Aurora kinase A revives dormant laryngeal squamous cell carcinoma cells via FAK/PI3K/Akt pathway activation

Abstract

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment.

Keywords: Akt; FAK; PI3K; aurora kinase A; laryngeal cancer.

Figure 1. Culturing T-Hep2 cells with 0.1% FBS for 48 h induced dormancy and reduced AURKA expression

A. T-Hep2 cell proliferation as measured by CCK8 assay at 0, 24, 48, 72, 96 and 120 h, T-Hep2 cells with 0.1% FBS showed stagnant growth at 48 h (**P<0.01). B. T-Hep2 cell population (%) after starvation (*P<0.05). C. T-Hep2 cell cycle as analyzed by flow cytometry T-Hep2 cells in 0.1% FBS were nearly stagnant in G0/G1 phase. D. Dormancy-related proteins were analyzed by western blotting P130 and E2F4 levels were high and P107 and Ki67 levels were low in serum-starved T-Hep2 cells. E. Co-IP was used to examine the E2F4-P130 complex F. p-AURKA and total AURKA in D-Hep2 cells and T-Hep2 cells as analyzed by western blotting, p-AURKA expression was low in D-Hep2 cells. G. Protein ratio in D-Hep2 and T-Hep2 cells (**P<0.01).

Figure 2. AURKA downregulation induced dormancy in T-Hep2 cells

A. Effects of VX680 on T-Hep2 cell proliferation as measured by CCK8 assay at 0, 24 and 48 h Treated T-Hep2 cells exhibited reduced proliferation (**P<0.01). B. T-Hep2 cell population (%) after VX680 treatment (*P<0.05). C. Effects of VX680 on T-Hep2 cell cycle were analyzed by flow cytometry, T-Hep2 cells were arrested in G0/G1 phase 48 h after VX680 treatment. D. AURKA IF staining in T-Hep2 cells with or without AURKA inhibitor. AURKA and P107 levels were reduced, while P130 expression was higher in treated cells. E. Effects of VX680 on dormancy-related protein levels as analyzed by western blotting P130 and E2F4 levels were increased while p-AURKA, P107 and Ki67 were decreased in treated cells at 48 h. F. Protein ratio of p-AURKA in T-Hep2 cells (*P<0.05, **P<0.01). G. Co-IP was used to examine the E2F4-P130 complex in treated cells at 48 h.

Figure 3. Migration, invasion and dormancy-related protein expression in D-Hep2 cells overexpressing AURKA

A. Dormancy-related proteins were analyzed by western blotting, p-AURKA, AURKA, P107 and Ki67 levels were increased and P130 and E2F4 were reduced. B. Protein ratio in D-Hep2 cells. C. D-Hep2 cell proliferation was analyzed via colony formation assay, D-Hep2/AURKA cells enhanced cellular proliferation compared with D-Hep2/vector cells and D-Hep2/parental cells (C/a) plated cell number was 1×10³, *P<0.05; (C/b) plated cell number was 2×10³, *P<0.01). D. Relative D-Hep2 cell colony numbers. E. D-Hep2 cell mobility was analyzed via wound-healing assay. F. D-Hep2 cells/AURKA almost reached the middle of the scratch at 48 h (*P<0.05, **P<0.01). G. Cell migration and invasion were analyzed by transwell assays. H. AURKA promoted cellular migration and invasion (*P<0.05, **P<0.01).

Figure 4. AURKA promotes D-Hep2 cell tumor metastasis in nude mice

A. T-Hep2 cells and D-Hep2 cells were inoculated into nude mice and pulmonary nodules were observed after 45 days (N=5/group). H&E stains of pulmonary nodules (100×). B. Pulmonary tissue and nodules were quantified by H&E staining from T-Hep2, D-Hep2 (**P<0.01). C. D-Hep2/parental cells, D-Hep2/vector cells and D-Hep2/AURKA cells were inoculated into nude mice and pulmonary nodules were observed after 45 days (N=5/group). H&E stains of pulmonary nodules (100×). D. Pulmonary tissue and nodules were quantified by H&E staining from D-Hep2/parental cells, D-Hep2/vector cells and D-Hep2/AURKA cells (**P<0.01).

Figure 5. Effects of AURKA on FAK, PI3K, Akt activity

A. Expression of FAK/PI3K/Akt pathway regulatory factors in T-Hep2 and D-Hep2 cells as analyzed by western blotting. These factors were overexpressed in T-Hep2 cells compared with D-Hep2 cells. B. Protein ratio in T-Hep2 and D-Hep2 cells (*P<0.05, **P<0.01). C. Effects of VX680 in T-Hep2 cells at 0, 24 and 48 h on the FAK/PI3K/Akt pathway as analyzed by western blotting. p-FAK (Tyr397), p-PI3K and p-Akt levels in treated T-Hep2 cells were lower than in untreated cells. D. Protein ratio in T-Hep2 cells (**P<0.01). E. Effects of AURKA upregulation in D-Hep2 cells on the FAK/PI3K/Akt pathway as analyzed by western blotting, p-FAK(Tyr397), p-PI3K and p-Akt levels were higher in transfected D-Hep2 cells compared to untransfected cells. F. Protein ratio in D-Hep2 cells (**P<0.01).

Figure 6. Effects of FAK/PI3K/Akt inhibition on dormancy-related protein expression

Effects of FAK A., PI3K B. or Akt C. inhibition in D-Hep2/AURKA cells on dormancy-related protein expression as analyzed by western blotting. P130 and E2F4 levels were increased, while P107 and Ki67 were decreased. Dormancy-related protein ratio in D-Hep2/AURKA cells treated with TAE226 D., Omipalisib E. or Triciribine F. Effects of FAK G. inhibition in D-Hep2/AURKA cells on the FAK/PI3K/Akt pathway as analyzed by western blotting, p-FAK (Tyr397), p-PI3K and p-Akt levels were decreased and p-FAK (Tyr861, Tyr925), FAK, p-AURKA, AURKA, PI3K and Akt were not changed. Effects of PI3K inhibition H. in D-Hep2/AURKA cells on the FAK/PI3K/Akt pathway as analyzed by western blotting, p-PI3K and p-Akt levels were decreased and p-FAK (Tyr397, Tyr861, Tyr925), FAK, p-AURKA, AURKA, PI3K and Akt were not changed. Effects of Akt inhibition I. in D-Hep2/AURKA cells on the FAK/PI3K/Akt pathway as analyzed by western blotting, p-Akt levels were decreased, but p-AURKA, AURKA, p-FAK (Tyr397, Tyr861, Tyr925), FAK, p-PI3K, PI3K and Akt were not changed. Protein ratio in D-Hep2/AURKA cells treated with TAE226 J., Omipalisib K. or Triciribine L. (*P<0.05, **P<0.01).

Figure 7. FAK/PI3K/Akt inhibition reduces D-Hep2/AURKA cell mobility, migration and invasion

Effects of FAK A., PI3K B. or Akt C. inhibition in D-Hep2/AURKA cells on cell mobility as assessed via wound healing assay. FAK, PI3K and Akt all promoted D-Hep2/AURKA cell mobility. D-Hep2/AURKA cell wound size with FAK D., PI3K E. or Akt F. Effects of FAK G., PI3K H. or Akt I. inhibition in D-Hep2/AURKA cells on cell migration and invasion as measured via transwell assay. FAK, PI3K and Akt each stimulated cell migration and invasion. Cell number in every field following FAK J., PI3K K. or Akt L. inhibition. (*P<0.05, **P<0.01).