The Hedgehog gene expression program regulates lipid metabolism in basal cell carcinoma and medulloblastoma
Misactivation of the Hedgehog (Hh) pathway can cause cancers such as medulloblastomas, the most common malignant brain tumor in children, and basal cell carcinomas, the most common cancer in the United States. Hedgehog signals are transmitted through primary cilia, where Hedgehog ligands bind to Patched1 and activate Smoothened through interactions with cilia-associated sterol lipids. The gene expression programs driving cellular responses to ciliary Hh signals are incompletely understood. Thus, to define Hh target genes, we performed RNA sequencing of cells after treatment with Hh ligands (Shh, Dhh, Ihh), cilia-associated lipids (7b,27-dihydroxycholesterol, 24(S),25-epoxycholesterol), or synthetic lipids or small molecules that activate Smoothened (20(S)-hydroxycholesterol, SAG). Treatment with Hh pathway agonists identified a core gene expression program comprised of 155 genes driving lipid synthesis, metabolism, signaling, adhesion, or angiogenesis. These datasets were integrated with RNA sequencing of Hh-human medulloblastomas (n=?), a Math1-Cre SmoM2 mouse genetic model of Hh-associated medulloblastoma (n=?), and human basal cell carcinomas (n=10) to ascertain how malignant Hh signaling differs from canonical Hh signaling. We discover a conserved response to ciliary Hh signals in human or mouse medulloblastomas, including known target genes such as Gli1 or Ptch1, and novel target genes such as Hsd11b1 or Retnla. Importantly, mechanistic studies reveal Hsd11b1 to be a putative negative regulator of Hh signaling that is dysregulated in malignancies. We further demonstrate Retnla to be a positive regulator of Hh signaling that drives expression of Hsd11b2, a druggable dependency underlying Hedgehog-associated medulloblastoma. Orthotopic implantation of neuroepithelial stem cells that overexpress either Hsd11b1 and Retnla demonstrate that tumors derived Hsd11ß1 overexpression are more primitive and less aggressive whereas Retnla overexpression forms tumors that are more differentiated and behave more aggressively. In sum, we illuminate the first comprehensive transcriptome of Hh signaling and highlight the intricate interplay between Hh signaling and lipid metabolism that Hh-dependent malignancies dysregulate to drive tumor progression. Overall design: Hedgehog signaling defined in vitro in NIH3T3 wildtype and Smoothened knockout cells. Treated with 2 oxysterol lipids, 2 artifical Hedgehog agonists, and 3 endogenous Hedgehog ligands, and 3 vehicle controls. Bulk RNA-sequencing performed therafter. This gene expression program was then compared to that of 9 human basal cell carcinomas and 9 normal skin samples. Lastly, Retnla and Hsd11b1 biology, which was discovered in this paper, was compared through orthotopic tumors seeded from neuroepithelial stem cells that either overexpress Hsd11b1 or Retnla.