Abstract: A large-scale gene expression study of melanoma metastases was performed to identify genes involved in late-stage tumor progression. Overall 248 genes, out of more than 47,000 tested, are differentially expressed when comparing peripheral areas (invasion front) with central tumor areas of melanoma metastases. As determined by gene ontology analysis, members of the STAT signaling pathway show significant enrichment. In particular, Stat1 is highly expressed in peripheral compared with central tumor areas. In line with this, stable knockdown of STAT1 in metastatic melanoma cells significantly impairs their migratory and invasive capacity in wounding and matrigel assays. Moreover, STAT1 knockdown affects the metastatic behavior of melanoma cells in a mouse model of melanoma metastasis. Taken together, these data suggest that Stat1 might play a role in late-stage melanoma progression. Interference with the Stat1 pathway could have therapeutic implications for late-stage melanoma patients.
Abstract: The development and progression of malignant tumours are often due to deregulated cell cycle control involving a plethora of different molecules. Among these, tumour suppressor proteins like p53 play a crucial role. p53 induces 14-3-3sigma, a multifunctional protein kinase inhibitor, centrally involved in cell cycle control and DNA damage repair after genotoxic stress. Recently, it has been shown that 14-3-3sigma is epigenetically silenced in a variety of tumours, which might contribute to tumour development and progression via impaired cell cycle control. In addition, p53, its inhibitor MDM2 and 14-3-3sigma form a signalling module in which 14-3-3sigma positively regulates the activity of p53 through feedback regulation. Here we present a mathematical model integrating the effects of 14-3-3sigma gene silencing, the dynamics of 14-3-3sigma induction and compartmentalisation by genotoxic stress and the role of interacting molecules p53 and MDM2. In vitro experiments with different melanoma cell lines were performed and our mathematical model was subjected to computer simulations to analyse different scenarios of activation depending on gene methylation status and DNA damage levels. Our analysis indicates that 14-3-3sigma expression is silenced by high gene methylation, but also that strong stimulation is necessary to induce 14-3-3sigma expression in cases of intermediate levels of gene methylation. More intriguingly, the model suggests that epigenetic silencing of 14-3-3sigma affects p53 dynamics in a synergistic way, such that the accumulative effect of partial downregulation of p53 expression and reduction of its nuclear fraction could affect drastically the activity of p53 as a transcription factor.
Abstract: In the present report, the possible role of a recently described functional polymorphism of the osteopontin (OPN) promoter at position -443 (-443T/C) for OPN expression in melanoma cells was addressed. As shown by real-time PCR analysis, melanoma metastases that were homozygous for the -443C allele expressed significantly higher levels of OPN mRNA compared with those that were either heterozygous (-443T/C) or homozygous for the -443T allele. In line with this, immunoblotting showed significantly enhanced baseline and bFGF-induced OPN protein expression in melanoma cell lines which were homozygous for the -443C allele, compared with cell lines with other allelic variants. Similar results were obtained in in vitro luciferase assays. Chromatin immunoprecipitation (ChIP) demonstrated binding of c-Myb to the -443 OPN promoter region, and binding could significantly be enhanced after bFGF stimulation. Moreover, as shown by electrophoretic mobility shift assays (EMSA), recombinant DNA-binding domain of c-Myb bound in a sequence-specific manner to this region. Finally, the role of c-Myb for OPN gene regulation via binding to the -443 promoter region could be further substantiated by ectopic overexpression of c-Myb in melanoma cells, using different reporter gene constructs. Taken together, it is demonstrated that the -443 promoter region exerts influence on OPN gene expression in melanoma cells, and differential binding of c-Myb transcription factor appears to play a major role in this process. These findings might be a feasible explanation for different OPN expression levels in metastatic tumors and may also have prognostic and therapeutic relevance.
Abstract: BACKGROUND: The family of 14-3-3 proteins plays an important role in cancer biology by interfering with intracellular signalling pathways and cell cycle checkpoints. The 14-3-3sigma isoform acts as a tumor suppressor and is often inactivated during tumor development. RESULTS: Here, we demonstrate enhanced CpG methylation of the 14-3-3sigma gene in lymph node and cutaneous melanoma metastases compared with primary tumors, associated with dramatically reduced mRNA expression. In line with this, treatment of different metastatic melanoma cell lines with 5-aza-2'-deoxycytidine (5-Aza-CdR), a potent inhibitor of cytosine methylation, significantly induces 14-3-3sigma protein expression. Additional treatment with histone deacetylase inhibitor 4-phenylbutyric acid (Pba) further enhances 14-3-3sigma expression. Induction of 14-3-3sigma expression by 5-Aza-CdR/Pba treatment leads to almost complete inhibition of cell proliferation, with cells predominantly arrested in G2-M. The antiproliferative effect of 5-Aza-CdR/Pba was reversed in 14-3-3sigma knockdown cells. Similarly, melanoma cell lines stably overexpressing 14-3-3sigma show dramatically reduced cell proliferation rates. Moreover, synchronous 14-3-3sigma stably overexpressing cells do not progress through cell cycle, but display a permanent increase in the population of 4n DNA containing cells. Interestingly, overexpression of 14-3-3sigma induces senescence of melanoma cells and is involved in melanoma cell senescence under genotoxic stress. Finally, 14-3-3sigma knockdown supports migratory capacity of melanoma cells in vitro, while 14-3-3sigma overexpression has opposing effects. CONCLUSION: Taken together, the present report indicates that epigenetic silencing of 14-3-3sigma might contribute to tumor progression in malignant melanoma via loss of cell cycle control, impaired cellular senescence program and support of migratory capacity.
Abstract: Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3(-/-)) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3(-/-) mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrP(Sc) and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3(-/-) compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrP(Sc) in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrP(Sc).
Abstract: A microRNA expression screen was performed analyzing 157 different microRNAs in laser-microdissected tissues from benign melanocytic nevi (n = 10) and primary malignant melanomas (n = 10), using quantitative real-time PCR. Differential expression was found for 72 microRNAs. Members of the let-7 family of microRNAs were significantly downregulated in primary melanomas as compared with benign nevi, suggestive for a possible role of these molecules as tumor suppressors in malignant melanoma. Interestingly, similar findings had been described for lung and colon cancer. Overexpression of let-7b in melanoma cells in vitro downregulated the expression of cyclins D1, D3, and A, and cyclin-dependent kinase (Cdk) 4, all of which had been described to play a role in melanoma development. The effect of let-7b on protein expression was due to targeting of 3'-untranslated regions (3'UTRs) of individual mRNAs, as exemplified by reporter gene analyses for cyclin D1. In line with its downmodulating effects on cell cycle regulators, let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells. Taken together, these findings not only point to new regulatory mechanisms of early melanoma development, but also may open avenues for future targeted therapies of this tumor.
Abstract: Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (deltaTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. DeltaTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73beta) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73beta significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73beta displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models (p < 0.05). In summary, our data show that Ad-mediated TA-p73beta gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo, suggesting a new chemosensitization strategy for malignant melanomas.
Abstract: Prion-induced chronic neurodegeneration has a substantial inflammatory component, and the activation of glia cells may play an important role in disease development and progression. However, the functional contribution of cytokines to the development of the gliosis in vivo was never systematically studied. We report here that the expression of interleukin-1beta (IL-1beta), IL-1beta-converting enzyme, and IL-1 receptor type 1 (IL-1RI) is up-regulated in a murine scrapie model. The scrapie-induced gliosis in IL-1RI(-/-) mice was characterized by an attenuated activation of astrocytes in the asymptomatic stage of the disease and a reduced expression of CXCR3 ligands. Furthermore, the accumulation of the misfolded isoform of the prion protein PrP(Sc) was significantly delayed in the IL-1RI(-/-) mice. These observations indicate that IL-1 is a driver of the scrapie-associated astrocytosis and possibly the accompanying amyloid deposition. In addition, scrapie-infected IL-1RI-deficient (IL-1RI(-/-)) mice showed a delayed disease onset and significantly prolonged survival times suggesting that an anti-inflammatory therapeutical approach to suppress astrocyte activation and/or glial IL-1 expression may help to delay disease onset in established prion infections of the central nervous system.
Abstract: The susceptibility of sheep to scrapie infection is influenced by prion gene alleles, which are modulated by polymorphic variations corresponding to amino acid positions 136, 154 and 173 of the prion protein (PrP). As no unquestioned report of a diseased sheep carrying homozygous alleles encoding alanine, arginine and arginine (PrPARR) at these sites has been published to date, sheep of this genotype are believed to be scrapie resistant. After the introduction of large-scale rapid testing for scrapie, a number of so-called 'atypical' scrapie cases have been found in Germany and elsewhere. Among those cases were two supposedly scrapie-resistant sheep. Brain samples from these animals tested positive for abnormal PrP (PrPSc) in one of four rapid tests available. Moreover, scrapie-associated fibril (SAF)-immunoblotting and immunohistochemistry, which are the generally accepted diagnostic techniques for scrapie, revealed prominent PrPSc deposition in the cerebellum. SAF immunoblotting also revealed PrPSc deposition in the obex, frontal cortex and brainstem. Transmission experiments to investigate the infectivity of scrapie propagated from these sheep have been initiated.
Abstract: The inhibition of CD40-CD40L interaction-mediated signalling was suggested as a therapeutic strategy for the treatment of Alzheimer's disease. Conversely, CD40-deficient neurons were reported to be more vulnerable to stress associated with ageing as well as nerve growth factor-beta and serum withdrawal. We studied the scrapie infection of CD40L-deficient (CD40L(-/-)) mice to see whether ablation of the CD40L gene would be beneficial or detrimental in this model of a neurodegenerative amyloidosis. CD40L(-/-) mice died on average 40 days earlier than wild-type control mice and exhibited a more pronounced vacuolation of the neuropil and an increased microglia activation. The experimental model indicates that a deficiency for CD40L is highly detrimental in prion diseases and reinforces the neuroprotective function of intact CD40-CD40L interactions. The stimulation of neuroprotective pathways may represent a possibility to delay therapeutically the disease onset in prion infections of the central nervous system.
Abstract: Fyn is a 59-kDa member of the Src family of tyrosine kinases synthesized on cytosolic polysomes and then targeted to the plasma membrane where it clusters in caveolae-like membrane microdomains. The cellular isoform of the prion protein (PrP) has also been identified to be a caveolar constituent and to participate in signal transduction events concerning cell survival and differentiation via recruitment of Fyn. We studied the scrapie infection of mice deficient for Fyn (Fyn(-/-)) to clarify the role of Fyn in an in vivo model of transmissible spongiforme encephalopathies. Fyn(-/-) mice died on average 9 days earlier than wild-type control mice, but no differences were seen regarding activation of astrocytes, vacuolization of the neuropil, and accumulation of misfolded prion protein. The experimental model suggests that a deficiency for Fyn is detrimental in prion diseases, although it has no major effect on the clinical course of an experimental prion infection of the CNS.
Abstract: The underlying pathomechanisms in prion infections of the central nervous system are still insufficiently understood. The identification of genes with altered expression patterns in the diseased brain may provide insight into the disease development on the molecular level, which ultimately leads to neuronal loss. To provide a detailed analysis of changes in the molecular level in prion disease pathology we used a large-scale gene array based approach, which covers more than 11,000 functionally characterised sequences and expressed sequence tags, for the analysis of gene expression profile alterations in the cortex, medulla, and pons of scrapie-infected mice. The study identified in total 114 genes with altered mRNA levels, the majority of which were previously not known to be affected by the disease. Overall the gene array data demonstrate the presence of a strong inflammatory reaction and stress response, and show similarities to gene expression patterns found in brains affected by Alzheimer's disease and aging, respectively.
Abstract: Prion infections of the central nervous system (CNS) are characterised by a reactive gliosis and the subsequent degeneration of neuronal tissue. The activation of glial cells, which precedes neuronal death, is likely to be initially caused by the deposition of misfolded, proteinase K-resistant, isoforms (termed PrP(res)) of the prion protein (PrP) in the brain. Cytokines and chemokines released by PrP(res)-activated glia cells may contribute directly or indirectly to the disease development by enhancement and generalisation of the gliosis and via cytotoxicity for neurons. However, the actual role of prion-induced glia activation and subsequent cytokine/chemokine secretion in disease development is still far from clear. In the present work, we review our present knowledge concerning the functional biology of cytokines and chemokines in prion infections of the CNS.
Abstract: A brain homogenate prepared from a terminally ill hamster infected with scrapie strain 263K was serially diluted and administered orally to groups of hamsters. The undiluted brain homogenate led to clinical scrapie in all animals inoculated. The attack rate decreased with dilutions of the homogenate, and subclinical infections were identified among the healthy survivors at 520 days post-infection by Western blotting. The number of animals succumbing to disease and the combined number of Western blot-positive survivors plus diseased hamsters were used to calculate the LD(50) and ID(50) of the inoculum. The model system represents an approximation to the transmission of TSEs such as new variant Creutzfeldt-Jakob disease (vCJD) via dietary exposure to the infectious agent and suggests that, due to the rather small difference between the calculated LD(50) and ID(50), the number of clinical cases will not be vastly exceeded by the number of subclinical carriers of the disease.
Abstract: Several lines of evidence suggest that immunisations may be helpful in the prophylaxis and treatment of neurodegenerative amyloidoses like Alzheimer's disease and prion infections. We used a synthetic prion protein-derived peptide (PrP105-125) and a recombinant PrP fragment (PrP90-230) as antigens for the active immunisation of mice, which were subsequently infected by dietary exposure to the scrapie agent. Immunisation with PrP105-125 prolonged the survival times significantly. In contrast, immunisation with PrP90-230 or adjuvants alone had no effect on the disease development. An epitope mapping of the antibodies raised against PrP90-230 revealed that reactivities against previously defined protective epitopes were either underrepresented or absent. These results point towards the possibility to prevent prion spread via the food chain by vaccinating humans or other species at risk to contract prion diseases.
