Science teams at Cold Spring Harbor Laboratory identify 13 new tumor-suppressor genes in liver cancer

November 19, 2008

Cold Spring Harbor, N.Y. - Over the years, hunting for cancer-related genes and understanding how they work has been an important, although time-consuming, exercise. At Cold Spring Harbor Laboratory (CSHL), five different research groups have now combined their expertise to speed up the rate of discovering cancer-related genes and validating their function in living animals.

The result of the collaborative effort is a large-scale, rapid, cost-effective genetic screen that in a preliminary test succeeded in uncovering 13 new tumor suppressors - genes that inhibit the activity of cancer genes. Tumor suppressors are important in cancer development generally, and specifically in liver cancer, where they frequently have been found to be missing in people who develop the illness.

These discoveries, published online ahead of print on Nov.17 and scheduled to appear in the November 26 issue of Cell, "are a huge step forward in understanding the genetics of cancer and open up a host of new strategies to improve its diagnosis and treatment," according to CSHL Professor Scott W. Lowe, Ph.D., the corresponding author of the study. Other authors include Scott Powers, Ph.D., Director of the Human Cancer Genome Center at CSHL, and CSHL Professors Gregory J. Hannon, Ph.D., W. Richard McCombie, Ph.D., and Michael Wigler, Ph.D.

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.

A rich and unexpected payoff

"The nature of these new genes is not obvious and we wouldn't have guessed their relationship to cancer if we hadn't followed this approach," says Lowe. "They may now allow us to make headway into poorly understood areas of cancer."

The newly identified tumor suppressor genes affect a wide array of cellular activities, including maintenance of cell structure, cellular metabolism, cell proliferation, and control of the levels of various tumor growth-enhancing proteins in the cell's nucleus. In one instance, the team's strategy also uncovered not genes in isolation but an entire network of genes that go awry in liver cancer. "Given that the cancer puzzle involves multiple genes in various combinations, we need to find all the hits that make the cell tip over the edge," says Lowe, explaining one advantage of his team's broad strategy.

Some of the genes identified might also lead to new strategies for cancer therapy. For example, some of the newly discovered tumor suppressor genes "code" for proteins that are secreted, which indicates that their ability to prevent cancer is dependent on their presence outside the cell. Reversing the loss of such proteins - by replenishing their levels via injections - is an easier fix than having to correct a defect in the genome via gene therapy.

In other words, the CSHL team's new strategy now makes it possible to rapidly filter from genomic information those genes that specifically impact cancer development in living animals, and thus focus follow-up studies on those that might be most clinically useful.
"An oncogenomics-based in vivo RNAi screen identifies new tumor suppressors in liver cancer" will appear in Cell on November 26. The complete citation is: Lars Zender, Wen Xue, Johannes Zuber, Camile P. Semighini, Alexander Krasnitz, Beicong Ma, Peggy Zender, Stefan Kubicka, John M. Luk, Peter Schirmacher, Richard W. McCombie, Michael Wigler, James Hicks, Gregory J. Hannon, Scott Powers, and Scott W. Lowe. The paper is available online at: doi:10.1016/j.cell.2008.09.061

Cold Spring Harbor Laboratory is a private, nonprofit research and education institution dedicated to exploring molecular biology and genetics in order to advance the understanding of and ability to diagnose and treat cancers, neurological diseases and other causes of human suffering.

Cold Spring Harbor Laboratory

---

---

	Tumor Suppressor Genes: Volume 2: Regulation, Function, and Medicinal Applications (Methods in Molecular Biology) by Wafik S. El-Deiry (Editor) The second volume of Tumor Suppressor Genes explores the cell biology and biochemical function of the tumor suppressor genes, as well as its physiological role in vivo. The authors detail the physical methods (NMR, microarray approaches, pot-translational structure analysis, analysis of regulation at the gene expression and protein signaling levels)used to understand the function of tumor suppressor genes. In vivo approaches discussed include studies in yeast, Drosophilia, mice, and human tumors. For both volumes: Leading physician scientists and academic researchers review all the known tumor suppressor genes, explain how they work, and describe how they were discovered and isolated. In many cases, the authors discuss specific genes that are frequently involved in hereditary or...
	Tumor Suppressor Genes by Katherine R. Polinsky (Editor) A tumour suppressor gene is a gene that reduces the probability that a cell in a multicellular organism will turn into a tumor cell. A mutation or deletion of such a gene will increase the probability of the formation of a tumor. Unlike oncogenes, tumor suppressor genes generally follow the 'two-hit hypothesis', which implies that both alleles that code for a particular gene must be affected before an effect is manifested. This is due to the fact that if only one allele for the gene is damaged, the second can still produce the correct protein. However, there are cases where mutations in only one allele will cause an effect. A notable example is the gene that codes for p53. Tumor suppressor genes, or more precisely, the proteins for which they code, either have a dampening or repressive...
	Tumor Suppressor Genes in Human Cancer (Cancer Drug Discovery and Development) by David E. Fisher (Editor) David Fisher, MD, PhD, and an authoritative panel of academic, cutting-edge researchers review and summarize the current state of the field. Describing the broad roles of tumor suppressors from a perspective based in molecular biology and genetics, the authors detail the major suppressors and the pathways they regulate, including cell cycle progression, stress responses, apoptosis, and responses to DNA damage. Leading-edge and forward-looking, Tumor Suppressor Genes in Human Cancer illuminates what is currently known of tumor suppressor genes and their regulation, work that is already beginning to revolutionize cancer target elucidation, drug discovery, and treatment design.
	Enzymes Involved in Heparan Sulfate Chain Elongation: Function of a Novel Family of Tumor Suppressors (Comprehensive Summaries of Uppsala Dissertations) by Thomas Lind (Author)
	New Developments in Metastasis Suppressor Research by Paul Jackson (Editor) The spread of cancer cells from their organ of origin to distant tissues is called metastasis. Cancer metastasis is the main cause of death from cancer, and in many cases is difficult to detect or treat. The process by which tumour cells become metastatic is complex and involves many stages, including detachment of cells from the main tumour mass, degradation of the surrounding extra-cellular matrix, invasion into nearby blood vessels, travel and survival through the circulatory system, attachment to a vessel wall, extra-vasation, degradation of the extra-cellular matrix into a distant tissue/organ, and the development of a novel blood supply. In order to accomplish this process, the cells acquire characteristics which are important for each stage.Recently, a class of genes known as...
	The p53 Tumor Suppressor Pathway and Cancer (Protein Reviews, Vol. 2) by Gerard P. Zambetti (Editor) The p53 tumor suppressor gene is mutated in approximately half of all human malignancies, including colon, lung, and breast cancers. It is well recognized that these mutations directly inactivate p53 tumor suppressor function. Furthermore, the p53 protein operates within a pathway and this pathway, including the mutations in p53, is likely inactivated by nearly every human tumor. In support of this hypothesis, 100% of mice that have been engineered such that they do not express p53 protein (knockout animals), develop highly malignant tumors by only 3-6 months of age. The importance of p53 in preventing human cancer is also evident by families in which a mutated p53 gene is inherited from a parent. Individuals who carry an inherited germline p53 mutation are associated with Li-Fraumeni...
	Oncogenes and Tumor Suppressor Genes in Human Malignancies (Cancer Treatment and Research) by C.C. Benz (Editor), E.T. Liu (Editor) This volume begins by reviewing selected malignancies in which the search for clinically relevant oncogenes has led to more focused studies on gain-of-function and loss-of-function genetic abnormalities, as well as autocrine and paracrine growth factor loops known to regulate tumor physiology and malignant cell behavior. Many of these genetic and functional abnormalities are shared by several different tumor types and are not uniformly present in all tumors of the same type. This observation brings up molecular questions about the tissue-specific determinants that underlie individual cancers and also gives added impetus to the suggestion that molecular abnormalities (referred to as tumor markers) be included among the histopathologic features used for clinical diagnosis and...
	Tumor Suppressor Genes (Immunology Series) by George Klein (Editor)
	Tumor Suppressor Genes: Volume 1: Pathways and Isolation Strategies (Methods in Molecular Biology) by Wafik S. El-Deiry (Editor) Leading physician scientists and academic researchers review all the known tumor suppressor genes, explain how they work, and describe how they were discovered and isolated. In many cases, the authors discuss specific genes that are frequently involved in hereditary or sporadic cancers. They also provide a detailed guide to using powerful molecular genetic, cytogenetic, proteomic, and cell biological strategies to discover and isolate novel tumor suppressor genes and their targets. For both volumes: Leading physician scientists and academic researchers review all the known tumor suppressor genes, explain how they work, and describe how they were discovered and isolated. In many cases, the authors discuss specific genes that are frequently involved in hereditary or sporadic cancers. They...
	Tumor Suppressors: Involvement in Human Diseases, Viral Protein Interactions and Growth Regulation (Molecular Biology Intelligence Unit) by John W. Ludlow (Author), Gary R. Skuse (Author)