title: Histone modification enhances the effectiveness of IL-13 receptor targeted immunotoxin in murine models of human pancreatic cancer.
abstract: BACKGROUND
  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.
  
  
  METHODS
  We examined epigenetic regulation of IL-13Rα2 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. In vitro cytotoxicity assays and in vivo 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.
  
  
  RESULTS
  We found that CpG sites in IL-13Rα2 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 in vitro 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.
  
  
  CONCLUSIONS
  We have identified a novel function of histone modification in the regulation of IL-13Rα2 in pancreatic cancer cell lines in vitro and in vivo. 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.
authors.affiliation: Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA.
authors.forename: Toshio
authors.initials: T
authors.lastname: Fujisawa
authors.forename: Bharat H
authors.initials: BH
authors.lastname: Joshi
authors.forename: Raj K
authors.initials: RK
authors.lastname: Puri
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D000922:Immunotoxins / Q000276:immunology*; D018793:Interleukin-13 / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D053660:Receptors, Interleukin-13 / Q000235:genetics* / Q000276:immunology*; D018808:Transcription Factor AP-1 / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D018076:DNA, Complementary; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D000922:Immunotoxins; D018793:Interleukin-13; D053660:Receptors, Interleukin-13; D018808:Transcription Factor AP-1; D020782:Matrix Metalloproteinases