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The Hedgehog (Hh) pathway regulates the growth of a subset of adult gliomas and better definition of Hh-responsive subtypes could enhance the clinical utility of monitoring and targeting this pathway in patients. Somatic mutations of the isocitrate dehydrogenase (IDH) genes occur frequently in WHO grades II and III gliomas and WHO grade IV secondary glioblastomas. Hh pathway activation in WHO grades II and III gliomas suggests that it might also be operational in glioblastomas that developed from lower-grade lesions. To evaluate this possibility and to better define the molecular and histopathological glioma subtypes that are Hh-responsive, IDH genes were sequenced in adult glioma specimens assayed for an operant Hh pathway. The proportions of grades II-IV specimens with IDH mutations correlated with the proportions that expressed elevated levels of the Hh gene target PTCH1. Indices of an operational Hh pathway were measured in all primary cultures and xenografts derived from IDH-mutant glioma specimens, including IDH-mutant glioblastomas. In contrast, the Hh pathway was not operational in glioblastomas that lacked IDH mutation or history of antecedent lower-grade disease. IDH mutation is not required for an operant pathway however, as significant Hh pathway modulation was also measured in grade III gliomas with wild-type IDH sequences. These results indicate that the Hh pathway is operational in grades II and III gliomas and glioblastomas with molecular or histopathological evidence for evolvement from lower-grade gliomas. Lastly, these findings suggest that gliomas sharing this molecularly defined route of progression arise in Hh-responsive cell types.
Published by Elsevier Ireland Ltd.
Pilocytic astrocytoma is commonly viewed as a benign lesion. However, disease onset is most prevalent in the first two decades of life, and children are often left with residual or recurrent disease and significant morbidity. The Hedgehog (Hh) pathway regulates the growth of higher WHO grade gliomas, and in this study, we have evaluated the activation and operational status of this regulatory pathway in pilocytic astrocytomas. Expression levels of the Hh pathway transcriptional target PTCH were elevated in 45% of tumor specimens analyzed (ages 1-22 years) and correlated inversely with patient age. Evaluation of a tissue array revealed oligodendroglioma-like features, pilomyxoid features, infiltration, and necrosis more commonly in specimens from younger patients (below the median patient age of 10 years). Immunohistochemical staining for the Hh pathway components PTCH and GLI1 and the proliferation marker Ki67 demonstrated that patients diagnosed before the age of 10 had higher staining indices than those diagnosed after the age of 10. A significant correlation between Ki67 and PTCH and GLI1 staining indices was measured, and 86% of Ki67-positive cells also expressed PTCH. The operational status of the Hh pathway was confirmed in primary cell culture and could be modulated in a manner consistent with a ligand-dependent mechanism. Taken together, these findings suggest that Hh pathway activation is common in pediatric pilocytic astrocytomas and may be associated with younger age at diagnosis and tumor growth.
Hedgehog pathway activity has been demonstrated in malignant glioma. However, its role in tumor growth has not been determined. Here we demonstrate that pharmacological inhibition of the Hedgehog pathway in established orthotopic malignant glioma xenografts confers a survival advantage. Pathway inhibition is measured in transplanted human tumor cells and not in host mouse brain. Correspondingly, survival benefit is observed only in tumors with an operational Hedgehog pathway. These data indicate that Hedgehog signaling regulates the growth of select malignant gliomas. We also demonstrate that Hedgehog pathway component and gene target expression segregate to CD133(+) tumor initiating cells. Treated mice eventually succumb to disease, thus, targeting the Hedgehog pathway in CD133(+) cells produces significant, but incomplete tumor regression. Therefore, our studies suggest that more complete tumor regression may require the inclusion of other therapeutic targets, including CD133(-) cells.
The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish have immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations, however, co-expressing a constitutively active human AKT1 resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myxoma. All tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and that the binary transgenic approach is a useful tool for studying interaction of oncogenes and oncogenic pathways in zebrafish.
Generation of distinct ventral neuronal subtypes in the developing spinal cord requires Shh signaling mediated by the Gli family of transcription factors. Genetic studies of Shh(-/-);Gli3(-/-) double mutants indicated that the inhibition of Gli3 repressor activity by Shh is sufficient for the generation of different neurons including motor neurons. In this study, we show that although ventral neural progenitors are initiated in normal numbers in Shh(-/-);Gli3(-/-) mutants, the subsequent appearance of motor neuron progenitors shows a approximately 20-hour lag, concomitant with a delay in the activation of a pan-neuronal differentiation program and cell cycle exit of ventral neural progenitors. Accordingly, the Shh(-/-);Gli3(-/-) mutant spinal cord exhibits a delay in motor neuron differentiation and an accumulation of a ventral neural progenitor pool. The requirement of Shh and Gli3 activities to promote the timely appearance of motor neuron progenitors is further supported by the analysis of Ptch1(-/-) mutants, in which constitutive Shh pathway activity is sufficient to elicit ectopic and premature differentiation of motor neurons at the expense of ventral neural progenitors. Taken together, our analysis suggests that, beyond its well established dorso-ventral patterning function through a Gli3-derepression mechanism, Shh signaling is additionally required to promote the timely appearance of motor neuron progenitors in the developing spinal cord.
The Hedgehog (Hh) pathway is important for skeletal patterning and morphogenesis during embryonic development. Papers by Ohba et al. and Mak et al. in this edition of Developmental Cell suggest that Hh signaling may exert delicate control over the activities of osteoclasts and osteoblasts, the cell types primarily responsible for bone resorption and formation.
The hedgehog (Hh) signaling pathway regulates progenitor cells during embryogenesis and tumorigenesis in multiple organ systems. We have investigated the activity of this pathway in adult gliomas, and demonstrate that the Hh pathway is operational and activated within grade II and III gliomas, but not grade IV de novo glioblastoma multiforme. Furthermore, our studies reveal that pathway activity and responsiveness is confined to progenitor cells within these tumors. Additionally, we demonstrate that Hh signaling in glioma progenitor cells is ligand-dependent and provide evidence documenting the in vivo source of Sonic hedgehog protein. These findings suggest a regulatory role for the Hh pathway in progenitor cells within grade II and III gliomas, and the potential clinical utility of monitoring and targeting this pathway in these primary brain tumors.
The sonic hedgehog (SHH) receptor Patched 1 (Ptch1) is critical for embryonic development, and its loss is linked to tumorigenesis. Germ line inactivation of one copy of Ptch1 predisposes to basal cell carcinoma and medulloblastoma in mouse and man. In many cases, medulloblastoma arising from perturbations of Ptch1 function leads to a concomitant up-regulation of a highly similar gene, Patched2 (Ptch2). As increased expression of Ptch2 is associated with medulloblastoma and other tumors, we investigated the role of Ptch2 in tumor suppression by generating Ptch2-deficient mice. In striking contrast to Ptch1-/- mice, Ptch2-/- animals were born alive and showed no obvious defects and were not cancer prone. However, loss of Ptch2 markedly affected tumor formation in combination with Ptch1 haploinsufficiency. Ptch1+/-Ptch2-/- and Ptch1+/-Ptch2+/- animals showed a higher incidence of tumors and a broader spectrum of tumor types compared with Ptch1+/- animals. Therefore, Ptch2 modulates tumorigenesis associated with Ptch1 haploinsufficiency.
Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.
Mutations affecting the transmembrane proteins Patched (Ptc) or Smoothened (Smo) that trigger ligand-independent activity of the Hedgehog (Hh) signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma. Despite extensive genetic studies demonstrating the importance of these receptor components in embryonic patterning and cancer, the mechanism by which Ptc regulates Smo is not understood. Here we report that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, we show that free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; we demonstrate that the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumour suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule.