Class I HDACs are mediators of smoke carcinogen-induced stabilization of DNMT1 and serve as promising targets for chemoprevention of lung cancer

Abstract

DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDAC) 1 to 3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter, and derepression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen-induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability. In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention.

Conflict of interest statement

Conflict of Interest: none

Figures

Figure 1. Phenotypic changes associated with passage of HBEC 3kt in tobacco related carcinogens

A: Passage of HBEC 3kt in carcinogen increases clonogenicity by soft agar colony formation assay. B: Immunoblots of passage 14, 25 and 31 of benz(a)pyrene and MNU exposed or vehicle treated bronchial epithelial cells reveals increases in DNMT1, G9a and HDAC1–3 protein levels, associated with a decrease in histone-H3 acetylation. Histograms indicate quantified values of fold change between carcinogen exposed and mock treated cells normalized to total H3 expression. C: quantitative rt-PCR for DNMT1, G9A and HDACs 1–3 reveals that DNMT1 protein is not regulated by changes in steady-state mRNA levels, while G9A and HDAC1–3 are. Values are relative expression between T31 and 3kt and normalized to 18S RNA expression. D: quantitative rt-PCR for SFRP2 shows transcriptional repression in carcinogen exposed cells, normalized to 18s expression.

Figure 2. DNMT1 stability is differentially affected in carcinogen exposed cells

A: Aphidicholine synchronized (5uM for 24hrs) 3kt and T31 cells reveals that DNMT1 is dysregulated throughout the cell cycle post carcinogen exposure (0hrs: G0/G1; 6hrs: G0/G1 12hrs: S/G2; 24hrs. S/G2) B: Aphidicholine synchronized stably transfected 3kt cells with either control vector (EV) or HDAC3 reveals that HDAC overexpression causes DNMT1 dysregulation throughout the cell cycle C: Stably expressed Full-length V5-tagged DNMT1 associates with GST-tagged HDAC1, HDAC2 and HDAC3 irrespective of DNaseI pre-incubation D: siRNA knockdown of HDAC1,2,3 (H1,H2,H3 respectively) decreases levels of DNMT1 in T31 cells, untreated 3kt and non-silencing siRNA (NS) transfected T31 serve as controls. Histograms show change in expression compared to non-silencing siRNA normalized to beta actin.

Figure 3. Modulating type I HDAC function alters DNMT1 stability

A: T31 cells were exposed to 1mM VPA for 24h in the absence of presence of the proteasome inhibitor MG-132 (2.5uM ×16h). VPA induced DNMT1 degradation was blocked by MG-132. B: Pull-down of N-terminally truncated DNMT1 by IMAC in siRNA treated T31 cells. Total Acetylated lysine band runs at same molecular weight at V5H6-DNMT1. Ubiquitination of DNMT1 is increased with HDAC knockdown and VPA exposure. All cells were treated for 16hrs with 2.5uM MG-132. C: Treatment of 3kt and T31 with 1mM VPA, 10nM 17AAG or a combination reveals that HSP90 driven DNMT1 stabilization is not a major component of DNMT1 stabilization in carcinogen exposed T31 cells. D: VPA induces loss of DNMT1in T31 cells independent of Set7 expression. Knockdown of SET7 did not show an appreciable loss of DNMT1. VPA treatment causes a significant reduction in DNMT1 levels with or without Set7 expression

Figure 4. Immunohistochemistry reveals upregulation of DNMT1 and type I HDACs in human lung tumors compared to matched normal tissue

A: Representative images of a single patient’s tumor (T) and matched normal tissue (N). B: Weighted indices of DNMT1, HDAC1, 2, or 3 in tumor versus normal tissue. 20 Patients’ tumors and matched normal tissues were scored and averaged. 2-tailed student’s t test was employed to check for significance, values indicated.

Figure 5. HDAC inhibition restores carcinogen transformed cells to a more normal phenotype

A: 24hour exposure of T31 cells to 1mM VPA reduced levels of DNMT1 and type I HDACs and increased global histone H3-Acetylation and H3K4 dimethylation. B: Soft agar colony formation assay. Carcinogen transformed cells (T30) were treated with 0.1mM or 0.5mM VPA for 3 weeks while seeded in soft agar. Clonogenecity of T30 cells were reduced by VPA. C: carcinogen-transformed T31 cells were passaged for 28 days in the presence of VPA. Bisulfite sequencing of the SFRP2 locus was performed on cells derived from day 0 and day 28. Long term VPA treatment of carcinogen transformed cells decreases methylation of SFRP2 promoter CpG island (p 1. This leads to stabilization of DNMT1. VPA exposure inhibits class I HDAC2, leading to DNMT1 degradation by an acetylation dependent pathway3. Smoke carcinogen exposure leads to epigenetic silencing of tumor suppressor genes (i.e. SFRP2)4. VPA can reverse this phenomena.5