Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

Patched-1 (Ptch1) is the primary receptor for Hedgehog (Hh) ligands and mediates both canonical and non-canonical Hh signalling. Previously, our lab identified that mice possessing a Ptch1 C-terminal truncation display blocked mammary gland development at puberty that is overcome by overexpression of activated c-src. Testing the hypothesis that this involves a direct interaction between Ptch1 and c-src, we identified through co-immunoprecipitation that Ptch1 and c-src associate in an Hh-dependent manner, and that the Ptch1 C-terminus regulates activation of c-src in response to Hh ligand. Since the effects of Ptch1 intracellular domain deletions on canonical Hh signalling are ill-defined, we assayed this through luciferase reporter assays and qRT-PCR. Transient assays revealed that the Ptch1 middle intracellular loop is required for response to ligand, while qRT-PCR from primary cells showed that C-terminal truncation impairs canonical Ptch1 function. Together, this indicates that the intracellular domains of Ptch1 mediate distinct canonical and non-canonical functions.

Patched-1 (Ptch1) is the primary receptor for Hedgehog (Hh) ligands and mediates both canonical and non-canonical Hh signalling. Previously, our lab identified that mice possessing a Ptch1 C-terminal truncation display blocked mammary gland development at puberty that is overcome by overexpression of activated c-src. Testing the hypothesis that this involves a direct interaction between Ptch1 and c-src, we identified through co-immunoprecipitation that Ptch1 and c-src associate in an Hh-dependent manner, and that the Ptch1 C-terminus regulates activation of c-src in response to Hh ligand. Since the effects of Ptch1 intracellular domain deletions on canonical Hh signalling are ill-defined, we assayed this through luciferase reporter assays and qRT-PCR. Transient assays revealed that the Ptch1 middle intracellular loop is required for response to ligand, while qRT-PCR from primary cells showed that C-terminal truncation impairs canonical Ptch1 function. Together, this indicates that the intracellular domains of Ptch1 mediate distinct canonical and non-canonical functions.

During embryonic development, the Hedgehog (Hh) signaling pathway plays an important role in the growth and patterning of numerous tissues and organs. In the developing retina, Hh signaling regulates the proliferation and differentiation of retinal progenitor cells (RPC) through mechanisms that are not completely understood. The principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3), which regulate the expression of target genes responsible for the effects of the Hh pathway on RPC. The network of genes targeted by this pathway in neural progenitor cells however, remains unknown. The objective of this thesis was to identify and characterize novel targets of Hh/Gli during retinal development. Using a computation approach, 390 genes were identified as having at least one conserved Gli binding motif within the vicinity of the coding sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5 retinal explants and that the induction of 25 of these were significantly different between the two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp, two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to determine a role for Sox8 during retinal development which could reflect compensation by the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient and required for Hh mediated induction in progenitor cell proliferation and cell fate determination. Therefore, in this thesis Hh target genes have been identified which could provide some insight into the mechanisms that are responsible for the cellular outcome of a response to the pathway.

Universiẗat, Diss., 2003--Dortmund.

Techn. University, Diss., 2005--Dresden.

BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for 15-20% of all pediatric brain tumors. In patients with MB, prognosis depends heavily on the molecular makeup of the tumor. New genomic approaches over the last decade have enabled researchers to sub-classify MB based on differences in the transcriptome: WNT, Sonic hedgehog (SHH), Group 3 (MYC-amplified), and Group 4 (heterogeneous). SHH tumors represent a third of all MB cases, and small-molecule inhibitors have already been developed that target SHH signaling. Most notably, vismodegib has shown great promise in the treatment of MB and other SHH-driven cancers by targeting Smoothened (SMO), an upstream regulator of GLI activity. However, most patients who had initially responded to the drug quickly acquired point mutations in SMO that led to treatment resistance. In addition, patients who harbored mutations downstream of SMO had no response to treatment and were found to be intrinsically resistant. Although most patients with SHH-MB can be cured, current treatments often require broad base therapies, such as radiation and chemotherapy, which can have harmful and long-lasting side effects. These observations underscore the need for less toxic, more targeted therapies that act at the level of the GLI family of transcription factors themselves. However, as transcription factors are generally considered undruggable, Dr. Robbins’ group at the University of Miami Miller School of Medicine sought to address this need by using focused screens of siRNAs or small molecules that target epigenetic GLI regulators. They identified several candidates that act as readers, writers, and/or erasers of protein acetylation and methylation and showed that a subset of these candidates act downstream of SMO to attenuate GLI signaling (data not yet published). Bromodomain- and Plant Homeodomain-linked Zinc Finger-containing Protein 1 (BRPF1) was one of these candidates and further analysis revealed that its knockdown reduced Gli1 expression by more than 50%. Recent studies link BRPF1 to cerebellar development and tumor formation in SHH-MB and may be suggestive of its role as a negative regulator. OBJECTIVES: We sought to compare basal levels of Brpf1 expression in normal versus MB in mice; to characterize Brpf1 knockdown versus overexpression in SHH cell lines; and to determine if BRPF1 merits further investigation as a candidate for future drug targeting therapies in MB and other SHH-driven cancers. METHODS: We used RT-qPCR and immunoblotting analysis to look at Brpf1 expression in Ptch+/- and adult wild-type mice. cDNA and protein samples were donated by colleagues in the lab. We also grew and maintained SHH Light2 cells in culture and then used these cells to carry out siRNA and plasmid DNA transfections. RNA extraction, RT-PCR, and RT-qPCR were used to examine transfection efficiency and its effect on Gli1 expression. RESULTS: Brpf1 levels were higher in SHH-MB compared to normal cerebellum. However, BRPF1 proteins were not detected in either normal or tumor samples. Brpf1 knockdown in Light2 cells correlated with an overall decrease in Gli1 expression while overexpression had no obvious affect on Gli1 expression. CONCLUSIONS: Our findings suggest that BRPF1 may function as a positive regulator of GLI activity. Recent studies verify this claim at least partially stating that BRPF1 acts as both a positive and negative regulator of gene expression depending on the context. Thus, before we can draw any final conclusions, more research is needed to look at BRPF1 in the specific context of the SHH pathway and developing cerebellum.

The Hedgehog (Hh) signalling pathway is essential for embryogenesis and regulating cellular homeostasis in the adult, however much remains unknown about the molecular mechanisms that control ligand expression. In 2002, Lee et al. demonstrated that conditional mutation of the Drosophila hyperplastic discs gene (hyd) resulted in increased hh levels, suggesting that Hyd can act as a negative regulator of hh gene expression. Based on this evidence, the aim of this project was to investigate the hypothesis that UBR5, the murine homologue of Hyd, acts as a novel regulator of Hh gene expression in mammals. To investigate this hypothesis in vivo, I utilized the developing mouse limb as a model system that is highly sensitive to abnormal Hh expression. Morphological analysis of Ubr5 limb mutant embryos did not reveal an obvious phenotype, however quantitative analysis of Ihh gene expression and its downstream targets at E13.5 demonstrated a significant decrease in levels. In addition, changes in the expression of Runx2 and Msx2 were detected. Therefore, these data indicate that UBR5 can act as a positive regulator of Ihh expression, in addition to regulating other factors involved in chondrogenesis. The role for UBR5 as a positive regulator of Hh expression was also supported by in vitro investigations, demonstrating that UBR5 is required for Shh expression in mouse embryonic stem cells. Morphological analysis of adult Ubr5 limb mutant mice revealed the presence of significantly shorter long bones. These observations support previous reports that interference with IHH during early chondrogenesis can negatively affect long bone growth in the adult. Interestingly, adult Ubr5 limb mutant mice also possess osteophytes, a feature typically observed in osteoarthritis (OA), in addition to sites of ectopic mineralization (EM) near tendons of the knee and ankle. Based on these observations and evidence from the literature, I hypothesize that in addition to the role for UBR5 as a positive regulator of Ihh expression in the bone, UBR5 also plays a role in ligament/tendon development and/or maintenance, whereby its loss results in defective ligaments/tendons that are incapable of stabilizing the joints of the limb, culminating in joint deterioration, as observed in OA, in addition to EM. However, further investigation is required to determine whether this is also related to deregulated Ihh. These experiments suggest that Ubr5 limb mutant mice could provide a novel mouse model in the study of OA and prompt the investigation of the potential role for EDD, the human homologue of UBR5, in OA initiation and progression.

During embryonic development, the Hedgehog (Hh) signaling pathway plays an important role in the growth and patterning of numerous tissues and organs. In the developing retina, Hh signaling regulates the proliferation and differentiation of retinal progenitor cells (RPC) through mechanisms that are not completely understood. The principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3), which regulate the expression of target genes responsible for the effects of the Hh pathway on RPC. The network of genes targeted by this pathway in neural progenitor cells however, remains unknown. The objective of this thesis was to identify and characterize novel targets of Hh/Gli during retinal development. Using a computation approach, 390 genes were identified as having at least one conserved Gli binding motif within the vicinity of the coding sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5 retinal explants and that the induction of 25 of these were significantly different between the two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp, two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to determine a role for Sox8 during retinal development which could reflect compensation by the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient and required for Hh mediated induction in progenitor cell proliferation and cell fate determination. Therefore, in this thesis Hh target genes have been identified which could provide some insight into the mechanisms that are responsible for the cellular outcome of a response to the pathway.