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The aim of this study was to evaluate whether the presence of simian virus-40 (SV40) is associated with increased release of vascular endothelial growth factor (VEGF) in human malignant mesothelioma (MM) cells. We studied nine cell lines derived from pleural effusion (PE) of patients with MM, and three different cultures of normal human mesothelial cells (NHMC) derived from pleural fluid of patients with congestive heart failure. NHMC were transfected with full length SV40 (NHMC-FL) or large T antigen (NHMC Tag) DNAs. High levels of VEGF were detected in conditioned media of each of two MM cells that tested positive for SV40 by PCR amplification and Southern blot hybridization and for Tag transcript by reverse transcription- polymerase chain reaction (RT-PCR) and immunoprecipitation. We also found that NHMC-FL released high amounts of VEGF. Conditioned media from SV40-positive MM cells and from FL-NHMC increased proliferation of human umbilical vein cells (HUVEC) and this effect was partially abrogated by adding specific blocking antibodies against VEGF. These results offer the first evidence that SV40 can cause VEGF release in SV40-positive MM cells and that entire viral genome is required for this effect.

Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946+/-14 pg/ml vs. 180+/-17 pg/ml; p

Several biochemical and clinical factors have been shown to correlate with survival in human malignant pleural mesothelioma (MM). Nevertheless, average survival of 4 to 10 months from diagnosis is sometimes not sufficient for full expression of these factors. Several studies have reported SV40 sequences in MM, suggesting a possible pathogenic role. We investigated whether the presence of these sequences had any effect on MM patient survival. For this study, we used polymerase chain reaction and Southern blot analysis to search for and identify SV40 DNA in biopsy samples from 83 MM patients. These cases were divided according to histology: 62/83 (74.7%) had epithelioid morphology (EMM) and 21/83 (25.3%) had either biphasic or sarcomatous morphology (B/SMM). SV40 positivity was significantly associated with B/SMM growth pattern (chi-squared test = 5.03, P = 0.025). Kaplan-Meier univariate analysis confirmed the independent effect of histology on MM survival (log-rank test = 13.9, P < 0.001) and showed a trend for increased survival in SV40-negative patients (log-rank test = 2.83, P = 0.09). Most importantly, Cox's regression model showed that SV40-positive status affected the predictive value of histology on patient survival. In particular, when SV40 expression was added to the B/SMM histotype, Cox's regression model showed a significant increase in hazard ratio (HR) with respect to SV40-negative B/SMM (HR = 4.25, 95% CI = 2.00–9.00, likelihood ratio test = 14.31, P < 0.001). We conclude that SV40 expression is significantly associated with B/SMM histology and represents an important prognostic cofactor when associated with the tumor subtype in MM patients. © 2000 Wiley-Liss, Inc.

Malignant mesothelioma of the pleura is divided in three morphological variants: epithelioid, sarcomatous, and biphasic. Histological similarities between epithelioid malignant mesothelioma (EMM) and lung adenocarcinoma are responsible for the difficult differential diagnosis. Monoclonal antibodies are useful for distinguishing the two neoplasms through immunohistochemical phenotyping, although many cases require ultrastructural characterization for definitive diagnosis. In this study, transmission electron microscopic observations of EMM were compared with those of peripheral adenocarcinoma of the lung (PAL). More specifically, the morphology of the basal lamina is described in 23 cases of EMM and 12 cases of PAL. Reduplication of the basal lamina (RBL) was found in 11 cases (48%) of EMM and in none of the PAL cases. The same cases were immunostained for type IV collagen and the localization of this basement membrane component corresponded to the areas where basal lamina was observed. Since RBL has been associated with neoplastic differentiation in other tumors, this novel feature in EMM needs to be evaluated in future prognostic studies in malignant mesothelioma of the pleura. Moreover, RBL expression in EMM may be an additional ultrastructural parameter used in the differential diagnosis between EMM and adenocarcinoma.

Aggressive tumor cells can show characteristics similar to normal embryogenic progenitor cells, and de-regulated embryonic developmental signaling networks have been linked to cancer initiation and progression. Thus, cell fate regulation in embryonic development and cell transformation during oncogenesis may share common signaling pathways. Several tumor types have been suggested to be initiated from stem-like cells. Exploring in tumors, the re-emergence of embryonic signaling pathways involved in stem cell self-renewal can produce new insight into the initiation and progression of cancer and may lead to development of new therapeutic strategies. The Nodal pathway is required for maintenance of undifferentiated embryonic stem cells. Nodal is a morphogen that belongs to the TGF-beta family. Its signaling can be regulated by co-factors such as the Epidermal Growth Factor - like member, Cripto-1 and another TGF-beta member, Lefty. Nodal and Cripto-1 are critical in embryonic development and is generally poorly detected in differentiated tissues. However, an increased expression has been detected in different human tumor types. The dismal prognosis of glioblastomas calls for increased biological knowledge and novel therapeutic targets. Gene expression studies in a glioblastoma xenograft model, where multicellular spheroids from glioblastoma patient biopsies were generated and implanted intracranially in nude rats and where highly infiltrative non-angiogenic tumors can switch to angiogenic tumors upon in vivo intracranial passaging, has revealed importance of gene ontology categories connected to development and negative regulators of differentiation. In this model we detected several significant overrepresented morphogenesis categories, and we hypothesized that increased activity in Nodal signaling also could be found in human glioblastomas. We assessed the gene expression of Nodal, Cripto-1 and Lefty in microarrays of infiltrative and angiogenic xenograft samples developed from four glioblastoma patients. The genes were expressed both in the infiltrative and angiogenic xenografts. Further we examined Nodal, Cripto-1 and Lefty expression in 199 primary glioblastomas by immunohistochemistry, and assessed their potential clinical relevance by analyzing the association between their protein expression and patient survival. A weak to moderate protein expression of Nodal and Lefty and a stronger expression of Cripto-1 was found in the patient samples. Interestingly a significant association between patient age, Cripto-1 expression and survival was demonstrated, where higher Cripto-1 scores were associated with poorer survival in younger patients. These findings suggest that Cripto-1 may represent a novel negative prognostic marker in younger glioblastoma patients and also a potential therapeutic target in this patient group. Citation Format: Berit B. Tysnes, Hege AA Sætran, Sverre J. Mørk, Naira V. Margaryan, Geir E. Eide, Rolf Bjerkvig, Luigi Strizzi, Mary J.C. Hendrix. Age-dependent association between protein expression of the embryonic stem cell marker Cripto-1 and survival of glioblastoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2013-2650

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. The current lack of effective therapies for treating metastatic melanoma exemplifies the desperate need for the identification of new targets and the development of new therapies. Nodal is an embryonic signaling molecule from the TGFβ superfamily that is not typically expressed in adult tissues but is reactivated in a number of aggressive cancers including melanoma, and breast and prostate carcinomas. In a study recently published by our group, we determined a connection between the Notch and Nodal pathways in aggressive melanoma. We identified a specific correlation between the expression of Notch4 and Nodal in aggressive melanoma cell lines and in advanced stage human melanomas. By inhibiting Notch4 expression or function, we established a link between Notch4 and Nodal signaling in promoting the aggressive phenotype of metastatic melanoma cells in vitro. Notch4 regulation of Nodal expression was identified as important in cellular plasticity, since inhibition of Notch4 function impaired the ability of aggressive cells to engage in vasculogenic mimicry (de novo formation of vascular-like networks by non-endothelial tumor cells). Notably, vascular-like network formation could be partially rescued by recombinant human Nodal, suggesting both pathways are important for this phenomenon. To extend our published observations, we utilized neutralizing antibodies to Notch4 and to Nodal, and compared gene expression profiles. From this, we identified a mechanism of feedback between signaling pathways that controls the expression of components of both pathways, including Nodal, Notch4, and the Notch ligand, Jagged2. Since Notch4 is enriched in the subpopulation of cells that form vascular-like networks in melanoma, we specifically analyzed the expression of angiogenesis signature genes previously associated with vascular-like network formation, and determined that inhibition of Notch4 or Nodal function downregulates a large group of these genes including members of the VEGF, angiopoietin, MMP, and cadherin families. We are currently investigating the hypothesis that the Notch4-Nodal signaling axis may be a master regulator of the vascular-like phenotype in aggressive melanoma cells. We suggest that inhibition of Notch4 and/or Nodal function may offer a potential molecular targeting strategy for metastatic melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5204. doi:1538-7445.AM2012-5204

The embryonic morphogen Nodal is a potent inducer of EMT and critical mediator of stem cell pluripotency in the developing embryo, but is not expressed in normal adult tissues. Intriguingly, our lab has recently shown that Nodal, a TGF-beta family member, is in fact secreted by melanoma and breast cancer cells and that Nodal expression positively correlates with tumor grade in human patient samples. Our current studies demonstrate a positive role for Nodal in regulating breast cancer cell renewal and aggressiveness. Specifically, short hairpin (shRNA) knockdown of Nodal expression in human breast cancer cell lines diminishes proliferation, invasion and clonogenicity of these cell lines in vitro and dramatically curtails tumor formation in a mouse xenograft model. Additionally, our data suggest that these effects may be caused, in part, by alterations in the expression of adhesion receptors known to function in tumor engraftment and metastasis. Taken together, our data suggest that Nodal signaling may facilitate the acquisition of self-renewal and migratory capabilities in breast cancer cells and represent a novel therapeutic target for disrupting these processes in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3353. doi:1538-7445.AM2012-3353

The diagnosis of melanoma is becoming more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity, phenotypic plasticity and the expression of embryonic related molecules and signaling pathways that are important for self renewal and pluripotency, present a challenge in targeting the most aggressive tumor cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways. In particular, the embryonic morphogen Nodal, belonging to the TGF-β superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is also expressed in aggressive melanoma, although unlike embryonic stem cells, melanoma does not express Lefty, a natural inhibitor of Nodal, thus allowing Nodal to be expressed in an unregulated manner. Further analysis revealed that Lefty is silenced in these tumor cells by DNA methylation. Down-regulation of Nodal expression in melanoma cells results in a significant reduction in clonogenicity and suppression of tumorigenesis. Additional translational studies indicate that antibodies against Nodal are capable of targeting and inducing apoptosis in human melanoma tumors lodged in the lungs of nude mice. Furthermore, our data show another stem cell signaling pathway, Notch, is expressed in these aggressive melanoma cells, and there is direct evidence demonstrating the molecular cross-talk between Nodal and Notch. Thus, these two prominent stem cell pathways contributing to melanoma cell plasticity may serve as promising dual therapeutic targets for clinical intervention.

Pleural malignant mesothelioma (MM) shows poor survival, regardless of tumour stage at diagnosis. MM is unresponsive to present treatment regimens and new protocols are desperately needed. The localised nature, the potential accessibility, and the relative lack of distant metastases make MM a particularly attractive candidate for somatic gene therapy. A common target for cancer gene therapy is the tumour suppressor protein p53. p53 does not seem to be mutated or deleted in MM, but it can be inactivated by binding to other proteins, like mdm2 and SV40 large T antigen. We tested the effects of a replication-deficient adenoviral vector carrying wild-type p53 cDNA in human MM cells. Our results show that >95% of MM cells were efficiently infected with 25 multiplicity of infection (MOI) of vector. Wild-type p53 was effectively expressed resulting in >80% inhibition of proliferation in MM cells. AdCMV.p53 infection induced apoptosis while controls did not show any evident morphological alterations. Ex vivo p53 gene transfer experiments inhibited tumourigenesis in nude mice. In vivo, direct intratumour injection of AdCMV.p53 arrested tumour growth and prolonged survival of treated mice. These results indicate that p53-gene therapy should be strongly exploited for clinical trials in MM patients.

The expression of angiogenic factors may represent useful markers for diagnosis and prediction of disease outcome. Basic fibroblast growth factor (b-FGF) is a potent angiogenic factor which promotes in vitro growth of endothelial cells and in vivo vessel formation. We investigated the expression of b-FGF in patients affected with malignant and non-malignant pleural diseases and presenting clinically with non-specific signs and symptoms. We also studied the relationships between the expression of b-FGF in patients with malignant pleural mesothelioma (MM) and tumour aggressiveness, assessed as tumour vessel density (TVD), or patient survival. Basic-FGF was measured by immunoassay in the serum and pleural effusions (PE) of 37 patients. Of these, MM was diagnosed in 15/37 patients while the remaining patients had either peripheral lung adenocarcinoma (PLA) or benign inflammatory pleural disease (BPD). The mean b-FGF level measured 8.5+/-6.1 pg/ml in the PE of the malignant group (MM + PLA) and 23.9+/-19.8 in the PE of the non-malignant group (BPD) (p=0.001). The mean b-FGF level was significantly lower in the PE of MM patients (6.9+/-5.2 pg/ml) compared to BPD patients (p=0.004). Linear regression analysis showed a significant inverse correlation (r=-0.59; p=0.041) between b-FGF levels found in MM PE and patient survival. A noteworthy relationship between high serum b-FGF levels and reduced survival was also observed (r=-0.57; p=0.052). Interestingly, both serum (r=0.48; p=0.114) and PE (r=0.26; p=0.413) b-FGF levels in MM patients correlated poorly with TVD. Our data indicate that b-FGF is significantly more expressed in non-malignant compared to malignant PE, this difference being particularly evident between MM and BPD. Our results also suggest that high b-FGF levels correlate with poor MM patient survival through mechanisms which may be independent of b-FGF angiogenic potential.

SummaryActivated platelets may engage in dynamic interplay with other blood cells. We examined the evidence for platelet activation and the formation of platelet-erythrocyte aggregates in chronic hemodialysis patients. Circulating activated platelets (P-selectin/CD63-positive platelets) were higher than normal controls (p