The APC tumor suppressor protein is associated with the regulation of Wnt signaling, however APC also controls other cellular processes including the regulation of cell adhesion and migration. The expression of full-length APC in SW480 colorectal cancer cells (SW480+APC) not only reduces Wnt signaling, but increases membrane E-cadherin and restores cell-cell adhesion. This report describes the effects of full-length, wild-type APC (fl-APC) on cell-cell adhesion genes and p120-catenin isoform switching in SW480 colon cancer cells: fl-APC increased the expression of genes implicated in cell-cell adhesion, whereas the expression of negative regulators of E-cadherin were decreased. Analysis of cell-cell adhesion-related proteins in SW480+APC cells revealed an increase in p120-catenin isoform 3A; similarly, depletion of APC altered the p120-catenin protein isoform profile. Expression of ESRP1 (epithelial splice regulatory protein 1) is increased in SW480+APC cells and its depletion results in reversion to the p120-catenin isoform 1A phenotype and reduced cell-cell adhesion. ESRP1 transcript is reduced in primary CRC and its expression correlates with the level of APC. Pyrvinium pamoate, which inhibits Wnt signaling, promotes ESRP1 expression. We conclude that re-expression of APC restores cell-cell adhesion gene and post-transcriptional regulatory programs leading to p120-catenin isoform switching and associated changes in cell-cell adhesion.

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

The majority of colon adenomas harbor genetic mutations in the APC gene. APC mutation leads to changes in Wnt signalling and cell-cell adhesion: as a consequence, intestinal crypt budding increases and the excess crypts accumulate to form adenomas that progress to colon cancer. When cultured with Wnt, R-spondin, EGF, Noggin, myofibroblast conditioned medium and Matrigel, crypts from normal mouse colon mucosa form crypt-producing organoids and can be passaged continuously. Under the same culture and passage conditions, crypts isolated from colon adenomas derived from Apcmin/+ mice typically grow as spheroidal cysts and do not produce crypts. The adenoma organoid growth requires EGF, but not Wnt, R-spondin or Noggin. However, when mouse colon adenoma spheroids are grown for more than 10 days in the presence of EGF, crypt formation occurs. EGF, EREG, β-cellulin, Neuregulin-1 or AREG are sufficient for initiating crypt formation, however, neuregulin-1 is more potent than the other EGF-family members. EGFR and ErbB2 inhibitors both prevent crypt formation in adenoma cultures. Either EGFR:ErbB2 or ErbB3:ErbB2 signalling is sufficient to initiate adenoma crypt budding and elongation. ErbB2 inhibitors may provide a therapeutic avenue for controlling and ablating colon adenomas.