Project description:Somatic L1 retrotransposition events have been shown to occur in epithelial cancers1-8. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases, but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds and many were present in multiple tumor sections implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth. We assessed the impact of somatic L1 insertions on the expression of the corresponding protein-coding genes by comparing protein abundance in the polyp with the highest number of somatic L1 insertions with that of its paired normal colon using mass spectrometry analysis. Of the 10 validated somatic insertions that were in protein coding regions in the polyp, two proteins – KIAA1217 and WARS2 – were downregulated in the adenoma >90% and >70%, respectively.
Project description:Increasing evidence shows the value of circulating tumour DNA (ctDNA) to detect cancer and monitor its progression. Somatic genomic structural variations (SVs) are promising personalized biomarkers for sensitive and specific detection of ctDNA in liquid biopsies. However, accurate, affordable, and fast identification of such SV biomarkers is challenging, which hinders routine use in the clinic. Here, we demonstrate a novel approach - termed SHARC - for rapid discovery of somatic SV breakpoints as personalized tumour biomarkers. SHARC combines low-coverage cancer genome sketching by using Oxford Nanopore portable sequencing with a random forest classification and a dedicated filtering pipeline to enrich for somatic SVs. Our method leverages the real-time and long-read capabilities of Nanopore sequencing to identify somatic SV breakpoints at nucleotide resolution from a tumour biopsy within two days. We applied SHARC to tumour samples of high-grade ovarian and prostate cancer and validated on average 10 somatic SVs per sample with the use of PCR mini-amplicons. Finally, we demonstrate that these somatic SV biomarkers can be used to detect tumour presence from liquid biopsies in a quantitative manner and we retrospectively monitored treatment response in patients with prostate cancer, demonstrating its potential benefit for clinical practice.
Project description:Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high frequency stimulation, suggesting it should play key roles in neurotransmission during periods of intense neuronal activity. However efforts in elucidating the physiological role of ADBE have been hampered by the lack of identified molecules which are unique to this endocytosis mode. To address this, we performed proteomic analysis on purified bulk endosomes, which are a key intermediate in ADBE. Bulk endosomes were enriched via two independent approaches, a classical subcellular fractionation method and isolation via magnetic nanoparticles. There was a 79 % overlap in proteins identified via the two protocols and these molecules formed the ADBE proteome. Bioinformatic analysis revealed a strong enrichment in cell adhesion, cytoskeletal and signalling molecules, in addition to expected SV and trafficking proteins. Network analysis identified rab GTPases as a central hub within the ADBE proteome. Subsequent investigation of a subset of these rabs revealed that constitutively active rab11 both facilitated ADBE and accelerated clathrin-mediated endocytosis. This first result suggests that the ADBE proteome will provide a rich resource for the future study of presynaptic function.
Project description:Glioblastoma recurrence originates from invasive cells that escape surgical resection, but their biology remains poorly understood. Here we generated three somatic mouse models recapitulating the main driver mutations of the human disease to characterise the infiltrative tumour margin. We find that, regardless of genetics, tumours are fuelled by highly proliferative glioma stem-like cells (GSCs) resembling active neural stem cells (NSCs), which recapitulate normal and injury-induced neurogenesis in both bulk and margin. Surprisingly, GSCs are evenly distributed across both regions, suggesting that invasive potential is uncoupled from stemness. However, tumour region influences fate choice, with margin cells progressing towards astrocyte-like, and bulk cells towards injured NSC-like (iNSCs) fates. iNSCs account for a significant proportion of dormant glioblastoma cells and are induced by interferon signalling within T-cell-rich niches that form selectively in the bulk. These findings identify key differences between bulk and margin and indicate that glioblastoma cell fate is under cell-extrinsic control.