Halofuginone Exerts Antiproliferative and Antiangiogenic Actions on Acute Promyelocytic Leukemia Cells through Modulation of the TGFβ Pathway.

The effects of TGFβ signaling in tumorigenesis is both cell type and context-dependent. Although this cytokine may behave as tumor suppressor in early stages of malignant transformation, tumor progression is often accompanied by altered TGFβ responsiveness and increased angiogenesis. Acute Promyelocytic Leukemia (APL) is a distinct subtype of Acute Myelogenous Leukemia characterized by rearrangements involving the PML and RARα genes on chromosomes 15 and 17, respectively. The expression of the PML/RARα oncoprotein leads to PML delocalization and functional impairment. Among its physiological roles, PML is a regulator of the TGFβ pathway, and the expression of PML-RARα has been associated with TGFβ resistance to differentiation and cell growth inhibition. Moreover, TGFβ is known to regulate Vascular Endothelial Growth Factor (VEGF) production and response. APL patients present increased bone marrow microvessel density, and the APL cell line NB4 was shown to secrete high levels of VEGF. Our aim was to test on APL the effect of Halofuginone (HF), an alkaloid that has been shown to inhibit TGFβ in other cell types. Cell cultures of NB4 and NB4-R2 cell lines, this latter resistant to ATRA, were treated with increasing doses of HF (6.25, 12.5, 25, 50, 100 ng/ml) and 10⁻⁶M of ATRA during 72 hours. Cell proliferation and apoptosis were accessed by flow cytometry using a simultaneous staining with bromodeoxyuridine and 7AAD. In NB4, there was significant cell growth inhibition with HF doses superior to 25 ng/ml (P <0.001). In addition, a 1.5 fold increase in apoptosis was seen with 100 ng/ml (P <0.001). In NB4-R2, cell growth inhibition was observed with 50 and 100 ng/ml and apoptosis with 100 ng/ml of HF (P < 0.001). HF was able to block the cell cycle progression at G1/S transition and, simultaneously, reduce Bcl2 protein expression in both cell lines. Concomitantly, mRNA expression of TGFβ target genes involved in cell cycle regulation was evaluated by real time PCR. Results showed the upregulation of p15, SMAD3, TGFβ and TGFβRI, and downregulation of c-MYC by treatment with high doses of HF (75 and 100 ng/ml). VEFG and TGFβ production was measured by ELISA in supernatants after 72 hours of culture. Significant reduction of VEGF levels was detected in samples treated with HF at doses higher than 25 ng/ml or with ATRA (P=0.018) and a decrease of TGFβ secretion was observed with 50 and 100 ng/ml of HF (P=0.026). Nuclear extracts from cell cultures treated as above were obtained, and western blot analysis showed that higher doses of HF (50 to 100 ng/ml) reduced TGFβ and Smad 4 expression. Our results indicate that HF was able to inhibit TGFβ at protein level and consequently to reduce VEGF production and thus may revert APL aberrant angiogenesis. As TGFβ transcription is at least in part auto-regulated, HF treatment was associated with an increase of TGFβ transcripts. These effects were independent of ATRA sensitivity, since both cell lines presented the same behavior. Although the disruption of TGFβ signaling itself is not sufficient to initiate malignant transformation, it may be a critical second step that contributes to leukemia progression. In this context, HF may have therapeutic potential in APL.