Functional characterization of CNOT3 variants identified in familial adenomatous polyposis adenomas

Abstract

Germline mutations in the tumor suppressor Adenomatous Polyposis Coli ( APC ) define Familial Adenomatous Polyposis (FAP), the genetic predisposition to developing adenomatous polyps. Recent sequencing of FAP adenomas have challenged established dogma that APC mutations alone represent the adenoma mutational landscape because recurrent somatic mutations in non-WNT pathway genes were also discovered. In particular, one of these novel genes, CNOT3 , presented E20K and E70K mutations that are predicted to be deleterious in silico . We utilized zebrafish embryos to determine if these mutations affect CNOT3 function and perform novel biology in an APC-dependent pathway in vivo . Human CNOT3 ( hCNOT3 ) and E20K mRNA injection rescued zebrafish cnot3a knockdown lordosis phenotype while E70K did not. In the FAP apc mcr zebrafish model, we show that ctbp1 , but not retinoic acid, regulates cnot3a expression. Injection of hCNOT3 and E20K , but not E70K , to homozygous apc mcr zebrafish initiated gut differentiation while cnot3a knockdown in wildtype embryos led to decreased intestinal development and differentiation. Finally, targeted sequencing of 37 additional FAP adenomas revealed CNOT3 mutations in 20% of these samples. Overall, our work supports a mechanism where CTBP1 regulates CNOT3 and that overall CNOT3 perturbation could work in concert with germline APC mutations in advancing adenomas to a more transformed state prior to progression to adenocarcinoma.

A signalling axis involving CNOT3, Aurora B and ERK promotes differentiation and survival of mesendodermal progenitor cells

“…The involvement of the NLS and adjacent Aurora B target sites in cancer is further supported by the recent identification of a Cnot3 -K286E mutation in 11% (4/37) of a cohort of pre-malignant adenomas from that were sequenced from Familial Adenomatous Polyposis (FAP) patients 48 (Figure 7C). Cnot3 -K286 is the first residue of the KKRGR nuclear localisation sequence that is located adjacent to the sites of Aurora B phosphorylation in mouse ESCs.…”

“…Mutations in the coding region of human Cnot3 have been observed in a number of cancers (Forbes et al, 2017), with genetic analysis providing evidence that Cnot3 mutations can have tumour suppressor (De Keersmaecker et al, 2013) and tumour promoting (Delacruz et al, 2019) effects. High levels of nuclear CNOT3 have also been observed in an aggressive colorectal cancer cell line (Cejas et al, 2017).…”

“…(4/37) of a cohort of pre-malignant adenomas from that were sequenced from Familial Adenomatous Polyposis (FAP) patients (Delacruz et al, 2019) ( Figure 8C). Cnot3-K286 is the first residue of the KKRGR nuclear localisation sequence that is located adjacent to the sites of Aurora B phosphorylation in mouse ESCs.…”

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“…Two of the most frequent mutations identified were CNOT3 E20K and CNOT3 E70K . Knockdown of cnot3a in apc MCR zebrafish embryos decreased intestinal development and differentiation (Delacruz et al, 2019). Additionally, introducing mutated human CNOT3 E20K mRNA into apc MCR embryos rescued intestinal differentiation while CNOT3 E70K mRNA did not, suggesting that E70K is an inactivating mutation.…”

“…Additionally, introducing mutated human CNOT3 E20K mRNA into apc MCR embryos rescued intestinal differentiation while CNOT3 E70K mRNA did not, suggesting that E70K is an inactivating mutation. CNOT3 E70K is commonly found in adenoma tissues and a variety of cancers, but the discovery of its inhibitory effect on intestinal differentiation is novel and suggests that CNOT3 E70K may contribute to the progression of adenomas to carcinomas (Delacruz et al, 2019).…”