The Antitumor Effect And Mechanism Of CK2Inhibitor CX-4945Alone And Combination With MEK Inhibitor PD-325901in Head And Neck Squamous Carcinoma Cells

Objective: We previously identified upregulated CK2as an upstream kinaseactivating NF-кB and modulating TP53mediated signaling pathwaysin promoting themalignant phenotype of head and neck squamous cell carcinoma (HNSCC). Here, weinvestigate the antitumor effects of CX-4945, a small molecule inhibitor of CK2,alone and in combination with MEK inhibitor PD-0325901(PD-901), in humanHNSCC models in vitro and in vivo.Design: The effects of CX-4945on cell proliferation were measured by MTTassay in vitro in a panel of9UM-SCC cell lines to determine half maximal inhibitoryconcentration (IC50). CX-4945inhibition of cell cycle and induction of cell deathwere measured by DNA flow cytometry. The effects of CX-4945on the activity ofNF-кB, BCL-XL, AP-1and TP53promoters were tested by the chemiluminescentreporter gene assay. The anti-tumor activity of orally administered CX-4945wasevaluated in SCID mice bearing UM-SCC1xenografts. The molecular changes afterCX-4945treatment were investigated using immunostainning and Western blot.Results: The IC50in9UM-SCC cell lines ranged from3.4μM to11.9μM.UM-SCC1and46were selected for further study, as UM-SCC1representing asubset with transcriptional reduced wtTP53, and UM-SCC46representing the subsetexpressing a mtTP53. CX-4945showed a concentration and time dependent cell cyclearrest in S and G2/M phases accompanied by a significant induction of sub-G0cells indicating cell death. CX-4945inhibited NF-кB and BcL-XL prosurvival reportergenes in both wtTP53(UMSCC1) and mtTP53(UMSCC46) cell lines, andconcurrently upregulated proapoptotic TP53, p21and prosurvival AP-1activity onlyin the wtTP53cell line. Correspondingly, in both cell lines, the phosphorylation ofAKT at S129, T308and S473were significantly reduced, while Erk1/2at Thr202andTyr204were increased only in UM-SCC1cells. In UM-SCC1xenograft model,CX-4945treatment significantly decreased PI3K/Akt/mTOR pathway activity asdetected by immunostaining of tissue specimens in both early (Day13) and late timepoints (Day33) after treatment. Conversely, TP53and Bax were significantlyincreased at early time points but not at late time points. The apoptosis (TUNEL)marker staining was significantly increased in tumor specimens, whileimmunostaining of p-Erk, c-Jun, FosL-1and proliferation (Ki67) were also increased.Combination of MEK inhibitor PD-0325901with CX-4945exhibited significantlysynergistic effects in vitro proliferation assay. No significant tumor volume reductionand improvement of survival was observed in CX4945alone. However, significantanti-tumor effects were observed in PD-901alone and in combination with CX-4945treated HNSCC xenograft mice.Conclusions: Inhibition of CK2via CX-4945revealed anti-proliferative andmodulation of PI3K/AKT, NF-кB, TP53and AP1activities in HNSCC cell lines invitro and in vivo. Activation of MEK-ERK-AP1pathway in cells with wtTP53leadsto drug resistance, and combinatory therapy could improve anti-tumor effectsobserved with CK2inhibition alone.