Role of mixed lineage kinase 3 in response of head and neck squamous cancer cell lines to epidermal growth factor receptor (EGFR) inhibition

6076 Background: More efficacious treatments of head and neck squamous cancers (HNSCC) are urgently needed. HNSCC overexpress EGFR, yet the efficacy of EGFR inhibitors is marginal, with minimal effect on patient survival. Mixed lineage kinases (MLKs) are members of a large family of mitogen-activated protein kinase (MAPK) kinase kinases (MAPKKKs) that activate MAPK pathways, involving JNK, ERK 1/2 and p38 cell signaling pathways. (Gallo KA, Johnson GL, Nat Rev Mol Cell Biol. 2002;3:663–72) We explored the role of MLK3 in HNSCC as a possible therapeutic target. Methods: The requirement of MLK3 for cell proliferation and survival of HNSCC cell lines UM-SCC-38 and -47 (HPV+) (obtained from TE Carey, U Michigan) was investigated using RNA interference (siRNA) and MLK3 inhibitor K252a to inhibit the expression and activity of MLK3. Cells were also exposed to EGFR inhibitor Compound 56 at various concentrations. Proliferation was assessed with MTT after 72 h exposure. The effect of MLK3 inhibition on relevant cell signaling pathways was assessed by immunoblotting with activation-specific phosphoantibodies directed against MAPKs, the prosurvival kinase Akt and apoptotic proteins. Results: MLK3 is overexpressed in malignant HNSCC cell lines compared to normal tonsil lysate, and its expression can be inhibited by siRNA or K 252a. Inhibition of MLK3 expression is associated with downregulation of phospho-Akt and with decreased proliferation. Compound 56 inhibits proliferation at lower concentrations in HPV+ cell line UM-SCC-47 (10 microM) compared with UM-SCC-38 (25 microM), inhibits expression of pErk, and decreases expression of pAkt. Combining siRNA or K252a and compound 56 showed less pAkt expression and increased apoptosis (caspase 3 or PARP cleavage) compared with compound 56 alone. Conclusions: MLK3 may have a role in avoiding apoptosis in HNSCC cells. Its inhibition may enhance the effects of EGFR inhibition in HNSCC. No significant financial relationships to disclose.