Browsing by Subject "Wnt"

The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.

In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury.

Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.

Activation of Wnt signaling pathways is critical to a variety of developmental events across all animal taxa. These highly evolutionarily conserved pathways are also important in the adult organism for maintaining homeostasis of self-renewing tissues. Because of its role in such important physiological processes, deregulation of Wnt signaling can have severe consequences; indeed, inappropriate activation of this pathway has been implicated in multiple human diseases, including cancer.

Upon binding their cellular receptors, canonical Wnt ligands, like Wnt 3A, stimulate the stabilization, accumulation, and nuclear translocation of a multifunctional cellular protein βcatenin, the consequence of which is induction of βcatenin-dependent transcription. This work describes the identification and characterization of two Wnt3A-stimulated intracellular signaling pathways activated in parallel to βcatenin stabilization: the RhoA pathway and the ERK pathway. These two auxiliary pathways do not affect βcatenin stability, accumulation, or subcellular localization; rather, they modulate βcatenin -dependent transcriptional activity through other mechanisms. As a result of their influence on βcatenin-dependent transcription, these pathways instruct cell fate decisions in C3H10T1/2 mesenchymal stem cells, in particular inhibition of adipogenesis and promotion of osteoblastogenesis.

Expression microarray analysis and biochemical and pharmacological techniques were used to further characterize the two Wnt3A-stimulated auxiliary pathways in C3H10T1/2 cells. Remarkably, each pathway influences βcatenin function via a novel mechanism. In the Wnt3A/RhoA pathway, Wnt3A-stimulated trimeric G proteins activate a RhoA-ROCK-SRF cascade. Activated SRF can cooperate with βcatenin to enhance the induction of Wnt3A target genes, like Ctgf, that also contain SRF binding sites within regulatory elements. In the Wnt3A/ERK pathway, Wnt3A transactivates the EGFR in a concentration-dependent manner, leading ultimately to ERK activation, which interacts with and promotes βcatenin/Tcf4 interaction and enhances induction of βcatenin/Tcf4 target genes.

These data emphasize the complexity of Wnt signaling and have intriguing implications regarding cross-regulation of the pathway, especially in stem cells. Also, since not all cells are capable of responding to Wnt3A by activation of these auxiliary pathways, this work identifies novel mechanisms that could underlie cell type-specific responses to Wnts and provides mechanistic insight into cellular responses to Wnt concentration gradients. Moreover, this work identifies novel transcriptional mechanisms important for promoting osteogenic cell fate specification, which could ultimately provide new therapeutic targets in disease states with bone loss or ineffective bone formation.

Brain tumors are an incredibly diverse group of neoplasms, as evidenced by their varied locations in the brain, histological characteristics, and genetic alterations. Brain tumor heterogeneity can be potentially explained by distinct oncogenic events or cells-of-origin, or by region-specific intrinsic or extrinsic factors. Brainstem Glioma (BSG) is a particularly deadly brain tumor, afflicting 200-300 children in the United States each year. High-grade BSG (also known as Diffuse Intrinsic Pontine Glioma, DIPG) cannot be surgically removed, and the standard treatment of radiation therapy provides only temporary relief from symptoms. The past 5 years has witnessed a dramatic increase in knowledge regarding the biological basis of this disease along with the realization that BSG is distinct from other more common types of glioma, such as cerebral cortex glioma (CG). It was the goal of this study to investigate the regional differences in gliomas arising in the brainstem versus the cerebral cortex, using mice as a model system, and to begin to understand the contributions of the various possible sources of heterogeneity.

In doing so, we have uncovered region-specific gene expression patterns in these two types of pediatric gliomas that are apparent even when the initiating genetic alterations and cell-of-origin are kept constant. Focusing on the paired box 3 (Pax3) gene, which is expressed at higher levels in BSG than CG, we have found that Pax3 expression not only characterizes mouse BSGs driven by PDGF signaling, Ink4aARF-loss, p53-loss, and H3.3-K27M expression, but also identifies a novel subset of human BSGs that are associated with PDGFRA alterations and wild type ACVR1 and that commonly harbor TP53 alterations and the H3.3-K27M mutation.

As Pax3 plays a pro-tumorigenic role in other types of cancer, we hypothesized that Pax3 expression contributes to the brainstem gliomagenesis process as well. By utilizing mouse models, we found that Pax3 inhibits apoptosis and promotes proliferation of Nestin-expressing brainstem progenitor cells in vitro and enhances PDGF-B-driven BSG in vivo. Furthermore, we speculate that Pax3 expression may be a marker for Wnt pathway activation in BSG, which is targetable via pharmacologic agents. Indeed, a subset of Wnt inhibitors tested effectively slowed the growth of BSG cells in vitro, however cross talk with the Shh pathway might indicate that dual Wnt and Shh inhibition is necessary.

In addition, the regional expression pattern of Pax3 in gliomas correlates with its expression in normal murine brain development, leading us to hypothesize that Pax3 progenitor cells in the neonatal brainstem can serve as a cell-of-origin for BSG. We discovered that targeting Pax3 progenitors with PDGF-B overexpression and Ink4aARF- or p53-loss induces high-grade BSG that physiologically resemble the human disease. This novel and distinct model of BSG may be utilized in the future for preclinical studies.

The identification of Pax3 as a regional marker of mouse and human BSG has led to the discovery of a novel subset of the human disease, the identification of a novel oncogene contributing to pathogenesis, and the characterization of a novel cell-of-origin with the potential to give rise to the disease. This information contributes significantly to the current understanding of the mechanisms and cellular origins of BSG, and will hopefully instruct future investigations into how to better treat this disease.

Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition.

First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression.

Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. In conclusion, SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.