Amplification of RICTOR has been suggested to drive PI3K/mTOR pathway activation in squamous cell lung cancer (SQLC), representing a potential predictive biomarker of response to targeted therapy. Here, we sought to perform a thorough validation of RICTOR as predictive biomarker in SQLC preclinical models before advancing potential intervention strategies to the clinic.
Three SQLC cell lines (H-1869, H-1703, SK-Mes-1) and tissue samples (97 SQLC patients) were subjected to targeted DNA sequencing analysis, FISH, qRT-PCR, and immunoblotting. The activity of PI3K/mTOR pathway inhibitors was examined by cell viability assays, while RNA interference (RNAi) using inducible systems was applied to evaluate the impact of RICTOR knock-down on cell growth and response to drugs.
RICTOR copy-number gain (CNG) was displayed in all cell lines (H-1869 harbored the highest CNG) and 28% of SQLC patients, as polysomy of the short arm of chromosome 5 as FISH (locus-specific, 5p-centromeric, and 5q-telomeric probes) revealed. Increasing levels of Rictor transcript/protein expression were detected in cell lines (H1869>SK-MES-1>H-1703). Therefore, we assessed causative link between RICTOR gene dosage and PI3K/mTOR pathway through genetic RNAi and pharmacological perturbation. We found that Rictor levels were not associated with increased activity of PI3K/mTOR axis or increased sensitivity to several pathway inhibitors (PF-05212384, AZD2014, MK-2206). Coherently, RICTOR silencing with inducible shRNA systems did not significantly affect cell growth and drug sensitivity of the three cell lines tested.
Here, we show that polysomy of 5p rather than amplification of RICTOR is evident in subset of SQLC patients and derived cell lines. While CNG of RICTOR corresponded to increased protein expression, no correlation was observed with PI3K/mTOR pathway activity and drug sensitivity in vitro. We complemented this with genetic perturbation analyses, whose results aligned with pharmacological findings and suggest that Rictor does not represent a predictive biomarker of response towards PI3K/mTOR directed therapy.
The authors.
Progetto AIRC (Fondazione AIRC per la Ricerca sul Cancro) IG 20583.
All authors have declared no conflicts of interest.