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

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. Overall design: EdU-seq assay followed by high throughput sequencing

癌症是一类由序贯突变驱动的多阶段疾病，但目前学界对多数癌症的致癌起始突变的分子机制仍尚未完全阐明。本研究以发育中的小脑为模型系统，阐明了儿童最常见的恶性脑肿瘤——髓母细胞瘤（medulloblastoma, MB）的肿瘤起始分子机制。音猬因子（Sonic hedgehog, SHH）通路的异常激活在髓母细胞瘤中十分常见，其中肿瘤抑制基因PTCH1（SHH信号通路的负调控因子）的突变是SHH亚型髓母细胞瘤（SHH-MB）最常见的起始致病事件。然而，作为发育性有丝分裂原的SHH，如何在其细胞起源——小脑颗粒层祖细胞（granule cerebellar progenitors, GCPs）中促进早期癌变，这一问题仍有待解析。本研究发现，GCPs在生理条件下暴露于Shh时，会产生一种独特的DNA复制应激（DNA replication stress），通过改变DNA复制动力学特征，同时提升复制起始位点激活（origin firing）频率与复制叉行进速度（fork velocity）。Shh可促进GCPs中DNA解旋酶的加载与激活，并增强依赖于Cdc7的复制起始位点激活水平。S期时长缩短，进而引发过度重组现象。这种升高的重组水平会引发拷贝数中性杂合性缺失（copy-number neutral loss of heterozygosity, cnLOH），该事件在PTCH1/ptch1基因座中频繁出现。此外，在髓母细胞瘤小鼠模型中，通过抑制Cdc7以降低复制起始位点激活频率，足以减少体细胞重组事件与癌前肿瘤的形成。综上，本研究证实：Shh诱导的组织特异性DNA复制应激可促进杂合性缺失（loss of heterozygosity, LOH）的发生；在肿瘤易感的Ptch1+/- GCPs中，该过程可导致该肿瘤抑制基因的功能丢失，从而作为早期致癌起始事件。实验设计：先开展EdU-seq检测，随后进行高通量测序。