Mechanistic Study Of ADAM12S Promoting The Proliferation And Metastasis Of Small Cell Lung Cancer Cells

A Disintegrin And Metalloproteinase 12(ADAM 12)is a member of the disintegrin protease family and is characterized by its proteolytic function.ADAM 12 has two splicing variants:a long transmembrane isoform ADAM12L and a short secreted variant ADAM12S.ADAM12 has been reported highly expressed not only in tumor tissues but also in serum and urine of small cell lung cancer(SCLC)patients and is negatively correlated to the overall patient survival rate.ADAM12S can promote the proliferation,migration,and invasion of SCLC cells.However,the underlying molecular mechanism is still elusive.Here we plan to identify AD AM12S regulated proteins and potential hydrolytic substrates of ADAM12S in SCLC cells,and explore the molecular mechanism by which ADAM12S promotes the proliferation and metastasis of SCLC cells.Part ?Quantitative proteomics and biochemical methods revealed a new mechanism by which ADAM12S promoted the proliferation and metastasis of SCLC cellsAims:To systematically identify the ADAM12S regulated proteins in SCLC cells,and explore the key proteins that affect the proliferation and metastasis of SCLC cells.Methods:qRT-PCR to detect the mRNA level of ADAM12S in SCLC cell lines.ADAM12S expression lentiviral plasmid was constructed based on the pLVX-IRES-ZsGreenl vector,and was used to pack lentivirus in HEK293T cells.H1688 cells were infected by ADAM12S/ZsGreen and ZsGreen expressing lentiviral particles and the GFP positive cells were sorted out by flow cytometer.CCK-8 growth assay,wound healing assay,transwell migration and Matrigel invasion assays were performed to study the effect of ADAM12S on the proliferation,migration,and invasion of H1688 cells.Quantitative proteomics based on stable isotope labeling by amino acids in cell culture(SILAC)technique was used to comprehensively screen the altered proteins in H1688 cells upon ADAM12S expression.Protein samples were mixed as the ratio of 1:1,separated by SDS-PAGE,and visualized by silver stain.After trypsin digestion,the resulting peptides were desalted and analyzed by Orbitrap mass spectrometer and MaxQuant software suite was used to search the human protein database to identify and quantify proteins in two samples.DAVID bioinformatics resources were used to reveal the biological functions and intracellular signaling pathways that the ADAM12-regulated proteins are participated in.Some identified proteins were validated by Western blotting(WB).qRT-PCR was performed to examine the Hexokinase I(HK1)mRNA level upon ADAM12S expression or depletion.c-Myc,Akt/ERK,and phosphorylated Akt/ERK were detected and quantified by WB.siRNA knockdown of ADAM12S results in the decrease of HK1 mRNA and protein level in H446 cells.HK1 knockdown was used to explore the effect of ADAM12S on the proliferation,migration,and invasion of SCLC cells.The stable HK1 knockdown H1688 cells were constructed by using shRNA lentivirus and were used to perform the soft agar colony formation assay.Glucose consumption and lactate production assays were performed in H1688 cell lines with ADAM12S expression with or without HK1 knockdown.Results:We identified 70 AD AM12S regulated proteins through triplicate quantitative proteomic analyses of samples from control and AD AM12S expressing cells.Bioinformatic analyses of ADAM12S regulated proteins showed that many proteins were involved in the tricarboxylic acid cycle,gluconeogenesis,malate metabolic process,and glucose metabolic process.WB results of caveolin-1(CAV1),cysteine and glycine-rich protein 1(CSRP1),aspartate aminotransferase(GOT1),annexin A6(ANXA6),glucose-6-phosphate 1-dehydrogenase(G6PD)and HK1 were consistent with mass spectrometry(MS)quantification.The protein and mRNA level of HK1 but not HK2 are increased in the ADAM12S expressing H1688 cells,whereas the protein level of HK1 is decreased in ADAM12S knockdown H446 cells.We observed significantly increased levels of glucose consumption and lactate production upon ADAM12S expression.When HK1 was knocked down,the increased consumption and lactate production were blocked.Meanwhile,HK1 knockdown completely or partially eliminates the enhanced proliferation,migration,invasion,and colony formation that are mediated by AD AM1 2S.In addition,we found that AD AM12S can enhance the protein and mRNA level of c-Myc as well as the phosphorylated Akt(Ser473).Conclusion:We used SILAC-based quantitative proteomics approach to identify the ADAM12S regulated proteins in SCLC cells.Seventy AD AM12S regulated proteins were obtained from MS analyses of three biological replicates.WB results confirmed that ADAM12S upregulated CAV1,CSRP1,GOT1,ANXA6,G6PD,and HK1.ADAM12S promoted the proliferation,migration,invasion,and colony formation of SCLC cells via upregulating the HK1,and as well as the activated HK1-dependent aerobic glycolysis.ADAM12S may upregulate HK1 through PI3K/Akt/c-Myc axis and then affect the proliferation and metastasis of SCLC cells.Part ? Identification of ADAM12S regulated glycoproteins in the secretome of SCLC cells using SPECS and label-free quantitative proteomicsAims:To systematically identify the ADAM12S regulated glycoproteins in the secretome of SCLC cells,and explore the potential hydrolytic substrates of ADAM12S that modulate the proliferation and metastasis of SCLC cells.Methods:SPECS(secretome protein enrichment with click sugars)was used to enrich the glycoproteins in the culture medium of H1688 cells expressing ZsGreen or ADAM12S and ZsGreen.Ac4ManNAz was added to the culture medium and azide was covalently labeled to glycoproteins,which were released into the extracellular secretome by secretion or proteolytic cleavage.The culture medium was collected and ultrafiltrated to remove Ac4ManNAz.The click chemistry reaction was performed to label biotin to the azido sugars by using Sulfo-DBCO-Biotin and the extra Sulfo-DBCO-Biotin was removed by ultrafiltration.Glycoproteins were purified with NeutrAvidin agarose resin.In these experiments,we first optimized the SPECS experimental procedures,and then used this method to enrich the glycosylated proteins.SDS-PAGE and silver stain were performed to separate and visualize proteins.After in-gel trypsin digestion,the tryptic peptides were analyzed by Orbitrap mass spectrometer and label-free quantification was performed by Proteome Discoverer software to identify the ADAM12S regulated glycoproteins.Results:Using the optimized SPECS method,we identified 67 secreted proteins in three biological replicates.Among them,17 proteins were predicted as the potential substrates of ADAM12S,including fibulin-2(FBLN2),growth differentiation factor 6(GDF6),vitamin K-dependent protein C(PROC),secreted phosphoprotein-1(SPP1),and melanoma cell adhesion molecule(MCAM or CD 146).We analyzed two most significantly regulated proteins,SPP1 and CD 146,whose cleavages may play important roles in the proliferation and migration of SCLC cells.