The Molecular Mechanisms Of EGFR-targeted Therapeutics And The Application Of Targeted Probe In Cancer Cell Classification

Epidermal growth factor(EGFR)is an important signal transduction molecule on the cell surface,which is related to cell growth,proliferation and differentiation.There are four members of the family,named EGFR,HER2,HER3 and HER4.These receptors bind to a variety of growth factors,and are activated through dimerization of intracellular kinases.Then,they transmit signals to downstream signaling molecules.The structural basis of EGFR activation had been extensively studied.It is known that the kinase dimerization,especially the specific,asymmetric dimerization of the kinase,is the key to the activation of the kinase.Overexpression and abnormal activity of this family of proteins can lead to cancer.The development of many small molecular kinase inhibitors and monoclonal antibodies had been applied in clinics,and many patients suffering from cancers driven by EGFR oncogenic mutation could benefit from these therapeutics.However,the underlying molecular mechanisms of how these therapeutics regulate EGFR functions is not completely clarified.In the study of this thesis,we used protein complementary assay to study the effects of these therapeutics on the hetero-dimerization of EGFR and HER2.We found that Lapatinib promotes EGFR/HER2 dimerization and is EGF-independent;Gefitinib and Herceptin can also promote EGFR/HER2 dimerization,but it relies on the participation of EGF.The mechanism of its dimerization varies.Using an EGFR-targeted fluorescent probe PDCy3,which was designed and characterized by our former lab members,we labeled the cells collected from the urine of prostate cancer patients and classified these cells by flow cytometry.We found that the probe was not sensitive to EGFR overexpressing.But in one sample with the highest sensitivity to the probe,the EGFR expression level was low but there were three missense mutations found in the EGFR kinase domain.Among these mutations,V745 M is an activating mutation,and two others are inactivating mutations.The cancer of this patient had progressed to the castration-resistant metastatic stage.Our experiments indicated that the PDCy3 probe is more sensitive to the activaing mutation of EGFR kinase.It might have potential in the application of tumor cell classification.