CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Generation of Knockout Cell Line with CRISPR/Cas9
2.3. siRNA Transfection
2.4. Toluidine Blue Staining
2.5. In Vivo Animal Studies
2.6. In Vivo Tumor Xenograft Formation
2.7. Tail Vein (Metastasis) Assay
2.8. Immunohistochemistry (IHC) Analysis
2.9. Immunofluorescence Staining
2.10. Western Blot Analysis
2.11. Co-Immunoprecipitation Experiments
2.12. TGF-β, EGFR and AKT Inhibition Studies
2.13. RNA Isolation and Quantitative Real-Time PCR
2.14. Generation of CD109 KO A431 Cells That Express EGFR (CD109KOEGFR)
2.15. Tumor Spheres Formation Assay
2.16. Statistical Analysis
3. Results
3.1. The Loss of CD09 Diminishes the Tumorigenicity and Abrogates the Metastatic Ability of SCC Cells In Vivo
3.2. CD109 Is Required for Maintaining the Stemness of SCC Cells, and the Loss of CD109 Diminishes the Cancer Stem Cell Population In Vitro in SCC Cells
3.3. CD109 Associates and Co-Localizes with EGFR, and Loss of CD109 Diminishes EGFR Expression in SCC Cells
3.4. The AKT Pathway, but Not the ERK1/2 and STAT3 Pathways, Is Inactivated in CD109 KO SCC Cells, While in Control SCC Cells, EGFR Kinase Inhibition Phenocopies AKT Inhibition in Inducing Cell Death and CD109KO-Like Mesenchymal State
3.5. GPI-Anchored CD109 Is Required for Maintaining EGFR Expression, Levels of Phospho-EGFR, and Phospho-AKT, and the Epithelial Phenotype in CD109KO SCC Cells
3.6. CD109 Is Required for EGF-Mediated Regulation of EGFR Levels, AKT Signaling, and to Maintain Stemness in SCC Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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human GAPDH: | forward primer | 5′-GACAACTTTGGTATCGTGGAAGG-3′ |
reverse primer | 5′-AGGGATGATGTTCTGGAGAGCC-3′ | |
human EGFR: | forward primer | 5′-AAACCGGACTGAAGGAGCTG-3′ |
reverse primer | 5′-CCCATTGGGACAGCTTGGAT-3′ | |
human CD109: | forward primer | 5′-CTGGAACACTGCCCTTCACA-3′ |
reverse primer | 5′-GTCCGGTTACACGTAGCTCA-3′ |
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Zhou, S.; Hassan, A.; Kungyal, T.; Tabariès, S.; Luna, J.L.R.G.; Siegel, P.M.; Philip, A. CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells. Cancers 2022, 14, 3672. https://doi.org/10.3390/cancers14153672
Zhou S, Hassan A, Kungyal T, Tabariès S, Luna JLRG, Siegel PM, Philip A. CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells. Cancers. 2022; 14(15):3672. https://doi.org/10.3390/cancers14153672
Chicago/Turabian StyleZhou, Shufeng, Amani Hassan, Tenzin Kungyal, Sebastien Tabariès, José Luis Ramírez García Luna, Peter M. Siegel, and Anie Philip. 2022. "CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells" Cancers 14, no. 15: 3672. https://doi.org/10.3390/cancers14153672