Tumor cell survival dependence on the DHX9 DExH-box helicase.

The NTP-dependent DExH/D-box helicase DHX9 is a key participant in a number of gene regulatory steps, including transcriptional, translational, and microRNA-mediated control, DNA replication and maintenance of genomic stability. DHX9 has also been implicated in tumor cell maintenance and drug response. Here we report that inhibition of DHX9 expression is lethal to human cancer cell lines and murine Eµ-Myc lymphomas. Using a novel conditional shDHX9 mouse model, we demonstrate that sustained and prolonged (6 months) suppression of DHX9 does not result in any deleterious effects at the organismal level. Body weight, blood biochemistry and histology of various tissues were comparable to control mice. Global gene expression profiling revealed that, although reduction of DHX9 expression resulted in multiple transcriptome changes, these were relatively benign and did not lead to any discernible phenotype. Our results demonstrate a robust tolerance for systemic DHX9 suppression in vivo and support the targeting of DHX9 as an effective and specific chemotherapeutic approach.

Nuclear translocation of IGF-1R via p150(Glued) and an importin-ß/RanBP2-dependent pathway in cancer cells.

Mounting evidence has shown that the insulin-like growth factor-1 receptor (IGF-1R) has critical roles in cancer cell growth. This has prompted pharmacological companies to develop agents targeting the receptor. Surprisingly, clinical trials using specific IGF-1R antibodies have, however, revealed disappointing results. Further understanding of the role of IGF-1R in cancer cells is therefore necessary for development of efficient therapeutic strategies. Recently, we showed that IGF-1R is sumoylated and translocated into the cell nucleus where it activates gene transcription. Several other studies have confirmed our findings and it has been reported that nuclear IGF-1R (nIGF-1R) has prognostic and predictive impact in cancer. To increase the understanding of IGF-1R in cancer cells, we here present the first study that proposes a pathway by which IGF-1R translocates into the cell nucleus. We could demonstrate that IGF-1R first associates with the dynactin subunit p150(Glued), which transports the receptor to the nuclear pore complex, where it co-localizes with importin-ß followed by association with RanBP2. Sumoylation of IGF-1R seems to be required for interaction with RanBP2, which in turn may serve as the SUMO E3 ligase. In the context of sumoylation, we provided evidence that it may favor nIGF-1R accumulation by increasing the stability of the receptor. Taken together, topographic and functional interactions between dynactin, importin-ß and RanBP2 are involved in nuclear translocation of IGF-1R. Our results provide new understanding of IGF-1R in cancer, which in turn may contribute to development of new therapeutic strategies.

Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3.

The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The messenger RNA (mRNA) cap-binding protein eIF4E is an oncoprotein that has an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eukaryotic translation initiation factor 4E (eIF4E) cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that transforming growth factor-beta (TGFß) induces eIF4E phosphorylation to promote the translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFß requires translational activation via the non-canonical TGFß signaling branch acting through eIF4E phosphorylation.

SS18-SSX fusion protein-induced Wnt/ß-catenin signaling is a therapeutic target in synovial sarcoma.

Synovial sarcoma is a high-grade soft tissue malignancy characterized by a specific reciprocal translocation t(X;18), which leads to the fusion of the SS18 (SYT) gene to one of three SSX genes (SSX1, SSX2 or SSX4). The resulting chimeric SS18-SSX protein is suggested to act as an oncogenic transcriptional regulator. Despite multimodal therapeutic approaches, metastatic disease is often lethal and the development of novel targeted therapeutic strategies is required. Several expression-profiling studies identified distinct gene expression signatures, implying a consistent role of Wnt/ß-catenin signaling in synovial sarcoma tumorigenesis. Here we investigate the functional and therapeutic relevance of Wnt/ß-catenin pathway activation in vitro and in vivo. Immunohistochemical analyses of nuclear ß-catenin and Wnt downstream targets revealed activation of canonical Wnt signaling in a significant subset of 30 primary synovial sarcoma specimens. Functional aspects of Wnt signaling including dependence of Tcf/ß-catenin complex activity on the SS18-SSX fusion proteins were analyzed. Efficient SS18-SSX-dependent activation of the Tcf/ß-catenin transcriptional complex was confirmed by TOPflash reporter luciferase assays and immunoblotting. In five human synovial sarcoma cell lines, inhibition of the Tcf/ß-catenin protein-protein interaction significantly blocked the canonical Wnt/ß-catenin signaling cascade, accompanied by the effective downregulation of Wnt targets (AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin) and the specific suppression of cell viability associated with the induction of apoptosis. In SYO-1 synovial sarcoma xenografts, administration of small molecule Tcf/ß-catenin complex inhibitors significantly reduced tumor growth, associated with diminished AXIN2 protein levels. In summary, SS18-SSX-induced Wnt/ß-catenin signaling appears to be of crucial biological importance in synovial sarcoma tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

Insulin-like growth factor-1 receptor acts as a growth regulator in synovial sarcoma.

Synovial sarcomas account for 5-10% of all soft tissue sarcomas and the majority of synovial sarcomas display characteristic t(X;18) translocations that result in enhanced transcription of the insulin-like growth factor-2 (IGF-2) gene. IGF-2 is an essential fetal mitogen involved in the pathogenesis of different tumours, leading to cellular proliferation and inhibition of apoptosis. Here we asked whether activation of IGF signalling is of functional importance in synovial sarcomas. We screened human synovial sarcomas for expression of IGF-2 and the phosphorylated IGF-1 receptor (IGF-1R), which mainly mediates the proliferative and anti-apoptotic effects of IGF-2. Since both the phosphatidylinositol 3'-kinase (PI3K)-AKT pathway and the MAPK signalling cascade are known to be involved in the transmission of IGF-1R signals, expression of phosphorylated (p)-AKT and p-p44/42 MAPK was additionally assessed. All tumours expressed IGF-2 and 78% showed an activated IGF-1R. All tumours were found to express p-AKT and 92% showed expression of activated p44/42 MAPK. To analyse the functional and potential therapeutic relevance of IGF-1R signalling, synovial sarcoma cell lines were treated with the IGF-1R inhibitor NVP-AEW541. Growth was impaired by the IGF-1R antagonist, which was consistently accompanied by a dose-dependent reduction of phosphorylation of AKT and p44/42 MAPK. Functionally, inhibition of the receptor led to increased apoptosis and diminished mitotic activity. Concurrent exposure of selected cells to NVP-AEW541 and conventional chemotherapeutic agents resulted in positive interactions. Finally, synovial sarcoma cell migration was found to be dependent on signals transmitted by the IGF-1R. In summary, our data show that the IGF-1R might represent a promising therapeutic target in synovial sarcomas.

Picropodophyllin induces downregulation of the insulin-like growth factor 1 receptor: potential mechanistic involvement of Mdm2 and beta-arrestin1.

The insulin-like growth factor 1 receptor (IGF-1R) is crucial for growth and survival of malignant cells. Experience in targeting IGF-1R in cancer models has shown that strategies promoting downregulation of the receptor are much more efficient in inducing apoptosis than those inhibiting the IGF-1R activity. Recently, we found that the cyclolignan picropodophyllin (PPP) inhibits phosphorylation of IGF-1R and activation of downstream signaling without interfering with the highly homologous insulin receptor (IR). Furthermore, PPP treatment caused strong regression of tumor grafts and prolonged survival of animals with systemic tumor disease. Here we demonstrate that PPP also downregulates the IGF-1R, whereas the IR and several other receptors were not affected. PPP-induced IGF-1R downregulation required expression of the MDM2 E3 ligase, which recently was found to ubiquitinate and cause degradation of the IGF-1R. In addition knockdown of beta-arrestin1, the adaptor molecule known to bridges MDM2 and IGF-1R, prevented downregulation of the receptor and significantly decreased PPP-induced cell death. All together these data suggest that PPP downregulates IGF-1R by interfering with the action of beta-arrestin1/MDM2 as well as the achieved receptor downregulation contributes to the apoptotic effect of PPP.

Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif.

AIMS/HYPOTHESIS: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. MATERIALS AND METHODS: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. RESULTS: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. CONCLUSIONS/INTERPRETATION: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Role of insulin-like growth factor 1 receptor signalling in cancer.

The insulin-like growth factor (IGF-1) signalling is highly implicated in cancer. In this signalling the IGF-1 receptor (IGF-1R) is unquestionable, the predominating single factor. IGF-1R is crucial for tumour transformation and survival of malignant cell, but is only partially involved in normal cell growth. This is in part due to the interactions with oncogenes. Recent findings suggest a close interplay with the p53/MDM2 pathway. Disturbances in components in the p53/MDM2/IGF-1R network may cause IGF-1R upregulation and growth advantage for the cancer cell. Targeting of IGF-1R is more and more seen as a promising option for future cancer therapy. Single chain antibodies and small molecules with selective effects on IGF-1R dependent malignant growth are of particular interest. Forthcoming clinical trials are welcome and will indeed be the only way to evaluate the impact of IGF-1R targeting in human cancer.

The insulin-like growth factor-1 receptor inhibitor PPP produces only very limited resistance in tumor cells exposed to long-term selection.

The cyclolignan PPP was recently demonstrated to inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), without affecting the highly homologous insulin receptor. In addition, PPP caused complete regression of xenografts derived from various types of cancer. These data highlight the use of this compound in cancer treatment. However, a general concern with antitumor agents is development of resistance. In light of this problem, we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select 10 malignant cell lines, with documented IGF-1R expression and apoptotic responsiveness to PPP treatment (IC50s less than 0.1 microM), only two survived an 80-week selection but could only tolerate maximal PPP doses of 0.2 and 0.5 microM, respectively. Any further increase in the PPP dose resulted in massive cell death. These two cell lines were demonstrated not to acquire any essential alteration in responsiveness to PPP regarding IGF-1-induced IGF-1R phosphorylation. Neither did they exhibit any increase in expression of the multidrug resistance proteins MDR1 or MRP1. Consistently, they did not exhibit decreased sensitivity to conventional cytostatic drugs. Rather, the sensitivity was increased. During the first half of the selection period, both cell lines responded with a temporary and moderate increase in IGF-1R expression, which appeared to be because of an increased transcription of the IGF-1R gene. This increase in IGF-1R might be necessary to make cells competent for further selection but only up to a PPP concentration of 0.2 and 0.5 microM. In conclusion, malignant cells develop no or remarkably weak resistance to the IGF-1R inhibitor PPP.

Role of insulin-like growth factor 1 receptor signalling in cancer.

The insulin-like growth factor (IGF-1) signalling is highly implicated in cancer. In this signalling the IGF-1 receptor (IGF-1R) is unquestionable, the predominating single factor. IGF-1R is crucial for tumour transformation and survival of malignant cell, but is only partially involved in normal cell growth. This is in part due to the interactions with oncogenes. Recent findings suggest a close interplay with the p53/MDM2 pathway. Disturbances in components in the p53/MDM2/IGF-1R network may cause IGF-1R upregulation and growth advantage for the cancer cell. Targeting of IGF-1R is more and more seen as a promising option for future cancer therapy. Single chain antibodies and small molecules with selective effects on IGF-1R dependent malignant growth are of particular interest. Forthcoming clinical trials are welcome and will indeed be the only way to evaluate the impact of IGF-1R targeting in human cancer.

Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission.

The fine tuning of both glutamatergic and GABAergic neurotransmission is to a large extent dependent upon optimal function of astrocytic transport processes. Thus, glutamate transport in astrocytes is mandatory to maintain extrasynaptic glutamate levels sufficiently low to prevent excitotoxic neuronal damage. In GABA synapses hyperactivity of astroglial GABA uptake may lead to diminished GABAergic inhibitory activity resulting in seizures. As a consequence of this the expression and functional activity of astrocytic glutamate and GABA transport is regulated in a number of ways at transcriptional, translational and post-translational levels. This opens for a number of therapeutic strategies by which the efficacy of excitatory and inhibitory neurotransmission may be manipulated.

Long-chain CoA esters activate human pancreatic beta-cell KATP channels: potential role in Type 2 diabetes.

AIMS/HYPOTHESIS: The ATP-regulated potassium (KATP) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the KATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent KATP channel activators in rodents, as studied in inside-out patches. METHODS: In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. RESULTS: Human KATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC50 of 0.7+/-0.1 micromol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell KATP channel currents increased approximately six-fold following addition of 1 micro mol/l LC-CoA ester. The presence of 1 micro mol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5+/-0.2 nS to 2.5+/-1.3 nS. CONCLUSION/INTERPRETATION: Taken together, these results show that LC-CoA esters are potent activators of the KATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate KATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions.

Gain of 17q in malignant fibrous histiocytoma is associated with a longer disease-free survival and a low risk of developing distant metastasis.

In this study, a panel of 39 primary malignant fibrous histiocytomas (MFH) of high malignancy grade were characterised for chromosomal alterations. The results were then evaluated in relation to the survival and the occurrence of recurrent disease during follow-up for an average period of 63 months. Chromosomal alterations detected by comparative genomic hybridisation (CGH) were recorded in 37 of the 39 cases analysed. The most frequent CGH abnormalities were gains of 17p, 20q, 16p, 17q, 1p31, 7q21, and 9cen-q22, and losses of 9p21-pter and 13q21-22. However, the patterns of CGH imbalances did not allow the identification of a single common event, suggesting that the key initiating event(s) is not a numerical imbalance. Patients with tumours harbouring a gain of 17q showed significantly longer overall and disease-free survival (P=0.001 and 0.008) as well as lower frequency of metastasis (P=0.018) during follow-up. Taken together, the findings suggest that the clinical outcome of MFH is associated with the genetic profiles of the primary tumours. Importantly, a subgroup of MFHs characterised by a low risk of developing metastasis and local recurrence is recognised based on their frequent gains of 17q by CGH.

Polariton parametric amplifier pump dynamics in the coherent regime.

We studied the pump coherent dynamics in a II-VI microcavity parametric amplifier, using angle-resolved four-wave mixing. The polariton parametric amplification is found to result in a strong quenching and saturation of the pump coherence lifetime above the threshold. For the polariton scattering processes that remain below the amplification threshold, we find an angle-dependent collision broadening associated with the efficiency of the polariton scattering towards the excitonic reservoir.

Characterization of lacZ complementation deletions using membrane receptor dimerization.

We describe the screening of lacZ deletions in mammalian cells and the discovery of a novel pair of lacZ deletions that can undergo alpha-complementation only when they are fused to peptides that interact with each other. The two lacZ deletions, delta N 11-75 and delta C 82-1023, were first characterized by fusing to two small interacting peptides and were then further analyzed by fusing to three membrane receptors (G protein-coupled receptors alpha 2cAR and D2DRL and receptor tyrosine kinase insulin receptor) that were known to form homodimers in the membrane. Histochemical and quantitative FACS assays demonstrated that the novel deletions have much lower level of association with each other, thus offering a much lower background in monitoring membrane protein interactions compared to previously published lacZ deletions. Furthermore, our method has the exciting potential to monitor simultaneously membrane receptor dimerization and localization to the cell surface of living cells.

Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker.

The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT-PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a 'therapeutic window' to be exploited in cancer immunotherapy.

Assessment and management of risks arising from exposure to cadmium in fertilisers--II.

A preliminary, illustrative human health risk assessment of exposure to cadmium in phosphate fertilisers was performed using typical UK data and a protocol previously developed for application by individual Member States in the European Union. The risk assessment indicated that the for the most pessimistic population exposures characterised by both extreme (97th percentile) cereal and potato consumption and high susceptibility to cadmium uptake, the estimated dose was under the WHO Provisional Tolerable Weekly Intake (PTWI) for fertiliser cadmium concentrations ranging from 15 to 100 mg Cd/kg P2O5 applied over 100 years. However, the low margin of safety for high risk groups and the uncertainties inherent in the overall risk assessment suggested that a prudent risk management strategy would involve maintenance of low levels of cadmium in fertilisers and/or conditions that permitted low accumulation of cadmium in soils. On this basis, two main risk reduction measures were developed and assessed: (a) imposition of limits on cadmium concentration in fertilisers; and (b) imposition of charges on levels of cadmium in phosphate fertilisers. An assessment of the economic impact of these risk reduction measures indicated that, at all price elasticities, the most significant impact in terms of changes in demand and changes in consumer expenditure on phosphate fertilisers will be seen with cadmium charges where no thresholds are defined. The impact on the consumer (i.e. farmer) will be an increase in spending of approximately US $4000 per year, which is considered significant, accompanied by a decrease in demand above 20%. If a threshold is set at 60 mg Cd/kg P2O5, the impact is significantly reduced, but stays relatively high compared to the other options. The analysis also indicates that the use of low-cadmium rock is the low cost option. At a likely rock price increase of approximately 5% and assuming a likely price elasticity of -0.2, the yearly costs to farmers will be approximately US $82 which is considered a minimal impact. In the worst case scenario (elasticity of -0.6 and a 10% increase in rock prices), the increase in spending by farmers will be of 3.9% or US $221 which is also considered to represent a minimal impact. At similar price elasticities, the use of decadmation technologies is predicted to be more costly than the use of low-cadmium rock but this option can still be considered as having a minimal impact on the consumer (increase in expenditure of 1.9-13.3% or US $106-748).

Characterization of hydrophobic prenyl groups of isoprenylated proteins in human cancer cells.

Extensive protease digestion of delipidated [3H]mevalonate (MVA)-labeled proteins, followed by HPLC separation of the products, is one approach to identify and study prenyl cysteines. Using this methodology three major [3H]MVA-labeled peaks appeared. Two of them represent farnesyl cysteine (FC) and geranylgeranyl cysteine (GGC). The third peak represents unknown products that are considerably more hydrophobic than FC and GGC, here designated HPC. Previously, we provided evidence that cysteine residues may also be modified by dolichyl groups. Dolichyl cysteines (DolC) belong to HPC. However, as shown in the present study, DolC only represents a minor portion of HPC. Data obtained from different sets of experiments, including [3H]GGOH-labeling and use of prenyl transferase inhibitors, suggest that HPC mainly involves CXC or CC residues with double-linked GG groups. In turn this points to the possibility that proteins modified by double GG groups are quite common, and may probably involve other proteins than the rab family of GTPases.