BMAL1 Suppresses Proliferation, Migration, and Invasion of U87MG Cells by Downregulating Cyclin B1, Phospho-AKT, and Metalloproteinase-9

Abstract

Several studies have shown that brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), an important molecule for maintaining circadian rhythms, inhibits the growth and metastasis of tumor cells in several types of cancer, including lung, colon, and breast cancer. However, its role in glioblastoma has not yet been established. Here, we addressed the function of BMAL1 in U87MG glioblastoma cells with two approaches—loss and gain of function. In the loss of function experiments, cell proliferation in U87MG cells transfected with small interfering RNA (siRNA) targeting BMAL1 was increased by approximately 24% (small interfering (si)-NC 0.91 ± 0.00 vs. si-BMAL1 1.129 ± 0.08) via upregulation of cyclin B1. In addition, cell migration and invasion of BMAL1 siRNA-treated glioblastoma cells were elevated by approximately 20% (si-NC 51.00 ± 1.53 vs. si-BMAL161.33 ± 0.88) and 209% (si-NC 21.28 ± 1.37 vs. si-BMAL1 44.47 ± 3.48), respectively, through the accumulation of phosphorylated-AKT (p-AKT) and matrix metalloproteinase (MMP)-9. Gain of function experiments revealed that adenovirus-mediated ectopic expression of BMAL1 in U87MG cells resulted in a 19% (Adenovirus (Ad)-vector 0.94± 0.03 vs. Ad-BMAL1 0.76 ± 0.03) decrease in cell proliferation compared with the control via downregulation of cyclin B1 and increased early and late apoptosis due to changes in the levels of BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and cleaved caspase-3. Likewise, cell migration and invasion were attenuated by approximately 24% (Ad-vector 55.00 ± 0.00 vs. Ad-BMAL1 41.83 ± 2.90) and 49% (Ad-vector 70.01 ± 1.24 vs. Ad-BMAL1 35.55 ± 1.78), respectively, in BMAL1-overexpressing U87MG cells following downregulation of p-AKT and MMP-9. Taken together, our results suggest that BMAL1 acts as an anti-cancer gene by altering the proliferation, migration, and invasion of glioblastoma cells. Therefore, the BMAL1 gene could be a potential therapeutic target in the treatment of glioblastoma.

Adjusting the Clock: the Importance of Circadian Rhythms in the Development of Glioblastomas and Its Intervention as a Therapeutic Strategy

“…Also, the expression of pro-apoptotic proteins was increased while the anti-apoptotic protein BCL-2 level decreased, suggesting that BMAL1 may operate as a tumor-suppressor in U-87MG cell cultures. Glioma migration and invasion were also reduced after ectopic expression of BMAL1, leading to downregulation of p-AKT and MMP-9 signaling pathways [215]. Similar to the observations described above, results obtained recently by Wagner and colleagues (2021) show that the downregulation of Bmal1 expression is associated with a more aggressive form of the tumor.…”

Proteomic Analysis Reveals That Placenta-Specific Protein 9 Inhibits Proliferation and Stimulates Motility of Human Bronchial Epithelial Cells

“…High CDK2 activity can phosphorylate breast cancer metastasis suppressor 1 (BRMS1) and suppress cell migration (55). Cell migration of BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1)-knockdown glioblastoma cells was elevated, accompanied with upregulation of cyclin B1 (56). Taken together, PLAC9 overexpression-induced cell motility probably relies on the combination of altered expression of CDK2, cyclin B1, c-Myc, and p21 Waf/cip1 , despite the unexpected changes in E-cadherin and vimentin levels.…”

Proteomic analysis reveals that Placenta-specific 9 induces cell proliferation and motility programs in human bronchial epithelial cells

“…High CDK2 activity could phosphorylates breast cancer metastasis suppressor 1 (BRMS1) then suppress cell migration [47]. Cell migration of BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1)-knockdown glioblastoma cells was elevated, accompanied with upregulation of cyclin B1 [48]. Taken together, Plac9 overexpression-induced cell motility probably relies on the coordination of altering expressions of CDK2, cyclin B1, c-Myc, p21 Waf/cip1 , despite the unexpected change E-cadherin and vimentin levels.…”