A study of the gene expression patterns after modulation of LGR5 cellular levels by siRNA knockdown or transgenic LEE011 overexpression shows that loss of LGR5 upregulates wnt response genes and key EMT pathway genes; conversely, overexpression of LGR5 favours cell-cell adhesion. These results highlight the importance of LGR5, not simply as marker of colorectal tumour cells, but as a regulator of wnt responses, cell motility and cell-cell adhesion. If overexpression of LGR5 in colorectal cancer cells is mediated by hyper-activated wnt pathway, what role does LGR5 play in wnt responses, and does expression of LGR5 contribute to the maintenance of ����cancer stemness����? To address the functional relevance of LGR5 expression in CRC cell lines, we reduced its expression in cells carrying a b-catenin mutation using inhibitory RNAs. We initially utilized lentiviral transduction of shRNA to LGR5. As controls, we used shRNAs directed to random sequences or to Msi-1. Musashi-1 is expressed in immature intestinal cells and is overexpressed in colorectal tumours, but is not a wnt-response gene. We used four separate shRNA constructs for each target gene: all were effective, and subsequent experiments were conducted using the most efficient shRNAs. Transduced cells were bulk selected in puromycin for two weeks to enrich for the shRNA-expressing cells, then switched to antibioticfree media for functional characterization. Knockdown efficiency was monitored by qRT-PCR and cell proliferation was assayed using MTT assays and colony INCB28060 formation in soft agar. Lentiviral delivery of shRNA to LGR5 or to Musashi-1 was effective in both cell lines and lead to a marked and specific reduction in expression of the target genes. The expression levels of the related genes LGR6 and Msi-2 were unaffected. We confirmed loss of LGR5 protein after knockdown using immunofluorescence, as LGR5 antibodies are not suitable for the detection of endogenous levels of this protein by Western Blot. Knockdown of either LGR5 or Msi-1 levels did not affect the growth of cells as adherent monolayers, however loss of LGR5 and Msi-1 had striking and opposing effects on the clonogenicity of the cells in soft agar. Knockdown of Musashi-1 lead to a reduction in the colony forming ability of both LIM1215 and LIM1899 cells, consistent with the loss of proliferation and tumour forming ability of the colorectal cell line HCT116 after downregulation of Msi-1 as reported by Sureban et al. In contrast, loss of LGR5 caused a reproducible and profound increase in the clonogenicity of both LIM1215 and LIM1899. These effects on colony formation were observed consistently in both cell lines and using two separate, LGR5-specific shRNA constructs. Selection of the cells in puromycin might have led to changes in the expression of genes other than LGR5, contributing to this surprising result.