R-4733

Presenilin1 (PS1)/γ‐secretase cleaves within the transmembrane domain of numerous receptor substrates. Mutations in PS1 have implications on the catalytic subunit of γ‐secretase decreasing its activity and becoming a potential causative factor for Familial Alzheimer’s Disease (FAD). This work studies the role of PS1/γ‐secretase on the processing, angiogenic signaling, and functions of VEGFR2 and the effects of PS1 FAD mutants on the γ‐secretase‐mediated epsilon cleavage of VEGFR2.

HEK293T cells expressing WT‐PS1 or PS1‐FAD mutants (M146V, I213T, E280A, G384A, L166P, E120K, A246E) were prepared using a lentivirus‐based transduction system and used in luminescence‐based experiments. PS1‐VEGFR2 co‐immunoprecipitation (Co‐IP) experiments were analyzed on WBs. Bimolecular fluorescence complementation (BiFc) assays were carried out in HEK293T cells overexpressing PS1 or VEGFR2 tagged with half complementary portions of the mVenus and visualized by fluorescence microscopy. The ClusPro web server was used for protein‐protein docking analyses. Anti‐PS1 siRNA technology was applied to downregulate PS1 in endothelial bEnd.3 cells.

Luciferase Assay shows that PS1 FAD mutants significantly decrease the γ‐secretase‐mediated epsilon cleavage of VEGFR2 when compared to WT‐PS1, leading to the production of significantly lower amount of the VEGFR2/CTF2 peptide indicating a loss of γ‐secretase proteolytic function. This loss of function is comparable to the effect of γ‐secretase inhibitor RO4929097. Co‐IP experiments show that PS1 forms complexes with VEGFR2 and BiFc experiments show that PS1 binds to VEGFR2. Docking analysis indicates that the binding is electrostatically favored. Co‐IP between the PS1‐FAD mutants and VEGFR2 show that FAD mutants do not have differential binding to the VEGFR2 compared to WT‐PS1, showing that the effect of these mutants on VEGFR2 cleavage is not due to altered binding. PS1 downregulation of endothelial cells indicates that PS1 plays a role in the regulation of the VEGFA‐induced phosphorylation of VEGFR2 at Y1175 and Y1054‐59 as well as p‐44/42 MAPK (Erk1/2) and in VEGFA‐induced tube formation in vitro.

PS1 FAD mutants decrease production of signaling peptide VEGFR2/CTF2 probably due to reduced γ‐secretase activity of FAD mutants. Interestingly FAD mutations do not seem to affect the PS1‐VEGFR2 association. Our data support that PS1 regulates downstream signaling and angiogenic functions of VEGFR2.

Choroid plexus carcinomas (CPCs) are extremely rare brain tumors and carry a dismal prognosis. Treatment options are limited and there is an urgent need to develop models to further research. In the present study, we established two CPC cell lines and performed multi-omics analyses. These cell lines serve as valuable models to propose new treatments in these rare but deadly brain tumors.

Multi-omic profiling including, (i) methylation array (EPIC 850 K), (ii) whole genome sequencing (WGS), (iii) CANCERPLEX cancer genome panel testing, (iv) RNA sequencing (RNA-seq), and (v) proteomics analyses were performed in CCHE-45 and NGT131 cell lines.

Both cell lines were classified as methylation class B. Both harbored pathogenic TP53 point mutations; CCHE-45 additionally displayed TP53 loss. Furthermore, alterations of the NOTCH and WNT pathways were also detected in both cell lines. Two protein-coding gene fusions, BZW2-URGCP, and CTTNBP2-ERBB4, mutations of two oncodrivers, GBP-4 and KRTAP-12-2, and several copy number alterations were observed in CCHE-45, but not NGT131. Transcriptome and proteome analysis identified shared and unique signatures, suggesting that variability in choroid plexus carcinoma tumors may exist. The discovered difference's importance and implications highlight the possible diversity of choroid plexus carcinoma and call for additional research to fully understand disease pathogenesis.

Multi-omics analyses revealed that the two choroid plexus carcinoma cell lines shared TP53 mutations and other common pathway alterations and activation of NOTCH and WNT pathways. Noticeable differences were also observed. These cell lines can serve as valuable models to propose new treatments in these rare but deadly brain tumors.