Stem Cell Mobilization and Egress of AML Blasts from the Bone Marrow Are Both Regulated by MT-MMP-Mediated Activation of MMP-2.

The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate secreted latent forms of MMPs and play a key role in cell migration and tumor cell invasion and metastasis. Previously we reported that mobilized peripheral blood (mPB) CD34⁺ cells as well as AML blasts secrete inactive (pro)MMP-2, in contrast to normal steady-state bone marrow (BM) CD34⁺ cells which do not. In this study we hypothesized that the egress from BM of normal hematopoietic stem/progenitor cells during G-CSF-induced mobilization or of AML blasts shares a common mechanism that involves activation of secreted proMMP-2 by MT-MMPs. We evaluated the expression of MT-MMPs (MT1-MMP, MT2-, MT3- MT4-, MT5-, MT6-) in normal and leukemic hematopoietic cells, as well as in BM stromal cells, and determined their role in proMMP-2 activation and migration. We found MT1-MMP expression (mRNA and protein) in mPB CD34⁺ cells and in the majority of AML samples (from 21 out of 26 patients) and upregulation of MT1-MMP by G-CSF in steady-state BM CD34⁺ cells. Moreover, we confirmed the secretion of proMMP-2 in media conditioned by hematopoietic (mPB CD34⁺, AML) cells and stromal cells. However, we detected the active form of MMP-2 in co-cultures of the hematopoietic (mPB CD34⁺, AML) cells with stromal cells, as well as in co-cultures of steady-state BM CD34⁺ cells stimulated with G-CSF with stroma. In cultures of stromal cells alone G-CSF had no effect on the expression of either MT1-MMP or MMP-2. To evaluate the role of MT-MMPs in proMMP activation, we examined the leukemic KG-1 cell line, which we found not to express any MT-MMPs, and did not detect active MMP-2 in co-cultures with stroma. On the other hand, primary AML samples that did not express MT1-MMP but expressed MT2-, MT4- or MT5-MMPs activated proMMP-2 in co-cultures with stroma. Moreover, the MT1-MMP inhibitor epigallocatechin-3-gallate significantly reduced trans-Matrigel migration of mPB CD34⁺ (by 50%) and AML cells (by 70–90%). Hence we suggest that in the BM microenvironment (i) MT1-MMP localized on the surface of hematopoietic cells activates MMP-2, inducing a highly proteolytic microenvironment, and (ii) MT1-MMP upregulation by G-CSF in BM CD34⁺ cells can result in CD34⁺ cell mobilization from BM. Similarly, the constitutive high expression of MT1-MMP and other MT-MMPs in AML blasts also contributes to MMP-2 activation in the BM microenvironment and may be conducive to the egress of AML blasts from the BM.