Pinpointing Tumor Suppressor Genes

Tumor suppressors are a crucial component of intracellular signaling networks that protect against uncontrolled cell proliferation. The benefits of such genes can be lost when DNA undergoes alterations, including mutations or deletions of entire stretches of chromosomes. A highly efficient genome-sequencing technique developed several years ago in the Wigler laboratory has made it possible to scan the genome of cancerous cells for, among other things, deleted portions of chromosomes where tumor suppressors are likely to reside.

Dr. Powers performed a genomic analysis of human liver cancer samples that provided a basis for the work of the combined teams. Powers' lab scanned the genomes of liver cancers from more than 100 patients to compile a list of deletions of chromosomal regions. These regions were hypothesized to be the location of most of the missing tumor suppressor genes. A comparison of this list with the genome sequence of a normal human cell revealed the identity of approximately 300 genes within the deleted chromosomal segments.

Chromosomal deletions aren't limited to cancer-related genes alone; any number of "passenger" or unrelated neighboring genes can inadvertently also be lost. The team therefore had to pinpoint the tumor suppressors among the 300 genes. "Genomic analysis of human tumors is important," Powers observed," but combining it with functional screening in mouse models is a notable step forward."

The usual route of characterizing a gene's function is to mutate it in mouse embryos and then create lines of mice that can then be examined for the mutation's effects. Lowe's group bypassed this step, which is time-consuming, by engineering mutations into the genome of adult mouse cells and then re-injecting these cells into adult mice. The team used a method honed by Dr. Hannon of introducing stable mutations into mouse cells via RNA interference, or RNAi, a technique in which small RNA molecules are introduced into cells to shut off specific genes.

RNA sequences that corresponded to all the 300 or so deleted genes were obtained from an RNAi "library" compiled by the Hannon lab. Lowe's team introduced these RNAi tools (known as "short-hairpin RNAs, or shRNAs) into progenitor cells that develop into mature liver cells, albeit ones engineered to over-produce a cancer gene product called Myc.

In cells with these Myc mutations, an additional "trigger" such as the shutting off of a tumor suppressor gene via RNAi would be sufficient to cause cancer. The engineered cells that carried a Myc mutation and an shRNA were injected into mice. Dramatically, those that received cells in which a tumor suppressor gene had been "silenced" by an shRNA developed tumors within a month.

The scientists homed in on the identity of the silenced tumor suppressors by simply isolating and analyzing the genetic material from the tumors. Their strategy identified 13 new tumor suppressor genes, most of which had not been linked to cancer before.