TGF-β1 Supports Leukemia Cell Survival Via Negative Regulation of FLI-1 Transcription Factor, ERK Inactivation and MMP-1 Secretion

Abstract 3543

Transforming growth factor β (TGF-β) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al., Br J Haematol.2008) and the anti-leukemic effects of action of TGF-β neutralizing antibody under TGF-β abundant and hypoxic bone marrow (BM) microenvironment (ASH, 2012). Friend leukemia virus integration 1 (FLI-1), a member of Ets transcriptional factors, plays a pivotal role in the regulation of extracellular matrix (ECM) genes, and is known to be negatively regulated through TGF-β1-dependent acetylation. We have recently reported that abnormal (high or low) expression of FLI-1 protein analyzed by reverse phase protein arrays is associated with inferior remission duration and reduced survival (Kornblau et al., Blood, 2011). Notably, among 195 proteins tested, FLI-1 expression correlated most with SMAD4, the common mediator in a family of SMAD proteins involved in TGFβ signaling. In this study, we investigated the molecular interactions between TGF-β1 and FLI-1 in AML cells and its functional role in TGF-β-mediated survival. In four AML cell lines, MV4;11, U937, NB4, and OCI-AML3, recombinant TGF-β1 (2ng/mL) induced the TGF-β downstream signaling targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and/or Smad2 phosphorylation, which was reversed by anti-TGF-β neutralizing antibody 1D11 (Genzyme). No consistent change of Smad4 expression was observed in TGF-β1 treated cells. Treatment with rhTGF-β1 inhibited serum starvation-induced apoptosis in MV4;11, U937 and NB4, but not in OCI-AML3 cells. The anti-apoptotic effect of TGF-β1 was associated with G₀/G₁ cell cycle arrest, which was effectively reversed by anti-TGF-β antibody 1D11.

In MV4;11, U937 and NB4 cells, in which rhTGF-β1 promoted cell survival, rhTGF-β1 downregulated expression levels of FLI-1 mRNA and/or protein. However, FLI-1 was upregulated by rhTGF-β1 in OCI-AML3 cells. Since FLI-1 activation is known to cause cell proliferation associated with Ras pathway activation, we investigated MAPK signaling downstream of Ras. Changes in ERK phosphorylation levels after rhTGF-β1 treatment were fully concordant with FLI-1, whereby phospho-ERK was downregulated in MV4;11, U937, and NB4 cells, and upregulated in OCI-AML3 cells. These effects were reversed by anti-TGF-β antibody 1D11. In turn, rhTGF-β1 induced Matrix metalloproteinase-1 (MMP-1) mRNA which inversely correlated with FLI-1 expression. (U937; 6.9 fold increase, OCI-AML3; 3.1 fold decrease). It has been reported that ERK signaling upregulates MMP-1 expression, and that FLI-1 downregulates MMP-1 promoter activity in human fibroblasts. MMP-1, being responsible for degradation of collagenous proteins of ECM, correlates with poor prognosis in leukemia. We also observed that rhTGF-β1 induced significant upregulation of anti-apoptotic Bcl-2 in MV4;11, U937, and NB4 cells, but not in OCI-AML3 cells.

In summary, TGF-β-induced FLI-1 downregulation and ERK inactivation may be implicated in pathological matrix remodeling via oncogenic MMP-1 transcription in TGF-β abundant BM microenvironment. These findings suggest that FLI-1 and MMP-1 contribute to chemoresistance and poor outcomes in AML and represent potentially targetable molecular aberrations in AML.