Abstract 93: Tissue-specific CRISPR in zebrafish identifies PVRL1 as a novel metastasis suppressor gene in melanoma
Authors: Ablain Julien , Dang Michelle , Rothschild Harriet , Zon Leonard I. 
Published: August 2018

Defining factors that lead to the spread of cancer will have great impact on designing new treatment options. To search for such modifiers of melanoma, we developed a vector system based on the CRISPR/Cas9 technology of genome editing that targets genes specifically in the melanocytes of a zebrafish melanoma model. As a proof of principle for this system, we showed that pten inactivation in Tg(mitfa:BRAFV600E), p53-/- zebrafish significantly accelerated tumor onset. This system allows us to interrogate the functional relevance of recurrent genetic alterations found in cancer patients by next generation sequencing. Here, we identified a frequent focal deletion on chr11q23.3 by GISTIC analysis of the TCGA cohort of primary human melanomas. This region contains a gene called PVRL1 that is homozygously lost in 8% of tumors. PVRL1 encodes nectin-1, a transmembrane protein involved in adherens junctions. To assess the role of PVRL1 in melanoma in vivo, we injected Tg(mitfa:BRAFV600E), p53-/- zebrafish embryos with our melanocyte-specific CRISPR vector directed against pvrl1. We detected tumors earlier in fish expressing a vector targeting pvrl1 compared to fish expressing a control vector (median: 18 vs 20 weeks). Deep sequencing of the pvrl1 CRISPR sites in tumors confirmed gene inactivation through frameshift insertions or deletions. To further assess the in vivo phenotype of pvrl1-deficient cells, we designed a transplantation assay in which we injected 300,000 primary zebrafish melanoma cells subcutaneously in the back of irradiated, transparent recipients. In this assay, pvrl1 CRISPR melanoma cells consistently migrated away from the injection site, and eventually invaded internal tissues, whereas control cells remained localized in the dorsal region of transplanted fish. Thus pvrl1 loss favors the spreading of melanoma cells. To investigate the function of PVRL1 at the molecular level, we established pvrl1 knock-out zebrafish melanoma cell lines in culture and realized that pvrl1 was required for attachment and sustained growth in vitro in the absence of zebrafish serum. Nectin-1 was up-regulated upon serum stress in both zebrafish and human lines, presumably to support cell survival via cell-cell interactions. Additionally PVRL1 overexpression in human melanoma cell lines increased cell aggregation in vitro. Finally, we found that nectin-1 expression was higher in primary melanomas compared to benign nevi, but lower in metastases compared to primary melanoma, in human tissue-microarrays. Collectively, these data suggest a model whereby PVRL1 expression controls a balance between growth and invasion. Cancer cells deficient for PVRL1 are less fit but more prone to detach from the tumor under serum stress conditions. These results establish PVRL1 as a new metastasis suppressor gene in melanoma and cast light on the role of cancer cell adhesion in tumor spreading.

Citation Format: Julien Ablain, Michelle Dang, Harriet Rothschild, Leonard I. Zon. Tissue-specific CRISPR in zebrafish identifies PVRL1 as a novel metastasis suppressor gene in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 93.