| Objective:Colorectal cancer(CRC)is one of the most common malignancies in the world.With the development of economic level,the incidence and mortality of colorectal cancer in Asian countries are gradually increasing.For patients with metastatic CRC(mCRC),chemotherapy was an important modality for combination therapy prior to 2004.With the FDA approval of Cetuximab in 2004 for the treatment of advanced metastatic colorectal cancer,targeted therapy has become an important means to improve the prognosis of advanced patients.Targeted therapeutic drugs work by blocking key factors in tumor proliferation pathways.Epidermal growth factor receptor(EGFR)is a transmembrane tyrosine kinase receptor belonging to the human epidermal growth factor receptor(EGFR)family.When it has been triggeried,it can lead to the proliferation of tumor cells,including inhibit apoptosis,promote the occurrence of invasion and metastasis.EGFR is an important therapeutic target for human tumors,including mCRC.Cetuximab,an EGFR monoclonal antibody,which is a mouse chimeric EGFR IgG1 monoclonal antibody that competitively binds to EGFR and exerts anti-tumor effects through a variety of pathways(such as blocking EGFR downstream signaling pathway,blocking cell cycle,down-regulating VEGF,and inducing cell death through ADCC effect). However,the efficacy of cetuximab monotherapy is limited,with a response rate of only 20% in mCRC.Primary and secondary drug resistance is the bottleneck in the clinical treatment of cetuximab.In colorectal cancer,BRAF mutation is a clear indicator of poor prognosis.It has been reported that BRAF gene mutation will lead to continuous activation of the downstream MAPK pathway and cannot be blocked by EGFR monoclonal antibody.Subgroup analyses of multiple clinical studies have shown that patients with BRAF mutations did not benefit from single anti-EGFR therapy.While vemurafenib,a small-molecule inhibitor targeting mutant BRAF,has the response rate of over 80% in patients with malignant melanoma,but the response rate in colorectal cancer patients with BRAF mutation was only 5%.Thus,the BRAF mutant colorectal cancer did not benefit from single-targeted therapy.Can benefit be made in strengthening the MAPK pathway through combined targeted therapy? In 2017 ASCO reported two studies on anti-EGFR combined with BRAF inhibitors in the treatment of BRAF mutant colorectal cancer,and all patients benefited from the combined targeted therapy.The complete mechanism of the combination of the two drugs to enhance the therapeutic sensitivity is still unclear.Therefore,in-depth study on the drug resistance and sensitization mechanism of colorectal cancer is of great significance to promote the precision treatment of colorectal cancer and to explore the best combined targeting model.At the same time,it is urgent to explore new high-efficiency and low-toxicity BRAF inhibitors in clinical practice.Although the latest data suggested that combined targeted therapies increases the sensitivity of cetuximab in BRAF mutant colorectal cancer,the total effective rate was still less than 20%.So there are still a large number of BRAF mutation patients who cannot be effectively treated.It is not clear whether BRAF mutations have other mechanisms other than the activation of the MAPK pathway leading to drug resistance.Therefore,it is necessary to explore the unknown mechanism of drug resistance.In recent years,immunotherapy has become a hot spot in the field of tumor therapy.Programmed cell death ligand 1(PD-L1)is a key immunomodulatory molecule that interacts with receptor pd-1 to inhibit the immune response of CD8+ cytotoxic T cells.At present,it is widely recognized that tumor cells can combine with pd-1 on lymphocyte surface through expression and secretion of PD-L1,promote apoptosis and depletion of activated T cells,and lead to tumor immune escape.In addition to acting through the PD-L1/T cell pd-1 axis,PD-L1 can also promote tumor proliferation in an immune-independent manner within tumor cells. Recently,it has been reported that Cetuximab is highly effective in patients with advanced head and neck squamous cell carcinoma with low expression of PD-L1.In addition,in head and neck squamous cell carcinoma,the blocking of pd-1-PD-L1 axis also had a synergistic anti-tumor effect on cetuximab combined with RT.These suggest that PD-L1 expression may be related to cetuximab resistance,but whether PD-L1 is related to the sensitivity of cetuximab to other tumors and the specific mechanism has not been clearly reported.This study will further explore the relationship between PD-L1 expression and the sensitivity of cetuximab in colorectal cancer,so as to maximize the role of cetuximab in the treatment of colorectal cancer.The purpose of this study was to investigate the in vivo and in vitro mechanism of enhancing the sensitivity of Cetuximab in colorectal cancer by combining BRAF inhibitors and blocking PD-L1.The mechanism of various proliferation and apoptosis pathways and immune checkpoint changes downstream of EGFR in this process was further discussed,and the results of this study will provide scientific basis for understanding the mechanism of drug resistance and clinical treatment of targeted colorectal cancer.Methods:1.Cell viability and proliferation were determined by MTT,EdU and colony formation assay.2.Cell kinase activity was determined by p-ERK PACE method.3.Detection of EGFR,p-EGFR,BRAF,ERK,p-ERK1/2,P38,p-p38,AKT,p-AKT,mTOR,p-mTOR,SRC,p-SRC,STAT3,p-STAT3,caspase-9,caspase-3,PARP,Actin,PD-L1,LEF-1,P70S6 K,p-P70S6 K,JNK,p-JNK,THRAP3 and GAPDH were expressed by Western blot method.4.In BALB/c thymic nude mice,a subcutaneous transplanted tumor mouse model of RKO/Caco-2 cells in colorectal cancer was established to study the effects of AZ304,Cetuximab and knockdown of PD-L1 to tumor proliferation in mice. 5.Immunohistochemical staining was used to detect the expressions of ki-67,p-ERK,p-EGFR and p-AKT in tumor tissue samples of nude mice.6.PD-L1,LEF-1 and THRAP3 siRNA were transfected instantaneously with Lipofectamine 2000 reagent to silence gene expression.7.QRT-PCR was used to verify the expressions of PD-L1 and LEF-1 mRNA.8.We constructed lentiviral expression vectors targeting silencing/overexpression of PD-L1 and BRAF genes,and established the stable transfected colorectal cancer cell lines.8.Immune co-deposition was used to detect the co-binding between proteins.9.Statistical processing: each experiment was repeated 3 times,and the data were expressed as mean standard deviation(s).SPSS 20.0 statistical software was used for t test,Spearman rank correlation test and Fisher’s precise probability calculation method.P< 0.05 was statistically significant.GraphPad Prism 6 software was used to calculate IC50,EC50 and GI50 values.Results:1.AZ304 is an effective dual inhibitor of BRAF/BRAF V600 E.In the cell-free assay,the IC50 of AZ304 on BRAF(WT),BRAF V600 E and CRAF was 79 nM,38 nM and 68 nM,respectively.AZ304 inhibited the phosphorylation of ERK in melanoma A375 cell line(BRAFV600E)and SK-MEL-31 cell line(BRAF WT)with EC50 of 65 nM and 60 nM,respectively.Western blot assay showed that AZ304 could induce a concentration dependent decrease of p-ERK in both BRAF mutant(BRAF MT)and BRAF wild(BRAF WT)tumor cell lines. In addition,AZ304 not only effectively inhibited the proliferation of BRAF mutant cells,but also had strong inhibitory activity against BRAF WT/RAS WT cell lines and BRAF WT/RAS MT cell lines in the cell proliferation inhibition test.These results suggest that AZ304 is a dual BRAF inhibitor with extensive and high efficacy.2.AZ304 effectively inhibited the proliferation activity of BRAF WT/MT colorectal cancer cell lines and down-regulated p-ERK,while Cetuximab could only inhibit the proliferation of BRAF WT colorectal cancer cell lines.MTT assay and EdU assay were used to detect the cell proliferation ability,and it was found that the proliferation activity of BRAF V600 E mutant colorectal cancer cell line(RKO,ht-29)and BRAF wild type colorectal cancer cell line(DiFi,caco-2)decreased with the increase of AZ304 concentration.Western blot assay was used to detect the changes of downstream ERK phosphorylation level after AZ304 acted on the two cell lines,and the results showed that AZ304 could effectively inhibit p-ERK in both BRAF MT/BRAF WT colorectal cancer cell lines.However,for the application of Cetuximab in BRAF MT/BRAF WT colorectal cancer cell lines at different concentrations,only BRAF WT cells showed a certain degree of proliferation inhibition,while BRAF MT cells were resistant to Cetuximab.These results suggest that AZ304 can effectively inhibit cell proliferation activity and significantly inhibit the phosphorylation of ERK in both BRAF mutant and BRAF wild-type colorectal cancer cell lines.3.AZ304 could increase the sensitivity of Cetuximab to colorectal cancer cell lines in vitro and in vivo.MTT assay and colony formation assay showed that the combination of both AZ304 and Cetuximab showed stronger inhibitory effect on cell proliferation in both BRAF MT and BRAF WT colorectal cancer cell lines.In addition,RKO(BRAF MT)and caco-2(BRAF WT)cell lines were used for subcutaneous tumorigenesis in nude mice,and the tumors were divided into four groups after reaching a certain volume: blank control,single AZ304,Cetuximab and the combination of the two drugs for treatment,respectively.The results suggested that both single drugs could partially inhibit the tumor proliferation of the two cell types in nude mice compared with the blank control group.The combined action of the two drugs inhibited the tumor of the two cell lines of nude mice more obviously,and caused the tumor regression of caco-2 nude mice to a certain extent.These results suggest that AZ304 can enhance the sensitivity of Cetuximab to both BRAF MT and BRAF WT colorectal cancer tumors in vitro and in vivo.4.AZ304 plays a key role in enhancing the sensitivity of Cetuximab to BRAF MT/BRAF WT colorectal cancer cell lines by inhibiting MAPK/ERK,PI3K/AKT/mTOR,and SRC/STAT3 proliferation pathways.We used Western blot to detect the key molecules of EGFR and its downstream signaling pathway.Compared with the control group,AZ304 single drug inhibited the MAPK/ERK,PI3K/AKT/mTOR,and SRC/STAT3 pathways in both BRAF MT(RKO,ht-29)and BRAF WT(DiFi,caco-2)cell lines to a certain extent.However,single drug Cetuximab only inhibited MAPK/ERK,PI3K/AKT/mTOR pathways in BRAF WT cell lines.Cetuximab had no inhibitory effect on the three downstream signaling pathways of BRAF MT cell lines. Compared with AZ304,the combination of the two drugs blocked the above three signaling pathways more completely.Notably,AZ304 single drug resulted in up-regulation of p-EGFR expression in all four cell lines,while combined with Cetuximab significantly reduced the phosphorylation level of EGFR.In addition,we further tested whether AZ304 could enhance the sensitivity of Cetuximab by inducing apoptosis.The caspase-9,caspase-3 and PARP cleavage bands were detected by Western blot assay.The results indicated that AZ304 alone could induce apoptosis to a certain extent,and the apoptosis induced by combined treatment was more significant.These results suggest that AZ304 enhances the sensitivity of Cetuximab in colorectal cancer by down-regulating three proliferation pathways and activating apoptosis pathways.5.The expression of PD-L1 was significantly up-regulated in BRAF mutant colorectal cancer cells,and PD-L1 resulted in decreased sensitivity of Cetuximab in colorectal cancer cell lines.We used flow cytometry,qRT-PCR and Western blot to detect the expression of pd-l1 on the cell surface and in general of 7 colorectal cancer cell lines in our laboratory under natural conditions,and found that the expression of pd-l1 in BRAF MT cells was significantly higher than that in other cells.MTT and colony formation experiments showed that silencing pd-l1 alone could inhibit the proliferation of BRAF MT cells to some extent,while the combined effect was more significant.Next,we used subcutaneous tumorigenesis in nude mice to verify the effect of pd-l1 expression changes on the activity of Cetuximab.Compared with the single group of pd-l1,the subcutaneous tumor proliferation and average tumor volume of the group of pd-l1 and Cetuximab were significantly reduced.These results suggest that inhibition of pd-l1 expression in BRAF MT colorectal cancer cells can increase the sensitivity of Cetuximab in colorectal cancer cells.6.BRAF V600 E mutation promotes the up-regulation of PD-L1 expression by up-regulating the transcription factor LEF-1.Illumina HiSeq sequencing was used to sequence the whole transcriptome of BRAF mutant RKO cell lines and wild-type DiFi cell lines.The differentially expressed mrnas were compared and analyzed to find mrnas that were upregulated by more than 3 times.The possible transcription factors of pd-l1 were predicted through the PROME official website,and the intersection with the sequencing results was selected to screen out the most significant transcription factor that may regulate pd-l1: lef-1.Further qrt-PCR and Western blot assays confirmed its up-regulated expression in BRAF mutant colorectal cancer cells.After "silencing" lef-1,the expression of pd-l1 was down-regulated by Western blot.These results suggest that lef-1 is abnormally expressed in BRAF mutant colorectal cancer cells and can be used as a transcription factor to promote the up-regulation of pd-l1 expression.7.PD-L1,through its binding with THRAP3,releases its inhibition of the PI3K/AKT pathway,resulting in decreased sensitivity of Cetuximab to colorectal cancer.We identified THRAP3 as the protein molecule most likely to bind to pd-l1 by immunocoip coupled mass spectrometry.THRAP3 binding to pd-l1 was also verified in cells.THRAP3 knockdown in BRAF mutant cells significantly increased the proliferation rate.Further transient knockout of pd-l1 and THRAP3 resulted in lower cell proliferation rate than that of THRAP3 silenced alone.Thus pd-l1 ACTS by binding to THRAP3 and inhibiting its antiproliferative activity.Western blot showed that the activity of PI3K/AKT pathway was inhibited after pd-l1 silencing,while the phosphorylation level of PI3K/AKT pathway was significantly up-regulated after THRAP3 silencing.Furthermore,the activation of PI3K/AKT by THRAP3 silencing was reversed by pd-l1 silencing.The above results suggest that pd-l1 promotes the proliferation of BRAF mutant colorectal cancer cells and leads to Cetuximab resistance by releasing the inhibitory effect of THRAP3 on the PI3K/AKT pathway.Conclusion:1.The BRAF inhibitor AZ304 has a wide range of anti-tumor activities,which can enhance the sensitivity of Cetuximab in both BRAF MT and BRAF WT colorectal cancer.2.AZ304 plays the role of sensitized Cetuximab through inhibiting MAPK/ERK,PI3K/AKT/mTOR,and SRC/STAT3 proliferation pathways.3.BRAF mutation promotes the transcriptional activation of PD-L1 through LEF-1,leading to the up-regulation of PD-L1 expression.4.PD-L1 actives the PI3K/AKT pathway through its binding with THRAP3,leading to the decreased sensitivity of Cetuximab to the effect of colorectal cancer. |
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