Abstract B43: Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas
Authors: Shimada Issei , Bandarigoda Somatilaka , Shelton John , Xuan Zhenyu , Rajaram Veena , Mukhopadhyay Saikat 
Published: May 2017

Co-regulated transcription of ribosomal protein genes (RPGs) is at the heart of efficient ribosome biogenesis; however, mechanisms that regulate RPG pathways in relation to normal vertebrate development and disease are not well understood. Granule progenitors in the external granule layer (EGL) of the developing mouse cerebellum show a sharp proliferative spike during the first two weeks after birth in a sonic hedgehog (Shh)-dependent manner. Medulloblastomas (MBs) constitute the most common malignant pediatric brain tumor originating in the cerebellum. In particular, Shh subtype MBs result from unregulated proliferation of granule progenitors, and understanding mechanisms underlying their proliferative capacity can provide insights into MB pathogenesis and therapeutics. Interestingly, increased ribosomal protein (RP) levels in MBs have been associated with poor patient survival. Here, we describe an unexpected role of global upregulation of the RPG pathway in normal cerebellar granule progenitor development and in pathogenesis of Shh subtype MBs.

Using unbiased RNA-seq analysis, we detected global transcriptional upregulation of RPG pathway in mouse cerebellum during postnatal development compared to adults. In situ hybridization and immunohistochemistry of individual RPs demonstrated increased transcript and protein levels of individual RPs specifically in the EGL, respectively. Increased RP levels in EGL progenitors resulted predominantly by transcriptional mechanisms, as opposed to post-mitotic purkinje neurons showing selective upregulation of RPG protein but not transcript levels. Translational analysis of nascent polypeptide chains by incorporation of O-propargyl-puromycin by flow cytometry and immunofluorescence revealed increased protein translation in EGL with respect to rest of the neonatal cerebellum, suggesting functional ribosomal biogenesis in addition to global upregulation of RPG pathway. To examine if reducing RP levels affect normal tissue homeostasis, we analyzed brain and cerebellum development in a hypomorphic allele of an individual RP (Rpl24 Bst/+). Although RPG pathway was transcriptionally downegulated in Rpl24 Bst/+ neonatal cerebellum with respect to wild type, gross cerebellar morphology and granule neuron numbers in adult animals were not affected. In addition, neurogenesis in the subventricular zone of the lateral ventricle and subgranular zone of the dentate gyrus of Rpl24 Bst/+ mice were not affected. Thus, partial reduction of RPs does not impact upon normal brain development.

Interestingly, Shh subtype MBs in mice demonstrated global upregulation of RPG transcripts and increased individual protein levels in comparison to postnatal cerebellum, which already has higher levels of RPG pathway compared to adults. Human Shh-MB samples also demonstrated higher RP levels with respect to normal cerebellar tissue. Analysis of RNA-seq datasets in TCGA suggested that global transcriptional upregulation of the RPG pathway is not a common feature of all human tumors, in particular only ~50% of tumor subtypes showed global upregulation. However, global upregulation of RPG pathway in Shh-MBs is remarkably significant both in the extent and severity of the increase. Interestingly, we observed reduced tumor size in mice with Rpl24 hypomorphism compared with control mice expressing only constitutively active Smoothened (SmoM2) in neural stem/progenitors, suggesting that the RPG pathway determines tumorigenic burden in Shh-MBs. Thus, unlike cerebellar progenitor development, Shh-MBs show dependency on global RPG transcription. Characterizing the RPG pathway in human MB subtypes with respect to patient prognosis and tumor recurrence, and/or therapeutic strategies targeting ribosomal biogenesis might be transformative in developing effective Shh-MB therapies.

Citation Format: Issei Shimada, Somatilaka Bandarigoda, John Shelton, Zhenyu Xuan, Veena Rajaram, Saikat Mukhopadhyay. Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B43.