KRAS-driven miR-29b expression is required for tumor suppressor gene silencing

Abstract

KRAS activation drives DNA methylation and silencing of specific tumor suppressor genes (TSGs). We previously showed that the ERK pathway induces transcriptional repression of TET1, which results in conversion of TSG promoters from a hydroxymethylated, active state to a hypermethylated and silenced state. Here we identified miR-29b as a KRAS-induced molecule that represses TET1 expression. In KRAS-transformed cells, ectopic miR-29b inhibition restores expression of TET1, thereby reactivating TSGs by reducing methylation and restoring hydroxymethylation. Mining gene expression data of lung cancer cell lines identified additional TSGs suppressed by KRAS signaling whose expression was restored by inhibition of miR-29b and re-expression of TET1. Because KRAS changes TSG promoters from hydroxymethylated to hypermethylated with miR-29b-dependent silencing of TET1, we demonstrate a model in which DNMT1 is present on target promoters prior to KRAS transformation. In addition, we propose miR-29b as a potential circulating biomarker and target for rational treatment of specific malignancies.

Keywords: DNA methylation; DNMT1; TET1; mir-29b; tumor suppressor gene.

Figure 1. Pharmacogenomic discovery of miR-29b as a TET1-targeting microRNA

(A) A Venn diagram summarizes identification of miR-29b as a predicted TET1-targeting microRNA whose expression depends on KRAS and MEK. (B) Hierarchical clustering analysis of miRNAs that depend on KRAS in HBEC3 and MEK in H1299 cells. (C) Validation of miR-29b expression in vector versus KRAS-transfected HBEC3 cells and DMSO versus PD98059-treated H1299 cells by qRT-PCR analysis. Data are presented as mean ± SD. ***p < 0.001 in comparison to control cells.

Figure 2. miR-29b antagonism restores TET1 expression in KRAS-transformed cell lines

(A) AM-29b restores expression of TET1, TET3 and DNMT3b mRNAs in HBEC3-KRAS and H1299 cells and normalized to NC. (B) AM-29b decreases global 5-mC levels in H1299 cells while 5-hmC levels were significantly elevated in both cell lines upon miR-29b inhibition. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison to NC cells.

Figure 3. miR-29b dependent transcriptional suppression of TSGs downstream of KRAS transformation

(A) Classification of lung cancer cell lines [4]. (B) Hierarchical clustering analysis of TSGs, whose mRNAs are depressed >1 log fold- change in Group 1 cell lines with respect to Group 2. (C) TSGs are consistently depressed in KRAS-transformed HBEC3 cells with respect to controls (upper panel). The same genes are re-expressed upon AM-29b transfection (lower panel). (D) Bioinformatically identified genes are almost universally reactivated by PD98059 and AM-29b in H1299 cancer cells. (E) A Venn diagram depicts the high overlap of TSGs silenced by KRAS transformation, reactivated by PD98059 and restored by AM-29b. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison to control cells.

Figure 4. Blocking miR-29b restores the methylation status of DAPK and MGMT in AM-29b treated HBEC3-KRAS cells

(A) AM-29b reactivates expression of the MGMT and DAPK TSGs. (B) AM-29b reduces hypermethylation of the MGMT and DAPK promoters. (C) AM-29b reverts specific KRAS-induced hypermethylation of MGMT and DAPK CpG islands. Nonmethylated and methylated CpGs are depicted as open and solid circles, respectively. Data are presented as mean ± SD. *p < 0.05; **p < 0.01 in comparison to control cells.

Figure 5. RAS and miR-29b-controlled TET1 chromatin occupancy controls the epigenetic status of MGMT and DAPK

(A) KRAS transformation depresses and miR-29b antagonism restores the 5-hmC status of MGMT and DAPK promoters. Open circles represent 5mC and C, filled circles represent 5-hmC, and X marks indeterminant sites. (B) KRAS transformation depresses and miR-29b antagonism restores TET1 occupancy of the MGMT and DAPK promoters. (C) In contrast to gene expression and 5-mC status which are regulated by KRAS and miR-29b, DNMT1 occupancy of MGMT and DAPK promoters is not regulated by KRAS or miR-29b. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001 in pairwise comparisons.

Figure 6. MEK-dependent miR-29b induction represses TET1 expression, thereby leading to RAS-dependent TSG hypermethylation and silencing

In contrast to earlier models, which proposed that KRAS drives DNMT1 transcription leading to TSG hypermethylation, our data indicate that KRAS drives miR-29 induction through the RAF-MEK-ERK pathway and that net hypermethylation depends on down-regulation of TET1. Moreover, TET1 and DNMT1 are both present on target gene promoters prior to KRAS activation.