Expression of the 67-kDa Laminin Receptor Is Associated with Increased AML Cells through the Enhanced GM-CSF/Stat5 Signaling

• Published in: Blood Vol. 110; no. 11; p. 2845
• Main Authors: Ando, Koji, Miyazaki, Yasushi, Sawayama, Yasushi, Horio, Kensuke, Matsuo, Emi, Yamasaki, Reishi, Inoue, Yoriko, Iwanaga, Masako, Imanishi, Daisuke, Tsushima, Hideki, Yoshida, Shinichiro, Taguchi, Jun, Hata, Tomoko, Tomonaga, Masao
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 16.11.2007
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V110.11.2845.2845

[Background] The 67-kDa laminin receptor (67LR) is a non-integrin cell surface receptor that mediates high-affinity interactions with laminin. Although 67LR was reported to interact with GM-CSF receptor (GM-CSFR) modulating the signaling pathway in hematopoietic cells, little report regarding its expression and the characteristics of AML. [Objective] We addressed whether the surface expression of 67LR be related to the characteristics of AML, and its molecular mechanisms. [Methods] The surface expression of 67LR on CD34-positive AML cells was assessed since it is widely expressed on the mature hematopoietic cells. After purification of CD34 positive cells, flowcytometric analysis of 67LR in 27 AML (M0 [1], M1 [3], M2 [11], M4 [6], M5 [2], M6 [2], MDS/AML [2]) was performed. A GM-CSF dependent TF-1 leukemia cell line (established from M6) was used for the in vitro analysis. cDNA or short interfering RNA of LR was transfected into TF-1 (TF-1LR and TF-1si). Using these cell lines, cell growth, colony formation, cell cycle distribution, and protein phosphorylation were tested. [Results] AML cases were divided into two groups by the surface-expression of 67LR: 9cases in the high-expression group (positive in >25% of cells, LR-H) and 18 in the low-expression group (LR-L).The median WBC was significantly higher in LR-H than LH-L (36,700 and 3,250/μl, respectively, p=0.015). Nucleated cell count of bone marrow was also higher in LR-H (p=0.01). Overall survival of LR-H was significantly poor than LR-L (20% and 54%, respectively, p=0.01). Since the expression of 67LR was related to the increased AML cells in clinical samples, we hypothesized that the expression of 67LR influenced the growth of leukemia cells. Using TF-1, the surface-expression of 67LR was modulated by the over expression or interfering of mRNA of 67LR. The surface expression of 67LR on wild type TF-1, TF-1LR and TF-1si was 64%, 92% and 36%, respectively. In WST1 assay, the expression of 67LR was related to the proliferation of TF-1 cells: TF-1LR proliferated rapidly than control (absorbance 1.71±0.06 and 1.24±0.10, respectively. p=0.0023), whereas TF-1si cells grew slowly (absorbance of TF-1si, 0.790±0.004; control, 1.149±0.052, p=0.0018). The number of colony in semi-solid media was related to the expression of 67LR: TF-1LR formed 300±10, the control cells made 91±8colonies/5000cells (p<0.001), and TF-1si made less colonies than control (46±3 and 76±6 colonies, respectively, p=0.002). The cells in S-phase increased in TF-1LR than control by BrdU assay (38±1% and 32±2%, respectively, p=0.01). These results demonstrated the surface expression of 67LR was associated with the enhanced cell growth in TF-1. Since 67LR was reported to interact with GM-CSFR, we tested the signaling though GM-CSFR. After 16 hours of serum-free culture, cells were treated with GM-CSF and FBS. The phosphorylation of both ERK1/2 and Stat5 was diminished in TF-1si than control. In contrast, that of Stat5 increased in TF-1LR than control (MFI of phspho-Stat5, 265±39 and 158±54, respectively, p=0.05). [Conclusion] These results suggested that the surface expression of 67LR contributed, at least, to the proliferation of AML through the enhancing the signaling of GM-CSF/Stat5 pathway. This may relate to the poor prognosis of LR-H group.