PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients
Abstract
:1. Introduction
2. Materials and Methods
2.1. Osteosarcoma Specimens and Tissue Samples
2.2. Immunohistochemical Staining and Scoring in the Tissue Microarray
2.3. Cell Lines, Transfection, and Reagents
2.4. Cell Proliferation Assay
2.5. In Vitro Trans-Chamber Migration and Invasion Assays
2.6. Western Blotting, Ubiquitination Analysis, and Immunoprecipitation
2.7. Immunofluorescence Staining
2.8. Quantitative Real-Time PCR with Reverse-Transcription Analysis
2.9. Tumorigenic Assay
2.10. Statistical Analysis
3. Results
3.1. The Expression of PAK4 and PD-L1 Are Associated with Shorter Survival of Osteosarcoma Patients
3.2. PAK4 Expression Is Associated with the Activity of Proliferation and Invasiveness of Osteosarcoma Cells
3.3. PAK4 Is Involved in Resistance to Doxorubicin of Osteosarcoma Cells
3.4. PAK4 Is Involved in the Stabilization of PD-L1
3.5. PAK4 Expression Is Associated with the Infiltration of Immune Cells in Tumor-Bearing Mice
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence | Product Size | Accession Number | |
---|---|---|---|---|
PAK4 | forward | 5′-GGACATCAAGAGCGACTCGAT-3′ | 113 | NM_001014831.3 |
reverse | 5′-CGACCAGCGACTTCCTTCG-3′ | |||
PD-L1 | forward | 5′-GGACAAGCAGTGACCATCAAG-3′ | 235 | NM_001267706.2 |
reverse | 5′-CCCAGAATTACCAAGTGAGTCCT-3′ | |||
FOXO3 | forward | 5′-CGGACAAACGGCTCACTCT-3′ | 150 | NM_001455 |
reverse | 5′-GGACCCGCATGAATCGACTAT-3′ | |||
CCND1 (Cyclin D1) | forward | 5′-GAGGAAGAGGAGGAGGAGGA-3′ | 236 | NM_053056.2 |
reverse | 5′-GAGATGGAAGGGGGAAAGAG-3′ | |||
P27 | forward | 5′-TCTACTGCGTGGCTTGTCAG-3′ | 240 | AB001740.1 |
reverse | 5′-CTGTATTTGGAGGCACAGCA-3′ | |||
BAX | forward | 5′-CCCGAGAGGTCTTTTTCCGAG-3′ | 155 | NM_138763 |
reverse | 5′-CCAGCCCATGATGGTTCTGAT-3′ | |||
BCL2 | forward | 5′-GGTGGGGTCATGTGTGTGG-3′ | 89 | NM_000657 |
reverse | 5′-CGGTTCAGGTACTCAGTCATCC-3′ | |||
SNAL1 (Snail) | forward | 5′-ACCCCACATCCTTCTCACTG-3′ | 217 | NM_005985.3 |
reverse | 5′-TACAAAAACCCACGCAGACA-3′ | |||
TGF-β1 | forward | 5′-CCCACAACGAAATCTATGACAA-3′ | 246 | NM_000660.7 |
reverse | 5′-AAGATAACCACTCTGGCGAGTG-3′ | |||
MMP2 | forward | 5′-GATACCCCTTTGACGGTAAGGA-3′ | 112 | NM_004530 |
reverse | 5′-CCTTCTCCCAAGGTCCATAGC-3′ | |||
MMP9 | forward | 5′-TGTACCGCTATGGTTACACTCG-3′ | 97 | NM_004994 |
reverse | 5′-GGCAGGGACAGTTGCTTCT-3′ | |||
GAPDH | forward | 5′-AACAGCGACACCCACTCCTC-3′ | 258 | NM_001256799.1 |
reverse | 5′-GGAGGGGAGATTCAGTGTGGT-3′ |
Characteristics | No. | PAK4 | PD-L1 | |||
---|---|---|---|---|---|---|
Positive | p | Positive | p | |||
Age, years | <30 | 24 | 12 (50%) | 0.217 | 9 (38%) | 0.533 |
≥30 | 8 | 6 (75%) | 4 (50%) | |||
Sex | Male | 20 | 13 (65%) | 0.198 | 8 (40%) | 0.926 |
Female | 12 | 5 (42%) | 5 (42%) | |||
TNM stage | IIA | 15 | 8 (53%) | 0.755 | 5 (33%) | 0.430 |
IIB, III, IV | 17 | 10 (59%) | 8 (47%) | |||
T category | T1 | 16 | 8 (50%) | 0.476 | 5 (31%) | 0.280 |
T2, T3 | 16 | 10 (63%) | 8 (50%) | |||
N category | N0 | 30 | 17 (57%) | 0.854 | 12 (40%) | 0.780 |
N1 | 2 | 1 (50%) | 1 (50%) | |||
M category | M0 | 27 | 15 (56%) | 0.854 | 11 (41%) | 0.975 |
M1 | 5 | 3 (60%) | 2 (40%) | |||
PD-L1 | Negative | 19 | 7 (37%) | 0.007 | ||
Positive | 13 | 11 (85%) |
Characteristics | No. | OS | RFS | ||
---|---|---|---|---|---|
HR (95% CI) | p | HR (95% CI) | p | ||
Age, years, ≥30 (vs. <30) | 8/32 | 3.071 (1.105–8.532) | 0.031 | 3.139 (1.128–8.739) | 0.029 |
Sex, male (vs. female) | 20/32 | 0.874 (0.298–2.567) | 0.807 | 0.768 (0.263–2.246) | 0.630 |
TNM stage, ≥IIB (vs. IIA) | 17/32 | 2.865 (0.983–8.349) | 0.054 | 2.912 (0.997–8.507) | 0.051 |
T category, T2 and T3 (vs. T1) | 16/32 | 3.009 (1.035–8.750) | 0.043 | 3.142 (1.079–9.150) | 0.036 |
N category, N1 (vs. N0) | 2/32 | 3.830 (0.441–33.245) | 0.223 | 2.047 (0.255–16.465) | 0.501 |
M category, M1 (vs. M0) | 5/32 | 3.297 (0.904–12.025) | 0.071 | 2.553 (0.703–9.277) | 0.155 |
PAK4, positive (vs. negative) | 18/32 | 7.646 (1.726–33.875) | 0.007 | 7.981 (1.801–35.369) | 0.006 |
PD–L1, positive (vs. negative) | 13/32 | 5.195 (1.768–15.260) | 0.003 | 5.157 (1.768–15.039) | 0.003 |
Characteristics | OS | RFS | ||
---|---|---|---|---|
HR (95% CI) | p | HR (95% CI) | p | |
Age, years, ≥30 (vs. <30) | 3.502 (1.221–10.045) | 0.020 | ||
M category, M1 (vs. M0) | 8.491 (1.622–44.448) | 0.011 | ||
PAK4, positive (vs. negative) | 6.888 (1.237–38.367) | 0.028 | ||
PD-L1, positive (vs. negative) | 2.978 (0.902–9.835) | 0.073 | 5.512 (1.863–16.309) | 0.002 |
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Zhang, J.; Song, Y.; Ahn, A.-R.; Park, H.S.; Park, S.-H.; Moon, Y.J.; Kim, K.M.; Jang, K.Y. PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients. Cells 2024, 13, 1444. https://doi.org/10.3390/cells13171444
Zhang J, Song Y, Ahn A-R, Park HS, Park S-H, Moon YJ, Kim KM, Jang KY. PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients. Cells. 2024; 13(17):1444. https://doi.org/10.3390/cells13171444
Chicago/Turabian StyleZhang, Junyue, Yiping Song, Ae-Ri Ahn, Ho Sung Park, See-Hyoung Park, Young Jae Moon, Kyoung Min Kim, and Kyu Yun Jang. 2024. "PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients" Cells 13, no. 17: 1444. https://doi.org/10.3390/cells13171444