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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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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).
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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).

Mentions: Immunoblotting has showed that Erk phosphorylation was increased in tumors overexpressing ADAM17 (Figure 5A-B, n = 2, Fisher’s exact test, p = 0.0034). To further validate these data, we used A431 carcinoma cell line knockdown for ADAM17 gene (Figure 5C) to analyze Erk phosphorylation state and it confirmed lower phosphorylation levels in Erk (Figure 5D-E, n = 2, Fisher’s exact test, p = 0.0001).


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)

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

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3928084&req=5

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).
Mentions: Immunoblotting has showed that Erk phosphorylation was increased in tumors overexpressing ADAM17 (Figure 5A-B, n = 2, Fisher’s exact test, p = 0.0034). To further validate these data, we used A431 carcinoma cell line knockdown for ADAM17 gene (Figure 5C) to analyze Erk phosphorylation state and it confirmed lower phosphorylation levels in Erk (Figure 5D-E, n = 2, Fisher’s exact test, p = 0.0001).

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