Adhesion G‐Protein‐Coupled Receptor L2 Expression is Lost in Colorectal Cancer but is Also Associated with Colonic Stem Cells

Abstract

Colorectal cancer (CRC) is driven by uncontrolled expansion and differentiation of crypt stem cells. We are testing the role of adhesion G‐protein‐coupled receptor L2 (ADGRL2), whose function has yet to be studied in the intestine, as a potential gatekeeper for proliferating colonic stem cells and renewal.MethodsTo assess receptor regulation during disease, mRNA levels were measured in matched normal and tumor specimens from human CRC. We corroborated findings by mining Gene Expression Omnibus (GEO) datasets and examined receptor modulation in response to growth arrest conditions (serum starvation and MAP kinase inhibition) in human CRC cell lines. We further interrogated ADGRL2 function using a stable inducible shRNA knockout line using SW480 cells and compared the gene expression of colonocyte markers in the induced ADGRL2 KO cells versus non‐induced cells. To assess possible inflammatory links, mice were given DSS colitis with 3% DSS in drinking water for 3 days and colons were collected on day 7 in the hyperproliferative recovery phase.ResultsADGRL2 was significantly and selectively reduced in tumors relative to adjacent matched normal tissue (5.14 fold, p < 0.0001). This was confirmed using two GEO datasets, one of which also revealed a pronounced loss in adenomas (3.82 fold, p < 0.0001). ADGRL2 was induced by growth arrest conditions in CRC cell lines, both by serum starvation (2.79 fold, p = 0.0115) and map kinase inhibition (5.59 fold, p = 0.0086). Knockdown in SW480 cells resulted in a loss of the colonic stem cell marker LGR5, while other markers for colonic cell types remained unchanged. Analysis of publicly available microarray data confirmed that ADGRL2 and LGR5 levels are associated in the colonic mucosa. Adgrl2 levels were suppressed in recovery from DSS colitis (1.74 fold, p < 0.0001), suggesting a role in restricting repair mechanisms.ConclusionsDownregulation during recovery from colonic injury, induction by antiproliferative stimuli, and association with LGR5 together suggest that ADGRL2 may have a role in orchestrating epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. The substantial loss of ADGRL2 in human CRC adenoma and adenocarcinoma raises the possibility that ADGRL2 functions as a tumor suppressor. Thus, while modulation of ADGRL2 levels may mediate epithelial repair following injury, chronic loss could give way to uncontrollable stem cell expansion and loss of crypt architecture, leading to dysplasia and tumorigenesis.Support or Funding InformationThis work was supported by: NIH R01DK095004 and a Senior Research Award from the Crohn’s and Colitis Foundation (MRF). Support for EB includes The Saban Research Institute Predoctoral Intramural Funding Grant and The Roy E. Thomas Foundation Graduate Scholarship. Support for CM includes an Undergraduate Summer Research Fellowship from the Crohn’s and Colitis Foundation.