PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer
Abstract
1. Introduction
2. Results
2.1. Atezolizumab Blocks the Epitope of PD-L1 and Does Not Alter PD-L1 mRNA and Protein Expression
2.2. Atezolizumab Downregulates Genes Promoting Cell Migration/Metastasis and EMT
2.3. Atezolizumab Downregulates Anti-Apoptotic Genes, Upregulates Pro-Apoptotic Genes, and Downregulates Genes Involved in Cell Growth and Proliferation
2.4. Atezolizumab Upregulates DNA Repair Genes and Downregulates Genes Related to Hypoxia
2.5. Atezolizumab Downregulates NF-kB, Akt, and CD40 Signaling Pathways
2.6. Functional and Network Analyses Identified Key Genes Associated with the Response of MDA-MB-231 to Atezolizumab
2.7. Validation of RNA-Seq Data by RT-qPCR
2.8. Atezolizumab Increases Necrosis/Apoptosis and Reduces Proliferation and Viability in MDA-MB-231 Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Flow Cytometry
4.3. Western Blot
4.4. RNA Extraction and Reverse Transcription
4.5. Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction
4.6. RNA Library Preparation and RNA-Seq
4.7. RNA-Seq Analysis
4.8. Gene Set Enrichment Analyses and Modeling of Gene Interaction
4.9. Death/Apoptosis Assay
4.10. BrdU Cell Proliferation Assay
4.11. MTT Cell Viability Assay
4.12. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer | Sequence |
---|---|
PD-L1 | Forward, 5′- TGGCATTTGCTGAACGCATTT -3′ |
Reverse, 5′- TGCAGCCAGGTCTAATTGTTTT -3′ | |
NFKBIB | Forward, 5′- CGACACCTACCTCGCTCAG -3′ |
Reverse, 5′- GTCGGAATCGGGGTACAAGG -3′ | |
COMMD6 | Forward, 5′- GGAAACTGGGTATGGCTGTGA -3′ |
Reverse, 5′- TGTGGAATCGTCATTTCAAAGCA -3′ | |
BTG2 | Forward, 5′- ACGGGAAGGGAACCGACAT-3′ |
Reverse, 5′- CAGTGGTGTTTGTAGTGCTCTG -3′ | |
RNF122 | Forward, 5′- ATTCCAGTGGTGTAACGGGTG -3′ |
Reverse, 5′- CCTGTGCCGAAGATGACCATA -3′ | |
NBL1 | Forward, 5′- CATGTGGGAGATTGTGACGCT-3′ |
Reverse, 5′- CCTCGTGACTAGGCTCCTTG -3′ | |
ICAM3 | Forward, 5′- GGAGTTCCTTTTGCGGGTG -3′ |
Reverse, 5′- TCAGAGCTGGGACAATCAGTA -3′ | |
TRAF5 | Forward, 5′- CCACTCGGTGCTTCACAAC -3′ |
Reverse, 5′- GTACCGGCCCAGAATAACCT -3′ | |
SNAI1 | Forward, 5′-TCGGAAGCCTAACTACAGCGA -3′ |
Reverse, 5′- AGATGAGCATTGGCAGCGAG -3′ | |
VIM | Forward, 5′- GACGCCATCAACACCGAGTT-3′ |
Reverse, 5′- CTTTGTCGTTGGTTAGCTGGT-3′ | |
CDH1 | Forward, 5′- CGAGAGCTACACGTTCACGG -3′ |
Reverse, 5′- GGGTGTCGAGGGAAAAATAGG -3′ | |
β-ACTIN | Forward, 5′- AGAGCTACGAGCTGCCTGAC -3′ |
Reverse, 5′- AGCACTGTGTTGGCGTACAG -3′ |
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Saleh, R.; Taha, R.Z.; Sasidharan Nair, V.; Alajez, N.M.; Elkord, E. PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer. Cancers 2019, 11, 1050. https://doi.org/10.3390/cancers11081050
Saleh R, Taha RZ, Sasidharan Nair V, Alajez NM, Elkord E. PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer. Cancers. 2019; 11(8):1050. https://doi.org/10.3390/cancers11081050
Chicago/Turabian StyleSaleh, Reem, Rowaida Z. Taha, Varun Sasidharan Nair, Nehad M. Alajez, and Eyad Elkord. 2019. "PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer" Cancers 11, no. 8: 1050. https://doi.org/10.3390/cancers11081050
APA StyleSaleh, R., Taha, R. Z., Sasidharan Nair, V., Alajez, N. M., & Elkord, E. (2019). PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer. Cancers, 11(8), 1050. https://doi.org/10.3390/cancers11081050