RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma in Xenopus tropicalis

Abstract

Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small-cell lung cancer (SCLC), choroid plexus tumors, and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional redundancy between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain insight into this in vivo redundancy. We show that while rb1 inactivation is sufficient to induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking tp53 mutant X. tropicalis line, we demonstrate increased malignancy of rb1/rbl1-mutant glioma towards glioblastoma upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, the tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental setup, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1, generating new insights in the functional redundancy within the retinoblastoma protein family in suppressing neuroendocrine pancreatic cancer and glioma/glioblastoma.

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Fig. 1: Tp53 mutant X. tropicalis develop hematological malignancy and sarcomas.
Fig. 2: Small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC) in X. tropicalis upon mosaic CRISPR/Cas9 genome editing of rb1 and rbl1 in the anterior endoderm.
Fig. 3: Rb1 and rbl1 crispants (ectodermal targeted) develop retinoblastoma, excessive black skin pigmentation and a spectrum of brain tumors.
Fig. 4: Choroid plexus (CP) tumors arising in rb1/rbl1 crispants differ in grade according to their tp53 genotype.
Fig. 5: Rbl1 functions as a tumor suppressor in glioblastoma, while tp53 inactivation underlies progression.

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Acknowledgements

Research in the authors’ laboratory is supported by the Research Foundation—Flanders (FWO-Vlaanderen) (grants G0A1515N and G029413N), by the Belgian Science Policy (Interuniversity Attraction Poles—IAP7/07) and by the Concerted Research Actions from Ghent University (BOF15/GOA/011). Further support was obtained by the Hercules Foundation, Flanders (grant AUGE/11/14) and the Desmoid Tumor Research Foundation. TN is funded by “Kom op tegen Kanker” (Stand up to Cancer), the Flemish cancer society and previously held PhD fellowship with VLAIO-HERMES during the course of this work. DT and MC have a PhD fellowship from the Research Foundation—Flanders (FWO-Vlaanderen). We thank the Xenopus laevis Resource for Immunobiology (Rochester, NY, NIH R24 AI059830) for the kind gift of monoclonal antibody AM20 (10A91, CD8). We are indebted to Tim Deceuninck for animal care, Kelly Lemeire for technical assistance with TUNEL staining. We would like to thank the VIB BioImaging Core, and in particular Chris Guerin, Eef Parthoens, and Anneke Kremer, for access to the instrument park, training, and support.

Author information

TN, DDi, and KV designed the study. DDi, TN, DT, and RN were involved in generation and phenotyping of the tp53 mutant X. tropicalis. TN performed genome engineering and phenotyping of all rb1/rbl1, rb1/rbl1/tp53, and rb1/rbl1/tp53/pten mutants. TN, LE, and DDe were involved in laser-capture microdissection and downstream analysis. DC and JvD performed pathological analysis. DT, DDi, and GvI performed flow cytometry experiments. CV performed X-ray imaging. SD provided technical assistance throughout the project. MC performed BATCH-GE analysis of targeted amplicon sequencing data. TN and KV wrote the paper.

Correspondence to Kris Vleminckx.

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Naert, T., Dimitrakopoulou, D., Tulkens, D. et al. RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma in Xenopus tropicalis. Oncogene 39, 2692–2706 (2020). https://doi.org/10.1038/s41388-020-1173-z

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