LGR5 expression is regulated by EGF in early colorectal adenomas and governs EGFR inhibitor sensitivity

Abstract

Background:LGR5 serves as a co-receptor for Wnt/β-catenin signalling and marks normal intestinal stem cells; however, its role in colorectal cancer (CRC) remains controversial. LGR5+ cells are known to exist outside the stem cell niche during CRC progression, and the requirement for epidermal growth factor (EGF) signalling within early adenomas remains to be fully elucidated.Methods:Epidermal growth factor and gefitinib treatments were performed in EGF-responsive LGR5+ early adenoma RG/C2 cells. 2D growth assays were measured using an IncuCyte. LGR5 or MEK1/2 silencing studies were executed using siRNA and LGR5 expression was assessed by qRT–PCR and immunoblotting. Ki67 level and cell cycle status were analysed by flow cytometry.Results:Epidermal growth factor suppresses expression of LGR5 at both the transcript and protein level in colorectal adenoma and carcinoma cells. Suppression of LGR5 reduces the survival of EGF-treated adenoma cells by increasing detached cell yield but also inducing a proliferative state, as evidenced by elevated Ki67 level and enhanced cell cycle progression. Repression of LGR5 further increases the sensitivity of adenoma cells to EGFR inhibition.Conclusions:LGR5 has an important role in the EGF-mediated survival and proliferation of early adenoma cells and could have clinical utility in predicting response of CRC patients to EGFR therapy.

Targeting LGR5 in Colorectal Cancer: therapeutic gold or too plastic?

“…Indeed, our most recent study demonstrated that LGR5 silencing significantly enhanced the sensitivity of human adenoma cells to the epidermal growth factor receptor (EGFR) inhibitor gefitinib. 51 In this study, LGR5 mRNA and protein expression was suppressed during EGF-mediated proliferation of adenoma cells. Taken together, such findings indicate that LGR5 expression may play a key role in mediating the survival and/or proliferative responses of tumours during malignant transformation.…”

“… 21 Interestingly, cetuximab in combination with LGR5 ablation exhibited the best synergy, in agreement with our recent study that demonstrated increased killing of human adenoma cells when LGR5 knockdown is combined with gefitinib (EGFR inhibitor). 51 However, in the Sato study, tumour regression appeared to be dependent on the prior ability of the agent to induce LGR5 mRNA expression (which oxaliplatin did not), and CRISPR/Cas9-based therapies currently remain some distance from the clinic.…”

“…LGR5 is a well-established target of Wnt/β-catenin signalling, 16 but evidence from our laboratory has also shown that the PGE2 and EGF signalling pathways can alter LGR5 expression, which impacts the proliferation/survival capacity of human colorectal adenomas. 50 , 51 Collectively, the overexpression of LGR5 in primary tissue, association with poor patient prognosis, and the pro-tumourigenic activity of LGR5 in in vitro functional studies would indicate a pro-oncogenic role for LGR5 in colorectal tumourigenesis.…”

HIF-2α upregulation mediated by hypoxia promotes NAFLD-HCC progression by activating lipid synthesis via the PI3K-AKT-mTOR pathway

“…To measure cell proliferation, a live real-time IncuCyte ZOOM was used as described [51, 52]. For the migration and invasion studies, HepG2 cells or steatotic HepG2 cells were suspended in 100 μl of serum-free medium and placed in a transwell chamber with or without Matrigel.…”

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells

“…Subsequently beads were washed five times prior to proteomic analyses or boiled for 95°C for 5 min following washes for immunoblotting. Immunoblotting was performed as previously described 13 using antibodies to total β-Catenin (as above), phosphorylated β-catenin (Ser33/37/Thr41), β-Catenin (Clone-5), E-Cadherin (Clone-36), GSK3β (Clone-7; BD), α-tubulin (DM1A), lamin A/C (4C11; Sigma), Axin1 (C76H11), Axin2 (76G6), TCF-4 (C48H11), LEF-1 (C12A5), Survivin (71G4B7), CyclinD1 (92G2; Cell Signaling Technology), active β-catenin (8E7, Merck-Millipore, Watford, UK) and c-MYC (9E10; Santa Cruz, Heidelberg, Germany). β-Catenin and LEF-1 densitometry were performed as described in the Online Supplementary Methods .…”