Mechanisms of Hedgehog signaling in glioblastoma

Project summary Glioblastomas are deadly tumors and account for approximately half of all malignant primary brain cancers. Glioblastoma stem cells exist in interchangeable cellular states that are influenced by the tumor microenvironment, and there is a paucity of information concerning the cellular mechanisms responsible for communicating between glioblastoma cells and the tumor stroma. The Hedgehog pathway, which directs gene expression programs that are essential for development and adult stem cell homeostasis, is critical for survival of glioblastoma stem cells. Paradoxically, a clinical trial suggested that pharmacologic inhibition of the Hedgehog pathway accelerates glioblastoma growth. These clinical data provide an opportunity to re-evaluate how the Hedgehog pathway functions in cancer, and suggest that the role of Hedgehog signaling in glioblastoma stem cells remains to be elucidated. This proposal focuses on understanding how inhibition of the Hedgehog pathway drives glioblastoma at the tumor, cellular, and molecular scale. To do so, our proposal incorporates a novel astrocyte organoid model of glioblastoma that facilitates live imaging of cellular interactions within the tumor microenvironment. When integrated with CRISPR interference, pharmacology, and mouse syngeneic and patient derived xenograft models of glioblastoma, our innovative organoid system will facilitate previously impossible experiments to understand glioblastoma biology. Based on our preliminary organoid, in vivo, and single cell RNA sequencing data presented in this application, we hypothesize that cancer cell-extrinsic Hedgehog signaling in the tumor microenvironment restrains glioblastoma stem cell-renewal and invasion by inducing differentiation factors and remodeling the extracellular matrix. We will test this hypothesis by defining how Hedgehog signals are transduced through the glioblastoma microenvironment; determining if cancer-cell extrinsic Hedgehog signaling restrains glioblastoma stem cells by inducing bone morphogenic protein signaling; and determining if Hedgehog signaling restrains glioblastoma cell invasion by inducing genes that regulate migration through the extracellular matrix. This approached is based on the premise that by studying the mechanisms of Hedgehog signaling in glioblastoma, we will discover new insights into how glioblastoma cells communicate with the tumor microenvironment. Indeed, we know surprisingly little about how interactions between glioblastoma cells and the tumor microenvironment influence cancer progression, and almost nothing about how astrocytes, which comprise the majority of cell types in the adult brain, influence glioblastoma invasion or tumorigenesis. Though the objective of this proposal is to broadly improve our understanding of the mechanisms of Hedgehog signaling in glioblastoma, a long-term goal of this research is to understand these processes well enough to consider targeted therapeutic strategies to improve survival from glioblastoma. Thus, this work will explain why Hedgehog pathway inhibition unexpectedly accelerates glioblastoma growth, elucidate targetable mechanisms, and provide the preclinical basis for new clinical trials in glioblastoma patients.