Tian Xu, Ph.D. - US grants

Studies on the regulation of cell proliferation are important to our understanding of developmental biology and tumorigenesis. The long-term goal of the proposed research is to learn the molecular mechanisms that negatively regulate cell proliferation. In order to address this question, we are identifying growth-constraining tumor suppressor genes and studying their functions in the developing imaginal tissues of Drosophila melanogaster. Several aspects of this system make it uniquely suited for analyzing mechanisms that negatively regulate cell proliferation: (1) mechanisms are known to exist in controlling cell proliferation in imaginal tissues; (2) a newly developed genetic technique provides an unprecedented opportunity to identify mutations affecting such mechanisms in mosaic animals, and components of such mechanisms have already been identified; (3) additional components can be identified by future genetic screens; and (4) standard molecular and genetic techniques can be used to characterize these components. The three key genes to be studied are lats, "93B" and "2-1", in which recessive mutations can cause dramatic overproliferation of mutant cells in mosaic animals. The lats gene encodes a novel protein kinase homolog with a putative SH3-binding site. The specific aims of this proposal are: (1) to continue the molecular and genetic characterization of the lats, "93B", and "2-1" genes; and (2) to identify genetically and characterize additional genes involved in the negative regulation of cell proliferation or that interact with lats. The genetic screens will identify a network of genes involved in controlling cell proliferation during development. The genetic and molecular characterizations proposed will define the relationships among these genes and begin to assign biochemical functions which will aid in understanding their interactions at the molecular level. In this manner, we hope to learn how cell proliferation is regulated during normal development and how mutations could lead to abnormal growth and tumorigenesis.

[unreadable] DESCRIPTION (provided by applicant): Studies on the regulation of cell proliferation and tissue size are important to our understanding of developmental biology and tumorigenesis. The long-term goal of the proposed research is to learn the molecular mechanisms that regulate cell proliferation and tissue size during development. We have performed genetic screens in mosaic fruit flies, Drosophila melanogaster, to identify overgrowth mutations. Molecular and genetic characterization of the identified genes have revealed several key regulators of cell proliferation, cell growth, and tissue size, including the lats tumor suppressor. Mutations in lats cause dramatic overproliferation and tumor growth in Drosophila. Two mammalian lats homologs, Latsi and Lats2, have been isolated from humans and mice, and at least one of them also functions as a tumor suppressor. Mice deficient for Latsi develop soft tissue sarcomas and ovarian tumors. Biochemical and genetic experiments have shown that the Lats family proteins are negative regulators of the major cell cycle regulators, CDKs. However, substrates for the Lats kinase are unknown. Several lats-interacting genes have been identified in genetic modifier screens, including Src family members. Interestingly, lats and its interacting genes not only affect cell proliferation, but also deregulate tissue or organ size in Drosophila, a process which is not well understood. The specific aims of this proposal are: (1) Characterizing the roles of Src64 and Src42 in the regulation of cell proliferation and tissue size in imaginal tissues and their relationships with dcsk and lats; and (2) Identifying substrates for the Lats kinase. We hope that defining the relationships among the lats-interacting genes and identifying substrates of the Lats kinase will provide insights into molecular mechanisms regulating cell proliferation and tissue size during development. Since conserved mammalian homologs of Lats and Src are known to be involved in tumorigenesis, it is further hoped that these experiments will also contribute to our understanding of mechanisms underlying human cancer biology.

DESCRIPTION (provided by applicant): Metastasis is the major cause of mortality for cancer patients. However, the genetic basis for tumor progression and metastasis is largely unknown. Given that multiple genetic alterations occur during tumor initiation and progression, it has been difficult to find families with inherited mutations for both tumorigenesis and metastasis. The genomes of malignant tumor cells are often destabilized, which makes it a daunting challenge to pinpoint the causative alterations for tumor progression. Furthermore, metastasis involves multiple tissues and it is particularly difficult to study the process in tissue culture cells. Finally, tumor progression in humans and mammals takes a long period of time, which adds additional complication for experimental research. We have developed a Drosophila model for metastasis and have performed a genome-wide screen to identify mutations promoting tumor progression and metastasis. About 100 mutations have been identified which collaborate with oncogenic Ras in promoting tumor progression and metastasis in flies. These fly tumors exhibit a full spectrum of metastatic phenotypes observed in human malignant cancers including loss of cell adhesion, degradation of basement membrane, migration, invasion, and secondary tumor formation. This fly tumor metastasis model and the identified mutations provide a unique opportunity to dissect metastatic behavior in vivo and the mechanism underlying such phenomenon. We propose the following specific aims: (1) Genetic and phenotypic characterization for the recovered metastasis-promoting mutations to identify the genes that disrupted by these alterations and to document the phenotypic consequence caused by these mutations;and (2) Study molecular mechanism underlying oncogenic cooperation that promotes tumor progression and metastasis. We hope that these studies will improve our understanding of the genetic basis for metastatic behavior. Given that many molecules and pathways are conserved from flies to humans, it is further hoped that these experiments will also contribute to our understanding of some aspects of tumor progression and metastasis in humans.

DESCRIPTION: (provided by applicant) The mouse shares 99% of the genes with humans and is an ideal research model for enhancing our understanding of human biology and disease and for testing potential therapeutics. Our ability to utilize the mouse model is limited by the number of mouse mutants and disease models (3000 - 4000) that are available to the research and pharmaceutical community. We have developed a new transposon system, called pB, which can be used to efficiently mutate a large number of genes in mice and other mammalian systems. This system has the following advantageous characters: 1. pB transposes efficiently in the mouse germline and can rapidly produce a large number of single pB insertion mutant strains;2 Disrupted genes can be easily identified by PCR and sequencing;3. pB insertions favor genes and have a wide genomic distribution;4. pB insertions disrupt gene function when inserted into genes, and produce phenotypes similar to mutants generated by traditional knockout methods;5. pB can carry large DNA fragments, e.g., an Act-RFP marker and a LacZ reporter, which can report gene expression patterns;6. The resulting heterozygous or homozygous mutant animals and their wild-type siblings can be visually distinguished from each other without PCR or Southern using reporters such as Act-RFP;7. Mutant animals are directly produced by simple breeding, which bypasses costly and challenging traditional techniques involving ES cells and surgery;8. Visible genetic markers are employed in the breeding scheme to further improve efficiency and reduce cost;9. All mutants are generated in an identical genetic background;10. pB insertions can be precisely excised to revert the mutant phenotypes. We propose to utilize this pB insertional mutagenesis system and the cost-effective animal facility in Shanghai, China to produce 100,000 independent single pB insertion mutant strains in C57BL/6 mice in five years. We will determine the insertion sites, establish a database, and generate a comprehensive mouse KO resource which consists of frozen mutant embryos or sperm for approximately 20,000 unique genes or genetic loci. We estimate that the costs for the production and characterization of an independent insertion strain and for the storage of that strain are approximately $148 and $760, respectively. This comprehensive mouse mutant resource will significantly aid our ability to understand biology and improve human health.

DESCRIPTION (provided by applicant): Using a Drosophila tumor model in a genetic screen, we previously completed a genome-wide screen for mutations that cooperate with oncogenic Ras in promoting tumor growth and metastasis. Characterization of several identified mutations revealed unexpected biology and pathways, and especially highlighted the importance of cell-cell interaction and signaling in mediating tumor development and progression. Disruption of cell junction or apical-basal polarity leads to JNK activation, which is essential for tumor cell survivl, basement membrane degradation, tumor cell migration, and the unexpected behavior of interclonal cooperation with oncogenic Ras in promoting tumor growth and metastasis. Indeed, the behavior of tumor cells has long been recognized to be highly influenced by its microenvironment, interaction with surrounding wild type cells, and the extracellular milieu such as components of the extracellular matrix. The focus of our research, thus, logically switches from identifying the involved genes in the current funding period to uncover the molecular, cellular, and developmental mechanisms underlying tumor and host cell interactions for tumor growth and metastasis. We propose the following specific aims: (1) Characterizing novel RasV12 cooperating mutations and mechanisms related to JNK activation. We will characterize several new mutations/genes identified in our screen with the aim to identify and study novel tumor suppressors and to understand how JNK is activated. Our efforts will be focused on characterizing novel RasV12 cooperating mutations/genes that have mammalian orthologs mutated in human cancers and those genes that could help us to understand the causal link between disruption of cell polarity and JNK signaling in tumor development; (2) Dissecting cell-cell interaction and signaling mechanisms in promoting tumor growth and metastasis. We will further dissect the signaling between tumor and surrounding wild type cells that promotes tumor development, and will also characterize a novel tumor suppressor, which defines a new mode of cell-cell interaction via unique multicellular epithelial contacts; and (3) Studying organ-specific metastasis. We have discovered that the fly tumors also exhibit organ-specific metastasis behavior, and will try to identify the molecular basis for this targeted migration and invasion. In summary, having identified causative mutations for epithelial tumors, we are now poised to utilize the power of the Drosophila model organism to explore and unravel the molecular mechanisms underlying intercellular signaling that is central to the understanding of cancer biology. PUBLIC HEALTH RELEVANCE: Cell-cell interaction is critical for maintaining proper epithelial architecture and integrity involved in the regulation of cellular proliferation, adhesion, cell shae, and apoptosis, all of which are aberrant in malignant tumors. Having identified causative mutations for epithelial tumors that share common defects in cell polarity and signaling pathways, we now have the opportunity to use fly tumor model to explore and unravel the molecular mechanisms underlying intercellular signaling that is central to understanding human tumor malignancy.