Silencing the intestinal GUCY2C tumor suppressor axis requires APC loss of heterozygosity

ABSTRACT

Most sporadic colorectal cancer reflects acquired mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, while germline heterozygosity for mutant APC produces the autosomal dominant disorder Familial Adenomatous Polyposis (FAP) with a predisposition to colorectal cancer. In these syndromes, loss of heterozygosity (LOH) silences the remaining normal allele of APC, through an unknown mechanism, as the initiating step in transformation. Guanylyl cyclase C receptor (GUCY2C) and its hormones, uroguanylin and guanylin, have emerged as a key signaling axis opposing mutations driving intestinal tumorigenesis. Indeed, uroguanylin and guanylin are among the most commonly repressed genes in colorectal cancer. Here, we explored the role of APC heterozygosity in mechanisms repressing hormone expression which could contribute to LOH. In genetic mouse models of APC loss, uroguanylin and guanylin expression were quantified following monoallelic or biallelic deletion of the Apc gene. Induced biallelic loss of APC repressed uroguanylin and guanylin expression. However, monoallelic APC loss in Apc^min/+ mice did not alter hormone expression. Similarly, in FAP patients, normal colonic mucosa (monoallelic APC loss) expressed guanylin while adenomas and an invasive carcinoma (biallelic APC loss) were devoid of hormone expression. Thus, uroguanylin and guanylin expression by normal intestinal epithelial cells persists in the context of APC heterozygosity and is lost only after tumor initiation by APC LOH. These observations reveal a role for loss of the hormones silencing the GUCY2C axis in tumor progression following biallelic APC loss, but not in mechanisms creating the genetic vulnerability in epithelial cells underlying APC LOH initiating tumorigenesis.

KEYWORDS:

• APC
• colorectal cancer
• FAP
• guanylin
• guanylyl cyclase C
• GUCA2A
• GUCA2B
• GUCY2C
• LOH
• loss of heterozygosity

Author contributions

A.M.P. conceived and designed the experiments. A.M.P. performed the biochemical experiments and analyzed the data. A.M.P., J.R.B., A.A.E. and J.A.R. performed mouse colony maintenance and genotyping. A.M.P. wrote the paper with supervision, review and editing by S.A.W. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We would like to acknowledge the University of Pennsylvania Transgenic and Chimeric Mouse Facility for the CRISPR/Cas9 design and technical development of the Guca2b and Guca2a knockout mouse lines. RNA expression results for the GUCY2C signaling axis in the various stages of colorectal cancer published here are in whole based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. Illustration was created with Biorender.com. S.A.W. is the Samuel MV Hamilton Professor at Thomas Jefferson University.

Disclosure of Potential Conflicts of Interest

S.A.W. is a member of the Board and Chair of the Scientific Advisory Board of, and A.E.S. is a consultant for, Targeted Diagnostics & Therapeutics, Inc. which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

Supported by grants to S.A.W. from NIH [1R01 CA204881, 1R01 CA206026, P30 CA56036], Department of Defense Congressionally Directed Medical Research Program W81XWH-17-PRCRP-TTSA, The Courtney Ann Diacont Memorial Foundation, and Targeted Diagnostic & Therapeutics, Inc., and to A.E.S. (Department of Defense Congressionally Directed Medical Research Program W81XWH-17-1-0299, PhRMA Foundation, the W.W. Smith Charitable Trust, and Margaret Q. Landenberg Foundation). A.M.P. and J.A.R. were supported by Ruth Kirschstein Individual Research Fellowship Awards [F31 CA225123 and F30 CA232469, respectively]. J.A.R and J.B. were supported by pre-doctoral fellowships from the PhRMA Foundation. A.Z. was supported by NIH institutional award T32 GM008562 for Postdoctoral Training in Clinical Pharmacology.