Introduction

Cancer stem cells (CSCs) are a subset of cells within a tumor with infinite replicative potential that can differentiate to drive tumor initiation and promote relapse. Targeting CSC populations has emerged as a promising anti-cancer therapeutic strategy. Antibody-drug conjugates (ADCs), which utilize monoclonal antibody-mediated specificity to hone cytotoxic payloads to cancer cells, are one therapeutic modality being employed to target CSCs. Our lab has targeted leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), a well-defined biomarker of normal stem cells and CSCs that is overexpressed in colorectal cancers (CRC). Anti-LGR5 ADCs demonstrate in vitro and in vivo efficacy in gastrointestinal cancer models, however, tumor relapse following anti-LGR5 ADC treatment is a major obstacle. 

FDA-approved epidermal growth factor receptor- (EGFR)-targeted therapy has been shown to increase LGR5 mRNA expression levels in patient-derived models of CRC and treatment with the FDA-approved EGFR signaling inhibitor gefitinib increased the number of Lgr5+ cells in normal mouse intestine. The thrust of this work, then, is to evaluate combination therapies targeting both LGR5 and EGFR to eliminate CSCs. We hypothesize that EGFR-targeted therapies can increase LGR5 expression levels in CSCs to enhance the efficacy of anti-LGR5 ADCs and improve CRC treatment. 

Methods

KRAS^mut DLD-1, LoVo, LS180, and SW620 and KRAS^WT LIM1215 CRC cells were treated with a panel of FDA-approved EGFR and HER2 inhibitors including cetuximab, nimotuzumab, gefitinib, lapatinib, and trastuzumab and the MEK1/2 inhibitor trametinib. EGF was used to test the effect of EGFR activation on LGR5 expression. LGR5 protein levels were measured via Western Blot. EGFR-directed siRNA was used to determine the effect of EGFR knockdown (KD) on LGR5 expression. DLD-1 cells were treated with cetuximab for 48 hours and subjected to immunoprecipitation with EGFR antibody-conjugated sepharose beads. CRC cell lines were treated with anti-LGR5 ADC for 4 days with and without 2-day cetuximab pre-treatment and viability was measured using CellTiter-Glo assay to assess the effect of EGFR therapy pre-treatment on anti-LGR5 ADC potency.

Results

EGFR-targeted therapies upregulated LGR5 protein expression in CRC cells in a time-dependent manner irrespective of KRAS mutation status. LGR5 levels were unchanged in EGFR-negative/HER2-low SW620 cells after treatment with EGFR-targeted therapies. EGFR KD or treatment with EGF or trametinib substantially decreased LGR5 levels. Additionally, EGFR and LGR5 were shown to physically interact with one another, and that this interaction is enhanced via cetuximab treatment. Lastly, pretreatment of LoVo cells with cetuximab significantly enhanced anti-LGR5 ADC potency, decreasing IC₅₀ values 4-7 fold.

Conclusion

This preliminary data shows that EGFR-targeted therapy is able to upregulate LGR5 expression and enhance anti-LGR5 ADC potency in CRC cell lines. In vivo studies examining the effects of combination treatment on tumor growth and relapse will help to assess the overall efficacy of pretreatment with EGFR-targeted therapies versus monotherapy. Future studies to determine the mechanism of EGFR regulation of LGR5 expression are ongoing. Taken together, our results support the growing body of evidence that combining ADCs with other systemic therapies may be more effective in treating cancer.