Identification of aurora kinase A as an unfavorable prognostic factor and potential treatment target for metastatic gastrointestinal stromal tumors

Abstract

Although imatinib mesylate (IM) has revolutionized the management of gastrointestinal stromal tumors (GISTs), drug resistance remains a challenge. Previous studies have shown that the expression of aurora kinase A (AURKA) predicts recurrence in patients with primary, surgically resected GISTs. The current study aimed to evaluate the significance of AURKA expression as an unfavorable prognostic marker for advanced GISTs, and provide evidence that AURKA could be a potential therapeutic target in GISTs. The prognostic significance of the expression of AURKA, along with other clinicopathological factors, was analyzed in a cohort of 99 IM-treated patients with advanced GISTs. The potential use of an inhibitor of AURKA as a therapeutic agent against GISTs was also tested in GIST cell lines. Among 99 enrolled patients, poor performance status, large tumor size, drug response, and AURKA overexpression were independent prognostic factors for poor progression-free survival (PFS). For overall survival (OS), only large tumor size and AURKA overexpression were identified as independent unfavorable factors. In an in vitro study, MLN8237, an AURKA inhibitor, inhibited growth of both IM-sensitive and IM-resistant GIST cells in a concentration-dependent manner, and exhibited synergistic cytotoxicity with IM in GIST cells. The inhibitory effect of MLN8237 in GIST cells could be attributed to the induction of G2/M arrest, apoptosis, and senescence. Our study shows that AURKA expression independently predicted poor PFS and OS in patients with advanced GISTs who were treated with IM. An AURKA inhibitor may have potential as a therapeutic agent for both IM-sensitive and IM-resistant GISTs.

Figure 1. Expression of AURKA in gastrointestinal stromal tumors (GISTs) and the association between AURKA expression and survival

(A) Representative photomicrographs of samples with low (0%–29% and 30%–59% staining was scored as 0 or 1, respectively) or high (60%–80% and 81%–100% staining was scored as 2 or 3, respectively) expression of AURKA (400x, scale bar = 50 μm). (B-C) Kaplan-Meier plots of (B) progression-free survival and (C) overall survival of 99 patients with GIST according to AURKA expression. The P values for survival comparison, obtained by the log-rank test, were all less than 0.05.

Figure 2. MLN8237 inhibits AURKA and induces mitotic arrest in gastrointestinal stromal tumor (GIST) cell lines

(A) Immunoblotting with an anti-phospho-Aurora A (Thr288) antibody revealed inhibition of AURKA autophosphorylation in GIST cell lines synchronized by exposure to nocodazole and then treated with MLN8237. (B) Immunoblotting of three AURKA-expressing GIST cell lines with an anti-phospho-HistoneH3 (Ser10) (pHisH3) antibody revealed a dose-dependent induction of pHisH3 after treatment with MLN8237 for six days.

Figure 3. MLN8237 induces G2/M arrest in gastrointestinal stromal tumor (GIST) cell lines

The DNA profiles of GIST882, GIST48, and GIST430 cells treated with dimethyl sulphoxide (DMSO) or MLN8237 for six days were evaluated by flow cytometry. The percentages of cells in the sub-G1, G0/G1, S, and G2/M phases, as well as the percentage of cells with a DNA content >4N are shown in different colors.

Figure 4

Molecular changes induced by MLN8237 in the p53 pathway were determined by western blotting using gastrointestinal stromal tumor (GIST) cell lines. MLN8237 induced a dose-dependent increase in the expression of p21 in all three cell lines. Dose-dependent up-regulation of p53 could also be seen in GIST48 and GIST430 cells. GIST882 is a p53-negative cell line.

Figure 5

Treatment with MLN8237 inhibits AURKA and results in growth inhibition and apoptosis in gastrointestinal stromal tumor (GIST) cell lines. The viability of GIST882, GIST48, and GIST430 cells after treatment with various concentration of MLN8237 (MLN) for six days was measured with (A) the TACS™ MTT cell proliferation assay (expressed as a percentage of viability under controlled culture conditions) and (B) a trypan blue exclusion assay (expressed as viable cell number). (C) Apoptosis assay. Three GIST cell lines were treated with various concentration of MLN8237 (MLN) for six days. The percentage of apoptotic cells was determined using Annexin V-FITC/propidium iodide (PI) staining. All data represent the mean ± SD of three independent experiments. *P < 0.05.

Figure 6

The viability of GIST882, GIST48, and GIST430 cells treated with MLN8237 (MLN) in combination with imatinib (IM) for six days was measured with the TACS™ MTT cell proliferation assay (expressed as a percentage of viability under controlled culture conditions). All data represent the mean ± SD of three independent experiments. *A combination index (CI) < 1.0 indicates a synergistic effect.

Figure 7. MLN8237 treatment induces cellular senescence in gastrointestinal stromal tumor (GIST) cell lines

(A) Representative photomicrographs of GIST48 cells before and after MLN8237 treatment. Upper panel: images of cells after staining for senescence-associated β-galactosidase (SA-β-gal) activity; lower panel: corresponding bright field images. Scale bar = 50 μm. Some SA-β-gal positive cells are indicated by arrows. (B) Treating GIST48 cells with MLN8237 for six days dose-dependently increased SA-β-gal staining. (C) Western blot analysis of phospho-p70 S6 kinase (p-p70S6K) and DEC1 expression relative to that of actin in GIST48 cells treated with MLN8237. (D) Flow cytometric analysis of the expression of DcR2 in GIST48 cells treated with MLN8237 for six days. (E) Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of interleukin-6 (IL-6) expression relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in GIST48 cells treated with MLN8237. ^*P < 0.05.