Resumen de Yes1 drives tumor growth and progression and predicts sensitivity to dasatinib in non-small cell lung cancer
The characterization of new genetic alterations is essential to assign effective personalized therapies and reduce mortality rates in non-small cell lung cancer (NSCLC). The aim of this project is the validation of YES1 amplification as a therapeutic target in lung cancer.
We first sought to analyze the prognostic role of YES1 protein expression in NSCLC patients. The analysis performed in two independent cohorts of NSCLC patients showed that high YES1 protein expression was an independent predictor of poor prognosis in patients with NSCLC.
Stable YES1 overexpression in lung cancer cell lines significantly induced proliferation in cells harboring YES1 amplification and high expression (HighYES1 cell lines: H1792, H2009, Calu-1 and H661). No effect on cell proliferation was observed in cells without YES1 amplification (LowYES1 cell lines: A549 and SW900). The effect of YES1 overexpression was next evaluated in vivo. H1792, H2009, A549 and SW900 cells overexpressing YES1 developed larger subcutaneous tumors than those transfected with the empty vector; Calu-1 showed an inverse correlation. Interestingly, both control and YES1 overexpressing A549 cells metastasized to the lungs with no significant differences; however, an induction of liver metastases in YES1-overexpressing cells was observed, which was not present in the liver of empty vector group. In the case of Calu-1 cells, metastatic growth in the lungs was induced in cells overexpressing YES1. Next, A549 cells overexpressing YES1 transduced with luciferase (A549-TM-YES1) were subcutaneously injected in immunodeficient mice, confirming our previous results by bioluminescence imaging. Moreover, intracardiac inoculation of A549-TM-YES1 cells induced an increase in total tumor burden as well as bone metastasis as compared with control cells.
YES1 downregulation by two specific siRNAs significantly reduced cell proliferation and survival in HighYES1 lung cancer cell lines, in contrast to the effect observed in LowYES1 cells. Accordingly, in LowYES1 cell lines, apoptosis was not affected by YES1 downregulation, in contrast with HighYES1 H2009, H1792 and H661 cells. In HighYES1 Calu-1 and HCC95 cell lines, YES1 specific knockdown did not induce apoptosis but led to increased autophagy levels. Furthermore, YES1 knockdown inhibited the invasive potential in HighYES Calu-1 and H1792 cell lines. All these results suggest that basal YES1 levels determine the anti-proliferative and anti-invasive effects of YES1 downregulation.
The downstream signaling events triggered by YES1 were also studied. Phosphorylation of AKT and S6K were downregulated in siRNA treated cells. Consequently, YES1 overexpression led to an increase in phospho-S6K. These data suggest that YES1 sustains mTOR pathway activity.
To evaluate the feasibility of YES1 as a therapeutic target, we tested the effect of dasatinib, a multitarget tyrosine kinase inhibitor. Dasatinib treatment inhibited proliferation in HighYES1 cell lines, whereas LowYES1 cells were more resistant to this treatment. Consistently, dasatinib significantly blocked 3D migration in HighYES1 Calu-1 and H1792 cells in a dose-dependent manner, whereas invasive capabilities of LowYES1 A549 cell line were not affected. We next evaluated the pharmacological effectiveness of dasatinib in vivo. In HighYES1 tumors, a significant decrease in subcutaneous tumor volume was observed in dasatinib-treated mice when compared with vehicle-treated controls. However, in the LowYES1 cell lines, differences in tumor growth were found only in one out of the two tested cell lines. Furthermore, we used NSCLC PDX to explore the efficacy of dasatinib. Thus, dasatinib significantly impaired tumor growth in HighYES1 PDX. Moreover, two out of three LowYES1 PDX did not respond to dasatinib treatment. These data suggest that YES1 status may be used as a stratification biomarker to select NSCLC patients who may respond and benefit from Src-family of kinases inhibitors.
