Functional characterization of beta-arrestin 1 in neuroendocrine tumor cell lines

C. Campana*^a (Ms), LJ. Hofland^a (Prof), PM. Van Koetsveld^a (Mr), D. Ferone^b (Prof), F. Gatto^c (Dr), AM. Iyer^a (Dr)

^a Erasmus Medical Center, Rotterdam, NETHERLANDS ; ^b University of Genova, Genova, ITALY ; ^c IRCCS Policlinico San Martino, Genova, ITALY

* c.campana@erasmusmc.nl

Introduction: Somatostatin receptors (SSTs) are G-protein-coupled receptors (GPCRs) with the SST subtype 2a (SST₂) representing a major therapeutic target in neuroendocrine tumors (NETs). β-arrestin 1 belongs to a family of intracellular proteins primarily known for their role in GPCR trafficking, including SST₂. However, emerging evidence points to GPCR-independent involvement of β-arrestins in cellular signaling and proliferation resulting in their investigation in cancer development and progression. However, the role of β-arrestin 1 in the development of NETs and in treatment response are poorly understood.

Material and Methods: CRISPR-Cas9 mediated knock-outs (KO) of the β-arrestin 1 gene (ARRB1) were generated using the human pancreatic NET (BON-1), gastrointestinal NET (GOT1), and bronchial carcinoid (H727) cell lines, characterized by different SST expression levels. After clonal selection of cells, the editing efficiency for ARRB1 was verified by Sanger sequencing followed by inference of CRISPR edits (ICE) analysis and Western Blot. Expression of SSTs and β-arrestin 2 (ARRB2) in ARRB1 KO models was evaluated by RT-qPCR. Furthermore, cell growth was evaluated by DNA quantification.

Results: Two ARRB1 KO clones were obtained for each cell line, confirmed by ICE analysis and Western Blot. We found that the KO models were stable over multiple cell passages. The two ARRB1 KO clones of H727 cells showed a longer doubling time (2.89 ± 0.32 and 3.33 ± 0.54 days) compared to 2 negative controls transfected with non-targeting guide RNAs (1.83 ± 0.18 and 2.16 ± 0.27 days, p<0.0001). Preliminary data also showed a significant upregulation (between 6.09 and 11.17 fold, p=0.017) of SST₂ mRNA expression in the ARRB1 KO clones of H727 cells compared to negative controls, while no differences were observed in ARRB2 and SST₅ mRNA expression.

Discussion: In H727 cells, a lower growth rate and the upregulation of SST₂ mRNA were observed after β-arrestin 1 KO, supporting the role of this molecule in cell proliferation. Experiments evaluating SST expression levels and cell growth rate in BON-1 and GOT1 ARRB1 KO clones, together with the impact of the ARRB1 KO on the response to somatostatin analogues, are ongoing.

The author has declared no conflict of interest.