Supplementary Table 1, Supplementary Figures 1-7, Supplementary Materials and Methods. Supplemental Table 1. Tumor, treatment, and outcome characteristics of human HNSCC lines Supplementary Figure 1. Boxplots of IC50s for 11 HPV- cell lines (red) and 8 HPV+ lines (cyan); Supplementary Figure 2. PF-384 effects on PI3K/mTOR, and MAPK/ERK pathways in UMSCC-46; Supplementary Figure 3 PF-384 effects on IKK-dependent phosphorylation of NF-κB p65 protein in UM-SCC-1 and 46; Supplementary Figure 4. Effects of PIK3CA knockdown on PI3K-AKT pathway and NF-κB signaling in UMSCC-1 and 46; Supplementary Figure 5. Effects of PF-384 and PD-901 treatment on weights of mice bearing UMSCC-1 xenografts; Supplementary Figure 6. Immunohistochemical staining of UMSCC-1 xenografts; Supplementary Figure 7. Schematic of PI3K/Akt/mTOR and MEK/ERK pathways and targets of PF-384 and PD-901
ARTICLE ABSTRACT
Purpose: Head and neck squamous cell carcinomas exhibit variable sensitivity to inhibitors of the PI3K/mTOR pathway, an important target of genomic alterations in this cancer type. The mitogen-activated protein kinase kinase (MEK)/ERK/activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) pathways are also frequently co-activated, but their roles in resistance mechanisms to PI3K/mTOR inhibitors and as therapeutic targets in head and neck squamous cell carcinoma (HNSCC) are not well defined.Experimental Design: We determined the IC50s of dual PI3K/mTOR inhibitor PF-05212384 (PF-384) by XTT assays in 14 HNSCC lines with PI3K/Akt/mTOR cascade alterations. In two resistant models, we further characterized the molecular, cellular, and in vivo attributes and effects of combining PF-384 with MEK inhibitor PD-0325901 (PD-901).Results: PF-384 IC50s varied between 0.75 and 133 nmol/L in 14 HNSCC lines with overexpression or mutations of PIK3CA, and sensitivity correlated with increased phospho-AKT(T308/S473). In resistant UMSCC-1 and -46 models, PF-384 increased G0-/G1-phase accumulation but weakly induced sub-G0 cell death. PF-384 inhibited direct targets of PI3K/mTOR, but incompletely attenuated co-activated ERK and UMSCC-1 xenograft growth in vivo. PD-901 strongly inhibited MEK/ERK targets, and the combination of PF-384 and PD-901 inhibited downstream NF-κB and AP-1 transactivation, and IL8 and VEGF production in vitro. PD-901 potently inhibited tumor growth alone and with PF384, enhanced antiproliferative, apoptotic, and anti-angiogenesis activity in vivo.Conclusions: PI3K/mTOR inhibitor PF-384 exhibits variable activity in a panel of HNSCC cell lines with differing PIK3CA expression and mutation status. MEK inhibitor PD-901 overcomes resistance and enhances antitumor effects observed with PF-384 in vivo. Clin Cancer Res; 21(17); 3946–56. ©2015 AACR.
