (>90%) of melanoma models harbored mutations in either BRAF or NRAS, significant differences in subcutaneous growth aggressiveness became obvious. Unsupervised clustering revealed that genomic alterations rather than gene expression data reflected this aggressive phenotype, while no association with histology, stage or metastatic site of the original melanoma was found. Genomic clustering allowed separation of melanoma models into two subgroups with differing local growth aggressiveness in vivo. Regarding genes expressed at significantly altered levels between these subgroups, a surprising correlation with the respective gene doses (>85% accordance) was found. Genes deregulated at the DNA and mRNA level included well-known cancer genes partly already linked to melanoma (RAS genes, PTEN, AURKA, MAPK inhibitors Sprouty/Spred), but also novel candidates like SIPA1 (a Rap1GAP). Pathway mining further supported deregulation of Rap1 signaling in the aggressive subgroup e.g. by additional repression of two Rap1GEFs. Accordingly, siRNA-mediated down-regulation of SIPA1 exerted significant effects on clonogenicity, adherence and migration in aggressive melanoma models. Together our data suggest that an aneuploidy-driven gene expression deregulation drives local aggressiveness in human melanoma.
PMCID: PMC3380575  PMID: 22535842
malignant melanoma; aneuploidy; local aggressiveness; xenograft; integrative genomics
5.  DNA Damage, Somatic Aneuploidy, and Malignant Sarcoma Susceptibility in Muscular Dystrophies 
PLoS Genetics  2011;7(4):e1002042.
Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD–pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD–gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.
Author Summary
All kinds of muscular dystrophies (MDs) are characterized by progressive muscle wasting due to life-long proliferation of precursor cells of myo- (muscle), fibro- (connective tissue), and lipogenic (fat) origin. Despite discovery of many MD genes over the past 25 years, MDs still represent debilitating, incurable diseases, which frequently lead to premature death. Thus, it is imperative to gain novel insights into the underlying MD pathomechanisms. Here, we show that different mouse models for the most common human MDs frequently develop skeletal musculature-associated tumors, presenting as complex sarcomas, consisting of myo-, lipo-, and fibrogenic compartments. Collectively, these tumors are characterized by profound genomic instability such as DNA damage, recurring mutations in cancer genes, and aberrant chromosome copy numbers. We also demonstrate the presence of these cancer-related aberrations in dystrophic muscles from MD mice prior to formation of visible sarcomas. Moreover, we discovered corresponding genomic lesions also in skeletal muscles from human MD patients, as well as stem cells cultured thereof, and show that genomic instability precedes muscle degeneration in MDs. We thus propose that cancer-like genomic instability represents a novel, unifying pathomechanism underlying the entire group of genetically distinct MDs, which will hopefully open new therapeutic avenues.
doi:10.1371/journal.pgen.1002042
PMCID: PMC3077392  PMID: 21533183
6.  Overexpression of Aurora-A in primary cells interferes with S-phase entry by diminishing Cyclin D1 dependent activities 
Molecular Cancer  2011;10:28.
Background
Aurora-A is a bona-fide oncogene whose expression is associated with genomic instability and malignant transformation. In several types of cancer, gene amplification and/or increased protein levels of Aurora-A are a common feature.
Results
In this report, we describe that inhibition of cell proliferation is the main effect observed after transient overexpression of Aurora-A in primary human cells. In addition to the known cell cycle block at the G2/M transition, Aurora-A overexpressing cells fail to overcome the restriction point at the G1/S transition due to diminished RB phosphorylation caused by reduced Cyclin D1 expression. Consequently, overexpression of Cyclin D1 protein is able to override the Aurora-A mediated G1 block. The Aurora-A mediated cell cycle arrest in G2 is not influenced by Cyclin D1 and as a consequence cells accumulate in G2. Upon deactivation of p53 part of the cells evade this premitotic arrest to become aneuploid.
Conclusion
Our studies describe that an increase of Aurora-A expression levels on its own has a tumor suppressing function, but in combination with the appropriate altered intracellular setting it might exert its oncogenic potential. The presented data indicate that deactivation of the tumor suppressor RB is one of the requirements for overriding a cell cycle checkpoint triggered by increased Aurora-A levels.
doi:10.1186/1476-4598-10-28
PMCID: PMC3068985  PMID: 21410931
7.  O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients 
Neuro-Oncology  2009;12(1):28-36.
O6-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (χ2 test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76–17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45–2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.
doi:10.1093/neuonc/nop003
PMCID: PMC2940563  PMID: 20150365
O6-Methylguanine DNA methyltransferase; glioblastoma multiforme; protein expression; temozolomide
8.  Seven Novel and Stable Translocations Associated with Oncogenic Gene Expression in Malignant Melanoma1 
Neoplasia (New York, N.Y.)  2005;7(4):303-311.
Abstract
Cytogenetics has not only precipitated the discovery of several oncogenes, but has also led to the molecular classification of numerous malignancies. The correct identification of aberrations in many tumors has, however, been hindered by extensive tumor complexity and the limitations of molecular cytogenetic techniques. In this study, we have investigated five malignant melanoma (MM) cell lines from at least three different passages using high-resolution R-banding and the recently developed methods of comparative genomic hybridization and multicolor or multiplex fluorescence in situ hybridization. We subsequently detected nine consistent translocations, seven of which were novel: dic(1;11)(p10;q14), der(9)t(3;9)(p12;p11), der(4)t(9;4;7) (q33::p15-q23::q21), der(14)t(5;14) (q12;q32), der(9) t(9;22)(p21;q11), der(19)t(19;20)(p13.3;p11), der(10) t(2;12;7;10)(q31::p12→pter::q11.2→q31::q21), der(19)t(10;19)(q23;q13), and der(20)t(Y;20)(q11.23; q13.3). Furthermore, using the human HG-U133A GeneChip, positive expression levels of oncogenes or tumor-related genes located at the regions of chromosomal breakpoints were identified, including AKT1, BMI1, CDK6, CTNNB1, E2F1, GPNMB, GPRK7, KBRAS2, LDB2, LIMK1, MAPK1, MEL, MP1, MUC18, NRCAM, PBX3, RAB22A, RAB38, SNK, and STK4, indicating an association between chromosomal breakpoints and altered gene expression. Moreover, we also show that growth of all five cell lines can be significantly reduced by downregulating CDK6 gene expression with small interfering RNA (siRNA). Because the majority of these breakpoints have been reported previously in MM, our results support the idea of common mechanisms in this disease.
PMCID: PMC1501156  PMID: 15967107
Melanoma; oncogenic gene expression; translocation; CDK6; siRNA

Results 1-8 (8)