DNMT3B has oncogenic activity but evidence suggests that it does not promote CIMP nor cooperate with activated BRAFV600E in human intestinal cancer. (RNA-Seq)

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 Phenotype (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells are thought to 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 multiple approaches. By analysis of human TCGA data, we confirmed that expression of DNMT3B is frequently upregulated in CRC and this is often linked to amplification of the DNMT3B gene. In vitro, we found that ectopic expression of DNMT3B antagonizes BRAFV600E-induced proliferation arrest and inflammatory gene expression, two hallmarks of the tumor suppressive senescence program triggered by oncogenes in primary cells. Moreover, DNMT3B over expression decreased survival in a mouse model of BRAFV600E-induced intestinal cancer. However, in primary human cells in vitro, activated BRAFV600E repressed expression of DNMT3B and failed to induce a CIMP phenotype. Finally, a closer analysis of human TCGA data revealed that BRAFV600E mutations and CIMP are both linked to a low expression of DNMT3B. We conclude that while both BRAFV600E and DNMT3B both harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumour promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer. Overall design: Expression profiling by high-throughput sequencing of BRAF oncogene induced senescent fibroblasts with/without ectopically expressed DNMT3B with relevant controls.

约10%的人类结直肠癌（colorectal cancer, CRC）与激活型BRAFV600E突变相关，此类肿瘤通常不携带APC突变，且常伴随CpG岛甲基化表型（CpG Island Methylator Phenotype, CIMP）。学界认为，正常肠上皮细胞可通过激活内在p53与p16依赖的肿瘤抑制机制（如细胞衰老），响应致癌性BRAFV600E以抵御癌症。反之，CIMP可通过表观遗传沉默p16等肿瘤抑制基因，帮助肿瘤绕过此类抑癌机制。此前已有多项研究提出，DNMT3B是人类结直肠癌中BRAFV600E诱导CIMP的关键介导因子。本研究通过多种实验方法对这一假说展开验证。通过分析癌症基因组图谱（The Cancer Genome Atlas, TCGA）公共数据，我们证实结直肠癌组织中DNMT3B的表达常出现上调，且该现象往往与DNMT3B基因扩增相关。体外实验结果显示，异位表达DNMT3B可拮抗BRAFV600E诱导的增殖停滞与炎症基因表达——这两类特征正是原代细胞中致癌基因触发的肿瘤抑制性衰老程序的标志性表型。此外，在BRAFV600E诱导的肠癌小鼠模型中，DNMT3B过表达会降低小鼠生存率。然而，在体外培养的原代人类细胞中，激活型BRAFV600E会抑制DNMT3B的表达，且无法诱导CIMP表型。最后，对TCGA数据的更精细分析显示，BRAFV600E突变与CIMP均与DNMT3B低表达呈相关性。综上，尽管BRAFV600E与DNMT3B在体外及体内均具备致癌潜能，且存在部分促进肿瘤发生的协同证据，但二者并未频繁协同促进CIMP及人类肠癌的发生。总体实验设计：通过高通量测序，对携带BRAF致癌基因诱导衰老的成纤维细胞（分别转染/未转染异位表达的DNMT3B）及其对应对照样本进行表达谱分析。