Histone modification enhances the effectiveness of IL-13 receptor targeted immunotoxin in murine models of human pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>Interleukin-13 Receptor α2 (IL-13Rα2) is a tumor-associated antigen and target for cancer therapy. Since IL-13Rα2 is heterogeneously overexpressed in a variety of human cancers, it would be highly desirable to uniformly upregulate IL-13Rα2 expression in tumors for optimal targeting.</p> <p>Methods</p> <p>We examined epigenetic regulation of <it>IL-13Rα2 </it>in a murine model of human pancreatic cancer by Bisulfite-PCR, sequencing for DNA methylation and chromatin immunoprecipitation for histone modification. Reverse transcription-PCR was performed for examining changes in IL-13Rα2 mRNA expression after treatment with histone deacetylase (HDAC) and c-jun inhibitors. <it>In vitro </it>cytotoxicity assays and <it>in vivo </it>testing in animal tumor models were performed to determine whether HDAC inhibitors could enhance anti-tumor effects of IL-13-PE in pancreatic cancer. Mice harboring subcutaneous tumors were treated with HDAC inhibitors systemically and IL-13-PE intratumorally.</p> <p>Results</p> <p>We found that CpG sites in <it>IL-13Rα2 </it>promoter region were not methylated in all pancreatic cancer cell lines studied including IL-13Rα2-positive and IL-13Rα2-negative cell lines and normal cells. On the other hand, histones at IL-13Rα2 promoter region were highly-acetylated in IL-13Rα2-positive but much less in receptor-negative pancreatic cancer cell lines. When cells were treated with HDAC inhibitors, not only histone acetylation but also IL-13Rα2 expression was dramatically enhanced in receptor-negative pancreatic cancer cells. In contrast, HDAC inhibition did not increase IL-13Rα2 in normal cell lines. In addition, c-jun in IL-13Rα2-positive cells was expressed at higher level than in negative cells. Two types of c-jun inhibitors prevented increase of IL-13Rα2 by HDAC inhibitors. HDAC inhibitors dramatically sensitized cancer cells to immunotoxin in the cytotoxicity assay <it>in vitro </it>and increased IL-13Rα2 in the tumors subcutaneously implanted in the immunodeficient animals but not in normal mice tissues. Combination therapy with HDAC inhibitors and immunotoxin synergistically inhibited growth of not only IL-13Rα2-positive but also IL-13Rα2-negative tumors.</p> <p>Conclusions</p> <p>We have identified a novel function of histone modification in the regulation of IL-13Rα2 in pancreatic cancer cell lines <it>in vitro </it>and <it>in vivo</it>. HDAC inhibition provides a novel opportunity in designing combinatorial therapeutic approaches not only in combination with IL-13-PE but with other immunotoxins for therapy of pancreatic cancer and other cancers.</p

Raft-Dependent Endocytosis of Autocrine Motility Factor/Phosphoglucose Isomerase: A Potential Drug Delivery Route for Tumor Cells

Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway.Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen and thymus cells was negligible both in vitro and following intravenous injection in vivo where AMF/PGI-FITC was selectively internalized by subcutaneous B16-F1 tumor cells.The raft-dependent endocytosis of AMF/PGI may therefore represent a tumor cell specific endocytic pathway of potential value for drug delivery to tumor cells

Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol-3-kinase-dependent in breast carcinoma cells

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells

Gene expression profiling identifies IL-13 receptor alpha 2 chain as a therapeutic target in prostate tumor cells overexpressing adrenomedullin

Human adrenomedullin (AM) is a 52 amino acid peptide, which shares homology with the calcitonin gene-related peptide. Overexpression of AM in the prostate carcinoma cell line PC-3 results in growth inhibition with a 20% (for human AM) and 35% (for rat AM) increase in doubling time compared to parental or mock-transfected cells. We demonstrate by gene expression profiling that AM overexpression results in the dysregulation of approximately 100 genes. Examples of such genes include many involved in the formation of the cytoskeleton, cell adhesion and the extracellular matrix, as well as regulators of the cell cycle and apoptosis, cytokines and transcription factors. Several genes related to cell growth arrest, such as GADD45, IGF-BP6 and RUNX-3, are upregulated by AM. Interestingly, interleukin-13 receptor alpha 2 (IL-13R alpha 2) transcripts were significantly increased in clones overexpressing AM, which was confirmed by semiquantitative RT-PCR analysis. In addition, PC-3 cells treated with AM showed an overexpression of IL-13R alpha 2, which was abolished when cells were preincubated with an anti-AM blocking antibody. When PC-3 cells overexpressing AM and the IL-13R alpha 2 were treated with the highly specific IL13-PE38 cytotoxin, which binds to this receptor, a concentration-dependent inhibition of protein synthesis was observed. The IC(50) (concentration of cytotoxin inhibiting protein synthesis by 50%) ranged from 1 to 4 ng/ml. This cytotoxicity was specific as it was neutralized by the excess of IL-13 and confirmed by clonogenic assays. This study describes a novel AM-induced mechanism of tumor sensitization through the upregulation of functional IL-13R alpha 2 chain, an ideal target for the highly specific recombinant chimeric cytotoxin IL13-PE38

Cysteamine Suppresses Invasion, Metastasis and Prolongs Survival by Inhibiting Matrix Metalloproteinases in a Mouse Model of Human Pancreatic Cancer

Background: Cysteamine, an anti-oxidant aminothiol, is the treatment of choice for nephropathic cystinosis, a rare lysosomal storage disease. Cysteamine is a chemo-sensitization and radioprotection agent and its antitumor effects have been investigated in various tumor cell lines and chemical induced carcinogenesis. Here, we investigated whether cysteamine has anti-tumor and anti-metastatic effects in transplantable human pancreatic cancer, an aggressive metastatic disease. Methodology/Principal Findings: Cysteamine’s anti-invasion effects were studied by matrigel invasion and cell migration assays in 10 pancreatic cancer cell lines. To study mechanism of action, we examined cell viability and matrix metalloproteinases (MMPs) activity in the cysteamine-treated cells. We also examined cysteamine’s anti-metastasis effect in two orthotopic murine models of human pancreatic cancer by measuring peritoneal metastasis and survival of animals. Cysteamine inhibited both migration and invasion of all ten pancreatic cancer cell lines at concentrations (,25 mM) that caused no toxicity to cells. It significantly decreased MMPs activity (IC50 38–460 mM) and zymographic gelatinase activity in a dose dependent manner in vitro and in vivo; while mRNA and protein levels of MMP-9, MMP-12 and MMP-14 were slightly increased using the highest cysteamine concentration. In vivo, cysteamine significantly decreased metastasis in two established pancreatic tumor models, although it did not affect the size of primary tumors. Additionally, cysteamin

Association of Forced Vital Capacity with the Developmental Gene <i>NCOR2</i>

Background Forced Vital Capacity (FVC) is an important predictor of all-cause mortality in the absence of chronic respiratory conditions. Epidemiological evidence highlights the role of early life factors on adult FVC, pointing to environmental exposures and genes affecting lung development as risk factors for low FVC later in life. Although highly heritable, a small number of genes have been found associated with FVC, and we aimed at identifying further genetic variants by focusing on lung development genes. Methods Per-allele effects of 24,728 SNPs in 403 genes involved in lung development were tested in 7,749 adults from three studies (NFBC1966, ECRHS, EGEA). The most significant SNP for the top 25 genes was followed-up in 46,103 adults (CHARGE and SpiroMeta consortia) and 5,062 chi

Genome-wide association analysis identifies six new loci associated with forced vital capacity

Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10−8) with FVC in or near EFEMP1, BMP6, MIR129-2–HSD17B12, PRDM11, WWOX and KCNJ2. Two loci previously associated with spirometric measures (GSTCD and PTCH1) were related to FVC. Newly implicated regions were followed up in samples from African-American, Korean, Chinese and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and the pathogenesis of restrictive lung disease

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