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Deciphering Mechanisms of Development and Cancer by Model Organism Genetics

Research Summary

Tian Xu's group develops new genetic approaches in Drosophila and mouse to decipher the molecular mechanisms of development and cancer.

Mechanisms Regulating Tumor Growth and Metastasis
Tumor progression and metastasis are the major causes of mortality for cancer patients. Because multiple genetic alterations contribute to tumor progression, and metastasis involves multiple tissues, it has been difficult to decipher mechanisms underlying the growth and spread of tumor cells. We have designed and performed genetic screens in Drosophila to interrogate its genome in somatic cells for mutations that can promote tumor growth and/or cause metastatic behavior. The cellular composition of these flies resembles that of cancer patients who are chimeric individuals carrying a small number of mutated somatic cells. This approach allows us to identify and examine the effects of mutations that are otherwise lethal. The Drosophila model also offers an opportunity to uncover developmental mechanisms that contribute to tumor growth and metastasis.

Our genome-wide genetic screens have identified more than 200 mutations that can cause noninvasive RasV12 tumors of the developing eye to exhibit metastatic behaviors. The screens also recovered more than 500 mutations that can promote tumor growth, and 1,900 mutations that suppress tumor growth. Many Drosophila homologs of human tumor suppressors, such as PTEN, TSC1, and TSC2, have been identified through the screen. Our genetic and biochemical analyses show that the evolutionarily conserved Drosophila Tsc1 and Tsc2 function together in the insulin/phosphoinositide 3-kinase (PI3K)/PTEN/Akt pathway downstream of Akt. Furthermore, the S6 kinase (S6K) also functions in the same pathway, and TSC (tuberous sclerosis complex) defects can be alleviated by reducing S6K activity. This evolutionarily conserved pathway and the suggestion of downstream potential therapeutic targets have led to clinical trials for TSC and brain cancer patients in the United States, England, and Germany. Given that the pathway is mutated in a large number of cancers and is involved in insulin signaling, the therapeutic implications of results from such trials will go beyond the TSC and brain cancer patient population.

The genetic screens have also identified other novel tumor suppressors—such as the Lats (large tumor-suppressor) gene family—that cause tumor development from insects to mammals. Analyses of LATS proteins revealed that they are a novel family of negative cyclin-dependent kinase (CDK) regulators and also regulate the cytoskeleton by modulating activities of molecules such as LIMK1. Further characterization of these tumor growth-promotion mutations will likely identify new tumor suppressors and mechanisms regulating growth.

Our analyses also show that these tumor suppressors regulate organ and individual size during development, suggesting that tumorigenesis might reflect an impairment of the evolutionarily conserved mechanism that controls organism size.

We have found that the metastasis-promoting mutations in collaboration with oncogenic Ras mutation produce fly tumors with a full spectrum of metastatic behaviors observed in human malignant cancers. These behaviors include uncontrolled growth, degradation of basement membrane (BM) induction of cell migration, the invasion of nearby tissues, and the formation of distinct secondary foci. Our analyses show that mutation of cell polarity genes, which control apicobasal polarity, promotes metastasis by activating JNK (c-Jun N-terminal kinase) and down-regulating the E-cadherin/?-catenin adhesion complex. Furthermore, JNK and Ras signaling cooperate in promoting tumor growth. The contribution of tumor-initiating alterations to the development of metastasis may explain why tumors of distinct origins have vastly different metastatic potentials.

Our work has also revealed that tumor cells hijack the normal invasive developmental process to achieve progression. Elements of the invasion machinery, including JNK-induced MMP (matrix metalloproteinase) expression, are shared by both the developmental invasion process (disc eversion) and tumor invasion processes. The degradation of BM is an early event during invasion processes; preventing BM degradation blocks both tissue and tumor invasion, indicating that modulation of BM is essential for developmental and tumor invasion. Moreover, although expression of TIMP, an MMP inhibitor, is sufficient to halt developmental invasion, inhibition of proteases by both TIMP and RECK is required to block tumor invasion. These data suggest that tumor cells have a more robust invasion mechanism and could acquire metastatic behavior by co-opting developmental invasion programs. This co-option may be a general feature contributing to the progression of tumors. Finally, although past efforts utilizing MMP inhibitors have not yielded much success, our results argue that BM modulation could be a critical target for cancer therapy.

piggyBac, a Powerful Genetic Tool for Deciphering Mammalian Biology and Disease
The approach of forward genetics allows scientists to follow diseases or phenotypes in human patients or in model organism mutants, identify the causative genes, and then investigate molecular mechanisms. This approach, one of the most productive methods in modern science, has played a pivotal role in our understanding of biology and disease. However, the lack of comparable forward genetics in mammals has impaired our ability to understand many aspects of mammalian biology and disease. The current approaches of gene inactivation in the mouse, the mammalian model of choice, have been limited to targeted gene knockout or chemical mutagenesis, which is either prohibitively expensive or inefficient.

For the past 10 years, we have been exploring methods to improve mammalian genetics. We recently modified piggyBac (PB), a DNA transposon from the cabbage looper moth Trichoplusia ni, and showed that it can efficiently transpose in human and mouse cells and in the mouse germline, providing a powerful tool for genetic manipulations in mammals and in mammalian tissue culture cells.

Transgenic mice are normally produced by injection of naked DNA molecules, which often form concatemers upon integration into the genome and often result in undesirable expression due to extra gene copies or gene silence. On the other hand, the mammalian PB system produces single-copy transgenes with stable expression and inherence. Furthermore, transgenic animals can be visually identified by marker genes, such as RFP (red fluorescent protein) carried in the PB vector. Thus, the mammalian PB system is a powerful new transgenic method, for mice as well as a variety of other animals in which an efficient transgenic method is currently unavailable.

We have developed a PB transposition system in mice that allows the use of a single PB element for insertional mutagenesis. The PB transposition events distribute widely throughout the mouse genome, with a bias for landing in transcription units, where they create loss-of-function mutations. This technology permits for the first time the efficient production of a genome-wide set of insertional mutants in the mouse. New mutants are made by simple breeding, which eliminates the costly and labor-intensive production of mutants by ES cell-based knockout technology.

The disrupted genes can be simply identified by PCR. PB contains the RFP marker, which permits determination of the genotypes of mice by their color, greatly simplifying phenotypic characterization, colony management, and breeding. The transposon also carries a LacZ gene, which reports the expression of the endogenous gene. Finally, PB insertions can be precisely excised from their original insertion sites in the genome, allowing the reversion of insertional mutations and the correlation of mutant phenotypes with the disrupted genes. We are using this system to produce the first genome-wide set of mouse mutants, which allows the use of forward genetic screens to decipher the genetic basis of mammalian biology and disease.

As of July 22, 2008

Scientist Profile

Investigator
Yale University
Cancer Biology, Genetics