Spatial proteomics reveals distinct protein patterns in a cortical migration disorder caused by LIN28A overexpression and WNT activation

Abstract

Developmental signalling pathways act in stage and tissue dependent relation. Mis-activation can drive tumour formation and other disorders. The RNA-binding protein LIN28A maintains stemness and is overexpressed in embryonal brain tumours like the Embryonal Tumour with Multilayered Rosettes (ETMRs). ETMRs are highly aggressive tumours and lack appropriate therapeutic targets. CTNNB1 mutations, which stabilise this protein and activate the WNT-pathway, have been frequently described in this tumour. To profoundly study this tumour entity, a suitable mouse model is needed. One established mouse model to study ETMRs, resembles the human tumour histologically and molecularly, but lacks the characteristic LIN28A overexpression. The aim of this study was to investigate if the interplay of LIN28A overexpression and the stabilised CTNNB1 protein are required for initiation of tumour formation. Furthermore, the aim was to analyse the role of these oncogenic factors in the cerebral cortex during development and investigate the cerebral cortex proteome in a spatially resolved manner to detect associated molecular profiles. Cre-loxP based conditional mouse models were established, displaying a sole LIN28A overexpression (GL), a sole activation of CTNNB1 (GB) or the combination of both oncogenic factors (GBL). GBL mice did not show brain tumour formation but a disturbed lamination and impaired migration in the cerebral cortex. LIN28A transiently increased the proliferation rate at E14.5, whereas the proliferation was reduced in the GB model at E18.5. In the GBL model no significant difference in proliferation was observed. A refined protocol for spatially resolved proteome analysis using the nanosecond infrared laser (NIRL)-ablation system for nano-volume sampling reached a spatial resolution of about 40 µm per layer for comprehensive analysis of the developing brain. The proteome patterns revealed imbalances of the extracellular matrix receptors RPSA and ITGB1 and LAMININ accompanied by a porous pial border and neuronal overmigration in GBL brains. The GBL model revealed additionally, reduced hypoglycosylation of α-DAG and misplaced Cajal-Retzius cells in deeper cortex regions. Co-activation of LIN28A and CTNNB1 was not sufficient for brain tumour formation but resulted in a neuronal migration disorder resembling the human Cobblestone lissencephaly type 2. Therefore, the GBL model might be used as a model to investigate this rare and severe neuronal migration disorder. Moreover, the analysis revealed a novel function of oncogenic LIN28A affecting the extracellular matrix integrity which might potentially play role in tumour progression or invasion.