Previously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ. We examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 −ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results. We have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 − GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13. These results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy.

Previously, we have demonstrated that interleukin‐4 receptor α (IL‐4Rα) is overexpressed on a variety of human cancers and can serve as target for IL‐4 immunotoxin comprised of IL‐4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL‐4Rα expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real‐time RT‐PCR and IHC/ISH techniques. We also examined the effect of IL‐4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two‐sided P‐values. We used Cochran–Armitage Statistics with exact two‐sided P‐values to examine the trend analysis of IL‐4Rα over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL‐4Rα is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL‐4 receptor complex, IL‐2RγC and IL‐13Rα1, were absent or weakly expressed. IL‐4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration‐dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL‐4Rα overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL‐4Rα immunostaining (≥2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ≤ 0.0001). Similarly, 9% stage I tumors were positive for IL‐4Rα (≥2+) compared to 84% stage II (P ≤ 0.0001) and 100% stages III–IV tumors (P ≤ 0.0001). IL‐13Rα1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL‐2RγC was not expressed. Ten normal bladder specimens demonstrated ≤1+ staining for IL‐4Rα and IL‐13Rα1 and no staining for IL‐2RγC. These results demonstrate that IL‐4Rα is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL‐4Rα may be a bladder tumor‐associated protein and a prognostic biomarker.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.

Adrenocortical carcinoma (ACC) is a rare but lethal malignancy without effective current therapy for metastatic disease. IL‐13‐PE is a recombinant cytotoxin consisting of human interleukin‐13 (IL‐13) and a truncated form of Pseudomonas exotoxin A (PE). The main objectives of this Phase I dose‐escalation trial were to assess the maximum‐tolerated dose (MTD), safety, and pharmacokinetics (PK) of IL‐13‐PE in patients with metastatic ACC. Eligible patients had confirmed IL‐13 receptor alpha 2 (IL‐13Rα2) expressions in their tumors. IL‐13‐PE at dose of 1–2 μg/kg was administered intravenously (IV) on day 1, 3, and 5 in a 4‐week cycle. Six patients received 1 μg/kg and two patients received 2 μg/kg of IL‐13‐PE. Dose‐limiting toxicity was observed at 2 μg/kg, at which patients exhibited thrombocytopenia and renal insufficiency without requiring dialysis. PK analysis demonstrated that at MTD, the mean maximum serum concentration (C_max) of IL‐13‐PE was 21.0 ng/mL, and the terminal half‐life of IL‐13‐PE was 30–39 min. Two (25%) of the eight patients had baseline neutralizing antibodies against PE. Three (75%) of the remaining four tested patients developed neutralizing antibodies against IL‐13‐PE within 14–28 days of initial treatment. Of the five patients treated at MTD and assessed for response, one patient had stable disease for 5.5 months before disease progression; the others progressed within 1–2 months. In conclusion, systemic IV administration of IL‐13‐PE is safe at 1 μg/kg. All tested patients developed high levels of neutralizing antibodies during IL‐13‐PE treatment. Use of strategies for immunodepletion before IL‐13‐PE treatment should be considered in future trials.

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.