Interleukin-13 Receptor alpha2 (IL-13Ralpha2) is a tumor-associated antigen and target for cancer therapy. Since IL-13Ralpha2 is heterogeneously overexpressed in a variety of human cancers, it would be highly desirable to uniformly upregulate IL-13Ralpha2 expression in tumors for optimal targeting.

We examined epigenetic regulation of IL-13Ralpha2 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-13Ralpha2 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.

We found that CpG sites in IL-13Ralpha2 promoter region were not methylated in all pancreatic cancer cell lines studied including IL-13Ralpha2-positive and IL-13Ralpha2-negative cell lines and normal cells. On the other hand, histones at IL-13Ralpha2 promoter region were highly-acetylated in IL-13Ralpha2-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-13Ralpha2 expression was dramatically enhanced in receptor-negative pancreatic cancer cells. In contrast, HDAC inhibition did not increase IL-13Ralpha2 in normal cell lines. In addition, c-jun in IL-13Ralpha2-positive cells was expressed at higher level than in negative cells. Two types of c-jun inhibitors prevented increase of IL-13Ralpha2 by HDAC inhibitors. HDAC inhibitors dramatically sensitized cancer cells to immunotoxin in the cytotoxicity assay in vitro and increased IL-13Ralpha2 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-13Ralpha2-positive but also IL-13Ralpha2-negative tumors.

We have identified a novel function of histone modification in the regulation of IL-13Ralpha2 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.

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