LB-100, a novel Protein Phosphatase 2A (PP2A) inhibitor, sensitizes malignant meningioma cells to the therapeutic effects of radiation.

Atypical and anaplastic meningiomas (AAM) represent 20% of all meningiomas. They are associated with poor outcomes due to their tendency to recur. While surgery and radiation (RT) are first line therapy, no effective systemic medical treatment has been identified. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in cell cycle regulation and DNA repair. Here, we examined radiosensitizing effects of LB-100, a novel inhibitor of PP2A against AAM as a novel treatment strategy. Three human-derived immortalized meningioma cell lines, IOMM-LEE, GAR, and CH-157, were used to investigate the radio-sensitizing potential of LB-100 in AAM. Survival fraction by clonogenic assay, immunofluorescence, cell cycle analysis and protein expression were evaluated in vitro. The antitumor effects of combining LB-100 with RT were verified in vivo by using intracranial orthotopic xenograft mouse model. Pharmacologic PP2A inhibition with LB-100 prior to RT enhanced the radiosensitivity of meningioma cells and reduced survival fraction in clonogenic assays. LB-100 increased DNA double-strand breakage (measured by γ-H2AX), mitotic catastrophe cell death, and G2/M cell cycle arrest in irradiated meningioma cells. Also, LB-100 decreased activation of STAT3 and expression of its downstream proteins. In vivo, LB-100 and RT combined treatment prolonged the survival of mice with xenografts compared to RT alone. Taken together, these results provide convincing preclinical data to support the use of LB-100 as a radiosensitizing agent for treatment of malignant meningioma. Its potential for clinical application deserves further investigation.

Computational analysis of the mesenchymal signature landscape in gliomas.

BACKGROUND: Epithelial to mesenchymal transition, and mimicking processes, contribute to cancer invasion and metastasis, and are known to be responsible for resistance to various therapeutic agents in many cancers. While a number of studies have proposed molecular signatures that characterize the spectrum of such transition, more work is needed to understand how the mesenchymal signature (MS) is regulated in non-epithelial cancers like gliomas, to identify markers with the most prognostic significance, and potential for therapeutic targeting. RESULTS: Computational analysis of 275 glioma samples from "The Cancer Genome Atlas" was used to identify the regulatory changes between low grade gliomas with little expression of MS, and high grade glioblastomas with high expression of MS. TF (transcription factor)-gene regulatory networks were constructed for each of the cohorts, and 5 major pathways and 118 transcription factors were identified as involved in the differential regulation of the networks. The most significant pathway - Extracellular matrix organization - was further analyzed for prognostic relevance. A 20-gene signature was identified as having prognostic significance (HR (hazard ratio) 3.2, 95% CI (confidence interval) = 1.53-8.33), after controlling for known prognostic factors (age, and glioma grade). The signature's significance was validated in an independent data set. The putative stem cell marker CD44 was biologically validated in glioma cell lines and brain tissue samples. CONCLUSIONS: Our results suggest that the differences between low grade gliomas and high grade glioblastoma are associated with differential expression of the signature genes, raising the possibility that targeting these genes might prolong survival in glioma patients.

Multivariate Analysis of Radiation Responsive Proteins to Predict Radiation Exposure in Total-Body Irradiation and Partial-Body Irradiation Models.

In the event of a radiological or nuclear attack, advanced clinical countermeasures are needed for screening and medical management of the exposed population. In such a scenario, minimally invasive biomarkers that can accurately quantify radiation exposure would be useful for triage management by first responders. In this murine study, we evaluated the efficacy of a novel combination of radiation responsive proteins, Flt3 ligand (FL), serum amyloid A (SAA), matrix metalloproteinase 9 (MMP9), fibrinogen beta (FGB) and pentraxin 3 (PTX3) to predict the received dose after whole- or partial-body irradiation. Ten-week-old female C57BL6 mice received a single whole-body or partial-body dose of 18 Gy from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by cardiac puncture at 24, 48, 72 h and 1 week postirradiation. Plasma protein levels were determined via commercially available ELISA assay. A multivariate discriminant analysis was utilized to generate best-fit dose prediction models for whole-body exposures using the selected biomarker panel and its potential application to partial-body exposures was examined. The combination of values from FL, SAA, MMP9, FGB and PTX3 between 24 h and 1 week postirradiation yielded novel dose-response relationships. For day 1 postirradiation, the best-fit model yielded a predictive accuracy of 81% utilizing FL alone. The use of additional proteins did not enhance the model accuracy whereas, at day 2 postirradiation, the addition of PTX3 and FGB to FL increased the accuracy to 100%. At day 3 the use of FL and PTX3 yielded a predictive accuracy of 93% and at day 7 use of FL and SAA had an accuracy of 90%. Dose prediction of partial-body exposures based on the TBI model had a higher predictive accuracy when the percentage of the body exposed to radiation increased. Our findings indicate that this novel combination of radiation responsive biomarker proteins are an efficient method for predicting radiation exposure and are more accurate when used in concert compared to using any single biomarker protein alone.

FOXM1 and STAT3 interaction confers radioresistance in glioblastoma cells.

Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo. Moreover, we observed high expression of Forkhead box protein M1 (FOXM1) in irradiated GBM cells both in vitro and in vivo. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells, intrigued us to validate it as a radiosensitizing target. Here we show that FOXM1 inhibition radiosensitizes GBM cells by abrogating genes associated with cell cycle progression and DNA repair, suggesting its role in cellular response to radiation. Further, we demonstrate that radiation induced stimulation of FOXM1 expression is dependent on STAT3 activation. Co-immunoprecipitation and co-localization assays revealed physical interaction of FOXM1 with phosphorylated STAT3 under radiation treatment. In conclusion, we hypothesize that FOXM1 regulates radioresistance via STAT3 in GBM cells. We also, show GBM patients with high FOXM1 expression have poor prognosis. Collectively our observations might open novel opportunities for targeting FOXM1 for effective GBM therapy.

Modulation of miR-21 signaling by MPS1 in human glioblastoma.

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-ß/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy.

Molecular profiling of MPS1 gene silencing in U251 glioma cell line.

Aneuploidy has been recognized as a common characteristic of cancers. Aneuploidy frequently results from errors of the mitotic checkpoint, the major cell cycle control mechanism that acts to prevent chromosome missegregation. Mutation of the genes that control chromosome segregation during mitosis may explain the high rate of chromosomal instability and aneuploidy, a characteristic of most solid tumors, including glioblastomas (GBM)[1, 2]. Monopolar spindle 1 (MPS1), is an essential spindle assembly checkpoint kinase that is overexpressed in several human cancers [3-5]. In our previous publication, we have shown the role of MPS1 kinase in DNA repair and enhanced radiosensitivity in GBM[6]. Here, we provide methodological and analytical details of that study, to compare mRNA expression profile of siMPS1-silenced U251 cells with untransfected control, and siRNA control (siNeg) at 6, 24, and 48 hours after transfection. The raw data of this study is deposited in Gene Expression Omnibus under the accession number GSE57091.

Serum Amyloid A as a Biomarker for Radiation Exposure.

There is a need for minimally invasive biomarkers that can accurately and quickly quantify radiation exposure. Radiation-responsive proteins have applications in clinical medicine and for mass population screenings after a nuclear or radiological incident where the level of radiation exposure and exposure pattern complicate medical triage for first responders. In this study, we evaluated the efficacy of the acute phase protein serum amyloid A (SAA) as a biomarker for radiation exposure using plasma from irradiated mice. Ten-week-old female C57BL6 mice received a 1-8 Gy single whole-body or partial-body dose from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by mandibular or cardiac puncture at 6, 24, 48 and 72 h or 1-3 weeks postirradiation. SAA levels were determined using a commercially available ELISA assay. Data was pooled to generate SAA µg/ml threshold values correlating plasma SAA levels with radiation dose. SAA levels were statistically significant over control at all exposures between 2 and 8 Gy at 24 h postirradiation but not at 6, 48 and 72 h or 1-3 weeks postirradiation. SAA levels at 1 Gy were not significantly elevated over control at all time points. Total-body-irradiated (TBI) SAA levels at 24 h were used to generate a dose prediction model that successfully differentiated TBI mice into dose received cohorts of control/1 Gy and ≥ 2 Gy groups with a high degree of accuracy in a blind study. Dose prediction of partial-body exposures based on the TBI model correlated increasing predictive accuracy with percentage of body exposure to radiation. Our findings indicate that plasma SAA levels might be a useful biomarker for radiation exposure in a variety of total- and partial-body irradiation settings.

Protein Phosphatase 2A Inhibition with LB100 Enhances Radiation-Induced Mitotic Catastrophe and Tumor Growth Delay in Glioblastoma.

Protein phosphatase 2A (PP2A) is a tumor suppressor whose function is lost in many cancers. An emerging, though counterintuitive, therapeutic approach is inhibition of PP2A to drive damaged cells through the cell cycle, sensitizing them to radiotherapy. We investigated the effects of PP2A inhibition on U251 glioblastoma cells following radiation treatment in vitro and in a xenograft mouse model in vivo. Radiotherapy alone augmented PP2A activity, though this was significantly attenuated with combination LB100 treatment. LB100 treatment yielded a radiation dose enhancement factor of 1.45 and increased the rate of postradiation mitotic catastrophe at 72 and 96 hours. Glioblastoma cells treated with combination LB100 and radiotherapy maintained increased γ-H2AX expression at 24 hours, diminishing cellular repair of radiation-induced DNA double-strand breaks. Combination therapy significantly enhanced tumor growth delay and mouse survival and decreased p53 expression 3.68-fold, compared with radiotherapy alone. LB100 treatment effectively inhibited PP2A activity and enhanced U251 glioblastoma radiosensitivity in vitro and in vivo. Combination treatment with LB100 and radiation significantly delayed tumor growth, prolonging survival. The mechanism of radiosensitization appears to be related to increased mitotic catastrophe, decreased capacity for repair of DNA double-strand breaks, and diminished p53 DNA-damage response pathway activity.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins.

UNLABELLED: To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. IMPLICATIONS: Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies.

Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes.

The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of haematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibit increased DNA repair, compared with non-reactive cells, in actively transcribed chromatin after irradiation. In mapping these repair sites, we identify misrepair events and repair hotspots that are unique to each state. The precise characterization of genomic regions susceptible to mutation in specific transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, in particular those where double-strand break induction is a cornerstone of clinical intervention.

Inhibition of polo-like kinase 1 in glioblastoma multiforme induces mitotic catastrophe and enhances radiosensitisation.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in the United States of America (USA) with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signalling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumour growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.

Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration.

Exosomes are nanometer-sized lipid vesicles released ubiquitously by cells, which have been shown to have a normal physiological role, as well as influence the tumor microenvironment and aid metastasis. Recent studies highlight the ability of exosomes to convey tumor-suppressive and oncogenic mRNAs, microRNAs, and proteins to a receiving cell, subsequently activating downstream signaling pathways and influencing cellular phenotype. Here, we show that radiation increases the abundance of exosomes released by glioblastoma cells and normal astrocytes. Exosomes derived from irradiated cells enhanced the migration of recipient cells, and their molecular profiling revealed an abundance of molecules related to signaling pathways important for cell migration. In particular, connective tissue growth factor (CTGF) mRNA and insulin-like growth factor binding protein 2 (IGFBP2) protein levels were elevated, and coculture of nonirradiated cells with exosomes isolated from irradiated cells increased CTGF protein expression in the recipient cells. Additionally, these exosomes enhanced the activation of neurotrophic tyrosine kinase receptor type 1 (TrkA), focal adhesion kinase, Paxillin, and proto-oncogene tyrosine-protein kinase Src (Src) in recipient cells, molecules involved in cell migration. Collectively, our data suggest that radiation influences exosome abundance, specifically alters their molecular composition, and on uptake, promotes a migratory phenotype.

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody, AMG102.

The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.

Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome.

The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface alpha5beta1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1alpha) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1alpha mediated transcriptional activity as well as HIF-1alpha mediated angiogenic sprouting of ECs. HIF-1alpha plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1alpha activities.

Launching a novel preclinical infrastructure: comparative oncology trials consortium directed therapeutic targeting of TNFalpha to cancer vasculature.

BACKGROUND: Under the direction and sponsorship of the National Cancer Institute, we report on the first pre-clinical trial of the Comparative Oncology Trials Consortium (COTC). The COTC is a novel infrastructure to integrate cancers that naturally develop in pet dogs into the development path of new human drugs. Trials are designed to address questions challenging in conventional preclinical models and early phase human trials. Large animal spontaneous cancer models can be a valuable addition to successful studies of cancer biology and novel therapeutic drug, imaging and device development. METHODOLOGY/PRINCIPAL FINDINGS: Through this established infrastructure, the first trial of the COTC (COTC001) evaluated a targeted AAV-phage vector delivering tumor necrosis factor (RGD-A-TNF) to alphaV integrins on tumor endothelium. Trial progress and data was reviewed contemporaneously using a web-enabled electronic reporting system developed for the consortium. Dose-escalation in cohorts of 3 dogs (n = 24) determined an optimal safe dose (5x10(12) transducing units intravenous) of RGD-A-TNF. This demonstrated selective targeting of tumor-associated vasculature and sparing of normal tissues assessed via serial biopsy of both tumor and normal tissue. Repetitive dosing in a cohort of 14 dogs, at the defined optimal dose, was well tolerated and led to objective tumor regression in two dogs (14%), stable disease in six (43%), and disease progression in six (43%) via Response Evaluation Criteria in Solid Tumors (RECIST). CONCLUSIONS/SIGNIFICANCE: The first study of the COTC has demonstrated the utility and efficiency of the established infrastructure to inform the development of new cancer drugs within large animal naturally occurring cancer models. The preclinical evaluation of RGD-A-TNF within this network provided valuable and necessary data to complete the design of first-in-man studies.

Tumor vasculature-targeted delivery of tumor necrosis factor-alpha.

BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor-alpha (TNF-alpha) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF-alpha, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF-alpha targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno-associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF-alpha (AAVP-TNF-alpha). The activity of AAVP-TNF-alpha was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP-TNF-alpha infection of human melanoma cells resulted in high levels of TNF-alpha expression. Systemic administration of targeted AAVP-TNF-alpha to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF-alpha, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF-alpha specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF-alpha is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy.

A subset of host B lymphocytes controls melanoma metastasis through a melanoma cell adhesion molecule/MUC18-dependent interaction: evidence from mice and humans.

Host immunity affects tumor metastasis but the corresponding cellular and molecular mechanisms are not entirely clear. Here, we show that a subset of B lymphocytes (termed B-1 population), but not other lymphocytes, has prometastatic effects on melanoma cells in vivo through a direct heterotypic cell-cell interaction. In the classic B16 mouse melanoma model, one mechanism underlying this phenomenon is a specific up-regulation and subsequent homophilic interaction mediated by the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Presence of B-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in melanoma cells, indicating that the same protein interaction exists in humans. These results suggest a new but as yet unrecognized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metastasis in a mouse model and in melanoma patients. Given that monoclonal antibodies against MUC18 are in preclinical development but the reason for their antitumor activity is not well understood, these translational results are relevant in the setting of human melanoma and perhaps of other cancers.

Endothelial monocyte activating polypeptide-II induced gene expression changes in endothelial cells.

In the current study we used microarray (MA) analysis to examine gene expression changes in human umbilical vein endothelial cells (HUVEC) exposed to the tumor-derived cytokine, endothelial monocyte-activating polypeptide-II (EMAP-II). HUVEC treated with EMAP-II for 0.5, 1, 2, 4 and 8 h, were analyzed using 10K cDNA arrays. Our results demonstrated that changes in gene expression of <0.5 and >2 fold were seen for 69 genes and the majority of gene changes occurred early. Validation of MA analysis for 10 genes by real time RT-PCR, demonstrated the gene changes to be consistent and specific to HUVEC when compared to human fibroblasts treated with EMAP-II. Among these genes, downregulated in ovarian cancer 1 (DOC1) gene was studied further because of its possible role in EMAP-II induced cytoskeletal remodeling. DOC1 expression was silenced using small interfering RNA. SiRNA to DOC1 completely abolished EMAP-II stimulated gene expression of DOC1. Silencing of DOC1 gene expression reversed the modulatory effect of EMAP-II on 4 other genes, suggesting that DOC1 might play a role in mediating some of the effects of EMAP-II on endothelial cells.

Early genetic mechanisms underlying the inhibitory effects of endostatin and fumagillin on human endothelial cells.

A tumor needs to initiate angiogenesis in order to develop its own blood supply, to grow, to invade, and to spread. Angiogenesis, under normal conditions, is a tightly regulated balance between endogenous pro- and antiangiogenic factors. In this study, we investigated, by microarray analysis, the effects of two known antiangiogenic agents (endostatin and fumagillin) on the gene expression profiles of human umbilical vein endothelial cells (HUVEC) in order to elucidate pathways common to the effects of these agents. We observed a majority of gene expression changes within 1 and 2 h of treatment. The genes demonstrating these early expression changes are involved in cell proliferation, gene transcription, and a number have unknown functions. We selected four genes (DOC1, KLF4, TC-1, ID1) from the microarray profile that showed a similar pattern of expression for both of the antiangiogenic agents we tested. We then used small interfering RNAs (siRNA) in an attempt to better understand the role of these selected genes in the inhibitory activity of these agents. Because the gene expression changes occurred within 1 and 2 h of treatment, these genes might be involved in the initial pathways of angiogenesis inhibition.