Transient Inhibition of PI3Kδ Enhances the Therapeutic Effect of Intravenous Delivery of Oncolytic Vaccinia Virus

Tumor-targeting oncolytic viruses such as vaccinia virus (VV) are attractive cancer therapeutic agents that act through multiple mechanisms to provoke both tumor lysis and anti-tumor immune responses. However, delivery of these agents remains restricted to intra-tumoral administration, which prevents effective targeting of inaccessible and disseminated tumor cells. In the present study we have identified transient pharmacological inhibition of the leukocyte-enriched phosphoinositide 3-kinase δ (PI3Kδ) as a novel mechanism to potentiate intravenous delivery of oncolytic VV to tumors. Pre-treatment of immunocompetent mice with the PI3Kδ-selective inhibitor IC87114 or the clinically approved idelalisib (CAL-101), prior to intravenous delivery of a tumor-tropic VV, dramatically improved viral delivery to tumors. This occurred via an inhibition of viral attachment to, but not internalization by, systemic macrophages through perturbation of signaling pathways involving RhoA/ROCK, AKT, and Rac. Pre-treatment using PI3Kδ-selective inhibitors prior to intravenous delivery of VV resulted in enhanced anti-tumor efficacy and significantly prolonged survival compared to delivery without PI3Kδ inhibition. These results indicate that effective intravenous delivery of oncolytic VV may be clinically achievable and could be useful in improving anti-tumor efficacy of oncolytic virotherapy

Identification of genetic alterations in pancreatic cancer by the combined use of tissue microdissection and array-based comparative genomic hybridisation

Pancreatic ductal adenocarcinoma (PDAC) is characterised pathologically by a marked desmoplastic stromal reaction that significantly reduces the sensitivity and specificity of cytogenetic analysis. To identify genetic alterations that reflect the characteristics of the tumour in vivo, we screened a total of 23 microdissected PDAC tissue samples using array-based comparative genomic hybridisation (array CGH) with 1 Mb resolution. Highly stringent statistical analysis enabled us to define the regions of nonrandom genomic changes. We detected a total of 41 contiguous regions (>3.0 Mb) of copy number changes, such as a genetic gain at 7p22.2–p15.1 (26.0 Mb) and losses at 17p13.3–p11.2 (13.6 Mb), 18q21.2–q22.1 (12.0 Mb), 18q22.3–q23 (7.1 Mb) and 18q12.3–q21.2 (6.9 Mb). To validate our array CGH results, fluorescence in situ hybridisation was performed using four probes from those regions, showing that these genetic alterations were observed in 37–68% of a separate sample set of 19 PDAC cases. In particular, deletion of the SEC11L3 gene (18q21.32) was detected at a very high frequency (13 out of 19 cases; 68%) and in situ RNA hybridisation for this gene demonstrated a significant correlation between deletion and expression levels. It was further confirmed by reverse transcription–PCR that SEC11L3 mRNA was downregulated in 16 out of 16 PDAC tissues (100%). In conclusion, the combination of tissue microdissection and array CGH provided a valid data set that represents in vivo genetic changes in PDAC. Our results raise the possibility that the SEC11L3 gene may play a role as a tumour suppressor in this disease

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A phylogenetic approach to assessing the significance of missense mutations in disease genes

The identification of deleterious mutations within candidate genes is a crucial step in the elucidation of the genetic bases of human disease. However, the significance of any base or amino acid change within a gene is unknown until detailed structural and functional analysis has been carried out. A potentially rapid way of identifying functionally important sites within a gene is to identify evolutionarily conserved regions. Mutations affecting such sites are assumed to be deleterious for the carrier. In this communication we generalize this approach and present a formal framework to assess whether a specific mutation is deleterious given sequence data from a set of homologues. We propose a score that takes into account the nature of the mutation, the conservation of the affected residue among the different species, and their phylogenetic relationships. Its performance is examined using published TP53 mutations and frequent polymorphic variants

Expression of inositol 1,4,5-trisphosphate receptors in mouse oocytes and early embryos: The type I isoform is upregulated in oocytes and downregulated after fertilization

A fertilization-induced increase in intracellular Ca2+ is responsible for initiating all of the events of egg activation. In mammals, the Ca2+ increase takes the form of a series of Ca2+ oscillations showing complex temporal and spatial properties. To understand the nature of these changes, we have investigated the expression patterns of the three isoforms of the inositol trisphosphate receptor (InsP(3)R) during oocyte maturation and preimplantation development. We find that mouse oocytes express mRNAs for all three InsP(3)R subtypes. Semiquantitative ratio reverse-transcriptase polymerase chain reaction shows that the type II isoform is the predominant message in mature oocytes, representing 67% of the InsP(3)R mRNA. In contrast, protein analysis reveals that the type I isoform accounts for all of the detectable InsP(3)R protein, despite representing only 20% of the InsP(3)R mRNA. The levels of InsP(3)R protein were examined to determine whether they correlated with the Ca2+ signaling events surrounding the fertilization process. Type I InsP(3)R protein increased during oocyte maturation and, in addition, within 8 h of fertilization underwent a dramatic decrease. During development to the blastocyst the level of type I InsP(3)R protein did not return to prefertilization levels and types II and III remained below our detection limit. The decrease in InsP(3)R protein after fertilization was found to correlate with a decrease in the sensitivity of InsP(3)-induced Ca2+ release. These studies show that the expression of InsP(3)R mRNA is developmentally regulated, that Ca2+ signaling at fertilization is mediated exclusively through the type I InsP(3)R, and that the InsP(3)R is downregulated after fertilization. (C) 1998 Academic Press

MHC class II molecules on pancreatic cancer cells indicate a potential for neo-antigen-based immunotherapy

MHC class II expression is a hallmark of professional antigen-presenting cells and key to the induction of CD4+ T helper cells. We found that these molecules are ectopically expressed on tumor cells in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC) and on several PDAC cell lines. In contrast to the previous reports that tumoral expression of MHC-II in melanoma enabled tumor cells to evade immunosurveillance, the expression of MHC-II on PDAC cells neither protected cancer cells from Fas-mediated cell death nor caused T-cell suppression by engagement with its ligand LAG-3 on activated T-cells. In fact and surprisingly, the MHC-II/LAG-3 pathway contributed to CD4+ and CD8+ T-cell cytotoxicity toward MHC-II-positive PDAC cells. By combining bioinformatic tools and cell-based assays, we identified a number of immunogenic neo-antigens that can be presented by the patients’ HLA class II alleles. Furthermore, CD4+ T-cells stimulated with neo-antigens were capable of recognizing and killing a human PDAC cell line expressing the mutated genes. To expand this approach to a larger number of PDAC patients, we show that co-treatment with IFN-γ and/or MEK/HDAC inhibitors induced tumoral MHC-II expression on MHC-II-negative tumors that are IFN-γ-resistant. Taken together, our data point to the possibility of harnessing MHC-II expression on PDAC cells for neo-antigen-based immunotherapy

Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the β4 integrin and the PI3k pathway

International audiencePancreatic ductal adenocarcinoma is a devastating disease, characterized by a rapid progression and poor treatment response. Using gene expression profiling of pancreatic cancer tissues, we previously identified periostin as a potential diagnostic and therapeutic target. In this study, we report the overexpression of periostin in a larger set of pancreatic cancer tissues and show that although the periostin transcript is exclusively expressed in tumour cells, the protein product is only detected in the extracellular matrix adjacent to cancer cells. Using an enzyme-linked immunosorbent assay (ELISA) assay, we show significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. We demonstrate that periostin promotes the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhances the survival of tumour cells exposed to hypoxic conditions. At the molecular level, we provide evidence that the alpha(6)beta(4) integrin complex acts as the cell receptor of periostin in pancreatic cancer cells and that interaction promotes phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) though activation of the PI3 kinase pathway, but not the RAS/MEK/ERK pathway. These findings suggest an important role of periostin in pancreatic cancer and provide a rationale to study periostin for diagnostic and therapeutic applications