Dual targeting of PI3K and MEK enhances the radiation response of K-RAS mutated non-small cell lung cancer.

Mahmoud Toulany,Birgit Fehrenbacher,Mari Iida,H Peter Rodemann,Deric L Wheeler,Wael Y Mansour,Simone Keinath,Martin Schaller,Katharina Mueck,Firdevs F Iyi

doi:10.18632/oncotarget.9670

Abstract

Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460.Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.

Highlights

The RAS isoforms (K-RAS, H-RAS and N-RAS) have point mutations in nearly 30% of all human tumors and are small molecular weight GTPases that couple extracellular signals to intracellular effector pathways [1]
We demonstrated that long-term (24 h) treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor PI-103 in tumor cells with constitutive K-Ras activity led to Akt reactivation [16]
The effect of long-term inhibition of PI3K by PI-103 (24 h treatment) on the phosphorylation of Akt (S473 and T308) and its substrate PRAS40 at T246 was compared in K-RASmut NSCLC cell lines A549 and H460 with those in K-RAS wild-type (K-RASwt) H661 cells

Summary

Introduction

The RAS isoforms (K-RAS, H-RAS and N-RAS) have point mutations in nearly 30% of all human tumors and are small molecular weight GTPases that couple extracellular signals to intracellular effector pathways [1]. It is known that K-RAS mutation leads to constitutive K-Ras activity and is associated with stimulated autocrine production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin [2]. The phosphatidylinositol 3-kinase (PI3K)/Akt and MEK/ERK pathways are the major effectors of oncogenic RAS involved in tumor cell www.impactjournals.com/oncotarget clonogenic activity. Constitutive activation of these pathways leads to resistance to EGFR molecular targeting strategies such as the anti-EGFR antibody cetuximab or the EGFR-tyrosine kinase (EGFR-TK) inhibitors gefitinib and erlotinib [3]. We demonstrated that, like the production of the EGFR ligand amphiregulin in K-RAS mutated (K-RASmut) tumor cells, [2, 9] stimulated amphiregulin secretion is obvious in head and neck squamous (HNSCC) tumor cells overexpressing K-RAS wild-type (K-RASwt). K-Ras hyperactivity induces Akt activation either through an EGFR/PI3K-dependent pathway [2, 10] or through H-RAS-dependent direct activation of the PI3K pathway [11, 12]

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Conclusion