My current interest is cancer prevention. When Nixon declared war on cancer in 1971 there were two probable explanations for how cancers arise: 1) They are the inevitable result of mutations that accumulate within the DNA of individual cells as cells divide over our lifetime, or (2) they are caused by external agents (pollutants, viruses etc) that act on our DNA and cells to increase mutations rates or otherwise induce cancer. In fact, it was assumed that both explanations would prove to be correct: the question was, what fraction of cancers are “inevitable” because of limitations of our biological repair systems, and what fraction are “preventable” if one could identify and avoid the major cancer-causing behaviors or carcinogens in our environment?
Over the past 4-plus decades there has been extraordinary progress in understanding both the genetic basis of cancer and the reasons why cancers arise. While the question of how much cancer is “inevitable” and how much is “preventable” is still difficult to quantify precisely, it is probable that about half the cancers that result in death in the US today could have been prevented if we had known 20 years ago what we now know and had been able to carry out effective prevention measures. (These numbers are conservative - many epidemiologists believe that at least 70% current cancer deaths worldwide are preventable, by which they mean that these cancers need not have arisen in the first place.)
Over the past 40 years my lab worked first on mechanisms by which retroviruses cause cancer in mice, later on the identification of genes required for early development in zebrafish. The latter led me back to cancer research after my lab identified genes that are embryonic-lethal in the fish but that predispose adult fish to cancer as heterozygotes. In the course of studying zebrafish tumors we ‘rediscovered’ an old observation about cancer that may explain why it is so hard to cure the disease: namely the genetic heterogeneity within a single tumor (1). Different cells within one (fish or human) tumor often have different numbers of chromosomes, and, as we now know from large scale DNA sequencing of human tumors, different combinations of other types of mutations as well.
Given how difficult it is to treat and cure cancer, I became interested in exploring cancer prevention. A sabbatical in the epidemiology department at MDAnderson led me to several interests that I am pursuing, including (1) confirmation of data indicating that 50% of US cancer deaths over the next 20 years could be prevented and investigating to what extent appropriate prevention measures are or aren’t underway; (2) Increasing the interest of cancer molecular biologists in prevention and early detection strategies along with the long-standing emphasis of the field on developing drugs to cure or treat more advanced cancers.
(Please note: The Hopkins lab is no longer accepting students or postdocs.)
(1) Zhang, GJ., Hoersch, S., Amsterdam, A., Whitaker, C., Lees, JA., Hopkins, N. (2010) Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers. Proc. Natl. Acad. Sci. USA 107:16940-5.
Identification of genes essential for early development in zebrafish, and the role of these genes in longevity and cancer predisposition in adult fish.
Five-day old zebrafish embryos: Normal (wild type) on left side, mutant on right side, lacks cartilage. Top: live. Middle: cartilage stained with alcian blue. Bottom: sectioned, cartilage stained purple. All viewed lateral (parasagittal section). (Hopkins lab).
Insertional Mutagenesis in Zebrafish
Large-scale forward genetic screens are a powerful approach to identifying the genetic basis of developmental processes. Such screens have long been central to studies of invertebrate animals, and more recently the approach has been applied to vertebrate animals, including zebrafish and mice. Forward screens are particularly suitable in the fish. This is because it is possible to breed and maintain large numbers of zebrafish in the lab, and because early developmental mutations are easy to identify in fish embryos since embryos develop outside the mother and are transparent for the first week of life.
Most screens in zebrafish have employed chemical mutagens or radiation to induce mutations. However, cloning genes mutated by these agents is tedious. Thus we developed a method of insertional mutagenesis for the zebrafish using mouse retroviral vectors. Retroviruses are excellent mutagens since when they infect cells, a DNA copy of their genome is inserted into the host cell genome at many different locations. If the DNA insertion occurs in a gene and disrupts it, the viral DNA serves as a tag for cloning the mutated gene. We found that mouse retroviral vectors can infect the fish germ line efficiently, that proviral insertions are mutagenic, and that the mutated genes can be cloned very rapidly using the viral tag. Using this technology, we carried out a large screen to identify mutants with developmental defects visible by 5 days post fertilization. By this time fish are already free-swimming larvae. Most mutations we identified are embryonic or larval lethals. About 1/3 of the mutants have relatively specific phenotypes, while about 2/3 have less specific defects that involve many cells in the embryo. The latter often result from mutations in genes required for cell viability, the former from genes required for the patterning, differentiation, or growth of specific organs and structures.
We isolated ~550 mutants. These represent mutations in ~400 different genes. We have cloned the genes mutated in 375 of the mutants, and these include lesions in 275 different genes. About 20% of the genes are novel and almost all have clearly identifiable human homologues. Compelling evidence indicates that this collection of mutants includes at least 25% of the genes essential for development of the 5-day old fish. Thus there are only about 1600 embryonic or larval lethals in zebrafish.
To identify genes that play essential roles in specific aspects of development we are re-screening (called “shelf-screening”) our mutant collection using specific assays. Specific screens include those for mutants with cystic kidneys, those with defects in development of the jaw and cartilage, hair cell function and lateral line, those with defects in forebrain patterning, cell cycle, nuclear coded mitochondrial genes, and liver growth. Each screen yields between a few up to 20 genes. In the case of kidney, the genes that were identified appear to comprise a pathway and this pathway appears to correspond to that for human cystic kidney. In collaboration with other labs, we are shelf screening the collection to identify the genes essential for about 20 other developmental processes.
In the course of maintaining the mutants, we noted that some lines display early mortality and develop tumors as heterozygous adults. In collaboration with the lab of Jackie Lees, we analyzed tumor spectrum and frequency in the colony as a whole. This allowed us to identify a set of lines that define a novel class of tumor suppressors. Together these studies demonstrate the power of forward insertional mutagenesis in zebrafish to identify genes important for vertebrate development and disease, and to assign function to many genes whose biological and biochemical functions were not previously known.
We are focusing on mutants with defects in genes required for cell cycle and organ growth and genes that predispose to cancer. We are pursuing the mechanism of action of the novel tumor suppressor genes we have identified to date.
Sadler KC, Amsterdam A, Soroka C, Boyer J, Hopkins N. A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease. Development. Aug;132(15):3561-72. Epub 2005 Jul 6. (2005)
Amsterdam A, Sadler KC, Lai K, Farrington S, Bronson RT, Lees JA, Hopkins N. Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol. May;2(5):E139. Epub 2004 May 11. (2004)
Golling, G., Amsterdam, A., Sun, Z., Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K., Farrington, S., Lin, S., Nissen, R. and Hopkins, N. Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nature Genetics 31: 135-140 (2002).
Chen, W., Burgess, S., Golling, G., Amsterdam, A., and Hopkins, N. High throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebrafish. Journal of Virology, 76:2192-2198 (2002).
Sun, Z. and Hopkins, N. vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain. Genes and Development, 15: 3217-3229 (2001).
Amsterdam, A., Burgess, S., Golling, G., Chen, W., Sun, Z., Townsend, K., Farrington, S., Haldi, M., and Hopkins, N. A large scale insertional mutagenesis screen in zebrafish. Genes and Development, 13: 2413-2724 (1999).