Fbxw11 Triggers a Dual Signaling Axis to Exert Proliferation Effects on Lymphocytic Leukemia

Dysregulation of signal conduction will break the balance of normal hematopoiesis and cause various blood diseases. Ubiquitin-proteasome system (UPS) takes part in both physiological and pathological processes by post-translational regulating intracellular signal transduction pathways. Fbxw11 as a constituent of the SCF (Skp1-Cul1-F box) E3 ubiquitin ligase complex, regulates various signal transduction pathways and its pathological roles in tumorigenesis have been suggested. However the role of Fbxw11 in the development of leukemia and the underlying mechanisms remain largely unknown.

To assess the clinical relevance of Fbxw11, the expression of Fbxw11 in AL patients was also studied by real-time PCR. The expression of Fbxw11 was higher in newly diagnosed ALL (p=0.0304) and relapsed ALL patients (p=0.0389) than that in healthy donors. However, results from a follow-up study demonstrated that the expression of Fbxw11 was dramatically decreased after chemotherapy achieving CR (p=0.0202). These results were conformed in the BloodSpot database.

To study in vitro effects of Fbxw11 on leukemia cells, the lymphocytic leukemia cell line, L1210, was stably transfected with Fbxw11 transcript variants and defined as L1210-control, L1210-Fbxw11a, L1210-Fbxw11c and L1210-Fbxw11d, respectively. Increased percentage of S/G2/M phase cells was detected in L1210 cells with high level Fbxw11 by PI stainingand Ki-67/hoechst33342 staining. Fbxw11 significantly promoted the growth of L1210 cells. The proliferation effects of Fbxw11 on the leukemia cells were also tested in vivo . Tumor growth was significantly higher in groups with high level Fbxw11 variants. Significantly higher rate of Brdu positive cells were detected by confocal microscopy in slides formed by L1210 cells expressing high level Fbxw11 variants than control slides .

To further investigate the pro-proliferation mechanism by Fbxw11 transcript variants, Agilent Sureprint G3 Mouse Gene Expression Microarray was performed after total RNA was isolated from control L1210 cells and those expressing high level Fbxw11 transcript variants. Categories of NF-κB and Wnt/β-catenin signaling pathway has high enrichment score in KEGG and IPA respectively. Categories of regulation of cell proliferation and positive regulation of T cell proliferation have high enrichment score in GO biological process. Cell cycle regulator, CyclinD1 and CyclinD3, which were regulated by multiple transcription factors including NF-κB and β-catenin, were significantly up-regulated in L1210 cells expressing high level Fbxw11 transcript variants at protein level. The expression of other NF-κB-regulated genes and β-catenin/TCF-regulated genes were also up-regulated in L1210 cells with high level Fbxw11 transcript variants, which was consistent with the array data. Activation of both NF-κB and β-catenin/TCF signaling pathways was detected in 293T cells expressing high level Fbxw11 transcript variants by dual luciferase reporter system. Furthermore, combination of low dose inhibitors for NF-κB and β-catenin/TCF pathways (ICG-001 and JSH-23) completely diminished pro-proliferation effects of Fbxw11 though the use of low dose either inhibitor had little effect.

Taken together, Fbxw11 was up-regulated in ALL patients. High level expression of Fbxw11 stimulated the proliferation of L1210 lymphocytic leukemia cells in vitro and promoted tumor formation in vivo through cell cycle regulation. Most importantly, activation of both NF-κB and β-catenin/TCF signaling pathways were involved in this process. This work highlights dual signaling activation and illustrates the mechanism of Fbxw11 on the development of lymphocytic leukemia and implys possible approaches for leukemia therapy.

The work was supported by the Grants 81300376, 81370634, 81570153, from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 and 17 JCZDJC35000 from the Tianjin Science and Technology Programs