Supplemental Figure 2: Knockdown of PRAS40 is insufficient to alter downstream signaling and therefore sensitivity to gefitinib or desIGF1. OSC19 cells were transfected with siRNA specific to PRAS40 (siRNA #1 and siRNA #2) or a scramble siRNA followed by treatment with desIGF1, gefitinib or the combination of both. (A) Immunoblot representation and quantification of PRAS40, phospho-S6 and S6 48 hours after siRNA transfection. (B) OSC19 cell viability as measured by CyQUANT 72 h following transfection and drug treatment. (C) Relative expression of TRAIL (left panel) and PINK (right panel) 48 hours after transfection and 6 hours post drug treatment as quantified by qPCR. Data represents results from at least 3 biological replicates. Asterisks represent significant changes with p<0.05 as determined by Tukey test.
Funding
NIH
NIDCR
UVA Cancer Center
UVA Department of Otolaryngology- Head and Neck Surgery Pilot Project
Ruth L. Kirschstein T32 Institutional Research Award
ARTICLE ABSTRACT
EGFR inhibitors have shown poor efficacy in head and neck squamous cell carcinoma (HNSCC) with demonstrated involvement of the insulin-like growth factor-1 receptor (IGF1R) in resistance to EGFR inhibition. IGF1R activates the PI3K–Akt pathway, which phosphorylates proline-rich Akt substrate of 40 kDa (PRAS40) to cease mTOR inhibition resulting in increased mTOR signaling. Proliferation assays separated six HNSCC cell lines into two groups: sensitive to EGFR inhibition or resistant; all sensitive cell lines demonstrated reduced sensitivity to EGFR inhibition upon IGF1R activation. Reverse phase protein microarray analysis and immunoblot identified a correlation between increased PRAS40 phosphorylation and IGFR-mediated resistance to EGFR inhibition. In sensitive cell lines, PRAS40 phosphorylation decreased 44%–80% with EGFR inhibition and was restored to 98%–196% of control by IGF1R activation, while phosphorylation was unaffected in resistant cell lines. Possible involvement of mTOR in this resistance mechanism was demonstrated through a similar pattern of p70S6K phosphorylation. However, addition of temsirolimus, an mTORC1 inhibitor, was insufficient to overcome IGF1R-mediated resistance and suggested an alternative mechanism. Forkhead box O3a (FOXO3a), which has been reported to complex with PRAS40 in the cytoplasm, demonstrated a 6-fold increase in nuclear to cytoplasmic ratio upon EGFR inhibition that was eliminated with concurrent IGF1R activation. Transcription of FOXO3a-regulated TRAIL and PTEN-induced putative kinase-1 (PINK1) was increased with EGFR inhibition in sensitive cell lines; this effect was diminished with IGF1R stimulation.
These data suggest PRAS40 may play an important role in IGF1R-based therapeutic resistance to EGFR inhibition, and this likely occurs via inhibition of FOXO3a-mediated proapoptotic gene transcription.
