Aurora kinase targeting in lung cancer reduces KRAS-induced transformation

Abstract

BackgroundActivating mutations in KRAS are prevalent in lung cancer and have been causally linked to the oncogenic process. However, therapies targeted to oncogenic RAS have been ineffective to date and identification of KRAS targets that impinge on the oncogenic phenotype is warranted. Based on published studies showing that mitotic kinases Aurora A (AURKA) and B (AURKB) cooperate with oncogenic RAS to promote malignant transformation and that AURKA phosphorylates RAS effector pathway components, the aim of this study was to investigate whether AURKA and AURKB are KRAS targets in lung cancer and whether targeting these kinases might be therapeutically beneficial.MethodsIn order to determine whether oncogenic KRAS induces Aurora kinase expression, we used qPCR and western blotting in three different lung cell-based models of gain- or loss-of-function of KRAS. In order to determine the functional role of these kinases in KRAS-induced transformation, we generated KRAS-positive A549 and H358 cells with stable and inducible shRNA-mediated knockdown of AURKA or AURKB and evaluated transformation in vitro and tumor growth in vivo. In order to validate AURKA and/or AURKB as therapeutically relevant KRAS targets in lung cancer, we treated A549 and H358 cells, as well as two different lung cell based models of gain-of-function of KRAS with a dual Aurora kinase inhibitor and performed functional in vitro assays.ResultsWe determined that KRAS positively regulates AURKA and AURKB expression. Furthermore, in KRAS-positive H358 and A549 cell lines, inducible knockdown of AURKA or AURKB, as well as treatment with a dual AURKA/AURKB inhibitor, decreased growth, viability, proliferation, transformation, and induced apoptosis in vitro. In addition, inducible shRNA-mediated knockdown of AURKA in A549 cells decreased tumor growth in vivo. More importantly, dual pharmacological inhibiton of AURKA and AURKB reduced growth, viability, transformation, and induced apoptosis in vitro in an oncogenic KRAS-dependent manner, indicating that Aurora kinase inhibition therapy can specifically target KRAS-transformed cells.ConclusionsOur results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0494-6) contains supplementary material, which is available to authorized users.

Cisplatin‐resistant cancer cells are sensitive to Aurora kinase A inhibition by alisertib

“…For the cap-dependent dual luciferase reporter assays, cells were transfected with 1 lg of a dual-renilla-fireflyluciferase pcDNA3-rLuc-PolioIRES-fLuc reporter (a kind gift from John Blenis, Harvard Medical School), to measure cap-dependent/cap-independent translation (Dos Santos et al, 2016). Cells were plated in six-well plates, transfected using Lipofectamine 2000 reagent (Life Technologies), and treated with MLN8237 for 24 h. The lysates were prepared in triplicate, and the dual luciferase reporter assay (Promega) was used following the manufacturer's protocol.…”

Sirt1 protects from K‐Ras‐driven lung carcinogenesis

“…We used the b-galactosidase reporter for the pulse experiment, and the Katushka reporter for the pulse + chase experiment. We also found in Sirt1-Tg mice several downregulated protein kinases with pro-tumorigenic roles: Aurora kinase B (Aurkb); protein kinase C-iota (Prkci), and MAP-kinase activating death domain (Madd) [55][56][57]. During the pulse phase, we observed that genes involved in antioxidant defenses were upregulated in the Sirt1-Tg mice, such as several members of the glutathione S-transferase family, whose deletion has been found associated with poor prognosis in lung tumors [50], or Sod3, which has been shown to be downregulated in lung tumors [51] (Fig 4D), suggesting that Sirt1 overexpression is associated with stronger oxidative defense.…”

“…The differentially expressed genes were grouped according to their function, and we focused our attention on the significantly altered genes between Sirt1-Tg and Sirt1-WT pneumocytes that were reported to play a role during tumorigenesis (red-marked bars in Fig EV4D and E). Interestingly, Aurkb has been shown to act as a target of K-Ras signaling, and its disruption reverted K-Ras-mediated tumorigenesis in lung cells [57]. Genes involved in carbohydrate and amino acid metabolism were also differentially regulated in Sirt1-Tg pneumocytes: increased glycogen phosphorylase (Pygm), that has been found decreased in lung tumors [52]; and decreased expression of the tumor inducers glutamine-fructose-6 phosphatase transaminase 1 (Gfpt1) [53] and hexokinase 2 (Hk2) [54].…”

Aurora‐A Kinase: A Potent Oncogene and Target for Cancer Therapy

“…For example, breakpoint cluster region‐Abelson kinase (BCR‐ABL) promotes chronic myeloid leukemia (CML) cells proliferation via activating Aurora‐A and Aurora‐B kinases . Aurora‐A and Aurora‐B are also important targets of KRAS in lung cancer when targeting Aurora kinase reduces KRAS‐induced transformation . Thus, uncontrolled cell‐cycle progression is involved in Aurora‐A‐promoted tumorigenesis.…”

“…42 Aurora-A and Aurora-B are also important targets of KRAS in lung cancer when targeting Aurora kinase reduces KRAS-induced transformation. 43 Thus, uncontrolled cell-cycle progression is involved in Aurora-A-promoted tumorigenesis.…”