HER family receptor activation and dimerisation in colorectal cancer and cancer-derived exosomes

• Main Author: Gao, Fangfei
• Format: Dissertation
• Language: English
• Published: UCL (University College London) 2021

The EGFR pathway is upregulated in several human cancers including colorectal and head and neck cancers. The anti-EGFR antibodies cetuximab and panitumumab are currently widely used in the management of metastatic colorectal cancer with limited duration of response and almost inevitable development of resistance. HER receptors are able to form alternative dimers and can therefore compensate the loss of function of one receptor during targeted therapies. Usage of pan-HER inhibitor such as AZD8931 has been trialed and may represent one way of overcoming resistance to anti-EGFR therapy. However the effects of anti-HER therapy in patients cannot be easily measured without tissue biopsy. Analysis of exosomes offers a potential platform for the longitudinal monitoring of HER signalling in the form of a liquid biopsy by examining their protein and miRNA contents. Foster Resonance Energy Transfer (FRET) assay using fluorescence lifetime imaging microscopy (FLIM) was used for quantitative analysis of HER dimerisation in colorectal cancer cells. Treatment with cetuximab resulted in increase in HER3 phosphorylation and HER2-HER3 heterodimerisation in the sensitive cell line LIM1215, but not in resistant line DLD1 (KRAS WT). Treatment with AZD8931 resulted in reduced phosphorylation in HER1, HER2 and HER3 in both cell lines, and similar HER dimerisation changes as with treatment with cetuximab. HER3 activation and HER2-HER3 dimer rewiring upon anti-HER therapy could contribute to treatment resistance in colorectal cancer cells. Cell-derived and circulating exosomes were isolated using differential ultracentrifugation, and characterised using Nanosight and immunostaining. Reverse phase protein array provided an alternative high-throughput platform to dot blot for exosomal protein analysis but requires further optimisation. Exosomal HER protein quantity reflected HER expression in vitro. In a cohort of patients with advanced colorectal cancer patients undergoing first line systemic therapy, circulating exosomal HER3 changes predicted treatment failure prior to radiological progression. In another cohort of patients receiving first line AZD8931, early changes in exosomal HER2-HER3 dimer may be predictive of response to anti-HER therapy. Furthermore, levels of exosomal miR-21 could be used for monitoring of treatment response in patients with colorectal cancer.