The Natural Chemopreventive Agent Sulforaphane Inhibits STAT5 Activity

Abstract

Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.

Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

“…Mouse-specific quantitative PCR primers used are listed in Supplementary Table S1. Data were normalized to mouse S9 or 36b4 ribosomal mRNAs and expressed as relative mRNA levels, like previously reported ( 4 , 36 , 40 , 41 , 43 , 67 ). Data are mean ± SD of the quantitative PCR performed in either duplicate or triplicate and are representative of at least two independent experiments.…”

“…ChIP of STAT5, RNA polymerase II, total histone H3, acetylated histones H3/H4 and Brd2 (Ba/F3 cells) was performed from whole cell extracts as previously described ( 36 , 40 , 43 ). Improved Brd2 detection in Ba/F3–1*6 cells by ChIP was achieved using an alternative protocol of nuclei fractionation and chromatin shearing, adapted from Métivier et al .…”

“…( 68 ) and Okada and Fukagawa ( 69 ) respectively, with the following modifications. Formaldehyde-crosslinked cells ( 36 , 40 , 43 ) were sequentially lysed following Métivier's protocol ( 68 ). Nuclei were resuspended in HDG150 buffer (20 mM HEPES pH 7.6, 150 mM KCl, 10% glycerol, 0.5 mM DTT, 10 mM NaF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride) and sonicated six times for 20 s on a Branson Sonifier 250 (output 3, 50% duty cycle).…”

“…MNase activity was stopped with 5 mM EGTA and the sheared nuclear lysate was diluted 5-fold in immunoprecipitation (IP) buffer ( 40 ). After centrifugation (3000 x g , 10 min at +4°C), the supernatant was further precleared against protein A-sepharose and ChIP conducted as previously described ( 36 , 40 , 43 ), using 3 μg of Brd2-specific antibody or of rabbit IgG as a control.…”

“…Inhibitors targeting STAT5 transcriptional activity, at a step subsequent to its binding to DNA, have been described. They include deacetylase inhibitors such as trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB), initially described by our group, and more recently the bromodomain inhibitor (+)-JQ1 and the natural compound sulforaphane ( 40 – 43 ). The mechanism of inhibition of STAT5 activity by these inhibitors remains unknown.…”