title: Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells.
abstract: Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.
authors: Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
authors: Putty-Reddy
authors: PR
authors: Sudhir
authors: Chein-Hung
authors: CH
authors: Chen
authors: Madireddy
authors: M
authors: Pavana Kumari
authors: Mei-Jung
authors: MJ
authors: Wang
authors: Chih-Chiang
authors: CC
authors: Tsou
authors: Ting-Yi
authors: TY
authors: Sung
authors: Jeou-Yuan
authors: JY
authors: Chen
authors: Chung-Hsuan
authors: CH
authors: Chen
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D014408:Biomarkers, Tumor; D001980:Bronchi / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D050556:Fatty Acid-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D039961:Semaphorins / Q000235:genetics / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; C493412:FABP5 protein, human; D050556:Fatty Acid-Binding Proteins; D006023:Glycoproteins; C117307:KRAS protein, human; C114487:PDCD4 protein, human; D020543:Proteome; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D039961:Semaphorins; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; C558170:semaphorin 4B, mouse; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins