Studies On The Mechanisms Of The Reciprocal Regulation Between Regulator Of Calcineurin 1 And NF-κB And The Inhibition Of Acute Leukemia Cells Viability

Nuclear factor-KB (NF-кB) is a family of transcription factors that share homology to the retroviral oncoprotein v-Rel. Abnormal levels of constitutively activated NF-кB have been detected in hematopoietic cancers including acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), Hodgkin's lymphoma, and in various solid tumors (1-4). The active form of NF-кB is known to have anti-apoptotic activity and is thereby considered to be a key survival factor for several types of cancer (5,6). NF-кB deficiency delays leukemia onset in the TEL-JAK2 transgenic mouse model of human T-ALL (7), and inhibition of the pathway efficiently restricts tumor growth both in the established mouse models of leukemia and human ALL cells (8). Inhibition of NF-кB has been found to be an important mechanism of action of steroids, nonsteroidal anti-inflammatory drugs, and natural and synthetic compounds that show therapeutic and preventive activity in cancer.The mammalian NF-кB families contain five members including NF-кB1 (p50), NF-кB2 (p52), RelA (p65), RelB and C-Rel, which form various homo-or heterodimeric complexes (9). Activation of NF-кB is tightly modulated by inhibitory subunits known as IкB (10). IкBs bind to NF-кB dimers and sterically block their nuclear localization sequences, thereby causing their cytoplasmic retention. When cells are stimulated by activators, such as tumor necrosis factor a (TNFa) and lipopolysaccharide (LPS) (11,12), IкBa are phosphorylated at two conserved serine residues (S32 and S36), which subsequently leads to its degradation by ubiquitin-proteasome pathway (13). Then NF-кB dimers are released and translocated into the nucleus, where they regulate NF-кB target genes'transcription (9). An alternative mechanism for NF-кB activation involves the phosphorylation of tyrosine (Y) residue 42 of IkB (14,15), with or without inducing the degradation of IкB (15-17).Firstly identified in the Down Syndrome Critical Region (18,19), the regulator of calcineurin 1 (RCAN1), inhibits calcineurin activity in vitro and in vivo via interacting with calcineurin subunit A (20-24). Interestingly, RCAN1 not only can repress NFAT signaling pathway via inhibition of calcineurin (25), but also can be activated by activators of calcineurin-NFAT pathway such as calcium ionophore, VEGF, angiotensin II, TNF-a, etc. (26,27), thereby forming a negative feedback loop in RCAN1 gene regulation. The tight regulation of RCAN1 gene expression by a negative feedback loop implies the crucial role of RCAN1 in the regulation of cellular functions. Recent studies demonstrated the involvement of RCAN1 in cancer. Single extra transgenic copy of RCAN1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway (28,29). Hence, it is reasonable to speculate that cyclosporin A and FK506, immunosuppressive drugs that specifically inhibit calcineurin (31), would also suppress tumor angiogenesis. Surprisingly, numerous clinical studies indicate that a significant increase in cancer incidence is a serious complication of transplant recipients receiving long-term immunosupressive therapy (30). The mechanism behind this increased rate of cancer is not yet understood; however, such studies indicate another oncogenic pathway other than inhibition of calcineurin in which RCAN1 may be involved. As NF-кB plays vital roles in cancer, we were interested to know if RCAN1 can interact with NF-кB in tumorigenesis.In the present study, we showed RCAN1 can inhibit NF-кB activity. The underlying mechanism is that RCAN1 can interact and decrease the phosphorylation of IкB at Y42. Enforced expression of RCAN1 by adenovirus markedly reduced cell viability in leukemic cell lines and in primary acute leukemia cells. Also interestingly, we found that RCAN1 gene is regulated by NF-кB signaling pathway. A NF-кB responsive element was identified in the RCAN1.4 promoter region. Our studies implied a novel therapeutic pathway for acute leukemia by activating RCAN1 and inhibiting NF-кB.