TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis

Abstract

Truncating mutations in the tumor suppressor gene adenomatous polyposis coli (APC) are the initiating step in the vast majority of sporadic colorectal cancers, and they underlie familial adenomatous polyposis (FAP) syndromes. Modeling of APC- driven tumor formation in the mouse has contributed substantially to our mechanistic understanding of the associated disease, but additional models are needed to explore therapeutic opportunities and overcome current limitations of mouse models. We report on a novel and penetrant genetic cancer model in Xenopus tropicalis, an aquatic tetrapod vertebrate with external development, diploid genome and short life cycle. Tadpoles and froglets derived from embryos injected with TAL effector nucleases targeting the apc gene rapidly developed intestinal hyperplasia and other neoplasms observed in FAP patients, including desmoid tumors and medulloblastomas. Bi-allelic apc mutations causing frame shifts were detected in the tumors, which displayed activation of the Wnt/β-catenin pathway and showed increased cellular proliferation. We further demonstrate that simultaneous double bi-allelic mutation of apc and a non-relevant gene is possible in the neoplasias, opening the door for identification and characterization of effector or modifier genes in tumors expressing truncated apc. Our results demonstrate the power of modeling human cancer in Xenopus tropicalis using mosaic TALEN-mediated bi-allelic gene disruption.

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

“…Secondly, due to larger evolutionary distance, some organ systems such as lungs and limbs are not conserved between zebrafish and humans whereas X. tropicalis does allow faithful modeling of disease in these (White et al ., ). Thirdly, the Xenopus fate maps allow targeting of specific tissue lineages and in line with this we have shown that targeting different blastomeres can lead to tumor development in distinct targeted organs (Figure a; Bauer et al ., ; Moody, ; Van Nieuwenhuysen et al ., ). Importantly, targeted injections also allow circumventing possible embryonic lethality associated with knockout of tumor suppressor genes (Clarke et al ., ; Moser et al ., ; Naert et al ., ; Van Nieuwenhuysen et al ., ).…”

“…To model FAP in X. tropicalis , we have generated a TALEN pair targeting the mutation cluster region of the apc gene (Van Nieuwenhuysen et al ., ). Although apc TALEN injected F0 animals did not develop genuine intestinal adenomas, neoplasia were readily observed in the small intestine, macroscopically, histologically, and by proliferating cell nuclear antigen immunohistochemistry.…”

Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling

“…Choi et al showed that F0 phenotype analysis and germline transmission of the TALEN-induced mutations are facilitated by injection into one cell of two-cell embryos. The Vleminckx lab used TALENs to create mutations in the X. tropicalis tumor suppressor gene adenomatous polyposis coli ( apc ), which is implicated as the initiating mutation in many colorectal cancers, including familial adenomatous polyposis (FAP) syndrome (Van Nieuwenhuysen et al, 2015). These studies demonstrated F0 tadpoles derived from embryos injected with apc -specific TALENs develop intestinal hyperplasia and other neoplasms commonly observed in FAP patients within 6 weeks providing a useful model for the rapid testing of chemical compounds to treat FAP.…”

“…Xenopus animals and oocytes are used extensively to understand normal organ function and disease in humans (Labonne and Zorn, 2015), including cardiac congenital heart disorders and heterotaxy (Boskovski et al, 2013; Duncan and Khokha, 2016; Fakhro et al, 2011; Kaltenbrun et al, 2011; Langdon et al, 2012; 2007), gastrointestinal and pancreatic diseases (Kofent and Spagnoli, 2016; Pearl et al, 2009; 2011; Salanga and Horb, 2015; Womble et al, 2016), endocrine functions and disorders (Buchholz, 2015), kidney disease (Lienkamp, 2016), lung development (Rankin et al, 2011; 2015; Wallmeier et al, 2014), cancer (Chernet and Levin, 2013; Cross and Powers, 2009; Hardwick and Philpott, 2015; Haynes-Gilmore et al, 2014; Van Nieuwenhuysen et al, 2015; Wylie et al, 2015), ciliopathies (Kim et al, 2010; Klos Dehring et al, 2013; Ma et al, 2014), orofacial defects (Dickinson, 2016), and neurodevelopmental disorders (Erdogan et al, 2016; Pratt and Khakhalin, 2013). Looking forward, Xenopus is poised to take advantage of the new developments in genomics and genome engineering to better understand the molecular mechanisms underlying human disease (Harland and Grainger, 2011; Labonne and Zorn, 2015).…”

Tumor Immunology Viewed from Alternative Animal Models—the Xenopus Story

“…The introduction of mutations by genome editing to disrupt specific Xenopus genes makes it possible to recapitulate phenotypes of certain human diseases, including cancers [42–44]. For example, TALENs-mediated disruption of the tumor suppressor gene adenomatous polyposis coli apc gene in X. tropicalis was reported to lead to familial adenomatous polyposis similar to mouse and human intestinal cancers [43]. Also, akin to pediatric tumors of retina, double knockouts of the retinoblastoma 1 ( rb1 ) and retinoblastoma-like 1 ( rbl1 ) by CRISPR/Cas9 technology initiated retinoblastoma in X. tropicalis [44].…”