ADAM17 mediates OSCC development in an orthotopic murine model

Abstract

Background: ADAM17 is one of the main sheddases of the cells and it is responsible for the cleavage and the release of ectodomains of important signaling molecules, such as EGFR ligands. Despite the known crosstalk between ADAM17 and EGFR, which has been considered a promising targeted therapy in oral squamous cell carcinoma (OSCC), the role of ADAM17 in OSCC development is not clear.

Method: In this study the effect of overexpressing ADAM17 in cell migration, viability, adhesion and proliferation was comprehensively appraised in vitro. In addition, the tumor size, tumor proliferative activity, tumor collagenase activity and MS-based proteomics of tumor tissues have been evaluated by injecting tumorigenic squamous carcinoma cells (SCC-9) overexpressing ADAM17 in immunodeficient mice.

Results: The proteomic analysis has effectively identified a total of 2,194 proteins in control and tumor tissues. Among these, 110 proteins have been down-regulated and 90 have been up-regulated in tumor tissues. Biological network analysis has uncovered that overexpression of ADAM17 regulates Erk pathway in OSCC and further indicates proteins regulated by the overexpression of ADAM17 in the respective pathway. These results are also supported by the evidences of higher viability, migration, adhesion and proliferation in SCC-9 or A431 cells in vitro along with the increase of tumor size and proliferative activity and higher tissue collagenase activity as an outcome of ADAM17 overexpression.

Conclusion: These findings contribute to understand the role of ADAM17 in oral cancer development and as a potential therapeutic target in oral cancer. In addition, our study also provides the basis for the development of novel and refined OSCC-targeting approaches.

Figure 1

ADAM17-HA (AD17-HA) has mature form in SCC-9 cells and increases shedding of HB-EGF in HEK293 cells. A: SCC-9 cells were harvested, lysed with SDS sample buffer and immunoblotting was performed using anti-HA antibody. Anti-GAPDH antibody was used as loading control. B: SCC-9 cells were harvested and lysed in the presence and absence of BB-2516 and 1,10-phenanthroline. Immunoblotting was performed using anti-HA antibody and anti-GAPDH antibody was used as loading control. C: HEK293 cells overexpressing ADAM17-HA have increased shedding activity. HEK293 cells stably expressing a chimerical construct of HB-EGF fused with alkaline phosphatase (AP) were transfected with pcDNA-ADAM17-HA and released AP was quantified in cell supernatants after PMA (50 ng/ml) treatment. Two independent experiments with 3 replicates were performed (n = 2, Student’s t-test, DMSO: p = 0.0038, PMA: p = 0.0066). D: Immunoblotting of HEK293 cells overexpressing ADAM17-HA, performed using anti-HA antibody and anti-GAPDH antibody was used as loading control.

Figure 2

ADAM17 regulates cellular viability, migration, adhesion and proliferation. A: SCC-9 cells stably expressing ADAM17-HA or FLAG-GFP were seeded in 96-well plates. After 7 days cell viability was measured by MTT assay. Three independent experiments were performed (n = 3, Student’s t-test, p = 0.0004). B: SCC-9 cells stably expressing ADAM17-HA or FLAG-GFP were seeded in serum free media in the upper chamber of 96-well transwell plates. EGF at concentration of 100 ng/ml was added in serum free media in the lower chamber (n = 3, Student’s t-test, p = 0.0316). C: SCC-9 cells stably expressing ADAM17-HA or FLAG-GFP were seeded in Matrigel coated 96-well plates. After 1 h, cells were stained and adhesion measured (n = 3, Student’s t-test, p = 0.0001). D: ADAM-17 knockdown decreased adhesion of A431 cells. A431/untreated (mock), A431/control (scrambled) and A431/shRNA ADAM-17 cells were seeded in Matrigel coated 96-well plates. After 1 h, cells were stained and the cell adhesion was measured (n = 3, distinct letters represent significant differences at p < 0.0003, ANOVA followed by Tukey test). E: ADAM-17 knockdown decreased proliferation of A431 cells. Proliferation assay was performed in A431/control (scrambled) and A431/shRNA ADAM-17 cells by measuring BrdU incorporation into DNA in the presence of 2% or 10% FBS (n = 1, quintuplicate, distinct letters represent significant differences at p < 0.05, ANOVA followed by Tukey test).

Figure 3

SCC-9 cells overexpressing ADAM17-HA increased tumor size and proliferation. (A) SCC-9 cells overexpressing ADAM17-HA (AD17-HA) induce increased size of tumors in Nude mice. Cells grown in tissue culture plates were trypsinized, resuspended in PBS and injected in the tongue of Nude mice. After 20 days, mice were sacrificed, tumors excised and measured (n = 3, Student’s t-test, p = 0.0467). (B) SCC-9 cells overexpressing ADAM17-HA (AD17-HA) induce higher proliferative activity by immunohistochemical expression of Ki-67. Positive cells were calculated by counting labeled nuclei (positive-cells) of six high power fields (magnification of 400×) from each case with the aid of the Image J software and expressing the data as percentage (n = 6, Student’s t-test, p < 0.0001).

Figure 4

Bioinformatic analysis of ADAM17-regulated proteome. (A) Clustering of significantly up- and down-regulated proteins in tumor samples compared with control, Student’s t-test, p < 0.05, obtained in Perseus software. (B) Global interaction network by IPA consists of 56 (28%) of 200 differential expressed proteins, up-regulated proteins (red) and down-regulated proteins (green), plus additional interacting molecules that were not identified in this study (white). The two top biological networks generated by IPA were merged to obtain a global view. Major hubs in the network were highlighted in blue.

Figure 5

Tumors induced by injection of SCC-9 cells overexpressing ADAM17 (AD17-HA) have increased Erk phosphorylation. A: The immunoblotting indicates the expression of ADAM17-HA (anti-HA), total Erk (anti-Erk), phosphorylated Erk (anti-phospho Erk) and as loading control (anti-GAPDH). B: Phosphorylation levels were calculated by band intensity using ImageJ software (n = 2, Fisher’s exact test p = 0.0034). C: Knockdown of ADAM17 expression by shRNA in A431 cell line. Relative ADAM17 mRNA levels comparing shRNA and scrambled and mock control-treated cells were determined by quantitative RT-PCR. Each bar represents the mean ± SE of three independent experiments. D: shRNA-ADAM17 cells have shown a decreased in Erk phosphorylation. The immunoblotting indicates the expression of total Erk (anti-Erk), phosphorylated Erk (anti-phospho Erk) and as loading control (anti-GAPDH). E: Phosphorylation levels were calculated by band intensity using ImageJ software (n = 2, Fisher’s exact test p = 0.0001).

Figure 6

EGFR shows increased activation in SCC-9 cells overexpressing ADAM17 (AD17-HA). Immunoblotting showing increased EGFR phosphorylation in SCC-9 ADAM17-HA. Immunoblotting was performed using anti-EGFR, anti-phospho EGFR and anti-GAPDH antibodies. Phosphorylation levels were calculated by band intensity using ImageJ software and the GAPDH normalized intensity values are presented under the blots.

Figure 7

Tumors induced by injection of SCC-9 cells overexpressing ADAM17 (AD17-HA) have increased collagenase activity. C = tumors induced with SCC-9 overexpressing FLAG-GFP. ADAM17 = tumors induced with SCC-9 overexpressing ADAM17-HA. A: Collagenase activity is increased in SCC-9 overexpressing ADAM17-HA. B: Collagenase activity levels of ~100 kDa band in Figure A were calculated by band intensity using ImageJ software (n = 3, Student’s t-test p = 0.0285). C: shRNA-ADAM17 A431 cell conditioned media have reduced collagenase activity. D: Collagenase activity levels of ~100 kDa band in Figure C were calculated by band intensity using ImageJ software (n = 2, Student’s t-test p = 0.0370).