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 apcmcr zebrafish model, we show that ctbp1, but not retinoic acid, regulates cnot3a expression. Injection of hCNOT3 and E20K, but not E70K, to homozygous apcmcr 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.
Highlights
Adenomatous Polyposis Coli (APC) is a 312 kDa protein that functions as a tumor suppressor by acting as a requisite scaffolding protein that stabilizes the β-catenin destruction complex
In our recent work to define a comprehensive genomic landscape of adenomas and at-risk mucosa, we found that patient-derived Familial Adenomatous Polyposis (FAP) adenomas do not just have the expected somatic APC mutations, and recurrent mutations in Wnt pathway genes and in novel genes previously not linked to progressing colon adenomas to adenocarcinomas
We previously reported that CCR4-NOT Subunit 3 (CNOT3) mutations occur in FAP adenoma [19]
Summary
Adenomatous Polyposis Coli (APC) is a 312 kDa protein that functions as a tumor suppressor by acting as a requisite scaffolding protein that stabilizes the β-catenin destruction complex. APC mutations mostly occur in a specific region, the APC mutation cluster region (MCR), and singularly define Familial Adenomatous Polyposis (FAP), a genetic condition predisposing to the development of colorectal adenomatous polyps and early onset colorectal adenocarcinoma [3, 4]. These APC mutations are thought to disrupt the role of APC in promoting β-catenin degradation to temper proliferative canonical Wnt signaling [1, 5], and in modulating retinoic acid-dependent intestinal development [6,7,8,9]. When the APC gene is excluded, one gene with frequent accumulating genomic alterations CCR4NOT Transcription Complex Subunit 3 (CNOT3), which was observed in 5 out of 25 sequenced FAP adenomas [13]
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