Xin-Yuan Fu - US grants

When this grant proposal was originally submitted over five years ago, the STAT proteins had just been identified, cloned and initially characterized through the studies of interferon systems. Aims of the original proposal were to investigate a transcription factor ISGF3, which involves STAT1 and STAT2 activated by interferon. However, in the past five years we have made significant progress that went far beyond what I proposed in the original proposal. We have shown that the STAT pathway involves not only in the interferon system, but also as a common pathway mediating essential signals for gene regulation in response to other polypeptide ligands, such as EGF. We have further shown that the STAT signaling pathway plays critical roles in negative regulation of the cell growth. This finding provides a molecular basis why cytokines and growth factors, which usually promote cell proliferation, may also negatively regulate cell growth under certain conditions. Recently, we have also shown, for the first time, that the activation of the PTK-STAT signaling pathway can cause induction of apoptosis. These novel and important findings have significantly advanced our understanding of the cellular and molecular regulatory mechanisms mediated by cytokines and other polypeptide ligands, providing a solid explanation, at the molecular level, of a long-time puzzle that cyokines and growth factors may have dual (negative and positive) functions in regulation of cell growth and survival. Here, I propose to continue our studies on the functional roles of the STAT signaling pathway. We will focus on molecular mechanisms of STAT-mediated differentiation and apoptosis of lymphocyte. I propose the following aims: 1) To reveal the molecular basis of apoptosis by default after growth factor withdrawal. We will use Ba/F3 cells derived from pro B-cell as the model system to investigate functions of STAT proteins in the negative control of cell growth and in the induction of apoptosis after IL-3 withdrawal. 2) To use embryonic fibroblasts isolated from STAT-1 or STAT3 null (minus/minus) mice to investigate STAT functions in the induction of cell death by default. The possible cross-interactions of different signaling pathways in control of survival or apoptosis will be studied. We will also investigate whether IFNs can act as a survival signal if the STAT pathway is blocked. 3) To analyze STAT functions in the regulation of intracellular pathways to differentiation or to apoptosis using transgenic mice expressing dominant-negative mutant of STAT proteins. In particular, we will choose B-cells as model systems. The functions of STAT proteins in determining cell death during the B-cell development will be investigated. 4) To search systematically for the target genes that are specifically regulated by STAT proteins during lymphocyte development. In particular, we will try to identify the genes which may be involved in the control of lymphocyte proliferation, differentiation and apoptosis.

DESCRIPTION: (adapted from the investigator's abstract) After Dr. Fu finished his postdoctoral studies with Dr. James Darnell at Rockefeller University, he started as a principal investigator in January 1992 at the Mt. Sinai School of Medicine in New York. Since last September he has been appointed as a member of the faculty in the Department of Pathology, Yale University. Yale has provided him with an excellent environment. His laboratory has established collaborations in laboratories with Drs. David Stern, Richard Flavell and Jordan Pober, etc., who are well established in cancer biology and immunology. The scientific communication and research at Yale Medical School is one of the best in the world. For his career goals, Dr. Fu has been a productive research scientist since his graduate studies in Dr. James Manley's laboratory at Columbia University, where he was the first to identify the alternative splicing factor, ASF. When he was a postdoctoral fellow with Dr. Darnell, he purified the key interferon- stimulated gene factor, ISGF3, and cloned p91 and p113, the first two members of the STAT factors. As an independent principal investigator, Dr. Fu made the key discoveries on a fundamental signaling pathway linking cell surface receptors to transcription factors. His laboratory first showed that a new class of transcription factors, termed STAT (signal transducer and activator of transcription) contained the SH2 (Src homology region 2), critical domain in tyrosine kinase-mediated signal transduction, and presented the earliest evidence that STAT directly interacts with cell surface receptors (Fu 1992; Fu and Zhang 1993). A novel direct signaling model has been established from this work. This pathway was first discovered in the interferon system and it was shown that activation of STAT factors is a general mechanism to induce gene expression by many tyrosine kinase-associated cytokine receptors. In the next five years Dr. Fu plans to continue studies on STAT factors, focusing on the detailed mechanism of STAT activation, specificities of different STAT factors, and roles of tyrosine kinase/STAT pathway in cancers. His long-term career goal is to reveal the basic mechanism of signal transduction, especially interactions and regulation of different signaling pathways at the cellular level as well as at the level of development. He will apply his knowledge of signal transduction to the treatment of cancer and other human diseases. In this application Dr. Fu proposes the following studies: 1) to reveal the detailed molecular mechanism of activation of STAT by IFNg. He will characterize the IFNg- induced different STAT 91 transcription complexes. This includes purification, characterization and cloning of cytoplasmic co-factors and a possible nuclear DNA-binding factor in the complex. The roles of these possible co-factors in maintaining the specificity of signaling will be determined. Dr. Fu believes that IFNg-induced transcriptional activation and DNA-binding specificities of different STAT complexes are the keys to understanding the important functions of IFNg immunoresponses. 2) to determine the functional roles of p91 in breast cancer cells, especially the cells which overexpress growth factors or their receptors. Phosphorylation states, cellular localization, DNA- binding activities and possible partners of p91 will be analyzed. To reveal the molecular mechanisms of the constitutive activation of p91 by growth factors in some cancer cells.

Fibroblast growth factor receptors (FGFRs) have crucial functions in differentiation, angiogenesis, cell migration and development. Mutations in FGFRs have been shown to cause dominantly inherited human skeletal abnormalities and other disorders. In particular, the achondroplasia class of chondrodysplasias is comprised of the most common genetic forms of dwarfism in humans. Its members, achondroplasia (ACH), hypochondroplasia (HCH) and thanatoporic dysplasia types I and II (TDI and TDII), are caused by distinct mutations of fibroblast growth factor receptor 3 (FGFR3) which retard skeletal growth and development. The molecular mechanism and mediators of these FGFR3-related growth abnormalities are unclear. We have shown that the mutant TDII FGFR3 has a constitutive tyrosine kinase activity that could specifically activate STAT1 both in vitro and in vivo. Furthermore, TDII FGFR3-induced STAT1 activation was correlated with translocation of STAT1 to the nucleus, expression of cell cycle inhibitor p21WAF1/CIP1 and cell growth arrest in tissue culture cells and in the cartilage cells from the TDII fetus. These results have shown, for the first time, that abnormal STAT activation and p21WAF1/CIP1 expression may be responsible for the TDIIFGFR3-caused bone disease. Based on these discoveries, it is necessary to expand our research work on the detailed molecular mechanisms of FGFR signal transduction, and to further reveal the complicated molecular basis of mutant FGFR- associated abnormalities. In this application, I propose the following specific aims: 1) To investigate the possible programmed cell death (apoptosis) induced by the expression of mutant FGFR3. To determine whether STAT1 activation and p21 expression are involved in the induction of apoptosis. 2) To reveal molecular mechanisms of STAT1 activation by the TDII receptor and search for other possible signaling molecules that may also play a role in the mutant FGFR3 function. 3) To study molecular mechanisms of developmental disorders caused by mutant ACH, HCH, and TDI receptors of FGFR3. We will determine whether the abnormal STAT activation is also involved in these disorders. We will also test whether STAT1 activation is one of the outcomes of constitutive activation of other tyrosine kinases. 4) To generate mouse models using TDII and ACH knock-in technique. These mice will be used for in vivo test for our hypothesis and for potential therapeutic studies. I believe that the experiments proposed in this application represent a novel and important dimension of research that will reveal a molecular basis for FGFR-related genetic disorders. The results from these studies will also contribute to the development of the molecular therapies for these disorders in the future.

DESCRIPTION: (adapted from the investigator's abstract) Cell growth and survival can be regulated by cytokine-induced signals. Nevertheless, the molecular mechanisms of signal transduction from cytokine receptors to mediators of cell growth and apoptosis are not well understood. STAT (signal transducer and activator of transcription) proteins can be activated by protein tyrosine kinases (PTK) in response to a variety of cytokines. They have shown that STAT activation is correlated with cell growth inhibition in response to EGF and IFN-g. Activated STAT proteins can regulate expression of some cyclin-dependent kinase (CDK) inhibitors, such as p21WAF1/CIP1, and induce expression of the ICE gene, which is involved in induction of apoptosis. Using a cell line which is deficient in STAT1, and a derived cell line into which STAT1a has been re-introduced, they demonstrate that STAT1 protein can cause cell growth suppression and apoptosis in response to IFN-g and EGF. On the basis of these results, Dr. Fu proposes a hypothesis that PTK-STAT signaling pathways can negatively regulate the cell growth and survival in response to cytokines. To further investigate functions of STAT proteins in cell growth control and apoptosis, they will do the following: 1) To create STAT deficient (knock-out) mice to investigate functions of STAT proteins in control of cell growth and survival in vivo. In particular, they will generate conditional and tissue specific STAT3 (-/-) mice using the LoxP-Cre system. 2) To utilize STAT3 knock-out mice, or cell lines derived from these mice, for analysis of functions of STAT3 in cell growth and apoptosis in response to several cytokines, including the IL-6 family of cytokines and EGF, PDGF, etc. 3) To analyze STAT6 functions in response to IL-4 using STAT6 (-/-) mice and derived cells. In particular, they will determine whether different forms of STAT6 have specific functions in growth inhibition or stimulation. 4) To analyze the functional roles of the STATs in cell cycle control, and possible cross-talks of the STAT pathway with the Ras-MAP kinase pathway and other mitogenic pathways during cell proliferation or apoptosis. Whether activation of STAT proteins exerts a general growth inhibitory effect on cell out-growth will also be determined. 5) To study the signaling pathways from PTK-STAT to induction of apoptosis in response to cytokines. Additionally, to study possible involvement of PTK-STAT pathways in cancer development and treatment. Possible STAT activation in other abnormal PTK activation systems will also be investigated. Dr. Fu believes that the experiments proposed in this proposal will explore a new field of STAT functions in cell growth control, apoptosis, cancer development, and will contribute to further understanding of the signal transduction mechanisms controlling both positive and negative regulations of a cell.

DESCRIPTION (provided by applicant): We propose a hypothesis that the Stat3 signaling plays essential roles in differentiation of precursor cells in mouse retina. The hypothesis suggests a molecular mechanism of STAT activation in response to cytokines or growth factors and transfer of the signal to the nucleus controlling cell determination and differentiation. This hypothesis is based on our recent findings, which demonstrated involvement of STAT proteins in cell growth control and in determining the dual functions of the cytokines (Cell, 70, 323; Cell, 74, 1135; Science, 272, 719; Nature, 386, 288; MCB, 17, 5328; Science, 283, 222; JBC, 275, 867). In particular, we have evidence showing that Stat3 can directly or indirectly regulate gene expression of key transcription factors for cell differentiation in retina, such as Hes1 or Otx2. We have shown that activation of Stat3 but not MAPK is necessary for CNTF-induced suppression of rod photoreceptor determination (Neuron, submitted). Recently we have successfully generated retina-specific Stat3 knock-out mice. More detailed examination and analysis of these mice are necessary and crucial for further understanding of molecular mechanisms of STAT-regulated cell differentiation in vivo. We will focus on molecular mechanisms of STAT-mediated cellular responses, characterize potential physiological and pathological roles of Stat3 protein, and furthermore clone new genes that are controlled by the Stat3 signal. Using a retinal explant system, STAT recombinant adenovirus, retina tissue-specific Stat3 knock-out animal, and mouse retina-specific DNA microarray, we will focus on the following specific aims 1) To reveal the molecular basis of Stat3 functions in response to growth factors or cytokines in mouse retina. 2) To investigate the molecular mechanisms of Stat3 in control of differentiation in distinct neuronal retina cells. 3) To study the Stat3 function in modulation of gene expression and to find novel genes that are controlled by Stat3 in retina. We believe that these experiments will explore a novel and exciting field of STAT functions in cell differentiation and pathogenesis of retina degeneration diseases. These studies will contribute to our understanding of the molecular mechanisms of gene regulation in response to cytokines and other extracellular factors in retina. The reagents generated or to be generated in this application will contribute to the whole community of retina research.

[unreadable] DESCRIPTION (provided by applicant): In this application we propose a novel strategy for development of effective tumor vaccines by modifying STAT3 activity in dendritic cells (DCs). This strategy is based on our recent progress in understanding roles of STAT proteins, STAT3 in particular, in DC function and differentiation. We have developed a unique animal model for studies of immune responses and immune tolerance using STAT3 deficient (STAT3-/-) dendritic cells and mice. We have found that STAT3 is specifically required for FltSL-mediated DC differentiation. However, the deficiency in dendritic cell differentiation in STAT3-/- mice could be partially rescued by GM-CSF, which activated other STAT proteins, STAT3 particularly. However, generation of plasmacytoid dendritic cells (pDC) was highly dependent on STAT3. We suggest that the lack of STAT3 activity and the loss of pDC may contribute to reduction of immune tolerance in the STAT3-/- mice. Our results suggest a critical mechanism of regulation of immune tolerance. Using this STAT3 deficient mouse model we will be able to study roles of STAT3 in dendritic cells as well as in lymphocytes, providing a powerful tool for dissecting mechanisms of both innate immunity and adaptive immunity systematically. Our KO mice are probably the only animal model system in which STAT3 is ablated during physiological development of dendritic cells. In this application we will study roles of STAT3 in antigen presentation and tumor vaccination. Our major hypothesis is that STAT3 regulates generation of tolerogenic dendritic cells and that inhibition of STAT3 activity will stimulate stronger immune responses that will facilitate development of an effective immune response in tumor vaccination. Specific Aim 1) To investigate whether STAT3 affects tumor antigen presentation in DCs and whether blocking of STAT3 in DC-facilitated vaccine will enhance anti-tumor immune response in vivo; Specific Aim 2) To investigate the possible mechanisms how STAT3 can modulate immune responses and the efficacy of DC-facilitated tumor vaccine. Specific Aim 3) To examine roles of STAT3 in a mouse model in which a prostate cancer could be developed due to tumor tolerance. We hypothesize that the loss of STAT3 in DCs will increase T cell responses against the tumor development. The interactions of these immune cells with tumor cells in presence or absence of STAT3 will be systematically examined. These proposed studies should reveal novel molecular and cellular mechanisms by which STAT3 controls innate and adaptive immune responses during vaccination. We believe that this project will provide novel rationale and concepts for development of therapeutics for human cancers as well as other immune disorders. [unreadable] [unreadable] [unreadable]