Expression of Homeobox Gene HLXB9 Mediated by Constitutive Active Signal Transduction Pathways Activates IL-6 in Hodgkin’s Disease Cell Lines.

Hodgkin’s disease (HD) is characterized clinically by rare neoplastic giant cells surrounded by a mass of infiltrating diverse lymphocytes indicating the importance of cell communication for this disease. A variety of cytokines have been detected in HD patients and cell lines, including IL-6 which plays an important role in pathogenesis, and high levels of which are associated with bad prognosis. Regulators of IL-6 expression known to be actively overexpressed in HD cells are NFkB and AP-1 proteins. Recently, the homeobox protein GBX2 has been described to activate IL-6 in prostate carcinoma. Hence, to investigate IL-6 regulation in HD cells, we screened a panel of HD cell lines (HDLM-2, KM-H2, L-1236, L-428, L-540, SUP-HD1) by RT-PCR for expression of EHG family homeobox genes comprising GBX2, GBX1, EN1, EN2 and HLXB9. We found HLXB9 to be expressed in 5/6 HD cell lines correlating with IL-6 expression while GBX2 expression was absent. Inhibitory treatment of HD cell lines HDLM-2 and KM-H2 with antisense oligonucleotides (AS) directed against HLXB9 reduced both HLXB9 and IL-6 expression as detected by RT-PCR. HD cell line L-540 expressed neither HLXB9 nor IL-6 and, in contrast to other HD cell lines, lacked constitutive NFkB activity. NFkB activation by TPA treatment induced IL-6 expression in L-540, while combination of TPA treatment with forced expression of full length HLXB9 significantly enhanced IL-6 expression in these cells. HELA cells (which expressed HLXB9 but not GBX2) transfected with an IL-6 promoter-driven reporter construct (pIL6-EGFP) yielded green cells (30%). Treatment of similarly transfected HELA cells with AS-HLXB9 or transfection of HELA with pIL6-EGFP lacking the putative HLXB9 binding site showed significantly fewer green cells (20% and 22%, respectively). Taken together, these results suggest that HLXB9 directly enhances IL-6 expression in HD cells and may contribute to their unique phenotype. Recently, we identified ectopic expression of HLXB9 following chromosomal juxtaposition with MYB in acute myeloid leukemia (manuscript submitted). In contrast, we failed to detect chromosomal rearrangements of HLXB9 or MYB in HD cell lines. Expression of GBX2 in chicken AML cells requires both MYB and an activated signal transduction pathway. To investigate such a mechanism in HD cell lines which we had confirmed to express MYB protein, we pharmacologically inhibited certain signal transduction pathways (MAPK, JAK-STAT and PI3K) showing that all three pathways contribute to HLXB9 expression as analyzed by RT/RQ-PCR. Because MAPK and JAK-STAT pathways were recently shown to be constitutively activated in HD we performed AKT/phospho-AKT analysis to demonstrate constitutively active PI3K pathway in 5/6 HD cell lines. Thus, constitutive activated signal transduction pathways (probably together with MYB) contribute to HLXB9 expression in HD cell lines. In summary, we identified ectopic expression of the homeobox gene HLXB9 activating IL-6 expression in HD cell lines. The PI3K pathway was found to be permanently active in HD cell lines contributing together with other constitutive active signal transduction pathways to HLXB9 expression.