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ADAM17 mediates OSCC development in an orthotopic murine model.

Simabuco FM, Kawahara R, Yokoo S, Granato DC, Miguel L, Agostini M, Aragão AZ, Domingues RR, Flores IL, Macedo CC, Della Coletta R, Graner E, Paes Leme AF - Mol. Cancer (2014)

Bottom Line: In this study the effect of overexpressing ADAM17 in cell migration, viability, adhesion and proliferation was comprehensively appraised in vitro.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas 13083-970, Brazil. adriana.paesleme@lnbio.cnpem.br.

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.

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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).
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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).

Mentions: SCC-9 cells overexpressing ADAM17-HA have been evaluated in viability, migration and adhesion assays. First, SCC-9 cells were seeded in 96-well plates and, after 7 days, cell viability was evaluated by MTT assay. SCC-9 cells overexpressing ADAM17-HA had increased viability compared with control (Figure 2A, n = 3, Student’s t-test, p = 0.0004).


ADAM17 mediates OSCC development in an orthotopic murine model.

Simabuco FM, Kawahara R, Yokoo S, Granato DC, Miguel L, Agostini M, Aragão AZ, Domingues RR, Flores IL, Macedo CC, Della Coletta R, Graner E, Paes Leme AF - Mol. Cancer (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928084&req=5

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).
Mentions: SCC-9 cells overexpressing ADAM17-HA have been evaluated in viability, migration and adhesion assays. First, SCC-9 cells were seeded in 96-well plates and, after 7 days, cell viability was evaluated by MTT assay. SCC-9 cells overexpressing ADAM17-HA had increased viability compared with control (Figure 2A, n = 3, Student’s t-test, p = 0.0004).

Bottom Line: In this study the effect of overexpressing ADAM17 in cell migration, viability, adhesion and proliferation was comprehensively appraised in vitro.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas 13083-970, Brazil. adriana.paesleme@lnbio.cnpem.br.

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.

Show MeSH
Related in: MedlinePlus