Introduction
Material and methods
Cell lines
Generation of stably transfected cells
Generation of stably ADAM17 knockdown A431 cell line
Orthotopic murine model of oral squamous cell carcinoma
Tumors size measurement
Proliferative activity of tumors by immunohistochemical expression of Ki-67
Protein extraction from tumors and in solution trypsin digestion
LC-MS/MS analysis and data analysis
Biological network analysis
Immunoblotting
Real time quantitative PCR
Functional assays
Analysis of ADAM17 activity by HB-EGF shedding on AP reporter assay
Cell viability assay by MTT
Transwell migration assay
Cell adhesion assay
Bromodeoxyuridine-labeling (BrdU) index
Collagenase activity in tumors and conditioned media
Statistical analysis
Results
SCC-9 cells overexpressing ADAM17 have higher sheddase activity on HB-EGF
SCC-9 overexpressing ADAM17 shows higher cell viability, migration and adhesion
ADAM17 knockdown promoted lower adhesion and proliferation in A431 cells
Tumors overexpressing ADAM17 have increased size and showed higher proliferative activity
MS-based proteomics and biological network analysis indicate up-regulated proteins in the Erk pathway
Function | Function annotation | p-value | Molecules | # Molecules |
---|---|---|---|---|
Cancer | Cancer | 1.57E-06 | ACADVL, APEX1, ATP2A1, ATP2A2, CYCS, DHX9, DPYSL2, DSC3, DSG1, DSG3, DSP, FABP5, FGB, FGG, FLII, G6PD, GOT1, HNRNPA1, HNRNPK, HSPA8, LAMA3, LAMB3, LRPPRC, MCM3, PARVA, PCNA, PGK1, PKM, RAN, RPS4X, RPS9, SFN, SOD2, STIP1, STK24, STMN1, TPM1, TPM2, TUBB2A | 39 |
Solid tumor | Solid tumor | 4.61E-06 | APEX1, ATP2A1, ATP2A2, DHX9, DPYSL2, DSC3, DSG1, DSG3, DSP, FABP5, FGB, FGG, FLII, G6PD, HNRNPA1, HNRNPK, HSPA8, LAMA3, LAMB3, LRPPRC, MCM3, PARVA, PCNA, PGK1, PKM, RPS9, SFN, SOD2, STIP1, STK24, STMN1, TPM1, TPM2, TUBB2A | 34 |
Carcinoma | Carcinoma | 9.78E-06 | APEX1, ATP2A1, ATP2A2, DHX9, DPYSL2, DSC3, DSG1, DSG3, DSP, FABP5, FGB, FGG, FLII, G6PD, HNRNPA1, HNRNPK, HSPA8, LAMA3, LAMB3, LRPPRC, MCM3, PARVA, PCNA, PGK1, PKM, RPS9, SFN, SOD2, STIP1, STMN1, TPM1, TPM2, TUBB2A | 33 |
Proliferation | Proliferation of cells | 2.00E-07 | ACTN1, APEX1, ATP2A2, DECR1, DSG3, DSP, FABP5, G6PD, HADHB, HNRNPA1, HNRNPF, HNRNPK, HSPA8, LAMA3, MCM3, PCNA, PDCD10, PGK1, PKM, PKP1, PRPS2, RAN, RPS3A, RPS4X, RPS9, SFN, SOD2, STK24, STMN1, TPM1, TPM2, TUBB2A | 32 |
Cell death | Cell death | 6.36E-05 | APEX1, ATP2A1, ATP2A2, CYCS, DECR1, DHX9, DSG1, DSG3, DSP, G6PD, HNRNPA1, HNRNPK, HSPA8, LAMA3, MSN, PARVA, PCNA, PDCD10, PKM, RAN, RPS3A, RUVBL2, SFN, SOD2, STIP1, STK24, STMN1, TPM1 | 28 |
Apoptosis | Apoptosis | 6.07E-06 | APEX1, ATP2A1, CYCS, DECR1, DHX9, DSG1, DSG3, DSP, G6PD, HNRNPA1, HNRNPK, HSPA8, LAMA3, MSN, PARVA, PCNA, PDCD10, PKM, RPS3A, RUVBL2, SFN, SOD2, STIP1, STK24, STMN1, TPM1 | 26 |
Necrosis | Necrosis | 1.96E-04 | APEX1, ATP2A1, ATP2A2, CYCS, DECR1, DSG1, DSG3, DSP, G6PD, HNRNPA1, HNRNPK, HSPA8, LAMA3, MSN, PARVA, PCNA, PKM, RAN, RPS3A, SFN, SOD2, STMN1, TPM1 | 23 |
Adenocarcinoma | Adenocarcinoma | 7.94E-03 | ATP2A1, DHX9, DSC3, DSG1, DSG3, DSP, FGB, FLII, HNRNPA1, HNRNPK, HSPA8, LAMA3, LAMB3, LRPPRC, PCNA, PGK1, PKM, RPS9, SOD2, TUBB2A | 20 |
Carcinoma | Carcinoma in lung | 7.96E-03 | APEX1, ATP2A1, DHX9, DSC3, DSG1, DSG3, DSP, FGB, FGG, HNRNPA1, HNRNPK, HSPA8, LAMA3, LRPPRC, PCNA, SOD2, STMN1, TUBB2A | 18 |
Carcinoma | Carcinoma in lung | 7.96E-03 | APEX1, ATP2A1, DHX9, DSC3, DSG1, DSG3, DSP, FGB, FGG, HNRNPA1, HNRNPK, HSPA8, LAMA3, LRPPRC, PCNA, SOD2, STMN1, TUBB2A | 18 |