Mechanisms Underlying The Synergistic Effect Between IRAK1 Kinase Inhibition And Cetuximab In The Treatment Colorectal Cancer

?Background?Clinical reports with the use of monoclonal anti-bodies directed against the ligand-binding site of the epidermal growth factor receptor(EGFR)have shown promising results in the treatment of colorectal cancer.However,due to the existence of primary and development of secondary drug resistance,the efficacy of these agents is usually endurable.It is now widely accepted that KRAS mutation contributes to cetuximab resistance in colorectal cancer,but mechanisms that underlie the resistance to cetuximab in KRAS wild type colorectal cancer is not well characterized.We have previously screened the effect of knocking down a penal of intracellular protein kinases on the response of colorectal cancer cells to cetuximab based on a bioinformatics datasheet and a synergistic effect between IRAK1 inhibition and cetuximab was observed.Considering that IRAK1 downstream of to TLR4 could activate PI3K/Akt and NF-KB et al,which has been reported to contribute to cetuximab resistance in colorectal cancer,we hypothesize that cetuximab administration activates IRAK1,which in turns activates PI3 K and NF-?B,resulting in cetuximab resistance.The current proposal was designed to verify the hypothesis.This study will further our understanding of the mechanisms involved in cetuximab resistance,help with the development of new targeting agents and regimen.?Objectives? To evaluate the relationship between cetuximab resistance and IRAK1 in colorectal cancer cells;to explore the mechanisms of synergistic effect of IRAK-Inh and cetuximab.?Methods? 1.Examining the expression of IRAK1 and p-IRAK1 by q RT-PCR or Western blot in normal colon epidermal cells and three KRAS wild colorectal cancer cells.WST-1 and Colonies form experiments were used to observe the proliferation of the three kinds of cancer cells.We used A nexxin V method to survey the change of apoptosis after treating by IRAK-Inh.2.Some related molecules were examined by Western blot in colon cancer cells with high-expressed IRAK1 and which were treated with IRAK-Inh or cetuximab or both of them.3.Immunohistochemistry were used to compare the differences of the expression of IRAK1 between tumor tissue and the adjacent normal tissue.And it was also used to illustrate the relationship between IRAK1 and the prognosis of the patients.4.Colon cancer cells were seeded into the nude mice to explore the anti-tumor effects of IRAK-Inh and cetuximab.?Results? 1.IRAK1 is highly expressed in colon cancer cells and the expression of which is associated with the proliferation and apoptosis of these cells.We used q RT-PCR and Western blot methods to examine the expression level of IRAK1 in m RNA and protein levels.We found that IRAK1 is higher in HT-29 and RKO than in the normal epithelial cells and Di Fi cells.The results of drug sensitive tests show that Di Fi is more sensitive to cetuximab than HT-29 and RKO.All of these results remind us of the relationship between IRAK1 and cetuximab.When we treated the drug resistance cell lines with IRAK-Inh and cetuximab,the growth rate of these cells were inhibited apparently.The apoptosis of HT-29 and RKO were increased obviously when they were added IRAK-Inh,at the same time the G0 phase were prolonged.2.TRAF6 is the substrate of IRAK1,and IRAK1 can affect the proliferation of colon cancer cells by regulating the activation of ERK and NF-?B.IRAK1 elevated with p-ERK and p-p65 after treating with cetuximab.On the contrary,p-IRAK1 and p-ERK and p-p65 were reduced by si IRAK1 and IRAK-Inh.The cell growth rate were up-regulated by transfected with sh-IRAK1,which is over expressed IRAK1,and p-ERK and p-p65 were added.TLR4 could be activated by cetuximab,and then IRAK1 were phosphorated,as the results of them the anti-tumor effects of cetuximab were decreased.3.The expression level of IRAK1 is closely related to the prognosis of colon cancer patients,and it is higher in tumor tissue than normal tissue.We bought a tissue array from Shanghai Xinchao,which contains the 5 years prognosis information of 90 patients.We found that expression level of IRAK1 is higher in tumor tissue than adjacent normal tissue by Immunohistochemistry and it is negative associated with the prognosis.4.IRAK-Inh could enhance the anti-tumor effect of cetuximab in nude mice.The mice were divided into 4 groups,DMSO,IRAK-Inh,cetuximab,and IRAK-Inh combined with cetuximab,when their tumor growth up to a certain size.Two weeks later those tumor were separated,and then we recognized that the tumor size were diminished obviously by using IRAK-Inh and cetuximab together.The change of some IRAK1 associated molecule is identical with our vitro experiments in RNA and protein of these tumors.?Conclusion? 1.The expression level of IRAK1 is negatively associated with the growth of colon cancer cells.2.TRAF6 is the substrate of IRAK1,and IRAK-Inh exerts the anti-tumor effect by inhibiting the activation of ERK and NF-?B.3.Cetuximab could suppress the activation of EGFR,and at the same time activate TLR4 and IRAK1,as the results of which the therapeutic effect of cetuxiamb were decreased.