IGFBP7 Gene Promoting Cell Proliferation in Acute Myeloid Leukemia Through Activation of AKT3 and CCND1

Abstract 1447

Insulin-like growth factor binding protein 7 (IGFBP7) has been ascribed properties of both tumor suppressor and enhancer of cell proliferation. In solid tumors the important role of IGFBP7 as a tumor suppressor was revealed in several studies.

In acute T-lymphoblastic leukemia (T-ALL), high IGFBP7 expression is associated with a more immature phenotype of early T-ALL, inferior survival, and predicts primary chemotherapy resistance. In a separate study, IGFBP7 acts as a positive regulator of ALL and bone marrow stromal cells growth, and significantly enhances in-vitro resistance to asparaginase. Higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (P=0.003) in precursor B-cell Ph negative ALL patients (n=147) treated with a contemporary polychemotherapy protocol.

In acute myeloid leukemia, the role of IGFBP7 is largely unknown. In our previous published study [Hu et al, 2011], we demonstrated that IGFBP7 overexpressed in majority of childhood AML (n=66) at diagnosis and upon relapsed, but not at remission stage. We now further explore its mechanism in promoting AML cells proliferation. Compared with control, transfection of full length IGFBP7 in K562 cells [V-BP7] resulted in 23% increased of proliferation in 48 hours. Cell cycle analysis by flow cytometry showed decreased G0/G1 phase and increased S phase in V-BP7 comparing with control, suggesting enhanced cell cycle progression. While transfection of IGFPB7 siRNA produced an opposite effect of reducing the cell growth in K562 cells.

In consistent with the nature of a secretory protein, the extracellular IGFBP7 level in the condition media from v-BP7 was significantly higher than that from vector control or parental K562 cells measured by ELISA. Incubation parental K562 cells in V-BP7 derived conditioned medium resulted in significant growth enhancement.

Gene expression profiling (GEP) was performed on V-BP7 in contrast to parental K562 cells. Genes which were up-regulated or down-regulated more than 2 folds were regarded as significant difference. Among 10 verified genes, AKT3 showed the highest extent of up-regulation and IGFBP7 siRNA transfection reduced its expression. Cyclin D1 (CCND1) expression was also significantly up-regulated and validated by RT-PCR and Western blot.

V-BP7 treated with an AKT inhibitor (Triciribine) at 2.5μM for 72 hours showed 50% reduction of cell viability. The cell cycle analysis indicated that triciribine reversed cell cycle progression in V-BP7, by increasing cells in G0/G1 phase and reducing cells in S phase. Western blot demonstrated that both phospho-AKT3 and CCND1 were down regulated after treatment with triciribine.

Using real time RT-PCR, we further identified that IGFBP7 and AKT3 expression were significantly correlated (p=0.001; r=0.255) in 39 newly diagnosed childhood AML.