NF-κB-dependent and -independent epigenetic modulation using the novel anti-cancer agent DMAPT

Abstract

The transcription factor nuclear factor-kappaB (NF-κB) is constitutively active in several cancers and is a target of therapeutic development. We recently developed dimethylaminoparthenolide (DMAPT), a clinical grade water-soluble analog of parthenolide, as a potent inhibitor of NF-κB and demonstrated in vitro and in vivo anti-tumor activities in multiple cancers. In this study, we show DMAPT is an epigenetic modulator functioning in an NF-κB-dependent and -independent manner. DMAPT-mediated NF-κB inhibition resulted in elevated histone H3K36 trimethylation (H3K36me3), which could be recapitulated through genetic ablation of the p65 subunit of NF-κB or inhibitor-of-kappaB alpha super-repressor overexpression. DMAPT treatment and p65 ablation increased the levels of H3K36 trimethylases NSD1 (KMT3B) and SETD2 (KMT3A), suggesting that NF-κB directly represses their expression and that lower H3K36me3 is an epigenetic marker of constitutive NF-κB activity. Overexpression of a constitutively active p65 subunit of NF-κB reduced NSD1 and H3K36me3 levels. NSD1 is essential for DMAPT-induced expression of pro-apoptotic BIM, indicating a functional link between epigenetic modification and gene expression. Interestingly, we observed enhanced H4K20 trimethylation and induction of H4K20 trimethylase KMT5C in DMAPT-treated cells independent of NF-κB inhibition. These results add KMT5C to the list NF-κB-independent epigenetic targets of parthenolide, which include previously described histone deacetylase 1 (HDAC-1) and DNA methyltransferase 1. As NSD1 and SETD2 are known tumor suppressors and loss of H4K20 trimethylation is an early event in cancer progression, which contributes to genomic instability, we propose DMAPT as a potent pharmacologic agent that can reverse NF-κB-dependent and -independent cancer-specific epigenetic abnormalities.

Main

Epigenetics is defined as heritable changes in gene expression mediated mostly by DNA methylation and histone tail modifications without changes in DNA sequence.¹ Epigenetic abnormalities in cancer lead to reprogramming of gene expression resembling embryonic stem cells, loss of tumor suppressors, or reactivation of oncofetal genes.^{2, 3} For example, enhanced histone 3 lysine 27 trimethylation (H3K27me3), mediated by a complex of proteins, including the histone methyltransferase EZH2, causes the silencing of tumor-suppressor genes.^{4, 5}

Similar to H3K27me3, histone 3 lysine 36 trimethylation (H3K36me3) has a diverse role in chromatin structure and function.⁶ H3K36me3 regulates transcription of active euchromatin, alternative splicing, DNA repair, and transmission of the memory of gene expression from parents to offspring.⁷ At least eight enzymes methylate H3K36. Nuclear receptor-binding SET domain protein 1 (NSD1; lysine methyltransferases 3B (KMT3B)) and NSD2 are involved in monomethylation and dimethylation, whereas SET domain containing 2 (SETD2; KMT3A) trimethylates H3K36.⁶ However, depletion of NSD1 in certain cells leads to loss of H3K36me3 and altered gene expression.^{8, 9}

NSD1 functions as a tumor suppressor in several cancers. For example, epigenetic silencing of NSD1 is observed in neuroblastoma.⁹ NSD1 mutation/deletion is observed in bladder cancer.¹⁰ NSD1 is one of the genes essential for anti-estrogen sensitivity in breast cancer.¹¹ NSD1 is also described as an oncogene in acute myeloid leukemia in which a chromosomal translocation creates nucleoporin 98 kDa (NUP98):NSD1 fusion protein, and the expression is driven by NUP98 regulatory elements.¹² NSD2 is an oncogene, whereas SETD2 is inactivated through mutation/deletion in multiple cancers.^{10, 13, 14} Interestingly, H3K36me3 and H3K27me3 on a gene may be mutually exclusive, suggesting distinct role of modulators of these modifications on gene expression.¹⁵

Loss of histone 4 lysine 20 trimethylation (H4K20me3) is a hallmark of cancer.¹⁶ The histone methyltransferases KMT5B (SUV4-20H1) and KMT5C (SUV4-20H2) mediate histone H4K20me3, whereas PHD finger protein 2 (PHF2) demethylates this residue.^{17, 18} NSD1 is also reported to trimethylate H4K20.⁹ H4K20me3 is necessary for the repressive pathway that induce pericentric heterochromatin required for G2/M arrest in response to DNA damage and recruitment of DNA repair complex.^{17, 19} In addition, loss of H4K20me3 is associated with telomere elongation and derepression of telomere recombination.²⁰ Depletion of KMT5B or KMT5C leads to increased telomere elongation, which is common in cancer.²⁰

Develo** drugs that reverse cancer-specific histone modifications is one of our goals. Towards this end, we targeted the transcription factor nuclear factor-kappaB (NF-κB), which is activated in cancers and regulates inflammation-associated epigenetic changes.^{21, 22} Using pharmacological and genomic approaches, we show upregulation of NSD1 and SETD2 upon NF-κB inhibition. Dimethylaminoparthenolide (DMAPT), the pharmacological inhibitor of NF-κB, additionally increased KMT5C and H4K20me3 independent of its NF-κB inhibition attribute.

Results

The effects of DMAPT on histone modifications

Parthenolide, an NF-κB inhibitor, reduces histone deacetylase 1 (HDAC-1) and DNA methyltransferase 1 independent of NF-κB inhibition.^{23, Supplementary Table S1.}

Apoptosis assay

Apoptosis was measured using Annexin V labeling using the Apoptosis Assay Kit from Invitrogen, and the number of apoptotic cells after 24 h of DMAPT treatment was measured by flow cytometry. Both floating and adherent cells were collected and stained with Alexa Fluor-488-conjugated Annexin V and propidium iodide. Annexin V-positive cells are apoptotic, propidium iodide-positive cells are necrotic and double-positive cells are necroapoptotic.

Histone extraction

Cells were lysed with Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100(v/v), 2 mM phenylmethylsulfonylfluoride (PMSF), 0.02% (w/v) NaN₃) and centrifuged at 6500 × g for 10 min at 4 °C to collect nuclei. The histones were subsequently extracted with 0.2 M HCl (Abcam histone extraction protocol, Cambridge, MA, USA).

Electrophoretic mobility gel shift assay

MDA-MB-231 and MEF cells were harvested in their exponential growth phase with or without TNFα (5 ng/ml, R&D Systems, Minneapolis, MN, USA) treatment for 15 min and assayed for NF-κB and SP-1 (as a control) DNA-binding activity as described previously.³⁹ Antibodies for supershift assays were purchased from Santa Cruz (c-Rel, cat. no. sc-070) and Millipore (p65, cat. no. 06-418; p50, cat. no. 06-886).

Statistical analysis

Results of qRT-PCR were analyzed using the GraphPad software (www.Graphpad.com). Analysis of variance was used to determine the P-values between mean measurements. A P-value of <0.05 was deemed significant.

Analysis of public databases for prognostic relevance of NSD1 and SETD2

Expression array data of various bladder cancer stages were obtained from NCBI GEO (GDS1479), and average±S.D. was calculated. NSD1 expression data were from a single affymetrix probe available in the data set, whereas average from three probes was used for SETD2. For breast cancer, analysis of TCGA data set⁵⁵ for NSD1 and SETD2 expression is presented although similar analysis using a public data set with gene expression pattern in tumors of 1809 breast cancer patients yielded similar results.⁴⁴