Van Nieuwenhuysen T et al. (2015), TALEN-mediated apc mutation in Xenopus tropical...

XB-ART-50857

Oncoscience 2015 May 19;25:555-66. doi: 10.18632/oncoscience.166.

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

Van Nieuwenhuysen T , Naert T , Tran HT , Van Imschoot G , Geurs S , Sanders E , Creytens D , Van Roy F , Vleminckx K .

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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.

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Species referenced: Xenopus tropicalis
Genes referenced: apc fap mapre1 pcna tyr

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???displayArticle.omims??? COLORECTAL CANCER; CRC [+]

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	Figure 2. Neoplastic phenotypes observed in apc TALEN mRNA injected tadpoles and frogsSmall intestine, cut open longitudinally, of wild type (WT) (A) and apc TALEN mRNA (40 pg) injected (B) adult frogs. In WT frogs, the epithelial lining of the duodenum is organized in parallel longitudinal folds. The intestines of apc TALEN injected animals are largely expanded and the intestinal folds are irregular and excessively undulated. Aberrant local protuberances are visible (black arrowhead). (C) Large fast growing tumors at the position where the hindlimbs emerge (red arrows). (D) Desmoid tumor, visible as a subcutaneous light colored mass (red arrow). (E) Epidermoid cysts (red arrows) originating at various positions in the tadpole skin. (F) Medulloblastoma visible as a large mass in the tadpole brain region.

	Figure 4. Histological analysis of hind limb, desmoid, epidermal and brain neoplasias of apc TALEN mRNA (40 pg) injected tadpoles and frogletsThe boxed regions in the left panels are shown at higher magnifications in the corresponding panels on the right. (A, B) Tumors associated with the developing hind limb are composed of uniform sheets of densely packed monotonously small, round cells with scant cytoplasm. (C, D) Desmoid tumor composed of spindle shaped or stellate fibroblastic cells interspersed with collagen deposits and with prominent vasculature. (E, F) Epidermoid cyst with neuroblastic elements, morphologically reminiscent of a malignant, poorly differentiated, primitive, embryonal neuroepithelial/neuroectodermal tumor (PNET). (G, H) Brain tumor with the histological characteristics of a medulloblastoma.
	Figure 1. TALEN mediated targeting of the MCR of the Xenopus tropicalis apc gene(A) Schematic representation of the apc protein, the region that was targeted by TALENs, and the TALENs used. (B) Deletions detected in the targeted region of apc gene in embryos injected with 300 pg of apc TALEN mRNA. The sequences in red indicate the apc TALEN recognition sites. (C) Bi-allelic mutation of the apc gene identified in desmoid and hindlimb tumors dissected from tadpoles having undergone apc TALEN mRNA (40 pg) injection. AA, Amino Acid; APC, Adenomatous Polyposis Coli; Arm, Armadillo repeats; MCR, Mutation Cluster Region; EB1 BD, End Binding protein 1 Binding Domain; HD, Homodimerization domain; MT BD, Microtubule Binding Domain.
	Figure 5. Active Wnt signalling in apc TALEN induced tumorsGFP expression is increased in tumors developing in apc TALEN treated embryos derived from a transgenic Wnt reporter line [23]. (A-C) Intense GFP expression reflecting Wnt activity in a hind limb associated tumor. The same tumor is shown 3, 4 and 7 weeks after apc TALEN injection. Active Wnt signalling is detected in the entire tumor in the initial stages (A) but gets restricted to undulated strands at the edge of the tumor in the later stages (B, C). (D) Brightfield and (E) fluorescent image of epidermoid cysts (red arrows) showing active Wnt signalling. Fluorescence of the neighboring skin is due to autofluorescence. (F) Brightfield and (G) fluorescent images of an exposed desmoid tumor (red arrowhead) with the skin partially removed showing active Wnt signaling in comparison to the neighboring exposed muscle (white arrows). The white arrowhead indicates autofluorescent skin overlying the tumor.
	Figure 6. Increased and mislocalized proliferation in apc TALEN induced tumorsHoechst (left panels) and PCNA (middle panels) double staining; magnified overlays in right panels. (A, B, C) In WT small intestine PCNA staining is primarily localized at the base of the folds and largely absent in the crests. (D, E, F) Small intestine of apc TALEN injected froglet showing the presence of PCNA staining along the entire trough-crest axis. (G, H, I) Epidermoid cysts showing a high number of proliferating PCNA positive cells. (J, K, L) Desmoid tumor displaying a large fraction of PCNA positive nuclei. (M, N, O) Hind limb-associated tumor with almost all nuclei staining positive for PCNA indicating a very high proliferation rate.
	Figure 7. Combined bi-allelic targeting of the tyr and apc genes in X. tropicalis tumorsINDELs found in the tyr gene in apc mutant tumors developing after co-injection of apc (40 pg) and tyr TALEN (300 pg) mRNAs. Note that in contrast to the INDELs observed in the apc locus (see Fig. 1C), deletions in the tyr locus can be in frame, e.g. δ3 or δ15.

References [+] :

Cathcart, COX-derived prostanoid pathways in gastrointestinal cancer development and progression: novel targets for prevention and intervention. 2012, Pubmed