NPM-ALK Oncogene Upregulates the Expression of Inducible Nitric Oxide Synthase in T-Cell Lymphoma Through STAT3/MicroRNA-26a-Dependent Mechanism

Abstract 1383

The oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma tumors. NPM-ALK promotes cellular survival and transformation, and initiates lymphomagenesis. To induce its effects, NPM-ALK interacts with a comprehensive network of oncogenes and tumor suppressor genes. Previous studies have identified upstream and downstream members of this network that modulate the effects of NPM-ALK. However, the exact mechanisms by which NPM-ALK induces its effects are not completely identified.

Nitric oxide (NO) is a gaseous molecule and a highly active free radical. It plays critical, yet versatile, roles in physiological cellular functions including survival, adhesion, migration, and angiogenesis. It also has important contributions to tumor progression and metastasis. NO is generated from L-arginine via 3 distinct isoforms of the enzyme NO synthase including the inducible form (iNOS). The expression and role of iNOS in NPM-ALK-expressing T-cell lymphoma is not known.

We found that iNOS mRNA and protein are expressed in the NPM-ALK-expressing T-cell lymphoma cell lines Karpas 299, DEL, and SR-786. In agreement with a functional role of iNOS in these cells, the selective iNOS inhibitor 1400W and the NO scavenger CPITO decreased the proliferation of the NPM-ALK-expressing T-cell lymphoma cell lines (p < 0.05). In addition, the NO donor SNAP recovered the decrease in the proliferation of these cell lines after treatment with the ALK inhibitors TAE684 and PF-2341066 (p < 0.05). Interestingly, iNOS protein was totally absent in normal human T lymphocytes in spite of a notable increase in iNOS mRNA. These observations suggested that the downregulation of iNOS protein in T lymphocytes vs. its high levels in the NPM-ALK-expressing T-cell lymphoma cell lines might occur posttranscriptionally. Because microRNA are major posttranscriptional regulators of protein expression, we set to analyze possible aberrancies in microRNA. First, we performed an array study comparing the expression of microRNA in T lymphocytes vs. NPM-ALK-expressing T cell lymphoma cell lines. Three web-based algorithms [TargetScan (http:// genes.mit.edu/targetscan/), miRanda (http:// www.microrna.org/microrna/home.do), and PicTar (http:// pictar.mdc-berlin.de/] identified microRNA-26a (miR-26a) to potentially bind with iNOS 3'-UTR. Importantly, statistical analysis of the array data showed that the expression of miR-26a is much more pronounced in T lymphocytes in comparison with the lymphoma cell lines (p < 0.00001), and real-time qPCR further confirmed these results. To examine the functional interactions between miR-26a and iNOS 3'-UTR, we performed a luciferase assay in 293T cells after transfection with iNOS 3'-UTR reporter gene. Wild type miR-26a, and not mutated miR-26a, induced a marked decrease in the luciferase activity of iNOS 3'-UTR reporter gene (p < 0.05). We then questioned whether NPM-ALK underlies the aberrancies in the expression of miR-26a and iNOS. Specific targeting of NPM-ALK by siRNA increased miR-26a and simultaneously decreased iNOS protein levels in the lymphoma cell lines. Next, we reasoned to examine how NPM-ALK induces its effects on this system. A transcription factor screening showed that miR-26a gene promoter, which is located within the promoter of a host gene, CTDSPL, contains 2 consensus sequences where it can potentially bind with STAT3, a major downstream target of NPM-ALK. Chromatin immunoprecipitation studies illustrated the binding between STAT3 and CTDSPL promoter. To analyze the functional interactions between STAT3 and miR-26a, a luciferase assay performed in NPM-ALK-expressing T-cell lymphoma cell lines showed that the downregulation of STAT3 by siRNA increased CTDSPL promoter activity. In addition, downregulation of STAT3 protein or the decrease in STAT3 phosphorylation by STAT3 or NPM-ALK siRNA, respectively, increased miR-26a levels and decreased iNOS protein expression in NPM-ALK-expressing T-cell lymphoma cell lines.

Our study proposes novel mechanisms by which NPM-ALK contributes to the survival of T-cell lymphoma. We are currently analyzing the expression of miR-26a and iNOS in primary human lymphoma tumors, and examining the effects of transfecting NPM-ALK-expressing T-cell lymphoma cell lines with miR-26a on cell proliferation, adhesion, and migration.