Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 23 (2), 100838

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation

Affiliations

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation

Douglas J MacKenzie et al. iScience.

Abstract

Approximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer.

Keywords: Biological Sciences; Cancer; Molecular Biology.

Conflict of interest statement

Declaration of Interests The authors have no competing interests to declare.

Figures

None
Figure 1
Figure 1
DNMT3B Is Frequently Amplified and Overexpressed in CRC (A) Violin plot showing distribution of DNMT3B expression levels in 207 CRC tumor samples. Mann-Whitney U test; p value = 5.785e-10. (B) DNMT3B relative copy number across 207 tumor samples. Lines at y axis −0.2 and 0.2 indicate the threshold for deletion and amplification, respectively. (C) Median centered log2 DNMT3B mRNA expression plot from 207 tumor samples (TCGA) annotated with BRAF mutation status. (D) The relationship between DNMT3B relative copy number and DNMT3B mRNA expression in 207 tumor samples.
Figure 2
Figure 2
DNMT3B Suppresses Features of OIS (A) Kinetic analysis assessing the impact of ectopic DNMT3B on markers of BRAFV600E-induced cell-cycle exit of primary IMR90 cells, determined by Western blot. MW markers in kDa. (B) Impact of ectopic DNMT3B on BRAFV600E-induced S-phase exit of primary IMR90 cells, determined by BrdU labeling. Each group n = 4, from days 3, 5, 7, and 10 post-infection. Middle line = median; box top/bottom = upper and lower quartiles; whiskers 90th (upper) & 10th (lower) percentile of data. Kruskal-Wallis test, p < 0.05. (C) Number of significantly differentially expressed genes between indicated pairwise comparisons of primary IMR90 cells expressing ectopic BRAFV600E and/or DNMT3B and/or empty vectors, determined from RNA-seq FPKM values (Cuffdiff, FDR corrected p value of <0.05). (D) Number of significantly up or down genes between indicated pairwise comparisons of primary IMR90 cells expressing ectopic BRAFV600E and/or DNMT3B and/or empty vectors, determined from RNA-seq FPKM values. (E) Heatmap of expression of significant changed proliferation-associated genes. (F) Heatmap of expression of significant changed SASP genes.
Figure 3
Figure 3
DNMT3B Accelerates Intestinal Cancer (A) Western blot showing doxycycline-inducible expression of Dnmt3b in small intestine (S) and colon (C) of R26-M2-rtTA;Col1a1-tetO-Dnmt3b1 and R26-M2-rtTA;WT mice. MW markers in kDa. (B) Number of small intestine tumors in VilCreErT2;LSL BrafV600E mice with and without ectopic Dnmt3b expression. Mean with error bars −/+ SEM. Mann Whitney test p = 0.98 (ns) n = 5 vs 5. (C) Representative small intestine tumor fromVilCreErT2;LSL BrafV600E mice, similar with or without Dnmt3b. (D) Representative immunohistochemistry images for Ki67 in VilCreErT2; LSLBrafV600E mice with and without ectopic Dnmt3b. Scale bars are 200 μM. (E) Representative immunohistochemistry images for p21 in VilCreErT2;LSLBrafV600E mice with and without ectopic Dnmt3b. Scale bars are 200 μM. (F) Kaplan-Meier curve comparing survival in VilCreErT2; LSLBrafV600E mice with and without intestinal Dnmt3b ectopic expression. “DNMT3B only” mice (“Col1a1-tetO-Dnmt3b1 +/−”) also shown. Log rank (Mantel-Cox) test, p < 0.0001. (G) Total small intestine tumor number in VilCreErT2; LSLBrafV600E; Dnmt3bwt/wt and VilCreErT2; LSLBrafV600E; Dnmt3bfl/fl mice. Mann Whitney test p = 0.42; n = 5 vs 5. Mean with error bars −/+ SEM. (H) Kaplan-Meier curve of survival in VilCreErT2;LSLBrafV600E mice with wild-type or floxed Dnmt3b. Log Rank (Mantel-Cox) test, p < 0.02.
Figure 4
Figure 4
Ectopic Expression of DNMT3B Does Not Induce CIMP (A) Western blot of lysates from human fetal lung fibroblasts (IMR-90) infected with lentivirus directing expression of BRAFV600E or corresponding empty vector. MW markers in kDa. (B) Scatterplot of CpG methylation in BRAFV600E-infected fibroblasts compared with proliferating “vector” controls (top), and replicative senescent (PD88) compared with proliferating controls (PD28) (bottom). (C) Smoothened methylation plots of CIMP panel genes in vector and BRAFV600E-expressing cells. Red and blue lines indicate the smoothened average percentage methylation at corresponding CpGs. Individual CpGs are indicated by black “ticks” along the x axis.
Figure 5
Figure 5
Human BRAFV600E/CIMP Tumors Express Low Levels of DNMT3B (A) Clustering of TCGA human CRC based on Illumina 450K DNA methylation array data (all CpGs showing variation >0.2 StdDev). BRAF mutation and DNMT3B amplification status are shown below (red = mutated/amplified). (B) CpG probes (y axis of the heatmap) clustered based on increasing DNMT3B copy number (x axis of the heatmap). (C) Relationship between DNMT3B copy number and methylation of CIMP biomarker genes (CACNA1G, RUNX3, IGF2, MLH1, NEUROG1, CRABP1, CDKN2A). Red line marks the boundary between non-amplified and amplified DNMT3B. Each black dot indicates the mean methylation score in the indicated probes in one patient sample. Y axis—mean methylation, x axis—relative copy number. (D) Boxplot of DNMT3B FPKM mRNA expression in human colorectal carcinoma TCGA patients subdivided by CIMP status. Mann Whitney test, p = 0.001. (E) Violin plot showing distribution of DNMT3B expression levels in 207 CRC tumor samples, separated according to BRAF WT or BRAFV600E mutant. p value = 0.0001.

Similar articles

See all similar articles

References

    1. Acosta J.C., O'loghlen A., Banito A., Guijarro M.V., Augert A., Raguz S., Fumagalli M., da Costa M., Brown C., Popov N. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell. 2008;133:1006–1018. - PubMed
    1. Braig M., Lee S., Loddenkemper C., Rudolph C., Peters A.H., Schlegelberger B., Stein H., Dorken B., Jenuwein T., Schmitt C.A. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature. 2005;436:660–665. - PubMed
    1. Cancer Genome Atlas Network Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–337. - PMC - PubMed
    1. Carragher L.A., Snell K.R., Giblett S.M., Aldridge V.S., Patel B., Cook S.J., Winton D.J., Marais R., Pritchard C.A. V600EBraf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16INK4a. EMBO Mol. Med. 2010;2:458–471. - PMC - PubMed
    1. Chen Z., Trotman L.C., Shaffer D., Lin H.K., Dotan Z.A., Niki M., Koutcher J.A., Scher H.I., Ludwig T., Gerald W. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature. 2005;436:725–730. - PMC - PubMed

LinkOut - more resources

Feedback