Abstract
Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.
Highlights
Transforming growth factor beta (TGFb) is a cytokine that regulates multiple cellular responses, including inhibition of cell proliferation and induction of differentiation, senescence, and apoptosis [1,2]
Siegel et al have shown that activation of TGFb delays the appearance of primary mammary tumors, and mice deficient in TGFb signaling are prone to earlier tumor development, suggesting that the tumor suppressor response of TGFb is important in the early stages of tumorigenesis
Our results demonstrate that HF treatment induces anti-proliferative and pro-apoptotic effects, up-regulates TGFb target gene expression, and significantly reduces the leukemic burden in vivo
Summary
Transforming growth factor beta (TGFb) is a cytokine that regulates multiple cellular responses, including inhibition of cell proliferation and induction of differentiation, senescence, and apoptosis [1,2]. Its actions are mediated by binding to the serine/ threonine kinase receptor TbRII that recruits and activates TbRI, which in turn phosphorylates downstream targets. These include the proteins SMAD2 and SMAD3, which translocate to the nucleus in a complex with the common mediator SMAD4 to regulate transcription of target genes [3,4]. Among the TGFb-mediated effects in premalignant cells are the suppression of c-Myc expression [5] and the induction of the cell cycle inhibitors p15 and p21 These actions imply a tumor suppressor role for TGFb, its effects are both celland context-dependent. Advanced disease is accompanied by increased expression and activation of the ligand but decreased TGFb responsiveness, facilitating tumor cell growth [7]
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