Cleo E. Rolle

Mechanisms of Immune Evasion by Gliomas

A major contributing factor to glioma development and progression is its ability to evade the immune system. This chapter will explore the mechanisms utilized by glioma to mediate immunosuppression and immune evasion. These include intrinsic mechanisms linked to its location within the brain and interactions between glioma cells and immune cells. Lack of recruitment of naïve effector immune cells perhaps accounts for most of the immune suppression mediated by these tumor cells. This is enhanced by increased recruitment of microglia which resemble immature antigen presenting cells that are unable to support T-cell mediated immunity. Furthermore, secreted factors like TGF-β, COX-2 and IL-10, altered costimulatory molecules and inhibition of STAT-3 all contribute to the recruitment and expansion of regulatory T cells, which further modulate the immunosuppressive environment of glioma. In light of these findings, multiple immunotherapeutic treatment modalities are currently being explored.

Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow-derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response.

Challenges in Clinical Design of Immunotherapy Trials for Malignant Glioma

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor. The traditional treatments for GBM, including surgery, radiation, and chemotherapy, only modestly improve patient survival. Therefore, immunotherapy has emerged as a novel therapeutic modality. Immunotherapeutic strategies exploit the immune systems ability to recognize and mount a specific response against tumor cells, but not normal cells. Current immunotherapeutic approaches for glioma can be divided into 3 categories: immune priming (active immunotherapy), immunomodulation (passive immunotherapy), and adoptive immunotherapy. Immune priming sensitizes the patients immune cells to tumor antigens using various vaccination protocols. In the case of immunomodulation, strategies are aimed at reducing suppressive cytokines in the tumor microenvironment or using immune molecules to specifically target tumor cells. Adoptive immunotherapy involves harvesting the patients immune cells, followed by ex vivo activation and expansion before reinfusion. This article provides an overview of the interactions between the central nervous system and the immune system, and discusses the challenges facing current immunotherapeutic strategies.

The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.

Biodistribution of an oncolytic adenovirus after intracranial injection in permissive animals: a comparative study of Syrian hamsters and cotton rats

Conditionally replicative adenoviruses (CRAds) are often evaluated in mice; however, normal and cancerous mouse tissues are poorly permissive for human CRAds. As the cotton rat (CR) is a semipermissive animal and the Syrian hamster (SH) is a fully permissive model for adenoviral replication, we compared them in a single study following intracranial (i.c.) injection of a novel glioma-targeting CRAd. Viral genomic copies were quantified by real-time PCR in brain, blood, liver and lung. The studies were corroborated by immunohistochemical, serological and immunological assays. CR had a multiple log higher susceptibility for adenoviral infection than SH. A similar amount of genomic copies of CRAd-Survivin-pk7 and human adenovirus serotype 5 (AdWT) was found in the brain of CR and in all organs from SH. In blood and lung of CR, AdWT had more genomic copies than CRAd-Survivin-pk7 in some of the time points studied. Viral antigens were confirmed in brain slices, an elevation of serum transaminases was observed in both models, and an increase in anti-adenoviral antibodies was detected in SH sera. In conclusion, CR represents a sensitive model for studying biodistribution of CRAds after i.c. delivery, allowing for the detection of differences in the replication of CRAd-Survivin-pk7 and AdWT that were not evident in SH.

A chimeric adenovirus with an Ad 3 fiber knob modification augments glioma virotherapy

Malignant gliomas remain refractory to treatment despite advances in chemotherapy and surgical techniques. Viral vectors developed to treat gliomas have had low transduction capabilities, limiting their use. Gliomas over‐express CD46, CD80, and CD86, all of which bind adenovirus serotype 3.

Pathobiological Implications of MUC4 in Non–Small-Cell Lung Cancer

Introduction: Altered expression of MUC4 plays an oncogenic role in various cancers, including pancreatic, ovarian, and breast. This study evaluates the expression and role of MUC4 in non–small-cell lung cancer (NSCLC). Methods: We used a paired system of MUC4-expressing (H292) and MUC4-nonexpressing (A549) NSCLC cell lines to analyze MUC4-dependent changes in growth rate, migration, and invasion using these sublines. We also evaluated the alterations of several tumor suppressor, proliferation, and metastasis markers with altered MUC4 expression. Furthermore, the association of MUC4 expression (by immunohistochemistry) in lung cancer samples with patient survival was evaluated. Results: MUC4-expressing lung cancer cells demonstrated a less proliferative and metastatic phenotype. Up-regulation of p53 in MUC4-expressing lung cancer cells led to the accumulation of cells at the G2/M phase of cell cycle progression. MUC4 expression attenuated Akt activation and decreased the expression of Cyclins D1 and E, but increased the expression of p21 and p27. MUC4 expression abrogated cancer cell migration and invasion by altering N- & E-cadherin expression and FAK phosphorylation. A decrease in MUC4 expression was observed with increasing tumor stage (mean composite score: stage I, 2.4; stage II, 1.8; stage III, 1.4; and metastatic, 1.2; p = 0.0093). Maximal MUC4 expression was associated with a better overall survival (p = 0.042). Conclusion: MUC4 plays a tumor-suppressor role in NSCLC by altering p53 expression in NSCLC. Decrease in MUC4 expression in advanced tumor stages also seems to confirm the novel protective function of MUC4 in NSCLC.

Sphingosine kinase 1 is required for mesothelioma cell proliferation: role of histone acetylation.

Background Malignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics. Methodology/Principal Findings In this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK1−/− null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts. Conclusions/Significance The lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target.

Proteomic characterization of non-small cell lung cancer in a comprehensive translational thoracic oncology database.

BackgroundIn recent years, there has been tremendous growth and interest in translational research, particularly in cancer biology. This area of study clearly establishes the connection between laboratory experimentation and practical human application. Though it is common for laboratory and clinical data regarding patient specimens to be maintained separately, the storage of such heterogeneous data in one database offers many benefits as it may facilitate more rapid accession of data and provide researchers access to greater numbers of tissue samples.DescriptionThe Thoracic Oncology Program Database Project was developed to serve as a repository for well-annotated cancer specimen, clinical, genomic, and proteomic data obtained from tumor tissue studies. The TOPDP is not merely a library--it is a dynamic tool that may be used for data mining and exploratory analysis. Using the example of non-small cell lung cancer cases within the database, this study will demonstrate how clinical data may be combined with proteomic analyses of patient tissue samples in determining the functional relevance of protein over and under expression in this disease.Clinical data for 1323 patients with non-small cell lung cancer has been captured to date. Proteomic studies have been performed on tissue samples from 105 of these patients. These tissues have been analyzed for the expression of 33 different protein biomarkers using tissue microarrays. The expression of 15 potential biomarkers was found to be significantly higher in tumor versus matched normal tissue. Proteins belonging to the receptor tyrosine kinase family were particularly likely to be over expressed in tumor tissues. There was no difference in protein expression across various histologies or stages of non-small cell lung cancer. Though not differentially expressed between tumor and non-tumor tissues, the over expression of the glucocorticoid receptor (GR) was associated improved overall survival. However, this finding is preliminary and warrants further investigation.ConclusionThough the database project is still under development, the application of such a database has the potential to enhance our understanding of cancer biology and will help researchers to identify targets to modify the course of thoracic malignancies.

Combined MET Inhibition and Topoisomerase I Inhibition Block Cell Growth of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)–induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC. Mol Cancer Ther; 13(3); 576–84. ©2013 AACR.