The monoclonal EGFR antibody cetuximab (C225/Erbitux™
) was FDA-approved for the treatment of primary HNC in combination with radiation in 2006, making it the first new drug for this cancer in over 45 years (2
). Despite widespread EGFR expression, cetuximab is only effective in a subset of HNC patients. We previously reported that GPCR ligands can transactivate EGFR via autocrine release of EGFR ligands (9
). In addition, GPCR ligands can activate the mitogenic MAPK pathway in the presence of EGFR inhibitors and combined targeting of individual GPCRs and EGFR resulted in enhanced survival in the presence of GPCR ligands in the presence of GPCR ligand inhibition (9
). In this study, we showed that GPCR ligand BK can enhance the phosphorylation of p70S6K in the presence of EGFR siRNA or cetuximab. In addition, we observed combined treatment with cetuximab and RAD001 improves anti-tumor response in both in vitro
and in vivo
GPCRs have been previously linked to the activation of p70S6K in different benign and malignant cell types. In bladder cancer, the GPCR ligand carbachol was shown to activate p70S6K in the presence of the EGFR TKI AG1478 (15
). In smooth muscle airway cells, thrombin and EGF were reported to synergistically activate p70S6K (16
). In addition, we identified that p70S6K phosphorylation was additively decreased by combined treatment with a GRPR inhibitor and EGFR TKI using RPPA analysis (10
). In the present study, we show for the first time that in the presence of EGFR downmodulating agents including cetuximab, GPCR ligands can induce p70S6K phosphorylation. We also observed that even in the absence of GPCR ligands, EGFR downmodulation results in increased p70S6K phosphorylation alone. Furthermore, the increased expression of phosphorylated p70S6K in post-cetuximab treated biopsies support p70S6K as plausible therapeutic target to overcome de novo and acquired resistance to EGFR inhibition.
While studies to date have not identified p70S6K activation as a feedback mechanism in response to EGFR downmodulation, several reports demonstrate that inhibition of EGFR results in activation of the insulin growth factor receptor pathway, which signals via the PI3K/Akt/p70S6K pathway (17
). IGF1R downmodulation has been shown to augment EGFR signaling. Furthermore, our antibody array showed that phospho-IRS (Ser312) was increased 2-fold in EGFR siRNA transfected cells () and it has been reported that mTOR/p70S6K regulates the phosphorylation at this site in the presence of insulin (19
). Therefore, it is possible that EGFR downmodulation in HNC leads to increased p70S6K phosphorylation, at least in part, via increased insulin signaling.
The effects of mTOR inhibitors have been shown to be promising in preclinical HNC models (20
). However, rapalogs are allosteric inhibitors and their activity results in a feedback loop that induces Akt phosphorylation (22
). However, dual PI3K/mTOR inhibitors(23
) may be more potent in overcoming the limited clinical efficacy of cetuximab. Although the combination of C225 and RAD001 did not result in tumor regression, this may be due to use of the subtherapeutic concentrations of cetuximab used. Therefore, it is possible that a combination of therapeutic doses of cetuximab and RAD001 will be more potent and result in tumor regression.
It is also important to note that RAD001 lacks specificity and also inhibits phoshporylation of 4EBP1 so it is not possible to distinguish precisely which target is most important using this rapalog. The results of the present study provide a biological basis for the ongoing clinical trial combining cetuximab and RAD001 in HNC patients (http://clinicaltrials.gov/ct2/show/NCT01009346
). Since GPCR ligands including BK and PGE2 in HNC are ubiquitous in the tumor microenvironment, p70S6K represents a promising therapeutic target, particularly in combination with EGFR blockade.