Kunxin Luo - US grants

DESCRIPTION (provided by applicant): Transforming growth factor-? (TGF?) regulates a wide variety of normal cellular processes including proliferation, survival, cell-matrix interaction, differentiation and plays a complex role during mammalian tumorigenesis. SnoN is a potent negative regulator of TGF? signaling through binding to and antagonizing the activity of the Smad proteins. It is a member of the Ski family of classically defined proto-oncogenes that when overexpressed, induces transformation of chicken and quail embryo fibroblasts. It is expressed in all adult cells and tissues at a low level but its expression is altered (up- or down-regulated) in many human cancer cells. Previous studies related to SnoN function mostly focused on its ability to promote oncogenic transformation in chicken embryo cells. Virtually nothing is known about its function in normal mammalian epithelial cells, and its role in mammalian tumorigenesis has not been well defined. The long-term goal of this proposal is to understand the function of SnoN and the SnoN/Smad interaction in regulation of cell proliferation, survival and senescence as well as mammalian tumorigenesis and to determine the molecular mechanisms underlying these processes. We will employ the mouse embryo fibroblasts (MEF) and MCF10A normal human mammary epithelial cell line to investigate the function of SnoN in normal mammalian cells. In an effort to determine the physiological significance of the SnoN/Smad interaction, we have isolated MEF from a strain of knock-in mice that express a mutant SnoN deficient in binding to the Smad proteins. These MEF cells display enhanced sensitivity to apoptotic stimuli and more interestingly, premature senescence, indicating that the SnoN/Smad interaction may regulate the apoptosis and senescence responses. We have also employed small-interference RNA approach in MCF10A cells and showed that SnoN promotes epithelial survival in a basement membrane-dependent manner. In this proposal, we would like to test the hypothesis that SnoN possess both anti-oncogenic and pro-oncogenic activities through regulation of cell senescence, survival and proliferation in both Smad-dependent and Smad-independent manner. The specific aims are: 1) To determine the molecular mechanism by which SnoN regulates cell senescence;2) To determine whether SnoN can function as a tumor suppressor through its ability to induce premature senescence;3) To determine the function of SnoN in normal human epithelial cells. These studies will allow us to understand the function of SnoN in normal mammalian cells and how deregulation of these activities facilitates tumorigenesis. PUBLIC HEALTH RELEVANCE: SnoN is a potent negative regulator of transforming growth factor-? (TGF-?) signaling, which play important but complex roles during mammalian tumorigenesis. It regulates many aspects of cellular functions including proliferation, differentiation, senescence and motility and contains both oncogenic and anti-oncogenic activities. Identification of the signaling pathways mediating each of these activities and deciphering the specificity of these pathways are crucial not only for understanding the mechanisms controlling malignant progression but also for development of novel cancer therapy that specifically targets the oncogenic activity of SnoN while preserving its anti-oncogenic activity.

DESCRIPTION (provided by applicant): The tightly regulated homeostatic mechanisms between hepatocyte proliferation and apoptosis that exist in normal liver tissue are disrupted in many liver diseases and during hepatocarcinogenesis. The transforming growth factor beta (TGFbeta) signaling pathway is a central component of the mechanisms by which cell growth and apoptosis are regulated in the liver. The long-term objective of this project is to understand the molecular mechanism by which TGFbetaa regulates hepatocyte proliferation and apoptosis. TGFbeta is a potent inducer of hepatocyte apoptosis both in vivo and in vitro. The apoptotic activity of TGFbeta can be antagonized by the action of insulin and its downstream signaling molecules. This proposal is designed to understand how TGFbeta pathway and insulin pathway cross talk to regulate the sensitivity of hepatocytes to TGFbeta-induced apoptosis. Smad proteins are critical mediators of TGFbeta signaling. In the absence of ligand, Smad2 and Smad3 are located in the cytoplasm. Upon binding of TGFbeta to its receptors and the subsequent activation of the receptor serine/threonine kinases, Smad2 and Smad3 become phosphorylated by the receptor kinases and then translocate to the nucleus, where they can form heteromeric complexes with Smad4 and activate transcription of TGFbeta responsive genes. Among these Smad proteins, Smad3 has been shown to mediate TGFbeta-induced apoptosis. In a search for molecules that interact with Smad3, the P.I. identified Akt, a critical molecule functioning in the cell survival pathway downstream of insulin. Akt can directly interact with Smad3, and this interaction is regulated by TGFbeta and insulin. Because expression of an activated Akt results in protection of cells from TGFbeta-induced apoptosis, we hypothesized that Akt may protect cells from TGFbeta-induced apoptosis by interacting with Smad3 and preventing it from mediating the apoptotic signals of TGFbeta. The following specific aims are designed to further characterize the interaction between Akt and Smad3 and uncover the physiological significance of this interaction. The specific aims are: 1) Mapping the amino acid residues in Akt and Smad3 required for their interaction. 2) Analysis of the role of the Smad3- Akt interaction in protection from TGFbeta-induced apoptosis. 3) Analysis of the molecular mechanism by which Akt inhibits TGFbeta-induced apoptosis. In particular, we will investigate whether Akt inhibits Smad3 through affecting its phosphorylation or through physical sequestration. These studies will allow us to have a better understanding of how TGFa and insulin signaling pathways cross talk to regulate the sensitivity to TGFbeta-induced apoptosis. Since this cross talk plays an important role in the control of liver size and disruption of this highly regulated process can result in liver diseases and hepatocarcinogenesis, these studies may contribute to a better understanding of the maintenance of normal liver homeostasis and the cause of liver diseases.

DESCRIPTION (provided by applicant): The transforming growth factor-b (TGFb) superfamily of cytokines, including TGFbetas and bone morphogenic proteins (BMPs), play important roles in the regulation of cell growth and vertebrate development. Smad proteins are critical mediators of TGFbeta and BMP signaling. Upon phosphorylation and activation by the TGFbeta or BMP receptor kinases, the Smad proteins translocate into the nucleus and regulate transcription of TGFbeta or BMP responsive genes. In the nucleus, the activities of the Smads are tightly regulated by both positive and negative cellular co-factors. The P.I. has previously identified a nuclear proto-oncoprotein Ski as an important negative regulator of TGFb and BMP signaling. The long-term goal of this proposal is to understand the function of Ski oncoprotein in development and transformation, in particular related to its ability to interact with the Smad proteins. Ski is a unique oncoprotein in that it can induce both oncogenic transformation as well as terminal differentiation of muscle precursors. However, the mechanism by which Ski regulates these two processes has not been defined. The P.I. has shown that in the nucleus, Ski interacts with the TGFbeta- as well as BMP- specific Smad complexes and represses their abilities to activate transcription of TGFbeta and BMP target genes. Since TGFbeta-Smad pathway has been shown to be an important tumor suppressor pathway and BMPs are critical regulators or embryonic development, we hypothesize that the ability to interact with the Smad proteins is required for both the transforming activity of Ski and its ability to modulate muscle and neuronal differentiation. This proposal is designed to test this hypothesis and carry out detailed functional analyses of the Ski-Smad interaction during mammalian epithelial carcinogenesis and embryonic development as well as to investigate the molecular mechanisms of Ski function. The specific aims are: 1) Analysis of the role of Ski in oncogenic transformation;2) Microarray analysis of Ski inducible genes;3) Analysis of the role of the Ski-Smad interaction in embryonic development. These studies will allow a mechanistic understanding of functions of the Ski oncoprotein as well as the role of Ski and the Ski-Smad interaction in mammalian epithelial carcinogenesis and embryonic development.

DESCRIPTION (provided by applicant): The transforming growth factor-b (TGFb) superfamily of cytokines, including TGFbetas and bone morphogenic proteins (BMPs), play important roles in the regulation of cell growth and vertebrate development. Smad proteins are critical mediators of TGFbeta and BMP signaling. Upon phosphorylation and activation by the TGFbeta or BMP receptor kinases, the Smad proteins translocate into the nucleus and regulate transcription of TGFbeta or BMP responsive genes. In the nucleus, the activities of the Smads are tightly regulated by both positive and negative cellular co-factors. The P.I. has previously identified a nuclear proto-oncoprotein Ski as an important negative regulator of TGFb and BMP signaling. The long-term goal of this proposal is to understand the function of Ski oncoprotein in development and transformation, in particular related to its ability to interact with the Smad proteins. Ski is a unique oncoprotein in that it can induce both oncogenic transformation as well as terminal differentiation of muscle precursors. However, the mechanism by which Ski regulates these two processes has not been defined. The P.I. has shown that in the nucleus, Ski interacts with the TGFbeta- as well as BMP- specific Smad complexes and represses their abilities to activate transcription of TGFbeta and BMP target genes. Since TGFbeta-Smad pathway has been shown to be an important tumor suppressor pathway and BMPs are critical regulators or embryonic development, we hypothesize that the ability to interact with the Smad proteins is required for both the transforming activity of Ski and its ability to modulate muscle and neuronal differentiation. This proposal is designed to test this hypothesis and carry out detailed functional analyses of the Ski-Smad interaction during mammalian epithelial carcinogenesis and embryonic development as well as to investigate the molecular mechanisms of Ski function. The specific aims are: 1) Analysis of the role of Ski in oncogenic transformation; 2) Microarray analysis of Ski inducible genes; 3) Analysis of the role of the Ski-Smad interaction in embryonic development. These studies will allow a mechanistic understanding of functions of the Ski oncoprotein as well as the role of Ski and the Ski-Smad interaction in mammalian epithelial carcinogenesis and embryonic development.

DESCRIPTION (provided by applicant): There are two primary objectives of this conference. The first is to facilitate and foster productive interactions among investigators working on different aspects of TGF-[unreadable] superfamily biology. In particular, the organizers intend to provide a forum for the development of collaborative relationships among investigators working on the biochemistry, molecular, cellular and developmental biology of TGF-[unreadable] superfamily members as it applies to normal development and human pathology. Of particular interest is to offer a venue for integrating biochemical knowledge about signal transduction mechanisms at the cellular and molecular level, with systems approaches that aim to describe how these factors coordinate developmental processes and contribute to various human disease states including, cancer, cardiovascular disorders, and musculoskeletal pathologies. The organizers will accomplish this goal through a diverse and interactive program of talks and poster sessions, organized by theme, and all held in an intimate setting with a limited number of participants. Many leaders in the various fields of TGF-[unreadable] biology have already been invited, and with their input, develop scientific sessions that highlight recent advances in different aspects of TGF-[unreadable] biology. Ample discussion time will be provided to allow development of productive scientific interactions. A second goal for this conference is to provide a forum to promote and encourage the development of women, minorities, and talented young scientists in this field. Women make up a significant fraction of the session chairs and invited speakers. The work of young investigators will be highlighted in a special short talk session. Based on five previous conferences with similar goals, the organizers anticipate successfully meeting their objectives. PUBLIC HEALTH RELEVANCE: The TGF-[unreadable] superfamily comprises the largest set of polypeptide growth factors in vertebrates and they regulate an extensive number of developmental, physiological and homeostatic processes. A great deal of recent work has gone into understanding the molecular mechanisms of TGF-[unreadable] signaling and the way in which aberrations in this signaling pathway lead to developmental abnormalities (birth defects) and human disease pathologies, including cancer, cardiovascular disorders, musculoskeletal pathologies, and psychiatric conditions to name a few. This conference covers a wide range of topics including the mechanism of TGF-[unreadable] signaling, structural determination of components of the pathway, and discussion of the involvement of these ligands in developmental and differentiation processes relevant to birth defects and human disease. These discussions have direct relevance for the development of effective therapeutic strategies to treat a wide number of disease states that are caused by, or exacerbated by, alterations in this signaling pathway.

DESCRIPTION (provided by applicant): The transforming growth factor 2 (TGF2) signaling controls many cellular processes including cell proliferation and apoptosis and is critical for maintaining tissue homeostasis. Aberrant levels of TGF2 signaling lead to defective tissue regeneration and various human diseases such as cancer. The long-term goal of this project is to understand how proper levels of TGF2 signaling activity are maintained and regulated in normal tissues and primary cells using a system biology approach. The goal of this application is to develop a comprehensive mathematical model that can be used to predict Smad signaling dynamics and behavior and to determine the function of SnoN-mediated negative feedback regulation of Smad signaling. We will employ primary hepatocytes from wild type and SnoN-null mice to perform quantitative measurement and establish a mathematical model, and further confirm the model in liver tissue sections. We choose to work with liver because TGF2 is known to be a potent inhibitor of hepatocyte proliferation and is also the critical negative regulator of liver regeneration. In primary hepatocytes, TGF2 signals through Smad2 and Smad4 proteins. Upon phosphorylation by the active TGFss receptor kinases, Smad2 homo-oligomerizes and heterooligomerizes with Smad4, leading to its accumulation in the nucleus. The heteromeric Smad2/Smad4 complex then interacts with other transcription factors to regulate expression of TGF2-responsive genes. The activities of the Smad proteins are regulated by positive and negative cellular co-factors. Through a system biology study, we have recently determined that SnoN is the most important negative regulator of Smad signaling in primary hepatocytes. SnoN binds to Smad2 and Smad4 and represses their ability to activate TGF2 target genes. Interestingly, SnoN itself is induced by TGF2, suggesting that it modulates TGF2 signaling in a negative feedback manner. Our preliminary study suggests that SnoN neither affects the duration nor the initial timing of Smad signaling, but rather decreases the level of target gene expression at high TGF2 concentrations. We hypothesize that the SnoN-mediated negative feedback functions to suppress cell-to-cell variability in Smad target gene expression and linearize dose-response behavior of TGF2 signaling, thereby maintaining the robustness of this signaling pathway. In this proposal we will combine mathematical modeling with quantitative measurements to test this hypothesis at both the cellular level, using primary hepatocytes, and at the organ level, using liver tissues from wild type and snoN mutant mice. Three specific aims have been proposed: 1) Analysis of the impact of SnoN on TGF2 signaling in primary hepatocytes. 2) Analysis of the effects of SnoN on Smad-dependent gene expression. 3) Modeling SnoN regulation of Smad signaling during liver damage and regeneration. This study will significantly advance our knowledge regarding the mechanism and regulation of this important signaling pathway and how de-regulation of this signaling activity results in carcinogenesis. PUBLIC HEALTH RELEVANCE: The transforming growth factor 2 (TGF2) signaling controls many cellular processes and is critical for maintaining tissue homeostasis. Aberrant levels of TGF2 signaling lead to defective tissue regeneration and various human diseases such as cancer and diabetes. The long-term goal of this project is to understand how proper levels of TGF2 signaling activity are maintained and regulated in normal tissues and primary cells using a system biology approach.

DESCRIPTION (provided by applicant): Basal-like breast cancer is the most aggressive subtype of human breast cancer with a higher malignant grade, increased metastatic tendency and poor prognosis. Current treatment options are extremely limited, and new targeted therapy is urgently needed. Recent advances suggest that basal breast cancer originate from the mammary luminal progenitor population, and human basal-like breast tumors and luminal progenitor cells share a similar molecular signature. The objective of this study is to understand the signaling events that regulate mammary luminal progenitor cell fate and how disruption of these regulatory program contributes to basal breast cancer progression, with an emphasis on how the crosstalk between TGF? and STAT5 signaling is coordinated by SnoN. The mammary luminal progenitor cells are derived from the mammary stem cells and are responsible for the massive epithelial expansion during the pregnancy/lactation cycles in the adult glands. Little is known about the molecules/pathways that control the establishment and maintenance of this luminal progenitor fate. Past studies have shown that epithelial expansion during pregnancy is regulated by growth factors and hormones, including TGF? and prolactin. While prolactin, signaling through STAT5, promotes alveologenesis and lactogenesis, TGF? functioning via the Smads, inhibits them. This negative effect of TGF? must be suppressed during late pregnancy to ensure proper alveologenesis. How TGF? signaling is suppressed and coordinated with /STAT5 pathway at this critical juncture has not been defined. SnoN is a critical negative regulator of TGF? signaling by binding to the Smads and repressing their transcription activity. We found that in the mammary gland, SnoN expression is sharply upregulated at late pregnancy and early lactation. Using mouse models with altered expression of SnoN as well as a 3D differentiation model system, we have identified a previously unrecognized role of SnoN in enabling alveologenesis and onset of lactation, likely through promoting Stat5 stability and activation. Since Stat5 is required for the establishment of luminal progenitor cells, we hypothesize that SnoN regulates alveologenesis by promoting luminal progenitor cell fate through co-ordinating the activities of STAT5 and TGF? signaling: by enhancing STAT5 expression and activation and at the same time suppressing Smad signaling. In basal breast cancer cells, aberrant regulation of SnoN and STAT5 expression may promote tumorigenesis. Two specific aims have been designed to test this hypothesis in vivo and in vitro. Aim I will determine the importance of SnoN regulation of STAT5 and Smads in mammary alveologenesis and tumorigenesis in vivo using mouse models. In aim 2, we will determine the molecular mechanism by which SnoN enhances STAT5 expression and activation. Our study may identify important determinant of luminal progenitor cell fate and key pathways that drive basal breast cancer progression. This may facilitate development of novel therapeutic drugs for the treatment of basal breast cancer.

The objective of this research is to determine how a human cell generates specific biological outcomes in response to a given hormone or growth factor. In animals, cells are exposed to a variety of regulatory hormones and growth factors that act as a signal to cause the cells to adopt different fates, including growth, survival, acquisition of specific functions such as producing insulin, or becoming a special cell type such as a neuron. These hormones or growth factors cannot enter cells and thus rely on a series of proteins inside the cell to transmit the signals into the cell nucleus and orchestrate the changes in gene expression, leading to specific biological outcomes. A key unanswered question is how these signaling proteins activate highly specific sets of genes in response to a hormone. This signaling specificity is essential for normal development and cell function. Disruption of this specificity results in defects in development and other diseases. This research project, a collaborative effort of the Luo lab at UC Berkeley and the Henis lab at Tel Aviv University, will dissect the molecular basis of an important signaling pathway to understand how a single signal can create different cellular outcomes, a long-standing puzzle in signal transduction. The project will also contribute to interdisciplinary education and training of postdoctoral fellows, undergraduate and graduate students, including an underrepresented minority graduate student. Finally, the project is expected to contribute to the broader scientific community through publication of the major findings and sharing of reagents.

The Hippo signaling pathway will be used as a model system for this project. Hippo signaling regulates multiple biological processes via two homologous transcription effectors, TAZ and YAP. Although they perform largely non-redundant functions, how this functional specificity is achieved is unknown. Recently a process called liquid-liquid phase separation (LLPS) has been discovered that allows intracellular proteins to form membraneless condensates inside the cells. Our preliminary study has shown that TAZ, but not YAP, forms nuclear condensates via LLPS together with its important functional partners. We propose that TAZ may employ LLPS to compartmenta¬lize its partner proteins that perform similar functions within a physically distinct domain, enabling efficient gene expression and pathway insulation to generate specificity. Biochemical and biophysical studies will be performed to define the molecular basis of TAZ LLPS and to determine the mechanisms of TAZ LLPS assembly. If successful, this research will provide a new paradigm to address the broad question of signaling specificity for multiple intracellular pathways and has many implications for fundamental cell biology.

This project is jointly funded by the Cellular Dynamics and Function program and the Genetic Mechanisms program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.