Dynamic Proteomic Changes And Therapeutic Targets In The Development Of Colorectal Cancer

Part 1: Construction of colorectal cancer protein database and proteomics of samples at different stagesPurpose:Collect specimens at different stages in the development of colorectal cancer(CRC)to construct a colorectal cancer protein database.Use PCT-DIA(Pressure Cycling Technology-Data-independent acquisition)technology to analyze the proteomics of the collected cohort samples.Methods:Collect fresh frozen tissue samples(FF)and formalin fixed and paraffin-embedded(FFPE)tissue samples,from surgical oncology department of the Second Affiliated Hospital of Zhejiang University School of Medicine.Samples includes precancerous lesions and other pathological stages colon.First prepare and obtain peptide from tissues,using mass spectrometry data dependent acquisition(DDA)quantitative technology to construct a colorectal cancer protein database,that is,a priori spectral library,and then design a cohort for each stage of the specimen to be tested.Dynamic randomized grouping was carried out based on gender,age,and pathological type,and quantitative analysis of proteomics was performed on the collected cohort samples using PCT-DIA technology.Results:For the construction of the colorectal cancer protein database,a total of 6 fresh tissue specimens were collected,including 1 case of normal epithelium at the resection margin of the colon,1 case of tubular adenocarcinoma,and 4 cases of mucinous adenocarcinoma.There were 9 cases of FFPE,including 2 cases of hyperplastic polyps,3 cases of adenoma,and 4 cases of mucinous adenocarcinoma.In the subsequent stages of CRC,a total of 85 related cases were collected,including 16 cases of colorectal cancer with distal normal margins(N),17 cases of hyperplastic polyps(P),22 cases of colorectal adenoma(A),and colorectal cancer.There were 30 cases of cancer(C),including 15 cases of tubular adenocarcinoma(C1)and 15 cases of mucinous adenocarcinoma(C2),totaling 85 cases each.2 sampling points were taken as biological replicates,and 27 cases were randomly selected for technical studies repeat.The DDA method was used to construct a library of 5 FF specimens and 9 FFPE specimens.The library contained 19,085 specific peptides,9,173 protein groups,and 6,229 specific proteins.The 85 corresponding samples were dynamically randomized and divided into 12 groups.Each group included 1 mouse liver(M)sample and a pool(P)sample containing some samples from the cohort.The quality control evaluation of the mass spectrometer instrument shows that the instrument is stable.Based on searching the self-built protein library mentioned above,the mass spectrometry data of the 85 cohort samples finally identified 4,830 specific proteins with biological repeatability r=0.813 and technical repeatability r=0.953.Conclusions:Constructed a high-quality CRC protein library belonging to our own laboratory.Using the PCT-DIA method can obtain protein data in FFPE samples on a large scale,with high biological reproducibility at the protein level.Part 2: Bioinformatics analysis of protein dynamic changes during the development of colorectal cancerAims:To perform corresponding bioinformatics analysis on the protein expression data of each stage of colorectal cancer obtained above.Analyze the characteristics and patterns of protein expression at each stage of CRC.Searching for key protein molecules in the progress of CRC.Methods:Use the Ingenuity Pathway Analysis(IPA)pathway analysis software produced by QIAGEN to perform core pathway and comparative analysis,use the R package "Mfuzz" for cluster typing,and Gene Ontology(GO)with the R package "cluster Profiler" for related pathway enrichment.Set analysis,other analysis software includes SPSS22.0,Graph Pad Prism,etc.Results:Each stage of CRC has a specific protein expression pattern.Comparing the difference proteins between the three pathological stages of P,A,C with the N stage,as the degree of malignancy increases,the number of different proteins also increases.There are also 99 protein differences between C1 and C2.Differential proteins increase rapidly in the pre-cancerous stage,but decline in the cancerous stage.Differential protein disease-related functions,from benign lesions to malignant development,increase in correlation with CRC,and CRC develops after moderately differentiated or even poorly differentiated adenocarcinoma,the protein molecules show more of liver cancer-related molecules.Comparing C1 and C2,it can be found that C2 has similar molecular characteristics to adenomas and has a higher degree of malignancy.In the analysis of specific metastatic factors,the changes in ECM(extracellular matrix)are the most concentrated,the adhesion of tumor cells is enhanced,and the angiogenesis in the cancer group is enhanced.Unsupervised Cluster analysis was performed on the dynamic changes of proteins at each stage,and six clusters of biological value were summarized,and it was found that ECM-related proteins were dominant in the rising pattern.It was found that Protocol-Lysine,2-Oxoglutarate 5-Dioxygenase 2(PLOD2),as a regulatory protein of ECM,increased with the progress of CRC,and has the possibility of being a potential target.Conclusion:Our analysis found that the joint changes of tumor and microenvironment promoted the progress of CRC.As the degree of malignancy increases,the protein expression pattern changes correspondingly.The secreted proteins(cytokines,etc.)gradually increase with the deterioration,affecting their own microenvironment,blood,and reaching various target organs(such as liver).The microenvironment in which cancer cells grow,PLOD2 is an important protein molecule in the progression of CRC,and has the potential to become a therapeutic target.Part 3: Clinical specimen verification of PLOD2 and exploration of its potential therapeutic valuePurpose:The aforementioned data analysis found that PLOD2 plays an important role in the progression of CRC.In this part,a variety of methods have been verified in new clinical specimens,combined with molecular experiments to explore its potential therapeutic value,and multi-omics analysis to explore the biological mechanism.Methods:Forty cases were randomly selected from the original cohort,and the mass spectrometry parallel reaction monitoring(PRM)technology was used for PLOD2 protein quantitative verification.Newly enrolled 4 cases were performed western blotting(WB)verification on colorectal cancer and cancer-negative resection margin tissues,and immunohistochemistry(IHC)was used to verify different stages of one familial adenomatous polyposis(FAP)patient.Tissue microarray(TMA)containing 79 resection margin tissues and 118 cancer tissues was used for immunohistochemical verification of the new cohort.CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 technology was applied to construct a PLOD2 knockout cell line model(PLOD2-KO)to explore the effects of PLOD2 on tumor cell proliferation,invasion,and metastasis.Mouse subcutaneous tumor models and patient-derived xenografts(PDX)were used to explore the effect of PLOD2 expression on tumor growth,and we test the potential treatment ability of the small molecule inhibitor of PLOD2,minoxidil.Finally,the transcriptome and proteome were subjected to dual-omics sequencing on the PLOD2-KO cell line to analyze the pathways of its influence.Results:In 40 samples from the original cohort,the quantitative results of PRM protein showed high consistency with the DIA data.Among the newly enrolled 4 pairs of tissues,WB verified that the expression of PLOD2 in tumor tissues was significantly higher than that in normal colon tissues.In tissue microarray,79 cases of resection margin tissues and 118 cases of cancer tissues were verified.In the resection margin tissues,68 cases(86.1%)had low expression of PLOD2,8 cases(10.1%)had expression,and only 3 cases had high expression(3.8%).In cancer tissues,54 cases(45.8%)had high expression of PLOD2,37 cases(31.4%)had high expression,and only 27 cases(22.9%)had low expression,and the expression level of PLOD2 in cancer tissues was related to the patient’s prognosis.In the FFPE tissue samples of FAP patients,it was found that intestinal epithelial tissues of different malignancies in the same patient,and the expression of PLOD2 increased with the increase of malignancy.CRISPR-Cas9 was successfully used to construct PLOD2 knock out(KO)models in HCT116 and HT-29 cell lines.Minoxidil was used as a PLOD2 inhibitor,and it was found that in the PLOD2-KO group and the inhibitor group,tumor cell clone formation was reduced,cell proliferation slowed down,and invasion and metastasis ability was weakened.Using the above cells to establish a mouse subcutaneous tumor model,it was found that the growth of subcutaneous tumors in the PLOD2-KO group and the inhibitor group was slower than that of the control group.In PDX models with different levels of PLOD2 expression,the tumor volume of PDX mice with high PLOD2 expression increased faster than that of PDX with low expression of PLOD2.The tumor growth of PDX mice with high PLOD2 expression was significantly slowed down after using minoxidil inhibitor,while There was no significant change in the tumor growth of PDX mice with low PLOD2 expression or not using minoxidil inhibitors.RNA-sequence and DIA-MS sequencing indicate that the expression of PLOD2 in colorectal cancer cell lines is closely related to cellular cholesterol metabolism and type I interferon secretion,suggesting that PLOD2 may change the related pathways.Conclusion:With the development of intestinal epithelial tissue from normal polyps and adenomas to malignant tumors,the expression of PLOD2 in the tissues gradually increases.PLOD2 can promote proliferation in CRC,and its specific mechanism may be related to cell metabolism and interferon secretion pathway.Its expression can be inhibited by minoxidil,and PLOD2 can be knocked out or inhibited to slow down tumor growth.