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Sonic hedgehog accelerates DNA replication to cause replication stress promoting cancer initiation in medulloblastoma (EdU-Seq)

ABSTRACT: Cancer is a multi-stage disease caused by sequential mutations; however, the molecular mechanism generating cancer-initiating mutations for many cancers is not well understood. Using the developing cerebellum as model system, here we delineate a molecular mechanism for tumor initiation in medulloblastoma (MB), the most frequent malignant pediatric brain tumor. Activation of the Sonic hedgehog (SHH) pathway is frequent in MB, with mutations in the tumor suppressor PTCH1 (a negative regulator of SHH signaling) being the most common initiating event for SHH-MB. However, how SHH as a developmental mitogen promotes early carcinogenesis in the cells of origin, granule cerebellar progenitors (GCPs), remains to be determined. Here we report that physiological exposure of GCPs to Shh causes a distinct form of DNA replication stress, altering DNA replication dynamics to increase both origin firing and fork velocity. Shh promotes DNA helicase loading and activation in GCPs, with increased levels of Cdc7-dependent replication origin firing. S-phase duration is reduced and hyper-recombination consequently occurs. Such elevated recombination causes copy-number neutral LOH, an event seen frequently at the PTCH1/ptch1 locus. Moreover, Cdc7 inhibition to attenuate origin firing in a MB mouse model is sufficient to reduce somatic recombination and preneoplastic tumor formation. We therefore establish that tissue-specific replication stress induced by Shh acts to promote LOH, which in tumor-prone Ptch1+/- GCPs can result in loss of this tumor suppressor, as an early cancer initiating event.

ORGANISM(S): Mus musculus

PROVIDER: GSE147409 | GEO | 2020/07/20

REPOSITORIES: GEO

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Project description:Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Since microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using two spontaneous mouse MB models with specific initiating mutations, Ink4c-/-; Ptch1+/- and Ink4c-/-; p53-/-. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, nine were encoded by the miR-17~92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that three miR-17~92 cluster miRNAs (miR-92, miR-19a and miR-20) were also overexpressed in human MBs with a constitutively activated SHH signaling pathway, but not in other forms of the disease. To test whether the miR-17~92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c-/-; Ptch1+/- mice. These, but not similarly engineered cells from Ink4c-/-; p53-/- mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17~92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17~92 cluster and the SHH signaling pathway in the development of MBs in mouse and man. Samples: purified CGNP-like mouse medulloblastoma cells; purified CGNPs from age day 6 (P6) mice; whole cerebellum from P6 mice; whole cerebellum from 1 month old mice. Each from three backgrounds (except tumors) - wt; Ptc+/-,Ink4c-/-; p53-/-,Ink4c-/-. Two to five biological replicates each.

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Sonic hedgehog accelerates DNA replication to cause replication stress promoting cancer initiation in medulloblastoma (EdU-Seq)

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