Overexpression of Mitochondrial IF1 Prevents Metastatic Disease of Colorectal Cancer by Enhancing Anoikis and Tumor Infiltration of NK Cells
Abstract
:1. Introduction
2. Results
2.1. Increased Expression of IF1 Is Associated with Increased Survival of CRC Patients
2.2. Transcriptomic Profiling of HCT116 Cells with Differential Expression of IF1
2.3. Proteomic Analysis of HCT116 Cells with Differential Expression of IF1
2.4. Phenotypic Analysis of HCT116 Cells with Differential Expression of IF1
2.5. In Vivo Tumorigenesis of HCT116 Cells with Differential Expression of IF1
2.6. IF1 Favors Anoikis of Colon Cancer Cells
2.7. IF1 Favors Infiltration and Cytotoxicity of NK Cells
3. Discussion
4. Materials and Methods
4.1. Patient Specimens and IF1 Quantification
4.2. Generation of Cell Lines, Cell Cultures and Spheroids
4.3. NK Cell Isolation and Labeling
4.4. Cellular Lysis and Western Blotting
4.5. Gene Array Hybridization
4.6. Gene Data Analysis
4.7. Quantification of mRNA
4.8. Proteomic Analysis and ITRAQ Labeling
4.9. Reverse Phase-Liquid Chromatography RP-LC-MS/MS and Data Analysis
4.10. Immunohistochemistry
4.11. Cellular Proliferation, Cell Death Assays and Cellular Respiration
4.12. Soft-Agar, Wound Healing, Invasion and Anoikis Assays
4.13. In Vivo Assays
4.14. Flow Cytometry Analyses of Spheroid Invasion
4.15. In-Vivo Confocal Microscopy of NK cell Invasion and Cytotoxicity
4.16. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Univariate Analysis | Overall Survival | Disease-Free Survival | ||
Variable | HR (95% CI) | p-Value | HR (95% CI) | p-Value |
Age (year) | ||||
≤70 | 1 (Reference) | 1 (Reference) | ||
>70 | 3.01 (0.97–9.27) | 0.055 | 3.67 (1.20–11.23) | 0.022 |
Gender | ||||
Female | 1 (Reference) | 1 (Reference) | ||
Male | 0.59 (0.22–1.53) | 0.280 | 0.58 (0.22–1.47) | 0.252 |
Grade | ||||
I | 1 (Reference) | 1 (Reference) | ||
II | 2.94 (0.38–22.62) | 0.300 | 3.56 (0.46–27.27) | 0.221 |
III | 7.094 (0.78–63.99) | 0.081 | 8.62 (0.95–78.21) | 0.055 |
Lymph nodes | ||||
No | 1 (Reference) | 1 (Reference) | ||
Yes | 4.62 (1.49–14.30) | 0.008 | 3.61 (1.28–10.23) | 0.015 |
CEA | ||||
≤5 µg/L | 1 (Reference) | 1 (Reference) | ||
>5 µg/L | 2.79 (1.06–7.36) | 0.038 | 2.64 (1.02–6.86) | 0.045 |
Metastasis | ||||
No | 1 (Reference) | 1 (Reference) | ||
Yes | 4.34 (1.57–11.94) | 0.004 | 3.87 (1.42–10.52) | 0.008 |
IF1 | ||||
Low | 1 (Reference) | 1 (Reference) | ||
High | 0.176 (0.06–0.49) | 0.001 | 0.19 (0.07–0.52) | 0.001 |
Multivariate Analysis | Overall Survival | Disease-Free Survival | ||
Variable | HR (95% CI) | p-value | HR (95% CI) | p-value |
Age (year) | ||||
≤70 | ------- | ------- | 1 (Reference) | |
>70 | ------- | ------- | 2.50 (0.76–8.21) | 0.131 |
Lymph nodes | ||||
No | 1 (Reference) | 1 (Reference) | ||
Yes | 3.18 (0.95–10.61) | 0.059 | 2.25 (0.71–7.13) | 0.165 |
CEA | ||||
≤5 µg/L | 1 (Reference) | 1 (Reference) | ||
>5 µg/L | 0.96 (0.17–5.20) | 0.960 | 1.31 (0.21–7.98) | 0.767 |
Metastasis | ||||
No | 1 (Reference) | 1 (Reference) | ||
Yes | 3.23 (0.55–18.77) | 0.191 | 1.94 (0.31–12.08) | 0.476 |
IF1 | ||||
Low | 1 (Reference) | 1 (Reference) | ||
High | 0.250 (0.08–0.76) | 0.015 | 0.32 (0.10–0.99) | 0.049 |
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González-Llorente, L.; Santacatterina, F.; García-Aguilar, A.; Nuevo-Tapioles, C.; González-García, S.; Tirpakova, Z.; Toribio, M.L.; Cuezva, J.M. Overexpression of Mitochondrial IF1 Prevents Metastatic Disease of Colorectal Cancer by Enhancing Anoikis and Tumor Infiltration of NK Cells. Cancers 2020, 12, 22. https://doi.org/10.3390/cancers12010022
González-Llorente L, Santacatterina F, García-Aguilar A, Nuevo-Tapioles C, González-García S, Tirpakova Z, Toribio ML, Cuezva JM. Overexpression of Mitochondrial IF1 Prevents Metastatic Disease of Colorectal Cancer by Enhancing Anoikis and Tumor Infiltration of NK Cells. Cancers. 2020; 12(1):22. https://doi.org/10.3390/cancers12010022
Chicago/Turabian StyleGonzález-Llorente, Lucía, Fulvio Santacatterina, Ana García-Aguilar, Cristina Nuevo-Tapioles, Sara González-García, Zuzana Tirpakova, María Luisa Toribio, and José M. Cuezva. 2020. "Overexpression of Mitochondrial IF1 Prevents Metastatic Disease of Colorectal Cancer by Enhancing Anoikis and Tumor Infiltration of NK Cells" Cancers 12, no. 1: 22. https://doi.org/10.3390/cancers12010022