- Identification of properties of tumor proagating cells that can be therapeutically targeted in mouse and human sarcomas
- Use of genetically modified mice to determine how novel members in the hedgehog-signaling cascade regulate bone development, growth, cartilaginous neoplasia and the development of osteoarthritis
- Study of interactions between hematopoietic and mesenchymal cells, and the role of novel proteins involved in this interaction in bone and cartilage development, repair, and the rejuvenation of fracture healing
- Discovery of novel therapies for desmoid tumor, a mesenchymal tumor also called aggressive fibromatosis, using cell culture and genetically modified mice
5R01CA251407-02: Targeting the metastasis initiating cell in undifferentiated pleomorphic sarcoma
Undifferentiated pleomorphic sarcoma (UPS) is a soft tissue sarcoma, with one of the worst prognosis. We will build on our discovery that UPS contains a small subpopulation of metastasis initiating cells (MCs) that are enhanced for their ability to form metastasis. Our proof of principle data showed that targeting genes differentially expressed in the MC population inhibits metastasis in UPS tumors established as xenografts in mice. In our preliminary data, we show that epigenetic changes distinguish the MC from the rest of the UPS cell populations. Furthermore, we found that individual cell populations in UPS produce secreted factors that influence behavior of other cell UPS cell populations, acting in a competitive manner. Our hypothesis is that the MC population is maintained by epigenetic changes that endow this subpopulation of cells with distinct properties that drive sarcoma metastasis. We will test this hypothesis by answering the following two questions: 1) What drives the MC population? Here we will build on our preliminary data suggesting that epigenetic events driven by the regulation of histone acetylation and methylation maintain the MC. The function of differentially expressed genes in the MC in regulating metastatic ability will be assessed using a lung organ on a chip assay and findings will be tested in-vivo in murine tumors. 2) Can pharmacologically targeting the MIC population be used to treat UPS? Here we will build on our gene expression data, CRISPER screens, and results from a high throughput drug screen to identify agents that target the MC. Pharmacologic agents and genetic approaches in murine tumors and human primary UPS tumors established as xenografts in immunodeficient mice to determine their effect on disease progression and metastasis. This proposed work utilizes unique mouse models of sarcoma and human tumors to test novel biologic processes related to cellular heterogeneity in sarcoma. As such, it will provide important biologic insights not only about UPS, but also about cell heterogeneity in cancer in general. In addition, it will lead to the development of new treatment approaches for UPS, a tumor with a poor outcome using currently available therapies.
The pace of bone repair slows with aging, increasing the chance of developing a delayed union or non-union. These complications are treated with surgical procedures causing significant morbidity and even mortality, especially in older adults. Here we will build on our previous work using heterochronic parabiosis (in which two mice of a different age share a blood supply) showing that exposure to a young circulation and young macrophage cells rejuvenates fracture repair in older mice. In our preliminary data we used cell lineage tracing analysis and parabiosis experiments to determine the developmental source of macrophage in fracture repair, and found these derived from a subpopulation of cells of yolk sac origin. Interestingly these cells reside in the spleen and are recruited through the circulation during bone repair. As mice age, this subpopulation of cells becomes depleted. In this proposal we study the role of this cell population and the factors they produce in the rejuvenation of fracture repair by undertaking the following aims: 1) Identify the role of macrophages derived from yolk sac progenitors in the rejuvenation of fracture repair. Heterochronic parabiosis in which these cells can be labeled or depleted will be investigated to define the contribution of young cells from this population of macrophage cells that can improve the quality of fracture repair in older animals. 2) Determine the function of genes expressed in unique macrophage subpopulations present in young mice in bone repair: We used single-cell RNA sequencing and found a unique subpopulation of macrophages cells present in bone repair in only young animals. Mice lacking genes which encode for secreted proteins in various macrophage populations will be used in heterochronic parabiosis to determine their contribution to the rejuvenation of fracture repair. 3) Define how specific macrophage populations and the proteins they secrete alter mesenchymal differentiation in fracture repair. Our prior work showed an important role for beta-catenin in mesenchymal cell differentiation and in fracture repair rejuvenation. Here we will use in-vitro approaches to determine how specific subpopulations of macrophage cells and the proteins they secrete alter mesenchymal cell differentiation in cells from young and old animals. There will be an initial focus on beta-catenin, but an unbiased approach will be used as well. This proposed work builds on our prior studies of rejuvenation by heterochronic parabiosis in fracture repair. It will address critical gaps in our knowledge about the mechanism responsible for the rejuvenation phenotype driven by heterochronic parabiosis. Our work will also identify a novel therapeutic approach to address a critical clinical problem in older patients, delayed fracture healing.
Osteoarthritis (OA) is a common degenerative process that is a major health problem in the US population. While it is known that muscle strength and exercise can modulate OA symptoms, the mechanism by which skeletal muscle alters OA pathogenesis is only partly elucidated. The conventional thought is that muscle activity directly affects the joint biomechanically by modulating joint loading and altering bone strength, attenuating OA severity. However, muscle also secretes factors that can have paracrine effects. How such factors affect the joint and how they change with exercise and in muscle pathologies is not known. Using cell culture studies, we found that human muscle cells exercised in culture produce secreted factors that alter the expression of important genes in OA severity in human articular explants. We then modified an approach that allows circulatory exchange between two mice using catheters rather than a standard parabiosis technique. This approach allows the study of the role of circulating factors in one animal on the severity of OA in another animal, while the animals have different exercise activity levels. Our preliminary data shows the downregulation of genes important in OA severity in a sedentary animal when its circulation is shared with an exercised animal. Here we will develop approaches to identify these factors and will characterize an in-vivo strategy to test the function of such factors in rodents. We will undertake the following aims: Building on our data showing that conditioned media from exercised muscle produces factors that inhibit expression of genes important in OA severity in osteoarthritic cartilage explants, we will further evaluate these paracrine effects for different exercise regimes and ages of the muscle cells. To identify differentially secreted proteins, we will use mass spectroscopy secretome analysis. Using our modified catheters based approach to exchange circulating blood, allowing animals to share a circulation while undertaking different physical activity regimens, we will determine how the duration of exercise, number of blood exchange procedures, and plasma or cell fractions alter the OA phenotype associated with a surgical joint injury. Expression of the most differentially regulated proteins by exercise identified in the first aim will be compared between the serum from exercised and quiescent mice using ELISA. Identified proteins could be targeted pharmacologically and as such, our data may identify a therapeutic approach that could be used to attenuate the severity of OA. Furthermore, this work will define the specific contribution of biochemical effects of exercise on OA severity.
More than 3% of the population develops an enchondroma (ECA), a benign tumor in bone composed of cells derived from the growth plate that can cause pain, deformity, and can be responsible for pathologic fractures. Enchondromas can progress to malignant chondrosarcoma (CSA). Mutations in genes encoding isocitrate dehydrogenase (IDH1 and 2) were identified in a large proportion of ECAs and CSAs. In our prior work, we found that IDH mutations inhibit growth plate chondrocyte differentiation, and chondrocyte-specific conditional Idh1 mutant mice develop ECAs. Mutant IDH uniquely produces the metabolite 2-hydroxyglutarate (2-HG), but we and others found that blocking the production of 2-HG pharmacologically does not alter CSA cell viability. While 2-HG has epigenetic effects that are likely important in tumor initiation, tumor maintenance must rely on other factors. Since IDH plays an important role in in metabolism, associated metabolic changes could drive the observed phenotype. We found high levels of glycogen in cells expressing a mutant IDH. Glycogen is also found in proliferating and pre-hypertrophic cells of the growth plate. In our previous work, we found that intracellular cholesterol biosynthesis was activated in IDH mutant chondrocytes and that it is also regulated in the growth plate, and its activity corelates with glycogen levels. This raises the possibility that intracellular cholesterol biosynthesis, which is activated by Sterol regulatory-element binding proteins (SREBP) transcription, also regulates glycogen. Our premise is that glycogen is an important energy source for pre- hypertrophic and hypertrophic growth plate chondrocytes and that glycogen stores are required to maintain the neoplastic phenotype in ECA and CSA. We also propose that glycogen depletion can suppress the neoplastic phenotype. In this proposal we will study what regulates glycogen in the growth plate, ECA and CSA, and determine the function of glycogen in these growth plate and neoplastic chondrocytes. To determine what regulates glycogen in the growth plate, ECA, and CSA, we prioritized genes known to regulate glycogen that were differentially regulated in the growth plate and by IDH mutations. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is one such gene which is differentially and interestingly, contains SREBP binding sites in its promoter region. Our preliminary data suggest that SREBP regulates PPP1R3C which then regulates glycogen. Our studies will use cell lines from human tumors and genetically modified mice that develop enchondromas to define the function of glycogen and PPP1R3C in the growth plate, ECA, and CSA. In addition, we will study how SREBP regulates PPP1R3C and glycogen. Glycogen synthase will be deleted genetically, or we will cells with drugs that inhibit glycogen synthesis and breakdown. This data will provide pre-clinical information on which to base novel therapies for ECA and CSA.