Wnt/β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in most colorectal cancers (CRC) through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction. Following Wnt ligand presentation, this complex is inhibited. However, a role for APC in this inhibition has not been shown. Here, we utilized Wnt3a-beads to locally activate Wnt co-receptors. In response, the endogenous β-catenin destruction complex reoriented toward the local Wnt cue in CRC cells with full-length APC, but not if APC was truncated or depleted. Non-transformed human colon epithelial cells displayed similar Wnt-induced destruction complex localization which appeared to be dependent on APC and less so on Axin. Our results expand the current model of Wnt/β-catenin signaling such that in response to Wnt, the β-catenin destruction complex: (1) maintains composition and binding to β-catenin, (2) moves toward the plasma membrane, and (3) requires full-length APC for this relocalization.

Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.

Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota

Somatic mutations in the Tp53 tumor suppressor gene are the most commonly seen genetic alterations in cancer, and germline mutations in Tp53 predispose individuals to a variety of early-onset cancers. Development of appropriate translational animal models that carry mutations in Tp53 and recapitulate human disease are important for drug discovery, biomarker development, and disease modeling. Current Tp53 mouse and rat models have significant phenotypic and genetic limitations and often do not recapitulate certain aspects of human disease. We used a marker-assisted speed congenic approach to transfer a well-characterized Tp53 mutant allele from an outbred rat to the genetically inbred Fischer 344 rat to create the F344-Tp53tm1(EGFP-Pac)Qly/Rrrc (F344-Tp53) strain. On the F344 genetic background, the tumor spectrum shifted, with the primary tumor types being osteosarcomas and meningeal sarcomas, compared to the hepatic hemangiosarcoma and lymphoma identified in the original outbred stock model. The Fischer model is more consistent with the early onset of bone and central nervous system sarcomas found in humans with germline Tp53 mutations. The frequency of osteosarcomas in F344-Tp53 homozygous and heterozygous animals was 57% and 36%, respectively. Tumors were highly representative of human disease radiographically and histologically, with tumors found primarily on long bones with frequent pulmonary metastases. Importantly, the rapid onset of osteosarcomas in this promising new model fills a current void in animal models that recapitulate human pediatric osteosarcomas and may facilitate studies to identify therapeutic targets.

The Wnt/β-catenin signaling pathway is deregulated in nearly all colorectal cancers (CRCs), predominantly through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC mutation is thought to allow a “just-right” amount of Wnt pathway activation by fine-tuning β-catenin levels. While at a much lower frequency, mutations that result in a β-catenin that is compromised for degradation occur in a subset of human CRCs. Here, we investigate whether one such “stabilized” β-catenin responds to regulatory stimuli, thus allowing β-catenin levels conducive for tumor formation. We utilize cells harboring a single mutant allele encoding Ser45-deleted β-catenin (β-catΔS45) to test the effects of Wnt3a treatment or APC-depletion on β-catΔS45 regulation and activity. We find that APC and β-catΔS45 retain interaction with Wnt receptors. Unexpectedly, β-catΔS45 accumulates and activates TOPflash reporter upon Wnt treatment or APC-depletion, but only accumulates in the nucleus upon APC loss. Finally, we find that β-catenin phosphorylation at GSK-3β sites and proteasomal degradation continue to occur in the absence of Ser45. Our results expand the current understanding of Wnt/β-catenin signaling and provide an example of a β-catenin mutation that maintains some ability to respond to Wnt, a possible key to establishing β-catenin activity that is “just-right” for tumorigenesis.

Wnt signaling is essential for intestinal homeostasis and is aberrantly activated in the majority of colorectal cancers (CRC). In over 80% of CRC, the tumor suppressor Adenomatous Polyposis Coli (APC) is mutated, resulting in expression of a truncated protein product. APC is a key component of the β‐catenin destruction complex, which maintains low cellular levels of β‐catenin but is inhibited following Wnt ligand presentation. The precise mechanism underlying β‐catenin destruction complex inhibition is not clear, nor is the exact role of APC in the complex. APC is primarily considered a core component of the destruction complex but has been shown to have other roles involving β‐catenin nuclear import/export and cytoskeletal functions. Here, we use Wnt3a beads to study the response of endogenous Wnt components to a local Wnt cue. Using three CRC cell lines, each with a different Wnt pathway status, we demonstrate that localized Wnt redistributes pathway components toward the Wnt source in the presence of full‐length, but not truncated APC. Further, use of the Wnt3a‐beads to perform protein pull‐down demonstrates that APC and β‐catenin both associate with the Wnt‐beads. APC depletion in nontransformed human colon epithelial cells diminishes this Wnt‐induced redistribution. Our results suggest revision of the current model as follows. In response to Wnt, the β‐catenin destruction complex: 1) maintains composition and binding to β‐catenin, 2) translocates to the plasma membrane, and 3) requires full‐length APC for this membrane trafficking. Currently, work is being performed to uncover mechanistic insights into the role of APC in destruction complex reorientation to a Wnt3a signal. Support or Funding Information: This study was supported by the National Science Foundation [grant number IOS‐1456538] and by the National Institutes of Health [P30CA168524].