FXR isoforms drive differentiation and differentially inhibit proliferation in colorectal cancer
The Farnesoid X Receptor (FXR) is a bile acid-activated transcription factor which is downregulated in colorectal cancer (CRC), and has been coined as a tumor suppressor in CRC based on mouse studies.  FXR is expressed as multiple isoforms of which a3 and a4 are predominantly expressed in the colon. These isoforms differ in a 12-bp region in the DNA binding domain, which is included in a3 and excluded in a4, due to alternative splicing. The 12bp exclusion results in additional alternative DNA binding and transcriptional profiles in the liver. To investigate the mechanistic and isoform-dependent underpinnings of the tumor suppressive action of FXR, we used human colon organoids in which mutations in four known CRC-driver genes; AKPS, KRAS, p53 and SMAD4 (AKPS), are engineered. We first confirmed that FXR expression is nearly absent already in early-stage CRC organoids, and show that this is due to constitutive Wnt signaling activation. Strikingly, lentiviral overexpression of FXRa4, not FXRa3, reduced proliferation in AKPS organoids in a ligand-independent manner. Transcriptomics indicated that FXRa4 selectively reduced EGFR signaling. Upon ligand addition, both FXRa3 and a4 AKPS organoids changed morphology and started budding, indicative of epithelial differentiation. Gene set enrichment analyses revealed that enterocyte, Paneth and goblet cell progenitor expression patterns were significantly enriched. In addition, electron microscopy showed the appearance of microvilli and increased nuclear circularity, indicative of differentiation towards columnar epithelium, in ligand-stimulated FXRa3 and FXRa4 AKPS organoids.  In conclusion, next to a ligand-dependent and isoform-independent effect on differentiation, we describe for the first time that FXR has isoform-dependent tumor-suppressive effects via inhibition of EGFR signaling. Overall design: To identify the effect of FXRa3 and FXRa4 overexpression and activation in an organoid model for colorectal cancer. FXRa3 and FXRa4 were overexpressed using lentiviral constructs and activated using obeticholic acid for 3 days. RNA was isolated and sequenced.