It’s funny how research can pay off in surprising ways. In this case, University of Wisconsin scientists were studying the tumor suppressor gene APC when they realized there was an unusual brand of monoallelic silencing going on.
Earlier studies have found that many early adenomas begin with the loss of function of the Adenomatous polyposis coli (APC) gene; most losing a copy APC and heterozygosity through homologous somatic recombination. Recent work from the Wisconsin team found tumors without copy number losses or gains in genomic material and that most tumors lose heterozygosity only on the short arm of chromosome 18. Discoveries made possible by new experimental approaches.
“We wanted to develop a straight forward assay or assays that would tell us if there could be changes in transcription driving tumor formation. Using a quantitative SNP Pyrosequencing assay combined with standard quantitative RT-PCR, we could determine if the gene was completely silenced, monoalleliclly silenced, or biallelically transcriptionally active. Combined with standard Sanger sequencing to look for secondary mutations we felt that we could get the most comprehensive assessment all possible events that could lead to tumor formation. This approach allowed us to detect monoallelic silencing in the absence of secondary mutation.” Explained lead author James Amos-Landgraf.
Amos-Landgraf went on to say, “There is a mixture of tumor and stromal cells (within a tumor) that makes any cancer specific events difficult to identify and analysis difficult to interpret. We designed experiments that could capture many different mechanisms of gene loss and silencing. By using F1 hybrids from two inbred lines we produced genetically identical offspring that were fully informative at all polymorphic loci. Then applying quantitative pyrosequencing in both DNA and RNA we were able to detect loss of heterozygosity at both the genetic and epigenetic level. This approach uncovered genetically stable tumors appear epigenetically unstable.” Here’s some of what they found with their new approach:
- Secondary APC mutations weren’t found in genomic DNA sequencing for the majority of tumors that maintained Apc heterozygosity.
- This heterozygosity of APC in genomic DNA apparently violates Knudson’s two-hit hypothesis (The idea that it takes two or more mutations to a genomic region to cause cancer), possibly representing a previously unrecognized pathway of early intestinal tumor development.
- Quantitative allele-specific assays of APC cDNA revealed two neoplastic pathways. The first group of tumors maintains heterozygosity of APC RNA expression and may involve haploinsufficiency for APC function. A second class of tumors shows monoallelic expression of the mutant APC allele, indicating a random or stochastic process of monoallelic epigenetic silencing of APC.
“I think the most remarkable epigenetic finding we saw is that monoallelic silencing can drive early tumor development. This appears to be a stochastic somatic event that occurs and is then fixed in the early adenoma.” Commented Amos-Landgraf.
So what’s next for Dr. Amos-Landgraf and colleagues? “Clearly, getting at the mechanism that is driving these early epigenetic events is important. We believe a whole genome approach in F1 material with both the monoallelic tumors and those that show no detectable changes will provide the most insight into driver versus passenger mutations and epigenetic events.”
We look forward to seeing what they dig up. Get filled in on monoallelic silencing in PNAS, January 2012.