S. K. Golden1, H. Jin1,3, V. Jain1, A. Ma1, A. Marin1, C. Matsumura1, R. Jaskula-Sztul1,3, S. Miyamoto2, H. Chen1,3 1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Oncology,Madison, WI, USA 3University Of Alabama,Surgery,Birmingham, Alabama, USA
Introduction: Medullary thyroid cancer (MTC) is a neuroendocrine malignancy arising from the calcitonin-producing C-cells of the thyroid gland. To date, no effective systemic therapies for MTC have been established. AB3 is a novel histone deacetylase inhibitor (HDACi) that has been shown to diminish MTC cell line proliferation by inducing apoptosis. Preliminary studies have suggested that phenotypic regulation by AB3 may be mediated by Notch3 signaling; however, AB3's broader effects on gene expression and its mode of Notch3 activation are not well understood.
Methods: The effects of AB3 treatment on transcription were investigated using a DNA microarray. Human MTC cell lines (MZ-CRC-1 and TT) were treated with 2 uM AB3 or DMSO vehicle control. The microarray results were filtered to remove non-coding genes, unnamed genes, and genes with an absolute fold change <1.5 or ANOVA p-value >0.05 in either cell line. Selected DNA microarray results were then validated by qRT-PCR. Lastly, a luciferase reporter construct harboring deletion fragments of the Notch3 upstream region was used to map AB3 responsive elements within the Notch3 promoter. Luciferase activity of the constructs and a promoter-less backbone were measured following treatment with AB3 (a Notch3 inducer) or vehicle control.
Results: 67,528 genes were assessed by DNA microarray after AB3 treatment. Of these, 2,243 showed differential expression after filtering. Genes affected by AB3 treatment included members of the nuclear factor-κB (NF-κB) pathway and its targets: NFKB1, ATM, PARP1, XIAP, IκBα, cyclin D1, and BCL2 (down-regulated), as well as SENP2 (up-regulated). These results were consistent with NF-κB pathway down-regulation and were validated by qRT-PCR. Interestingly, qRT-PCR showed substantial increases in Notch 1-3 transcription, including a 33 fold increase in Notch3. Deletion mapping identified the potential AB3-responsive elements within the human Notch3 promoter as a 31-bp fragment located 109 to 140 nucleotides upstream of the Notch3 start codon.
Conclusion: Down-regulation of the NF-κB pathway by AB3 was demonstrated by DNA microarray and validated by qRT-PCR. NF-κB is a major stress-responsive transcription factor whose activation is associated with intrinsic cancer cell resistance to chemo- and radiation therapy. Understanding the mechanism by which AB3 down-regulates NF-κB could provide insight into improving HDACi-based therapies. Furthermore, we describe for the first time the potential HDACi binding site within the Notch3 promoter, which could determine the mechanism(s) of HDACi regulation of Notch signaling.
