Regulation of Transcriptional Activity of Merkel Cell Polyomavirus Large T-Antigen by PKA-Mediated Phosphorylation.

Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.

The Merkel Cell Polyomavirus T-Antigens and IL-33/ST2-IL1RAcP Axis: Possible Role in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is a causal factor in Merkel cell carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT) and small T-antigen (sT). Cytokines produced by tumor cells play an important role in cancer pathogenesis, and viruses affect their expression. Therefore, we compared human cytokine and receptor transcript levels in virus positive (V+) and virus negative (V-) MCC cell lines. Increased expression of IL-33, a potent modulator of tumor microenvironment, was observed in V+ MCC cell lines when compared to V- MCC-13 cells. Transient transfection studies with luciferase reporter plasmids demonstrated that LT and sT stimulated IL-33, ST2/IL1RL1 and IL1RAcP promoter activity. The induction of IL-33 expression was confirmed by transfecting MCC-13 cells with MCPyV LT. Furthermore, recombinant human cytokine domain IL-33 induced activation of MAP kinase and NF-κB pathways, which could be blocked by a ST2 receptor antibody. Immunohistochemical analysis demonstrated a significantly stronger IL-33, ST2, and IL1RAcP expression in MCC tissues compared to normal skin. Of interest, significantly higher IL-33 and IL1RAcP protein levels were observed in MCC patient plasma compared to plasma from healthy controls. Previous studies have demonstrated the implication of the IL-33/STL2 pathway in cancer. Because our results revealed a T-antigens-dependent induction of the IL-33/ST2 axis, IL-33/ST2 may play a role in the tumorigenesis of MCPyV-positive MCC. Therefore, neutralizing the IL-33/ST2 axis may present a novel therapeutic approach for MCC patients.

Reciprocal transactivation of Merkel cell polyomavirus and high-risk human papillomavirus promoter activities and increased expression of their oncoproteins.

BACKGROUND: Approximately 15% of human cancers are attributed to viruses. Numerous studies have shown that high-risk human polyomaviruses (HR-HPV) and Merkel cell polyomavirus (MCPyV) are two human tumor viruses associated with anogenetal and oropharyngeal cancers, and with Merkel cell carcinoma, respectively. MCPyV has been found in HR-HPV positive anogenetal and oropharyngeal tumors, suggesting that MCPyV can act as a co-factor in HR-HPV induced oncogenesis. This prompted us to investigate whether the oncoproteins large T-antigen (LT) and small antigen (sT) of MCPyV could affect the transcriptional activity HPV16 and HPV18 and vice versa whether HPV16 and HPV18 E6 and E7 oncoproteins affected the expression of MCPyV LT and sT. Reciprocal stimulation of these viral oncoproteinscould enhance the oncogenic processes triggered by these tumor viruses. METHODS: Transient co-transfection studies using a luciferase reporter plasmid with the long control region of HPV16 or HPV18, or the early or late promoter of MCPyV and expression plasmids for LT and sT, or E6 and E7, respectively were performed in the HPV-negative cervical cancer cell line C33A, in the keratinocyte cell line HaCaT, and in the oral squamous cell carcinoma cell line HSC-3. Transfections were also performed with deletion mutants of all these promoters and with mutants of all four oncoproteins. Finally, the effect of E6 and E7 on LT and sT expression in the MCPyV-positive Merkel cell carcinoma cell line WaGa and the effect of LT and sT on the expression of E6 and E7 was monitored by Western blotting. RESULTS: LT and sT stimulated the transcriptional activity of the HPV16 and HPV18 LCR and v.v. E6 and E7 potentiated the MCPyV early and late promoter in all cell lines. Induction by E6 and E7 was p53- and pRb-independent, and transactivation by LT did not require DNA binding, nuclear localization and HSC70/pRb interaction, whereas sT stimulated the HPV16/18 LCR activity in a PP2A- and DnaJ-independent manner. CONCLUSIONS: These results indicate that the co-infection of MCPyV may act as a co-factor in the initiation and/or progression of HPV-induced cancers.

Promoter activity of Merkel cell Polyomavirus variants in human dermal fibroblasts and a Merkel cell carcinoma cell line.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is a human polyomavirus that establishes a life-long harmless infection in most individuals, with dermal fibroblasts believed to be the natural host cell. However, this virus is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. Several MCPyV variants with polymorphism in their promoter region have been isolated, but it is not known whether these differences affect the biological properties of the virus. METHODS: Using transient transfection studies in human dermal fibroblasts and the MCC cell line MCC13, we compared the transcription activity of the early and late promoters of the most commonly described non-coding control region MCPyV variant and six other isolates containing specific mutation patterns. RESULTS: Both the early and late promoters were significantly stronger in human dermal fibroblasts compared with MCC13 cells, and a different promoter strength between the MCPyV variants was observed. The expression of full-length large T-antigen, a viral protein that regulates early and late promoter activity, inhibited early and late promoter activities in both cell lines. Nonetheless, a truncated large T-antigen, which is expressed in virus-positive MCCs, stimulated the activity of its cognate promoter. CONCLUSION: The promoter activities of all MCPyV variants tested was stronger in human dermal fibroblasts, a cell line that supports viral replication, than in MCC13 cells, which are not permissive for MCPyV. Truncated large T-antigen, but not full-length large T-antigen stimulated viral promoter activity. Whether, the difference in promoter strength and regulation by large T-antigen may affect the replication and tumorigenic properties of the virus remains to be determined.

Rho-associated kinase is a therapeutic target in neuroblastoma.

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3ß-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model.

BACKGROUND: Immunochemotherapy, the combined use of immunotherapy and chemotherapy, has demonstrated great promise in several cancers. LTX-315 is an oncolytic peptide with potent immunomodulatory properties designed for the local treatment of solid tumors. By inducing rapid immunogenic cell death through the release of danger-associated molecular pattern molecules (DAMPs), LTX-315 is capable of reshaping the tumor microenvironment, turning "cold" tumors "hot" through a significant increase in tumor-infiltrating lymphocytes. METHODS: We investigated the potential of LTX-315 to be used in combination with standard-of-care chemotherapy (doxorubicin, brand name CAELYX®) against triple-negative breast cancer in an orthotopic 4 T1 mammary fat pad model. Tumor growth curves were compared using one-way ANOVA analysis of variance and Tukey's multiple comparisons test, and animal survival curves were compared using the log-rank (Mantel-Cox) test. We considered p values ≤0.05 to indicate statistical significance. RESULTS: We found that LTX-315 displayed a strong additive antitumoral effect when used in combination with CAELYX®, and induced immune-mediated changes in the tumor microenvironment, followed by complete regression in the majority of animals treated. Furthermore, imaging techniques and histological examination showed that the combination induced strong local necrosis, followed by an increase in the infiltration of CD4+ and CD8+ immune cells into the tumor parenchymal tissue. CONCLUSIONS: Our data demonstrate that LTX-315 is a promising combination partner with CAELYX® for the treatment of triple-negative breast cancer.

MicroRNAs as Potential Biomarkers in Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive type of skin cancer associated with a poor prognosis. This carcinoma was named after its presumed cell of origin, the Merkel cell, which is a mechanoreceptor cell located in the basal epidermal layer of the skin. Merkel cell polyomavirus seems to be the major causal factor for MCC because approximately 80% of all MCCs are positive for viral DNAs. UV exposure is the predominant etiological factor for virus-negative MCCs. Intracellular microRNA analysis between virus-positive and virus-negative MCC cell lines and tumor samples have identified differentially expressed microRNAs. Comparative microRNA profiling has also been performed between MCCs and other non-MCC tumors, but not between normal Merkel cells and malignant Merkel cells. Finally, Merkel cell polyomavirus encodes one microRNA, but its expression in virus-positive MCCs is low, or non-detectable or absent, jeopardizing its biological relevance in tumorigenesis. Here, we review the results of microRNA studies in MCCs and discuss the potential application of microRNAs as biomarkers for the diagnosis, progression and prognosis, and treatment of MCC.

Secretomic analysis of extracellular vesicles originating from polyomavirus-negative and polyomavirus-positive Merkel cell carcinoma cell lines.

Extracellular vesicles or exosomes constitute an evolutionarily conserved mechanism of intercellular signaling. Exosomes are gaining an increasing amount of attention due to their role in pathologies, including malignancy, their importance as prognostic and diagnostic markers, and their potential as a therapeutic tool. Merkel cell carcinoma (MCC) is an aggressive form of skin cancer with a poor prognosis. Because an effective systemic treatment for this cancer type is currently not available, an exosome-based therapy was proposed. However, comprehensive secretome profiling has not been performed for MCC. To help unveil the putative contribution of exosomes in MCC, we studied the protein content of MCC-derived exosomes. Since approximately 80% of all MCC cases contain Merkel cell polyomavirus (MCPyV), the secretomes of two MCPyV-negative and two MCPyV-positive MCC cell lines were compared. We identified with high confidence 164 exosome-derived proteins common for all four cell lines that were annotated in ExoCarta and Vesiclepedia databases. These include proteins implicated in motility, metastasis and tumor progression, such as integrins and tetraspanins, intracellular signaling molecules, chaperones, proteasomal proteins, and translation factors. Additional virus-negative and virus-positive MCC cell lines should be examined to identify highly representative exosomal proteins that may provide reliable prognostic and diagnostic biomarkers, as well as targets for treatment in the future. Data are available via ProteomeXchange with identifier PXD004198.

The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis.

BACKGROUND: Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood. METHODS: Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice. RESULTS: FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice. CONCLUSION: Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma.

Effect of Perioperative Dexamethasone and Different NSAIDs on Anastomotic Leak Risk: A Propensity Score Analysis.

BACKGROUND: Perioperative use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with risk of anastomotic leak (AL). However, concomitant use of other drugs could infer a bias in risk assessment. Thus, we aimed to interrogate the risk of AL associated with NSAIDs and steroids used perioperatively. METHODS: This study includes a consecutive series of patients having surgery involving an intestinal anastomosis from Jan 2007 to Dec 2009. Data records included demographic, perioperative, and surgical characteristics; AL rates; and use of NSAIDs and steroids. Risk of leak were estimated using unadjusted and multivariable (propensity score)-adjusted logistic regression models and reported as odds ratios (ORs). RESULTS: A total of 376 patients underwent 428 operations of which 67 (15.7 %) had AL. With no medication receivers as reference, the OR for leak when adjusted for age, sex, and propensity score was 1.07 (p = 0.92) for ketorolac, 1.63 (p = 0.31) for diclofenac and 0.41 (p = 0.19) for dexamethasone. Risk was increased for malignancy (OR 1.88, p = 0.023), use of a vasopressor (OR 2.52, p = 0.007), blood transfusions (OR 1.93, p = 0.026), and regular use of steroids (OR 7.57, p = 0.009). CONCLUSIONS: Other factors than perioperative drugs are crucial for risk of AL. Perioperative dexamethasone was associated with a nonsignificant reduced risk of AL.

Human articular chondrocytes express functional leukotriene B4 receptors.

Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho-specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro-inflammatory cytokines, growth factors and metalloproteinases by LTB4-stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4-promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high-affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified.

Complete regression and systemic protective immune responses obtained in B16 melanomas after treatment with LTX-315.

Malignant melanoma is the most aggressive and deadliest form of skin cancer due to its highly metastatic potential, which calls for new and improved therapies. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising potential as novel anticancer agents. Structure-activity relationship studies on the CAP bovine lactoferricin allowed us to de novo design short chemically modified lytic anticancer peptides. In the present study, we investigated the in vivo antitumor effects of LTX-315 against intradermally established B16 melanomas in syngeneic mice. Intratumoral administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by complete regression of the tumor in the majority of the animals. LTX-315 induced the release of danger-associated molecular pattern molecules such as the high mobility group box-1 protein in vitro and the subsequent upregulation of proinflammatory cytokines such as interleukin (IL) 1ß, IL6 and IL18 in vivo. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells both intradermally and intravenously. Together, our data indicate that intratumoral treatment with LTX-315 can provide local tumor control followed by protective immune responses and has potential as a new immunotherapeutic agent.

LTX-315 and adoptive cell therapy using tumor-infiltrating lymphocytes generate tumor specific T cells in patients with metastatic soft tissue sarcoma.

LTX-315 is an oncolytic peptide that elicits both local and systemic immune responses upon intratumoral injection. In the present pilot trial, we treated patients with metastatic soft tissue sarcoma with the combination of LTX-315 and adoptive T-cell therapy using in vitro expanded tumor-infiltrating lymphocytes. Six heavily pretreated patients were included in the trial and treated with LTX-315 of which four patients proceeded to adoptive T-cell therapy. Overall, the treatment was considered safe with only expected and manageable toxicity. The best overall clinical response was stable disease for 208 days, and in this patient, we detected tumor-reactive T cells in the blood that lasted until disease progression. In three patients T-cell reactivity against in silico predicted neoantigens was demonstrated. Additionally, de novo T-cell clones were generated and expanded in the blood following LTX-315 injections. In conclusion, this pilot study provides proof that it is feasible to combine LTX-315 and adoptive T-cell therapy, and that this treatment can induce systemic immune responses that resulted in stabilization of the disease in sarcoma patients with otherwise progressive disease. Further optimization of the treatment protocol is warranted to increase clinical activity. ClinicalTrials.gov Identifier: NCT03725605.

LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Low-dose aspirin delays an inflammatory tumor progression in vivo in a transgenic mouse model of neuroblastoma.

Tumor-associated inflammation is a driving force in several adult cancers and intake of low-dose aspirin has proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. The present study employs the transgenic TH-MYCN mouse model for neuroblastoma (NB) to evaluate inflammatory patterns paralleling tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Spontaneously arising abdominal tumors were monitored for tumor-associated inflammation ex vivo at various stages of disease and homozygous mice received daily low-dose aspirin (10mg/kg) using oral gavage or no treatment, from 4.5 to 6 weeks of age. Using flow cytometry, a transition from an adaptive immune response predominated by CD8(+) T cell in early neoplastic lesions, towards enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells, dendritic cells and tumor-associated macrophages, was detected during tumor progression. An M1 to M2 transition of tumor-associated macrophages was demonstrated, paralleled by a deterioration of dendritic cell status. Treatment with low-dose aspirin to mice homozygous for the TH-MYCN transgene significantly reduced the tumor burden (P < 0.01), the presence of tumor-associated cells of the innate immune system (P < 0.01), as well as the intratumoral expression of transforming growth factor-ß, thromboxane A2 (P < 0.05) and prostaglandin D2 (P < 0.01). In conclusion, tumor-associated inflammation appears as a potential therapeutic target in NB and low-dose aspirin reduces tumor burden in the TH-MYCN transgenic mouse model of NB, hence warranting further studies on aspirin in high-risk NB.

ERBB3 is a marker of a ganglioneuroblastoma/ganglioneuroma-like expression profile in neuroblastic tumours.

BACKGROUND: Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4). METHODS: Expression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses. RESULTS: We identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132-240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells. CONCLUSIONS: Conclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted.

Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target?

Neuroblastoma is the most common and deadly tumor of childhood, where new therapy options for patients with high-risk disease are highly warranted. Human cytomegalovirus (HCMV) is prevalent in the human population and has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumor cell immune evasion. Here we show that the majority of primary neuroblastomas and neuroblastoma cell lines are infected with HCMV. Our analysis show that HCMV immediate-early protein was expressed in 100% of 36 primary neuroblastoma samples, and HCMV late protein was expressed in 92%. However, no infectious virus was detected in primary neuroblastoma tissue extracts. Remarkably, all six human neuroblastoma cell lines investigated contained CMV DNA and expressed HCMV proteins. HCMV proteins were expressed in neuroblastoma cells expressing the proposed stem cell markers CD133 and CD44. When engrafted into NMRI nu/nu mice, human neuroblastoma cells expressed HCMV DNA, RNA and proteins but did not produce infectious virus. The HCMV-specific antiviral drug valganciclovir significantly reduced viral protein expression and cell growth both in vitro and in vivo. These findings indicate that HCMV is important for the pathogenesis of neuroblastoma and that anti-viral therapy may be a novel adjuvant treatment option for children with neuroblastoma.

Phosphorylation of Human Polyomavirus Large and Small T Antigens: An Ignored Research Field.

Protein phosphorylation and dephosphorylation are the most common post-translational modifications mediated by protein kinases and protein phosphatases, respectively. These reversible processes can modulate the function of the target protein, such as its activity, subcellular localization, stability, and interaction with other proteins. Phosphorylation of viral proteins plays an important role in the life cycle of a virus. In this review, we highlight biological implications of the phosphorylation of the monkey polyomavirus SV40 large T and small t antigens, summarize our current knowledge of the phosphorylation of these proteins of human polyomaviruses, and conclude with gaps in the knowledge and a proposal for future research directions.

Microsomal prostaglandin E synthase 1 determines tumor growth in vivo of prostate and lung cancer cells.

There is strong evidence for a role of prostaglandin E(2) (PGE(2)) in cancer cell proliferation and tumor development. In PGE(2) biosynthesis, cyclooxygenases (COX-1/COX-2) convert arachidonic acid to PGH(2), which can be isomerized to PGE(2) by microsomal PGE-synthase-1 (MPGES-1). The human prostate cancer cell line DU145 expressed high amounts of MPGES-1 in a constitutive manner. MPGES-1 expression also was detectable in human prostate cancer tissues, where it appeared more abundant compared with benign hyperplasia. By using shRNA, we established stable and practically complete knockdown of MPGES-1, both in DU145 cells with high constitutive expression and in the non-small cell lung cancer cell line A549, where MPGES-1 is inducible. For microsomes prepared from knockdown clones, conversion of PGH(2) to PGE(2) was reduced by 85-90%. This resulted in clear phenotypic changes: MPGES-1 knockdown conferred decreased clonogenic capacity and slower growth of xenograft tumors (with disintegrated tissue structure) in nude mice. For DU145 cells, MPGES-1 knockdown gave increased apoptosis in response to genotoxic stress (adriamycin), which could be rescued by exogenous PGE(2). The results suggest that MPGES-1 is an alternative therapeutic target in cancer cells expressing this enzyme.